Research ArticleBeneficial Effects of Fractions of Nardostachys jatamansi onLipopolysaccharide-Induced Inflammatory Response

Gi-Sang Bae1 Kwang-Ho Heo2 Sun Bok Choi34 Il-Joo Jo34 Dong-Goo Kim34

Joon-Yeon Shin34 Seung-Hee Seo5 Kyoung-Chel Park3 Dong-Sung Lee1 Hyuncheol Oh16

Youn-Chul Kim16 Ho-Joon Song34 Byung-Cheul Shin2 and Sung-Joo Park134

1 Hanbang Body-Fluid Research Center Wonkwang University Iksan Jeonbuk 540-749 Republic of Korea2Department of Rehabilitation Medicine of Korean Medicine Spine amp Joint Center Pusan NationalUniversity Korean Medicine Hospital Yangsan 626-770 Republic of Korea

3 Department of Herbology School of Oriental Medicine Wonkwang University Iksan Jeonbuk 540-749 Republic of Korea4 BK21 plus team Professional Graduate School of OrientalMedicineWonkwangUniversity Iksan Jeonbuk 540-749 Republic of Korea5 Department of Beauty Dongshin University Naju Jeonnam 252-17 Republic of Korea6College of Pharmacy Wonkwang University Iksan 570-749 Republic of Korea

Correspondence should be addressed to Sung-Joo Park parksj08wkuackr

Received 19 November 2013 Revised 28 February 2014 Accepted 3 March 2014 Published 30 March 2014

Academic Editor MinKyun Na

Copyright copy 2014 Gi-Sang Bae et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

It has been previously shown that Nardostachys jatamansi (NJ) exhibits anti-inflammatory properties against lipopolysaccharide(LPS) challenges However the potency of NJ constituents against LPS-induced inflammatory responses has not been examined Inthis present study we determinedwhichNJ extract fractions exhibit inhibitory effects against LPS-induced inflammatory responsesAmong the NJ fractions NJ-1 NJ-3 NJ-4 and NJ-6 inhibited LPS-induced production of NOThe NJ-3 NJ-4 and NJ-6 fractionsalso inhibited the production of cytokines such as IL-1120573 IL-6 and TNF-120572 However NJ-1 NJ-3 NJ-4 andNJ-6 showed differentialinhibitory mechanisms against LPS-induced inflammatory responses NJ-1 NJ-3 and NJ-4 inhibited LPS-induced activation of c-jun NH

2-terminal kinase (JNK) and p38 but did not affect activation of extracellular signal-regulated kinase (ERK) or NF-120581B On

the other hand NJ-6 inhibited activation of MAPKs and NF-120581B In addition in vivo experiments revealed that administration ofNJ-1 NJ-3 NJ-4 and NJ-6 reduced LPS-induced endotoxin shock with NJ-6 especially showing a marked protective effect Takentogether these results provide the evidence for the potential of selective NJ fractions against LPS-induced inflammation Thus itwill be advantageous to further isolate and determine single effective compounds from these potent fractions

1 Introduction

Inflammation is a complex immunologic response to harmfulstimuli including various pathogens irritants and infections[1] Infectious agents such as bacteria and proinflammatorycytokines can activate macrophages immune cells that arecritically involved in the regulation of innate immunitythrough certain receptors [2] The interaction between Toll-like receptor (TLR)-4 and the ligand lipopolysaccharide(LPS) induces an intracellular signaling cascade that acti-vates the mitogen-activated protein kinase (MAPK) familyextracellular signal-related kinase (ERK) p38 c-junNH

2-ter-

minal kinase (JNK) and key proinflammatory transcription

factors such as nuclear factor-kappa B (NF-120581B) [3] Subse-quent to TLR-4-LPS interactionmacrophages release variousinflammatory mediators such as nitric oxide (NO) andproinflammatory cytokines including interleukin (IL)-1120573 IL-6 and tumor necrosis factor alpha (TNF-120572) [4]

Nardostachys jatamansi (NJ) is widely used as a bittertonic and antispasmodic [5] It has been reported thatthe root of NJ contains various sesquiterpenes includingjatamansic acid and jatamansone lignans and neolignansWe previously reported that aqueous extract of NJ is effectivein protecting against inflammatory challenges [6ndash10] par-ticularly against LPS-induced inflammation and endotoxinshock [7] In particular one fraction of NJ extract (fraction

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 837835 11 pageshttpdxdoiorg1011552014837835

2 Evidence-Based Complementary and Alternative Medicine

NJ-110 MeOH

NJ-220 MeOH

NJ-340 MeOH

NJ-460 MeOH

NJ-580 MeOH

NJ-6100 MeOH

500mL 500mL 500mL 500mL 500mL 500mL1240mg 791mg 4907mg 4162mg 2731mg 678mg

Nardostachys jatamansi roots (NJ)water extract (38 g)

RPC-18 column chromatography (5 times 20 cm)

Scheme 1 The scheme of NJ fractions

4) demonstrated a protective effect against cerulein-inducedacute pancreatitis [11]However althoughmany of our studieshave shown the anti-inflammatory activities of NJ it is not yetknown which particular compound of NJ has the potentialto inhibit LPS-induced inflammation Therefore to take onestep closer to actual bioactive compounds from NJ we usedNJ fractions which have many bioactive compounds In thisstudy we performed in vitro and in vivo analyses to examinewhether the fractions of NJ have potential inhibitory effectsagainst LPS-induced inflammation in murine peritonealmacrophages and in an animal model of LPS-induced endo-toxin shock Moreover to elucidate a potential molecularanti-inflammatory mechanism we examined the activationof MAPKs and NF-120581B

2 Materials and Methods

21 Chemicals and Reagents RPMI-1640 fetal bovine serum(FBS) penicillin and streptomycin were obtained fromGibco BRL (Grand Island NY USA) Enzyme-linkedimmunosorbent assay (ELISA) antibodies for detection ofmouse IL-1120573 IL-6 and TNF-120572 were purchased from RampDSystems (Minneapolis MN USA) LPS from Escherichia coli055B5 was purchased from Sigma-Aldrich Chemical (StLouis MO USA) Antibodies against total and phospho-specific MAPKs (ERK 12 JNK and p38) were obtainedfrom Cell Signaling Technology (Beverly MA USA) I120581-B120572monoclonal antibody and peroxidase-conjugated secondaryantibody were purchased from Santa Cruz BiotechnologyInc (Santa Cruz CA USA) Prestained sodium dode-cyl sulfate-polyacrylamide gel electrophoresis markers wereobtained from Bio-Rad (Hercules CA USA) Trizol reagentand polymerase chain reaction (PCR) kits were purchasedfrom Invitrogen Corporation (Carlsbad CA USA)

22 Plant Materials The roots of NJ were purchased froma standard commercial source (Omni Herb Seoul Korea)The herbrsquos identity was confirmed at Wonkwang UniversityVoucher specimens (number 02-03-29) were deposited atthe College of Oriental Medicine Herbarium of WonkwangUniversityTheNJ roots were prepared by decocting the driedprescription of herbs (100 g) with boiling distilled water (1 L)for approximately 2 h The water extract was frozen at minus80∘Cand freeze dried to be powdered (735 g 735 ww)

23 Preparation of NJ Fractions The water extract (38 g)was subjected to octadecyl functionalized silica gel flash

column (5times 20 cm 63ndash200 120583mparticle size) chromatography(Scheme 1) Elution was carried out by stepwise gradient with500mL aliquots of MeOH in H

2O (starting from 10 and

followed by 20 up to 100 at 20 increments) yielding6 fractions (NJ1 124 g NJ2 791mg NJ3 4907mg NJ44162mg NJ5 2731mg and NJ6 678mg)

24 HPLC Sample Preparation and HPLC Conditions ForHPLC analysis the chromatographic system consisted of apump (3000HPLC pump Dionex Association USA) a UVdetector (Photodiode array detector Dionex Association)and an autosampler (Waters Association USA) A hydro-sphere C

18column (46mm times 250mm 5120583m) was used The

mobile phase was composed of (A) water and (B) acetonitrilewith an applied gradient of 10 B increasing to 60 B in50min and 60B increasing to 100B in 2min and then thesystemwas equilibrated for 8min with the 100 B conditions(total 60min) Detection of the peaks was performed at254 nm and sensitivity was set at 05 AUFs The injectionvolume was 20120583L and the flow rate was 10mLmin Astandard solution was prepared by dissolution in distilledmethanol (10120583g10mL) The solution of biological activefraction was filtered through a 045120583m membrane filter andsubjected to HPLC (Scheme 2)

25 Peritoneal Macrophage Culture Thioglycollate (TG)elicited peritoneal macrophages were harvested 4 d afterintraperitoneal injection of 3mL TG Peritoneal lavage wasperformed using 8mL of RPMI supplemented with 10 heat-inactivated FBS After incubation for 3 h nonadherent cellswere removed and the medium was changed for adherentcells Cells were then treated with LPS in the absence orpresence of NJ fractions

26 MTT Assay Cell viability was assayed using a mod-ified colorimetric technique that is based on the abilityof live cells to convert the tetrazolium compound 3-(45dimethylthiazol)-25-diphenyl tetrazolium bromide (MTT)into purple formazan crystalsMTT (5mgmL)was dissolvedinKrebs-Henseleit buffer (115mMNaCl 36mMKCl 13mMKH2PO4 25mMNaHCO

3 1M CaCl

2 and 1MMgCl

2) Fifty

microliters of the mixture was added to each well After30min of incubation at 37∘C the suspension was removedand formazan crystals were dissolved in 200 120583L of dimethylsulfoxide Aliquots of cells were seeded in wells of a 96-wellplate in duplicate and assayed at 540 nmusing amicro-ELISA

Evidence-Based Complementary and Alternative Medicine 3

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Scheme 2 The HPLC of NJ biological fractions

plate reader The number of NJ fraction-treated viable cellswas expressed as a percentage of the control (untreated cells)maintained for the same time period

27 Measurement of NO Concentration Murine peritonealmacrophages (2 times 105 cellswell) were pretreated with NJfractions for 1 h and then stimulated with LPS (500 ngmL)for 24 h To measure NO concentration 100 120583L aliquots wereremoved from conditioned media and incubated with anequal volume of Griess reagent at room temperature for10min The absorbance at 540 nm was then measured

28 ELISA Murine peritoneal macrophages (1 times 106cellswell) were stimulated with 500 ngmL of LPS andorvarious concentrations of NJ fraction for 24 h Culture super-natantswere collected and stored atminus80∘Cuntil use Cytokinelevels in supernatants were determined using a commercialsystem (RampD Systems) according to the manufacturerrsquosinstructionsTheELISAwas devised by coating 96-well plateswith antibodies specific for IL-1120573 IL-6 and TNF-120572 Coatedplates were washed with PBS containing 005 Tween-20 Allreagents used in this assay were incubated overnight at 4∘CRecombinant IL-1120573 IL-6 and TNF-120572 were diluted and usedas a standard Serial dilutions starting at 20 ngmL were usedto establish the standard curve Assay plates were exposedsequentially to biotinylated mouse IL-1120573 IL-6 and TNF-120572avidin peroxidase and a substrate solution of 221015840-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) containing30 hydrogen peroxide The plates were read at 405 nm

29 RNA Quantification Total cellular RNA was isolatedusing Trizol (Invitrogen Carlsbad CA USA) according tomanufacturerrsquos instructions Total RNA (1 120583g) was convertedto cDNA by 200 units of reverse transcriptase and 500 ng ofoligo-dT primer in 50mM Tris-HCl (pH 83) 75mM KCl3mMMgCl

2 10mM DTT and 1mM dNTPs at 42∘C for 1 h

The reaction was stopped by heating at 70∘C for 15min

210 Real-Time RT-PCR TaqMan quantitative RT-PCR wasperformed with a 7700 sequence detection system accord-ing to the manufacturerrsquos instructions (Applied BiosystemsFoster City CA USA) For each sample triplicate testreactions and a control reaction lacking reverse transcrip-tase were analyzed for expression of the gene of interestand results were normalized to those for the housekeep-ing gene hypoxanthine-guanine phosphoribosyltransferase(HPRT) Arbitrary expression units were calculated by divid-ing expression of the gene of interest by ribosomal pro-tein HPRT mRNA expression PCR was performed at anannealing temperature of 60∘C with 40 amplification cyclesThe commercial forward reverse and probe oligonucleotideprimers formultiplex real-timeTaqManPCRwere purchasedfrom ABI (Applied Biosystems Foster City CA USA)

211 Western Blot Whole cell lysates were prepared byboiling peritoneal macrophages in a sample buffer (625mMTris-HCl (pH 68) 2 sodium dodecyl sulfate 20 glyceroland 10 2-mercaptoethanol) Proteins in the cell lysateswere then separated by 10 SDS-PAGE and transferred to

4 Evidence-Based Complementary and Alternative Medicine

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Figure 1 Cytotoxic effects of NJ fractions on murine peritoneal macrophages The effects of NJ fractions on cell viability were measured byMTT assay Peritoneal macrophages were incubated with or without NJ fractions at indicated doses (120583gmL) for 24 h The values are mean plusmnSE of three independent experiments

119875 lt 0001 versus saline

a nitrocellulose membraneThemembrane was then blockedwith 5 skim milk in PBS-Tween-20 for 2 h at room temper-ature and incubated overnight with antibodies against phos-phorylated ERK 12 phosphorylated JNK phosphorylatedp38 and I120581-B120572 After washing three times each blot wasincubated with a secondary antibody for 1 h and immunore-active bands were visualized using an enhanced chemilumi-nescence detection system (Amersham Piscataway NJ USA)according to the manufacturerrsquos recommended protocol

212 Immunostaining Peritoneal macrophages were platedin a chamber slide and incubated with 500 ngmL of LPSfor 1 h at 37∘C The cells were fixed in 4 paraformaldehydefor 30min at RT and washed three times with PBS Thecells were treated with 01 Triton X-100 for 15min at roomtemperature After washing the cells were incubated with ablocking serum for 1 h and incubated overnight with a 1 100dilution of primaryNF-120581Bp65 antibody (SantaCruzBiotech-nology Santa Cruz CA USA) The cells were then washedand incubated with a 1 2000 dilution of Alexafluor 568-conjugated goat anti-mouse IgG (Invitrogen USA) for 2 h ina dark room For nuclear staining the cells were incubatedwith a 1 1000 dilution of DAPI for 5minThe slide was finally

washed and mounted for microscopic examination Stainingwith NF-120581B p65 antibody exhibited red fluorescence whichwas detected by fluorescence microscopy

213 Animal Model of LPS-Induced Endotoxin Shock Endo-toxin shock was induced in 6- to 8-week-old female C57BL6mice by intraperitoneal (ip) injection of bacterial endotoxin(LPS from E coli serotype O55B5 375mgkg) At 1 h afterNJ fraction administration LPS (375mgsdotkgminus1) was injectedintraperitoneally Survival was monitored for 120 h Animaluse and relevant experimental procedures were conductedin accordance with the National Institutes of Health (NIH)Guidelines for the Care and Use of Laboratory Animals Allexperiments were approved by the Animal Care Committeeof Wonkwang University

214 Statistical Analysis The results are expressed as themean plusmn SE of independent experiments Two-way ANOVAwas used to analyze the statistical significance of resultsamong groups If results were found to be statistically sig-nificant post hoc analysis was performed using the Dun-can method as a multiple comparison among groups All

Evidence-Based Complementary and Alternative Medicine 5

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LPS LPS LPS

NJ-1 minus minus 10 20 50 100NJ-2 minus minus 10 20 50 100NJ-3 minus minus

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Figure 2 Effects of NJ fractions on LPS-induced NO production Peritoneal macrophages were pretreated with NJ fractions at indicateddoses (120583gmL) for 1 h and then stimulated with LPS (500 ngmL) for 24 h The effects of NJ fractions on LPS-induced NO production weremeasured by Griess assay The values are mean plusmn SE of three independent experiments lowast119875 lt 005 versus saline +119875 lt 005 versus LPS

statistical analyses were performed using SPSS version 100statistical analysis software

3 Results

31 Cytotoxicity of NJ Fractions in Peritoneal MacrophagesThe first approach to study the biological activity of anycompound or plant extract is to ensure lack of effect oncellular metabolism To determine whether fraction of NJaffects cell viability peritoneal macrophages were incubatedfor 24 h with varying concentrations of extract (120583gmL) andcell viability was evaluated by MTT assay NJ-1 NJ-2 NJ-3 and NJ-6 did not demonstrate any significant cytotoxiceffects on peritoneal macrophages However at a relativelyhigher dose NJ-4 and NJ-5 showed significant cytotoxicity( 119875 lt 0001) (Figure 1) Therefore because higher levels

of NJ-4 (ge100 120583gmL) and NJ-5 (ge50120583gmL) could affectcellular metabolism we excluded data of NJ-4 and NJ-5 atthese concentrations

32 Effects of NJ Fractions on LPS-Induced NO Produc-tion Next to examine the anti-inflammatory effect of NJfractions we measured NO production Murine peritonealmacrophages were pretreated with the indicated concentra-tions of NJ fractions for 1 h and then stimulated with LPS(500 ngmL) for 24 h As shown in Figure 2 LPS stimulationincreased NO production However pretreatment with NJ-1 NJ-3 NJ-4 and NJ-6 significantly inhibited LPS-inducedproduction of NO but not with NJ-2 and NJ-5 (Figure 2)

33 Effects of NJ Fractions on LPS-Induced ProinflammatoryCytokine Production To examine the effects of NJ fractions

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Figure 3 Effects of NJ fractions on LPS-induced cytokine productions Peritoneal macrophages were treated with NJ fractions (120583gmL) for1 h and then stimulated with LPS at indicated time pointsThe supernatants were harvested and the levels of (a) IL-1120573 (b) IL-6 and (c) TNF-120572were measured by ELISA The values are mean plusmn SE of three independent experiments lowast119875 lt 005 versus saline +119875 lt 005 versus LPS

on LPS-induced proinflammatory cytokine production peri-toneal macrophages were pretreated with NJ fractions at theindicated concentrations for 1 h and then stimulated with500 ngmL of LPS for 24 h In particular we treated cells withNJ-1 NJ-3 NJ-4 and NJ-6 fractions which demonstratedinhibition of NO production As shown in Figures 3 and 4protein and mRNA levels of IL-1120573 IL-6 and TNF-120572 wereincreased by LPS stimulation However pretreatment withNJ-3 NJ-4 and NJ-6 inhibited the production of IL-1120573 IL-6and TNF-120572 significantly but not with NJ-1 fraction (Figures 3and 4)

34 Effects of NJ Fractions on the Activation of MAPKs andNF-120581B To examine inhibitory mechanism(s) of NJ fractionson production of NO and cytokines MAPK and NF-120581B were

measured The cells were pretreated with NJ-1 (100 120583gmL)NJ-3 (100 120583gmL) NJ-4 (50 120583gmL) and NJ-6 (100 120583gmL)for 1 h and then stimulatedwith 500 ngmLof LPS for 0 15 30or 60min Pretreatment withNJ-1 NJ-3 or NJ-4 significantlyinhibited the LPS-induced activation of JNK and p38 but notERK 12 (Figures 5(a)ndash5(c)) In addition pretreatment withNJ-6 significantly inhibited the LPS-induced activation ofERK 12 JNK and p38 (Figure 5(d)) To determine whetherNJ fractions inhibit LPS-inducedNF-120581B activation we exam-ined degradation of I120581-B120572 and NF-120581B p65 translocationTheNJ-1 NJ-3 andNJ-4 fractions did not inhibit the degradationof I120581-B120572 (Figures 6(a)ndash6(c)) However NJ-6 inhibited thedegradation of I120581-B120572 and translocation of NF-120581B p65 intonucleus suggesting that NJ-6 may influence LPS-inducedNF-120581B activation (Figures 6(d) and 6(e))

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Figure 4 Effects of NJ fractions onmRNA levels of LPS-induced cytokine expression Peritoneal macrophages were treated with NJ fractions(120583gmL) for 1 h and then stimulated with LPS at indicated time points Cells were collected for real-time RT-PCR Total RNA (1 120583g) wasprepared for the real-time RT-PCR of (a) IL-1120573 (b) IL-6 and (c) TNF-120572 The values are mean plusmn SE of three independent experimentslowast

119875 lt 005 versus saline +119875 lt 005 versus LPS

35 Effects of NJ Fractions on LPS-Induced Endotoxin ShockTo verify effects of NJ fractions in vivo we used a model LPS-induced endotoxin shock NJ fractions (NJ-1 NJ-3 NJ-4 orNJ-6) were injected at the indicated doses After 3 h a lethaldose of LPS (375mgkg) was administered intraperitoneallyThe survival rate was recorded every 12 h after LPS treat-ment Generally LPS-injected septic mice died within 48 hConsistent with in vitro experiments NJ fractions exhibitedstrong anti-inflammatory activities in vivo as shown bysignificant inhibition of LPS-induced death in the treatedmice (Figure 7) In particular NJ-4 and NJ-6 showed themost dramatic protection against LPS-induced endotoxinshock these fractions prevented nearly all harmful effects ofLPS (Figures 7(c) and 7(d))

4 Discussion

Recently our laboratory reported the beneficial effects of NJon various diseases such as endotoxin shock pancreatitisand diabetes [6ndash10] Particularly in endotoxin shock NJshowed a marked preventive effect and strong therapeuticpotential against LPS challenge [7] Although many of ourstudies have indicated the benefits of NJ on inflamma-tory diseases which compounds of NJ could exhibit anti-inflammatory properties are unknown Thus in this paperbringing more bright insight to identify the constituent com-pounds from NJ we selected and assorted effective fractionsthat would possess anti-inflammatory potential Overall weobtained six fractions from NJ Using the fourth fraction

8 Evidence-Based Complementary and Alternative Medicine

Time (min)LPS

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Figure 5 Effects of NJ fractions on MAPKs activation ((a)ndash(d)) Peritoneal macrophages were treated with NJ fractions (100120583gmL) for 1 hand then stimulated with LPS (500 ngmL) at indicated time points Cells were harvested for western blot Total cellular proteins (20120583g) wereresolved by SDS-PAGE transferred to PVDF membrane and detected with phospho-specific ERK 12 (4244 kDa) JNK (4654 kDa) andp38 (43 kDa) antibody ERK JNK and p38 were used as a loading control A representative western blot of three experiments is shown

we evaluated the protective effects on cerulein-induced acutepancreatitis (AP) However the sixth fraction remained to beexamined for potential to prevent an inflammatory responseto stimuli such as LPS Therefore in this study we used sixfractions isolated from NJ to examine the anti-inflammatoryactivity against LPS on murine macrophages

Generally sepsis is a life threatening condition caused bythe bodyrsquos response to bacterial products such as endotoxinwhich is often present with severe fever shock and respira-tory damage [12] Partially due to this phenomenon sepsisis often regarded as an uncontrolled inflammatory response[12] In the context of sepsis TNF-120572 IL-1120573 IL-6 and IL-8 arethemost frequently altered cytokines [13]When injected intoanimals these cytokines recapitulate many clinical and labo-ratory features of sepsis supporting the concept that sepsisrepresents a ldquocytokine stormrdquo Thus regulation of cytokinesmay prove valuable for the prevention and treatment ofsepsis In this study just as the total extract of NJ inhibitedthe production of cytokines [7] fractions 3 4 and 6 also

inhibited the production of cytokines (Figures 3 and 4)These results suggest that the ability of NJ to inhibit cytokineproduction may originate from fractions 3 4 andor 6

Using the fractions that were found to be effectiveagainst LPS we examined activation of MAPKs and NF-120581Bas possible regulatory mechanisms Generally it has beenreported that the production of proinflammatory mediatorsis modulated by the MAPKs and NF-120581B pathways Threemajor groups of MAPKs include the ERK JNK and p38which are all activated by phosphorylation [14ndash16] NF-120581Bactivation occurs mainly through the degradation of I120581Bthereby leading to subsequent release of NF-120581B dimers [17]Subsequently NF-120581B dimers translocate from the cytoplasmto the nucleus and activate the transcription of multiple 120581B-dependent target genes [17] In this study we examine thephosphorylation of MAPKs and degradation of I120581-B120572 NJ-1NJ-3 and NJ-4 inhibited the phosphorylation and activationof JNK and p38 but not ERK12 However NJ-6 inhibitednot only the activation of ERK12 JNK and p38 but also

Evidence-Based Complementary and Alternative Medicine 9

Time (min)LPS

60301506030150NJ-1 + LPS

120573-Actin

I120581-B120572

(a)

Time (min)LPS

60301506030150NJ-4 + LPS

120573-Actin

I120581-B120572

(b)

Time (min)LPS

60301506030150NJ-3 + LPS

120573-Actin

I120581-B120572

(c)

Time (min)LPS

60301506030150NJ-6 + LPS

120573-Actin

I120581-B120572

(d)

DAPI Merge

None

LPS

NJ-6

NF-120581B p65

(e)

Figure 6 Effects of NJ fractions onNF-120581B activation ((a)ndash(d)) Peritonealmacrophages were treatedwithNJ fractions (100120583gmL) for 1 h andthen stimulated with LPS (500 ngmL) at indicated time points The cells were harvested for western blot Total cellular proteins (20 120583g) wereresolved by SDS-PAGE transferred to PVDF membrane and detected with I120581-B120572 (40 kDa) antibody 120573-Actin was used as loading controlA representative western blot of three experiments is shown (e) Peritoneal macrophages were treated with NJ fractions (100120583gmL) for 1 hand then stimulated with LPS (500 ngmL) for 1 h Then nuclear translocation of NF-120581B p65 was examined using immunostaining NF-120581Bp65 was shown as red and the nucleus was shown as blue (DAPI) The values are means plusmn SE of three independent experiments lowast119875 lt 005versus saline +119875 lt 005 versus LPS

10 Evidence-Based Complementary and Alternative Medicine

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-1 (01mgkg) + LPS

NJ-1 (1mgkg) + LPSNJ-1 (10mgkg) + LPS

(a)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-3 (01mgkg) + LPS

NJ-3 (1mgkg) + LPSNJ-3 (10mgkg) + LPS

(b)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-4 (01mgkg) + LPS

NJ-4 (1mgkg) + LPSNJ-4 (10mgkg) + LPS

(c)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-6 (01mgkg) + LPS

NJ-6 (1mgkg) + LPSNJ-6 (10mgkg) + LPS

(d)

Figure 7 Effect of NJ fractions on LPS-induced endotoxin shock ((a)ndash(d)) Endotoxin shock was induced in 6ndash8-week-old female C57BL6mice as described in Section 2 NJ fractions were administered intraperitoneally to mice at 01 1 or 10mgkg 1 h before LPS injection ThenLPS (375mgkg) was injected to induce endotoxin shock The survival rates of endotoxin shock mice were monitored for survival for 120 hThis figure shows data for 10 mice per group

degradation of I120581-B120572 These results suggest that inhibitionof NO and cytokine production by NJ fractions is primarilythrough MAPKs or NF-120581B pathways

In this study we isolated two beneficial fractions of NJThe first one was NJ-4 which could show similar effectsto NJ total extract NJ-4 inhibited cytokine production andLPS-induced endotoxin shock similar to NJ total extract [7]Furthermore the regulatory mechanisms were similar NJtotal extract inhibited only MAPKs but NJ-4 inhibited theJNK and p38 Secondly NJ-6 showed dramatic inhibitionof LPS-induced inflammatory response Indeed NJ-6 inhib-ited the cytokine production and LPS-induced endotoxinshock dramatically However NJ-6 differed from total extractmechanistically NJ-6 inhibited the degradation of I120581-B120572and translocation of NF-120581B p65 which was not regulatedby the NJ total extract Further isolation of the NJ-4 andNJ-6 fractions could potentially yield a single compounddemonstrating inhibitory activity resembling that of NJ totalextract

In conclusion this study demonstrated which fractionsof NJ exhibit inhibitory activity against LPS-induced inflam-mation Among the 6 fractions isolated NJ-1 NJ-3 NJ-4 andNJ-6 showed the inhibition of NO and NJ-3 NJ-4 and NJ-6 showed the inhibition of cytokines In addition NJ-4 andNJ-6 exhibited dramatic prevention against LPS-endotoxinshock These results suggest that NJ-4 and NJ-6 may beeffective and beneficial candidates to ameliorate LPS-inducedinflammation

Abbreviations

ELISA Enzyme-linked immunosorbent assayERK Extracellular signal-regulated kinaseFBS Fetal bovine serumIL InterleukinJNK c-jun NH

2-terminal kinase

LPS LipopolysaccharideMAPKs Mitogen-activated protein kinases

Evidence-Based Complementary and Alternative Medicine 11

MTT 3-(45 Dimethylthiazol)-25-diphenyltetrazolium bromide

NF Nuclear factorNJ Nardostachys jatamansiNO Nitric oxidePCR Polymerase chain reactionTG ThioglycollateTLR Toll-like receptorTNF Tumor necrosis factor

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Gi-Sang Bae and Kwang-Ho Heo equally contributed to thiswork

Acknowledgments

This research was supported by a Basic Science ResearchProgram through theNational Research Foundation of Korea(NRF) fundedby theMinistryofEducation ScienceandTech-nology (NRF-2012R1A1A2040916 2012R1A5A2A35671257and 2012M2B2B1055246)

References

[1] C-S Sung and C-S Wong ldquoCellular mechanisms of neuroin-flammatory pain the role of interleukin-1120573rdquo Acta Anaesthesio-logica Taiwanica vol 45 no 2 pp 103ndash109 2007

[2] F O Martinez L Helming and S Gordon ldquoAlternative activa-tion of macrophages an immunologic functional perspectiverdquoAnnual Review of Immunology vol 27 pp 451ndash483 2009

[3] J D Schilling H M Machkovech L He A Diwan and J ESchaffer ldquoTLR4 activation under lipotoxic conditions leads tosynergistic macrophage cell death through a TRIF-dependentpathwayrdquo Journal of Immunology vol 190 no 3 pp 1285ndash12962013

[4] Y Ben-Neriah andM Karin ldquoInflammation meets cancer withNF-120581B as the matchmakerrdquo Nature Immunology vol 12 no 8pp 715ndash723 2011

[5] B C Bose Y Vijayvargiya and J N Bhatnagar ldquoNardostachysjatamansiDC a phyto-chemical study of its active constituentsrdquoIndian Journal of Medical Sciences vol 11 no 10 pp 799ndash8021957

[6] G-S Bae H-J Park D-Y Kim et al ldquoNardostachys jatamansiprotects against cerulein-induced acute pancreatitisrdquo Pancreasvol 39 no 4 pp 520ndash529 2010

[7] G-S Bae S-W Seo M-S Kim et al ldquoThe roots of Nar-dostachys jatamansi inhibits lipopolysaccharide-induced endo-toxin shockrdquo Journal of Natural Medicines vol 65 no 1 pp 63ndash72 2011

[8] G S Bae K C Park B S Koo et al ldquoThe inhibitory effectsof Nardostachys jatamansi on alcoholic chronic pancreatitisrdquoBMB Reports vol 45 no 7 pp 402ndash407 2012

[9] G S Bae K C Park B S Koo et al ldquoThe beneficial effectsof Nardostachys jatamansi extract on diet-induced severe acutepancreatitisrdquo Pancreas vol 42 no 2 pp 362ndash363 2013

[10] M-Y Song U-J Bae B-H Lee et al ldquoNardostachys jatamansiextract protects against cytokineinduced 120573-cell damage andstreptozotocin-induced diabetesrdquo World Journal of Gastroen-terology vol 16 no 26 pp 3249ndash3257 2010

[11] G S Bae M S Kim K C Park et al ldquoEffect of biologicallyactive fraction of Nardostachys jatamansi on cerulein-inducedacute pancreatitisrdquo World Journal of Gastroenterology vol 18no 25 pp 3223ndash3234 2012

[12] R S Hotchkiss and I E Karl ldquoThe pathophysiology andtreatment of sepsisrdquoThe New England Journal of Medicine vol348 no 2 pp 138ndash150 2003

[13] T S Blackwell and J W Christman ldquoSepsis and cytokinescurrent statusrdquo British Journal of Anaesthesia vol 77 no 1 pp110ndash117 1996

[14] H B Jijon W J Panenka K L Madsen and H G Par-sons ldquoMAP kinases contribute to IL-8 secretion by intestinalepithelial cells via a posttranscriptional mechanismrdquo AmericanJournal of PhysiologymdashCell Physiology vol 283 no 1 pp C31ndashC41 2002

[15] M Sanchez-Hidalgo A R Martın I Villegas and C Alarconde la Lastra ldquoRosiglitazone a PPAR120574 ligand modulates signaltransduction pathways during the development of acute TNBS-induced colitis in ratsrdquo European Journal of Pharmacology vol562 no 3 pp 247ndash258 2007

[16] F Scaldaferri M Sans S Vetrano et al ldquoThe role of MAPKin governing lymphocyte adhesion to and migration acrossthemicrovasculature in inflammatory bowel diseaserdquo EuropeanJournal of Immunology vol 39 no 1 pp 290ndash300 2009

[17] S Chae L Eckmann Y Miyamoto C Pothoulakis M KarinandM FKagnoff ldquoEpithelial cell I120581B-kinase120573has an importantprotective role in Clostridiumdifficile toxinA-inducedmucosalinjuryrdquo Journal of Immunology vol 177 no 2 pp 1214ndash12202006

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

NJ-110 MeOH

NJ-220 MeOH

NJ-340 MeOH

NJ-460 MeOH

NJ-580 MeOH

NJ-6100 MeOH

500mL 500mL 500mL 500mL 500mL 500mL1240mg 791mg 4907mg 4162mg 2731mg 678mg

Nardostachys jatamansi roots (NJ)water extract (38 g)

RPC-18 column chromatography (5 times 20 cm)

Scheme 1 The scheme of NJ fractions

4) demonstrated a protective effect against cerulein-inducedacute pancreatitis [11]However althoughmany of our studieshave shown the anti-inflammatory activities of NJ it is not yetknown which particular compound of NJ has the potentialto inhibit LPS-induced inflammation Therefore to take onestep closer to actual bioactive compounds from NJ we usedNJ fractions which have many bioactive compounds In thisstudy we performed in vitro and in vivo analyses to examinewhether the fractions of NJ have potential inhibitory effectsagainst LPS-induced inflammation in murine peritonealmacrophages and in an animal model of LPS-induced endo-toxin shock Moreover to elucidate a potential molecularanti-inflammatory mechanism we examined the activationof MAPKs and NF-120581B

2 Materials and Methods

21 Chemicals and Reagents RPMI-1640 fetal bovine serum(FBS) penicillin and streptomycin were obtained fromGibco BRL (Grand Island NY USA) Enzyme-linkedimmunosorbent assay (ELISA) antibodies for detection ofmouse IL-1120573 IL-6 and TNF-120572 were purchased from RampDSystems (Minneapolis MN USA) LPS from Escherichia coli055B5 was purchased from Sigma-Aldrich Chemical (StLouis MO USA) Antibodies against total and phospho-specific MAPKs (ERK 12 JNK and p38) were obtainedfrom Cell Signaling Technology (Beverly MA USA) I120581-B120572monoclonal antibody and peroxidase-conjugated secondaryantibody were purchased from Santa Cruz BiotechnologyInc (Santa Cruz CA USA) Prestained sodium dode-cyl sulfate-polyacrylamide gel electrophoresis markers wereobtained from Bio-Rad (Hercules CA USA) Trizol reagentand polymerase chain reaction (PCR) kits were purchasedfrom Invitrogen Corporation (Carlsbad CA USA)

22 Plant Materials The roots of NJ were purchased froma standard commercial source (Omni Herb Seoul Korea)The herbrsquos identity was confirmed at Wonkwang UniversityVoucher specimens (number 02-03-29) were deposited atthe College of Oriental Medicine Herbarium of WonkwangUniversityTheNJ roots were prepared by decocting the driedprescription of herbs (100 g) with boiling distilled water (1 L)for approximately 2 h The water extract was frozen at minus80∘Cand freeze dried to be powdered (735 g 735 ww)

23 Preparation of NJ Fractions The water extract (38 g)was subjected to octadecyl functionalized silica gel flash

column (5times 20 cm 63ndash200 120583mparticle size) chromatography(Scheme 1) Elution was carried out by stepwise gradient with500mL aliquots of MeOH in H

2O (starting from 10 and

followed by 20 up to 100 at 20 increments) yielding6 fractions (NJ1 124 g NJ2 791mg NJ3 4907mg NJ44162mg NJ5 2731mg and NJ6 678mg)

24 HPLC Sample Preparation and HPLC Conditions ForHPLC analysis the chromatographic system consisted of apump (3000HPLC pump Dionex Association USA) a UVdetector (Photodiode array detector Dionex Association)and an autosampler (Waters Association USA) A hydro-sphere C

18column (46mm times 250mm 5120583m) was used The

mobile phase was composed of (A) water and (B) acetonitrilewith an applied gradient of 10 B increasing to 60 B in50min and 60B increasing to 100B in 2min and then thesystemwas equilibrated for 8min with the 100 B conditions(total 60min) Detection of the peaks was performed at254 nm and sensitivity was set at 05 AUFs The injectionvolume was 20120583L and the flow rate was 10mLmin Astandard solution was prepared by dissolution in distilledmethanol (10120583g10mL) The solution of biological activefraction was filtered through a 045120583m membrane filter andsubjected to HPLC (Scheme 2)

25 Peritoneal Macrophage Culture Thioglycollate (TG)elicited peritoneal macrophages were harvested 4 d afterintraperitoneal injection of 3mL TG Peritoneal lavage wasperformed using 8mL of RPMI supplemented with 10 heat-inactivated FBS After incubation for 3 h nonadherent cellswere removed and the medium was changed for adherentcells Cells were then treated with LPS in the absence orpresence of NJ fractions

26 MTT Assay Cell viability was assayed using a mod-ified colorimetric technique that is based on the abilityof live cells to convert the tetrazolium compound 3-(45dimethylthiazol)-25-diphenyl tetrazolium bromide (MTT)into purple formazan crystalsMTT (5mgmL)was dissolvedinKrebs-Henseleit buffer (115mMNaCl 36mMKCl 13mMKH2PO4 25mMNaHCO

3 1M CaCl

2 and 1MMgCl

2) Fifty

microliters of the mixture was added to each well After30min of incubation at 37∘C the suspension was removedand formazan crystals were dissolved in 200 120583L of dimethylsulfoxide Aliquots of cells were seeded in wells of a 96-wellplate in duplicate and assayed at 540 nmusing amicro-ELISA

Evidence-Based Complementary and Alternative Medicine 3

40

30

20

10

0

0 10 20 30 40 50 60

(mV

)

Time (min)

NJ-1

60933

85667

0 10 20 30 40 50 60

60

50

40

30

20

10

0

(mV

)

Time (min)

NJ-3

61000

85833

0 10 20 30 40 50 60

200

150

100

50

0

Time (min)

(mV

)

NJ-4

255333

(a) (b)

(c) (d)

0 10 20 30 40 50 60

0

200

150

100

50(m

V)

Time (min)

NJ-6

224833

262833

Scheme 2 The HPLC of NJ biological fractions

plate reader The number of NJ fraction-treated viable cellswas expressed as a percentage of the control (untreated cells)maintained for the same time period

27 Measurement of NO Concentration Murine peritonealmacrophages (2 times 105 cellswell) were pretreated with NJfractions for 1 h and then stimulated with LPS (500 ngmL)for 24 h To measure NO concentration 100 120583L aliquots wereremoved from conditioned media and incubated with anequal volume of Griess reagent at room temperature for10min The absorbance at 540 nm was then measured

28 ELISA Murine peritoneal macrophages (1 times 106cellswell) were stimulated with 500 ngmL of LPS andorvarious concentrations of NJ fraction for 24 h Culture super-natantswere collected and stored atminus80∘Cuntil use Cytokinelevels in supernatants were determined using a commercialsystem (RampD Systems) according to the manufacturerrsquosinstructionsTheELISAwas devised by coating 96-well plateswith antibodies specific for IL-1120573 IL-6 and TNF-120572 Coatedplates were washed with PBS containing 005 Tween-20 Allreagents used in this assay were incubated overnight at 4∘CRecombinant IL-1120573 IL-6 and TNF-120572 were diluted and usedas a standard Serial dilutions starting at 20 ngmL were usedto establish the standard curve Assay plates were exposedsequentially to biotinylated mouse IL-1120573 IL-6 and TNF-120572avidin peroxidase and a substrate solution of 221015840-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) containing30 hydrogen peroxide The plates were read at 405 nm

29 RNA Quantification Total cellular RNA was isolatedusing Trizol (Invitrogen Carlsbad CA USA) according tomanufacturerrsquos instructions Total RNA (1 120583g) was convertedto cDNA by 200 units of reverse transcriptase and 500 ng ofoligo-dT primer in 50mM Tris-HCl (pH 83) 75mM KCl3mMMgCl

2 10mM DTT and 1mM dNTPs at 42∘C for 1 h

The reaction was stopped by heating at 70∘C for 15min

210 Real-Time RT-PCR TaqMan quantitative RT-PCR wasperformed with a 7700 sequence detection system accord-ing to the manufacturerrsquos instructions (Applied BiosystemsFoster City CA USA) For each sample triplicate testreactions and a control reaction lacking reverse transcrip-tase were analyzed for expression of the gene of interestand results were normalized to those for the housekeep-ing gene hypoxanthine-guanine phosphoribosyltransferase(HPRT) Arbitrary expression units were calculated by divid-ing expression of the gene of interest by ribosomal pro-tein HPRT mRNA expression PCR was performed at anannealing temperature of 60∘C with 40 amplification cyclesThe commercial forward reverse and probe oligonucleotideprimers formultiplex real-timeTaqManPCRwere purchasedfrom ABI (Applied Biosystems Foster City CA USA)

211 Western Blot Whole cell lysates were prepared byboiling peritoneal macrophages in a sample buffer (625mMTris-HCl (pH 68) 2 sodium dodecyl sulfate 20 glyceroland 10 2-mercaptoethanol) Proteins in the cell lysateswere then separated by 10 SDS-PAGE and transferred to

4 Evidence-Based Complementary and Alternative Medicine

0

20

40

60

80

100

10 20 50 1000

20

40

60

80

100

10 20 50 1000

20

40

60

80

100

10 20 50 100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

NJ-2NJ-1 NJ-3minus minus

10 20 50 100 10 20 50 100NJ-5 NJ-6minus minus

minus

10 20 50 100NJ-4 minus

Figure 1 Cytotoxic effects of NJ fractions on murine peritoneal macrophages The effects of NJ fractions on cell viability were measured byMTT assay Peritoneal macrophages were incubated with or without NJ fractions at indicated doses (120583gmL) for 24 h The values are mean plusmnSE of three independent experiments

119875 lt 0001 versus saline

a nitrocellulose membraneThemembrane was then blockedwith 5 skim milk in PBS-Tween-20 for 2 h at room temper-ature and incubated overnight with antibodies against phos-phorylated ERK 12 phosphorylated JNK phosphorylatedp38 and I120581-B120572 After washing three times each blot wasincubated with a secondary antibody for 1 h and immunore-active bands were visualized using an enhanced chemilumi-nescence detection system (Amersham Piscataway NJ USA)according to the manufacturerrsquos recommended protocol

212 Immunostaining Peritoneal macrophages were platedin a chamber slide and incubated with 500 ngmL of LPSfor 1 h at 37∘C The cells were fixed in 4 paraformaldehydefor 30min at RT and washed three times with PBS Thecells were treated with 01 Triton X-100 for 15min at roomtemperature After washing the cells were incubated with ablocking serum for 1 h and incubated overnight with a 1 100dilution of primaryNF-120581Bp65 antibody (SantaCruzBiotech-nology Santa Cruz CA USA) The cells were then washedand incubated with a 1 2000 dilution of Alexafluor 568-conjugated goat anti-mouse IgG (Invitrogen USA) for 2 h ina dark room For nuclear staining the cells were incubatedwith a 1 1000 dilution of DAPI for 5minThe slide was finally

washed and mounted for microscopic examination Stainingwith NF-120581B p65 antibody exhibited red fluorescence whichwas detected by fluorescence microscopy

213 Animal Model of LPS-Induced Endotoxin Shock Endo-toxin shock was induced in 6- to 8-week-old female C57BL6mice by intraperitoneal (ip) injection of bacterial endotoxin(LPS from E coli serotype O55B5 375mgkg) At 1 h afterNJ fraction administration LPS (375mgsdotkgminus1) was injectedintraperitoneally Survival was monitored for 120 h Animaluse and relevant experimental procedures were conductedin accordance with the National Institutes of Health (NIH)Guidelines for the Care and Use of Laboratory Animals Allexperiments were approved by the Animal Care Committeeof Wonkwang University

214 Statistical Analysis The results are expressed as themean plusmn SE of independent experiments Two-way ANOVAwas used to analyze the statistical significance of resultsamong groups If results were found to be statistically sig-nificant post hoc analysis was performed using the Dun-can method as a multiple comparison among groups All

Evidence-Based Complementary and Alternative Medicine 5

0 0

5

10

15

20

25

10 20 50 100

5

10

15

20

25

0

5

10

15

20

25

0

5

10

15

20

25

0

5

10

15

20

25

0

5

10

15

20

25

LPS LPS LPS

LPS LPS LPS

NJ-1 minus minus 10 20 50 100NJ-2 minus minus 10 20 50 100NJ-3 minus minus

10 20 50 100NJ-6 minus minus10 20 50 100NJ-5 minus minus10 20 50 100NJ-4 minus minus

Nitr

ite (120583

M)

Nitr

ite (120583

M)

Nitr

ite (120583

M)

Nitr

ite (120583

M)

Nitr

ite (120583

M)

Nitr

ite (120583

M)

lowast lowast lowast lowast lowast lowast

lowastdagger lowastdagger

lowastdaggerlowastdagger

lowast

lowastdagger

lowastlowast lowastlowast lowast lowast

lowastdagger

lowast

lowastdaggerlowastdaggerlowastdaggerlowastdagger

lowastdagger

lowastdagger lowastdagger

lowastdaggerlowastdagger

lowastdagger

Figure 2 Effects of NJ fractions on LPS-induced NO production Peritoneal macrophages were pretreated with NJ fractions at indicateddoses (120583gmL) for 1 h and then stimulated with LPS (500 ngmL) for 24 h The effects of NJ fractions on LPS-induced NO production weremeasured by Griess assay The values are mean plusmn SE of three independent experiments lowast119875 lt 005 versus saline +119875 lt 005 versus LPS

statistical analyses were performed using SPSS version 100statistical analysis software

3 Results

31 Cytotoxicity of NJ Fractions in Peritoneal MacrophagesThe first approach to study the biological activity of anycompound or plant extract is to ensure lack of effect oncellular metabolism To determine whether fraction of NJaffects cell viability peritoneal macrophages were incubatedfor 24 h with varying concentrations of extract (120583gmL) andcell viability was evaluated by MTT assay NJ-1 NJ-2 NJ-3 and NJ-6 did not demonstrate any significant cytotoxiceffects on peritoneal macrophages However at a relativelyhigher dose NJ-4 and NJ-5 showed significant cytotoxicity( 119875 lt 0001) (Figure 1) Therefore because higher levels

of NJ-4 (ge100 120583gmL) and NJ-5 (ge50120583gmL) could affectcellular metabolism we excluded data of NJ-4 and NJ-5 atthese concentrations

32 Effects of NJ Fractions on LPS-Induced NO Produc-tion Next to examine the anti-inflammatory effect of NJfractions we measured NO production Murine peritonealmacrophages were pretreated with the indicated concentra-tions of NJ fractions for 1 h and then stimulated with LPS(500 ngmL) for 24 h As shown in Figure 2 LPS stimulationincreased NO production However pretreatment with NJ-1 NJ-3 NJ-4 and NJ-6 significantly inhibited LPS-inducedproduction of NO but not with NJ-2 and NJ-5 (Figure 2)

33 Effects of NJ Fractions on LPS-Induced ProinflammatoryCytokine Production To examine the effects of NJ fractions

6 Evidence-Based Complementary and Alternative Medicine

0

30

60

90

120

150

180

210

0

30

60

90

120

150

180

210

0

30

60

90

120

150

180

210

0

30

60

90

120

150

180

210

10 20 50 100NJ-1 minus minus 10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus

IL-1120573

(pg

mL)

IL-1120573

(pg

mL)

IL-1120573

(pg

mL)

IL-1120573

(pg

mL)

lowastlowastlowastlowastlowastlowast lowast lowastlowast lowast lowast lowast lowast

lowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdagger

(a)

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

IL-6

(ng

mL)

IL-6

(ng

mL)

IL-6

(ng

mL)

IL-6

(ng

mL)

lowast lowast lowast lowast lowastlowast

lowast

lowastlowastlowast lowast lowast lowast lowast

lowastdagger

lowastdaggerlowastdagger

lowastdaggerlowastdagger

lowastdagger

(b)

0

1

2

3

4

0

1

2

3

4

0

1

2

3

4

0

1

2

3

4

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

LPSLPSLPS LPS

TNF-120572

(ng

mL)

TNF-120572

(ng

mL)

TNF-120572

(ng

mL)

TNF-120572

(ng

mL)

lowastlowastlowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastdagger

lowastdagger

lowastdagger lowastdagger

lowastdagger

(c)

Figure 3 Effects of NJ fractions on LPS-induced cytokine productions Peritoneal macrophages were treated with NJ fractions (120583gmL) for1 h and then stimulated with LPS at indicated time pointsThe supernatants were harvested and the levels of (a) IL-1120573 (b) IL-6 and (c) TNF-120572were measured by ELISA The values are mean plusmn SE of three independent experiments lowast119875 lt 005 versus saline +119875 lt 005 versus LPS

on LPS-induced proinflammatory cytokine production peri-toneal macrophages were pretreated with NJ fractions at theindicated concentrations for 1 h and then stimulated with500 ngmL of LPS for 24 h In particular we treated cells withNJ-1 NJ-3 NJ-4 and NJ-6 fractions which demonstratedinhibition of NO production As shown in Figures 3 and 4protein and mRNA levels of IL-1120573 IL-6 and TNF-120572 wereincreased by LPS stimulation However pretreatment withNJ-3 NJ-4 and NJ-6 inhibited the production of IL-1120573 IL-6and TNF-120572 significantly but not with NJ-1 fraction (Figures 3and 4)

34 Effects of NJ Fractions on the Activation of MAPKs andNF-120581B To examine inhibitory mechanism(s) of NJ fractionson production of NO and cytokines MAPK and NF-120581B were

measured The cells were pretreated with NJ-1 (100 120583gmL)NJ-3 (100 120583gmL) NJ-4 (50 120583gmL) and NJ-6 (100 120583gmL)for 1 h and then stimulatedwith 500 ngmLof LPS for 0 15 30or 60min Pretreatment withNJ-1 NJ-3 or NJ-4 significantlyinhibited the LPS-induced activation of JNK and p38 but notERK 12 (Figures 5(a)ndash5(c)) In addition pretreatment withNJ-6 significantly inhibited the LPS-induced activation ofERK 12 JNK and p38 (Figure 5(d)) To determine whetherNJ fractions inhibit LPS-inducedNF-120581B activation we exam-ined degradation of I120581-B120572 and NF-120581B p65 translocationTheNJ-1 NJ-3 andNJ-4 fractions did not inhibit the degradationof I120581-B120572 (Figures 6(a)ndash6(c)) However NJ-6 inhibited thedegradation of I120581-B120572 and translocation of NF-120581B p65 intonucleus suggesting that NJ-6 may influence LPS-inducedNF-120581B activation (Figures 6(d) and 6(e))

Evidence-Based Complementary and Alternative Medicine 7

0

50

100

150

200

0

50

100

150

200

0

50

100

150

200

0

50

100

150

200

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

Rela

tive I

L-1120573

Rela

tive I

L-1120573

Rela

tive I

L-1120573

Rela

tive I

L-1120573 lowast

lowast

lowast

lowast

lowast

lowastlowast lowast lowast lowast lowast

lowastdagger

lowastdaggerlowastdaggerlowastdagger

lowastdagger

lowastdagger

lowastdaggerlowastdagger

lowastdagger

(a)

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

Rela

tive I

L-6

Rela

tive I

L-6

Rela

tive I

L-6

Rela

tive I

L-6

lowast lowast lowast lowast lowast lowastlowastlowast lowast

lowast

lowast

lowastdagger

lowastdaggerlowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdaggerlowastdagger

lowastdagger

(b)

0

5

10

15

20

0

5

10

15

20

0

5

10

15

20

0

5

10

15

20

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

LPSLPSLPSLPS

lowast lowast lowast lowast

lowast

lowastlowast

lowast

lowastlowastlowastlowastlowastlowast

lowastdagger

lowastdaggerlowastdagger

lowastdagger

lowastdaggerlowastdagger

Rela

tive T

NF-120572

Rela

tive T

NF-120572

Rela

tive T

NF-120572

Rela

tive T

NF-120572

(c)

Figure 4 Effects of NJ fractions onmRNA levels of LPS-induced cytokine expression Peritoneal macrophages were treated with NJ fractions(120583gmL) for 1 h and then stimulated with LPS at indicated time points Cells were collected for real-time RT-PCR Total RNA (1 120583g) wasprepared for the real-time RT-PCR of (a) IL-1120573 (b) IL-6 and (c) TNF-120572 The values are mean plusmn SE of three independent experimentslowast

119875 lt 005 versus saline +119875 lt 005 versus LPS

35 Effects of NJ Fractions on LPS-Induced Endotoxin ShockTo verify effects of NJ fractions in vivo we used a model LPS-induced endotoxin shock NJ fractions (NJ-1 NJ-3 NJ-4 orNJ-6) were injected at the indicated doses After 3 h a lethaldose of LPS (375mgkg) was administered intraperitoneallyThe survival rate was recorded every 12 h after LPS treat-ment Generally LPS-injected septic mice died within 48 hConsistent with in vitro experiments NJ fractions exhibitedstrong anti-inflammatory activities in vivo as shown bysignificant inhibition of LPS-induced death in the treatedmice (Figure 7) In particular NJ-4 and NJ-6 showed themost dramatic protection against LPS-induced endotoxinshock these fractions prevented nearly all harmful effects ofLPS (Figures 7(c) and 7(d))

4 Discussion

Recently our laboratory reported the beneficial effects of NJon various diseases such as endotoxin shock pancreatitisand diabetes [6ndash10] Particularly in endotoxin shock NJshowed a marked preventive effect and strong therapeuticpotential against LPS challenge [7] Although many of ourstudies have indicated the benefits of NJ on inflamma-tory diseases which compounds of NJ could exhibit anti-inflammatory properties are unknown Thus in this paperbringing more bright insight to identify the constituent com-pounds from NJ we selected and assorted effective fractionsthat would possess anti-inflammatory potential Overall weobtained six fractions from NJ Using the fourth fraction

8 Evidence-Based Complementary and Alternative Medicine

Time (min)LPS

60301506030150NJ-1 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(a)

LPSTime (min) 60301506030150

NJ-3 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(b)

LPSTime (min) 60301506030150

NJ-4 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(c)

LPSTime (min) 60301506030150

NJ-6 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(d)

Figure 5 Effects of NJ fractions on MAPKs activation ((a)ndash(d)) Peritoneal macrophages were treated with NJ fractions (100120583gmL) for 1 hand then stimulated with LPS (500 ngmL) at indicated time points Cells were harvested for western blot Total cellular proteins (20120583g) wereresolved by SDS-PAGE transferred to PVDF membrane and detected with phospho-specific ERK 12 (4244 kDa) JNK (4654 kDa) andp38 (43 kDa) antibody ERK JNK and p38 were used as a loading control A representative western blot of three experiments is shown

we evaluated the protective effects on cerulein-induced acutepancreatitis (AP) However the sixth fraction remained to beexamined for potential to prevent an inflammatory responseto stimuli such as LPS Therefore in this study we used sixfractions isolated from NJ to examine the anti-inflammatoryactivity against LPS on murine macrophages

Generally sepsis is a life threatening condition caused bythe bodyrsquos response to bacterial products such as endotoxinwhich is often present with severe fever shock and respira-tory damage [12] Partially due to this phenomenon sepsisis often regarded as an uncontrolled inflammatory response[12] In the context of sepsis TNF-120572 IL-1120573 IL-6 and IL-8 arethemost frequently altered cytokines [13]When injected intoanimals these cytokines recapitulate many clinical and labo-ratory features of sepsis supporting the concept that sepsisrepresents a ldquocytokine stormrdquo Thus regulation of cytokinesmay prove valuable for the prevention and treatment ofsepsis In this study just as the total extract of NJ inhibitedthe production of cytokines [7] fractions 3 4 and 6 also

inhibited the production of cytokines (Figures 3 and 4)These results suggest that the ability of NJ to inhibit cytokineproduction may originate from fractions 3 4 andor 6

Using the fractions that were found to be effectiveagainst LPS we examined activation of MAPKs and NF-120581Bas possible regulatory mechanisms Generally it has beenreported that the production of proinflammatory mediatorsis modulated by the MAPKs and NF-120581B pathways Threemajor groups of MAPKs include the ERK JNK and p38which are all activated by phosphorylation [14ndash16] NF-120581Bactivation occurs mainly through the degradation of I120581Bthereby leading to subsequent release of NF-120581B dimers [17]Subsequently NF-120581B dimers translocate from the cytoplasmto the nucleus and activate the transcription of multiple 120581B-dependent target genes [17] In this study we examine thephosphorylation of MAPKs and degradation of I120581-B120572 NJ-1NJ-3 and NJ-4 inhibited the phosphorylation and activationof JNK and p38 but not ERK12 However NJ-6 inhibitednot only the activation of ERK12 JNK and p38 but also

Evidence-Based Complementary and Alternative Medicine 9

Time (min)LPS

60301506030150NJ-1 + LPS

120573-Actin

I120581-B120572

(a)

Time (min)LPS

60301506030150NJ-4 + LPS

120573-Actin

I120581-B120572

(b)

Time (min)LPS

60301506030150NJ-3 + LPS

120573-Actin

I120581-B120572

(c)

Time (min)LPS

60301506030150NJ-6 + LPS

120573-Actin

I120581-B120572

(d)

DAPI Merge

None

LPS

NJ-6

NF-120581B p65

(e)

Figure 6 Effects of NJ fractions onNF-120581B activation ((a)ndash(d)) Peritonealmacrophages were treatedwithNJ fractions (100120583gmL) for 1 h andthen stimulated with LPS (500 ngmL) at indicated time points The cells were harvested for western blot Total cellular proteins (20 120583g) wereresolved by SDS-PAGE transferred to PVDF membrane and detected with I120581-B120572 (40 kDa) antibody 120573-Actin was used as loading controlA representative western blot of three experiments is shown (e) Peritoneal macrophages were treated with NJ fractions (100120583gmL) for 1 hand then stimulated with LPS (500 ngmL) for 1 h Then nuclear translocation of NF-120581B p65 was examined using immunostaining NF-120581Bp65 was shown as red and the nucleus was shown as blue (DAPI) The values are means plusmn SE of three independent experiments lowast119875 lt 005versus saline +119875 lt 005 versus LPS

10 Evidence-Based Complementary and Alternative Medicine

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-1 (01mgkg) + LPS

NJ-1 (1mgkg) + LPSNJ-1 (10mgkg) + LPS

(a)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-3 (01mgkg) + LPS

NJ-3 (1mgkg) + LPSNJ-3 (10mgkg) + LPS

(b)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-4 (01mgkg) + LPS

NJ-4 (1mgkg) + LPSNJ-4 (10mgkg) + LPS

(c)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-6 (01mgkg) + LPS

NJ-6 (1mgkg) + LPSNJ-6 (10mgkg) + LPS

(d)

Figure 7 Effect of NJ fractions on LPS-induced endotoxin shock ((a)ndash(d)) Endotoxin shock was induced in 6ndash8-week-old female C57BL6mice as described in Section 2 NJ fractions were administered intraperitoneally to mice at 01 1 or 10mgkg 1 h before LPS injection ThenLPS (375mgkg) was injected to induce endotoxin shock The survival rates of endotoxin shock mice were monitored for survival for 120 hThis figure shows data for 10 mice per group

degradation of I120581-B120572 These results suggest that inhibitionof NO and cytokine production by NJ fractions is primarilythrough MAPKs or NF-120581B pathways

In this study we isolated two beneficial fractions of NJThe first one was NJ-4 which could show similar effectsto NJ total extract NJ-4 inhibited cytokine production andLPS-induced endotoxin shock similar to NJ total extract [7]Furthermore the regulatory mechanisms were similar NJtotal extract inhibited only MAPKs but NJ-4 inhibited theJNK and p38 Secondly NJ-6 showed dramatic inhibitionof LPS-induced inflammatory response Indeed NJ-6 inhib-ited the cytokine production and LPS-induced endotoxinshock dramatically However NJ-6 differed from total extractmechanistically NJ-6 inhibited the degradation of I120581-B120572and translocation of NF-120581B p65 which was not regulatedby the NJ total extract Further isolation of the NJ-4 andNJ-6 fractions could potentially yield a single compounddemonstrating inhibitory activity resembling that of NJ totalextract

In conclusion this study demonstrated which fractionsof NJ exhibit inhibitory activity against LPS-induced inflam-mation Among the 6 fractions isolated NJ-1 NJ-3 NJ-4 andNJ-6 showed the inhibition of NO and NJ-3 NJ-4 and NJ-6 showed the inhibition of cytokines In addition NJ-4 andNJ-6 exhibited dramatic prevention against LPS-endotoxinshock These results suggest that NJ-4 and NJ-6 may beeffective and beneficial candidates to ameliorate LPS-inducedinflammation

Abbreviations

ELISA Enzyme-linked immunosorbent assayERK Extracellular signal-regulated kinaseFBS Fetal bovine serumIL InterleukinJNK c-jun NH

2-terminal kinase

LPS LipopolysaccharideMAPKs Mitogen-activated protein kinases

Evidence-Based Complementary and Alternative Medicine 11

MTT 3-(45 Dimethylthiazol)-25-diphenyltetrazolium bromide

NF Nuclear factorNJ Nardostachys jatamansiNO Nitric oxidePCR Polymerase chain reactionTG ThioglycollateTLR Toll-like receptorTNF Tumor necrosis factor

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Gi-Sang Bae and Kwang-Ho Heo equally contributed to thiswork

Acknowledgments

This research was supported by a Basic Science ResearchProgram through theNational Research Foundation of Korea(NRF) fundedby theMinistryofEducation ScienceandTech-nology (NRF-2012R1A1A2040916 2012R1A5A2A35671257and 2012M2B2B1055246)

References

[1] C-S Sung and C-S Wong ldquoCellular mechanisms of neuroin-flammatory pain the role of interleukin-1120573rdquo Acta Anaesthesio-logica Taiwanica vol 45 no 2 pp 103ndash109 2007

[2] F O Martinez L Helming and S Gordon ldquoAlternative activa-tion of macrophages an immunologic functional perspectiverdquoAnnual Review of Immunology vol 27 pp 451ndash483 2009

[3] J D Schilling H M Machkovech L He A Diwan and J ESchaffer ldquoTLR4 activation under lipotoxic conditions leads tosynergistic macrophage cell death through a TRIF-dependentpathwayrdquo Journal of Immunology vol 190 no 3 pp 1285ndash12962013

[4] Y Ben-Neriah andM Karin ldquoInflammation meets cancer withNF-120581B as the matchmakerrdquo Nature Immunology vol 12 no 8pp 715ndash723 2011

[5] B C Bose Y Vijayvargiya and J N Bhatnagar ldquoNardostachysjatamansiDC a phyto-chemical study of its active constituentsrdquoIndian Journal of Medical Sciences vol 11 no 10 pp 799ndash8021957

[6] G-S Bae H-J Park D-Y Kim et al ldquoNardostachys jatamansiprotects against cerulein-induced acute pancreatitisrdquo Pancreasvol 39 no 4 pp 520ndash529 2010

[7] G-S Bae S-W Seo M-S Kim et al ldquoThe roots of Nar-dostachys jatamansi inhibits lipopolysaccharide-induced endo-toxin shockrdquo Journal of Natural Medicines vol 65 no 1 pp 63ndash72 2011

[8] G S Bae K C Park B S Koo et al ldquoThe inhibitory effectsof Nardostachys jatamansi on alcoholic chronic pancreatitisrdquoBMB Reports vol 45 no 7 pp 402ndash407 2012

[9] G S Bae K C Park B S Koo et al ldquoThe beneficial effectsof Nardostachys jatamansi extract on diet-induced severe acutepancreatitisrdquo Pancreas vol 42 no 2 pp 362ndash363 2013

[10] M-Y Song U-J Bae B-H Lee et al ldquoNardostachys jatamansiextract protects against cytokineinduced 120573-cell damage andstreptozotocin-induced diabetesrdquo World Journal of Gastroen-terology vol 16 no 26 pp 3249ndash3257 2010

[11] G S Bae M S Kim K C Park et al ldquoEffect of biologicallyactive fraction of Nardostachys jatamansi on cerulein-inducedacute pancreatitisrdquo World Journal of Gastroenterology vol 18no 25 pp 3223ndash3234 2012

[12] R S Hotchkiss and I E Karl ldquoThe pathophysiology andtreatment of sepsisrdquoThe New England Journal of Medicine vol348 no 2 pp 138ndash150 2003

[13] T S Blackwell and J W Christman ldquoSepsis and cytokinescurrent statusrdquo British Journal of Anaesthesia vol 77 no 1 pp110ndash117 1996

[14] H B Jijon W J Panenka K L Madsen and H G Par-sons ldquoMAP kinases contribute to IL-8 secretion by intestinalepithelial cells via a posttranscriptional mechanismrdquo AmericanJournal of PhysiologymdashCell Physiology vol 283 no 1 pp C31ndashC41 2002

[15] M Sanchez-Hidalgo A R Martın I Villegas and C Alarconde la Lastra ldquoRosiglitazone a PPAR120574 ligand modulates signaltransduction pathways during the development of acute TNBS-induced colitis in ratsrdquo European Journal of Pharmacology vol562 no 3 pp 247ndash258 2007

[16] F Scaldaferri M Sans S Vetrano et al ldquoThe role of MAPKin governing lymphocyte adhesion to and migration acrossthemicrovasculature in inflammatory bowel diseaserdquo EuropeanJournal of Immunology vol 39 no 1 pp 290ndash300 2009

[17] S Chae L Eckmann Y Miyamoto C Pothoulakis M KarinandM FKagnoff ldquoEpithelial cell I120581B-kinase120573has an importantprotective role in Clostridiumdifficile toxinA-inducedmucosalinjuryrdquo Journal of Immunology vol 177 no 2 pp 1214ndash12202006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

40

30

20

10

0

0 10 20 30 40 50 60

(mV

)

Time (min)

NJ-1

60933

85667

0 10 20 30 40 50 60

60

50

40

30

20

10

0

(mV

)

Time (min)

NJ-3

61000

85833

0 10 20 30 40 50 60

200

150

100

50

0

Time (min)

(mV

)

NJ-4

255333

(a) (b)

(c) (d)

0 10 20 30 40 50 60

0

200

150

100

50(m

V)

Time (min)

NJ-6

224833

262833

Scheme 2 The HPLC of NJ biological fractions

plate reader The number of NJ fraction-treated viable cellswas expressed as a percentage of the control (untreated cells)maintained for the same time period

27 Measurement of NO Concentration Murine peritonealmacrophages (2 times 105 cellswell) were pretreated with NJfractions for 1 h and then stimulated with LPS (500 ngmL)for 24 h To measure NO concentration 100 120583L aliquots wereremoved from conditioned media and incubated with anequal volume of Griess reagent at room temperature for10min The absorbance at 540 nm was then measured

28 ELISA Murine peritoneal macrophages (1 times 106cellswell) were stimulated with 500 ngmL of LPS andorvarious concentrations of NJ fraction for 24 h Culture super-natantswere collected and stored atminus80∘Cuntil use Cytokinelevels in supernatants were determined using a commercialsystem (RampD Systems) according to the manufacturerrsquosinstructionsTheELISAwas devised by coating 96-well plateswith antibodies specific for IL-1120573 IL-6 and TNF-120572 Coatedplates were washed with PBS containing 005 Tween-20 Allreagents used in this assay were incubated overnight at 4∘CRecombinant IL-1120573 IL-6 and TNF-120572 were diluted and usedas a standard Serial dilutions starting at 20 ngmL were usedto establish the standard curve Assay plates were exposedsequentially to biotinylated mouse IL-1120573 IL-6 and TNF-120572avidin peroxidase and a substrate solution of 221015840-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) containing30 hydrogen peroxide The plates were read at 405 nm

29 RNA Quantification Total cellular RNA was isolatedusing Trizol (Invitrogen Carlsbad CA USA) according tomanufacturerrsquos instructions Total RNA (1 120583g) was convertedto cDNA by 200 units of reverse transcriptase and 500 ng ofoligo-dT primer in 50mM Tris-HCl (pH 83) 75mM KCl3mMMgCl

2 10mM DTT and 1mM dNTPs at 42∘C for 1 h

The reaction was stopped by heating at 70∘C for 15min

210 Real-Time RT-PCR TaqMan quantitative RT-PCR wasperformed with a 7700 sequence detection system accord-ing to the manufacturerrsquos instructions (Applied BiosystemsFoster City CA USA) For each sample triplicate testreactions and a control reaction lacking reverse transcrip-tase were analyzed for expression of the gene of interestand results were normalized to those for the housekeep-ing gene hypoxanthine-guanine phosphoribosyltransferase(HPRT) Arbitrary expression units were calculated by divid-ing expression of the gene of interest by ribosomal pro-tein HPRT mRNA expression PCR was performed at anannealing temperature of 60∘C with 40 amplification cyclesThe commercial forward reverse and probe oligonucleotideprimers formultiplex real-timeTaqManPCRwere purchasedfrom ABI (Applied Biosystems Foster City CA USA)

211 Western Blot Whole cell lysates were prepared byboiling peritoneal macrophages in a sample buffer (625mMTris-HCl (pH 68) 2 sodium dodecyl sulfate 20 glyceroland 10 2-mercaptoethanol) Proteins in the cell lysateswere then separated by 10 SDS-PAGE and transferred to

4 Evidence-Based Complementary and Alternative Medicine

0

20

40

60

80

100

10 20 50 1000

20

40

60

80

100

10 20 50 1000

20

40

60

80

100

10 20 50 100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

NJ-2NJ-1 NJ-3minus minus

10 20 50 100 10 20 50 100NJ-5 NJ-6minus minus

minus

10 20 50 100NJ-4 minus

Figure 1 Cytotoxic effects of NJ fractions on murine peritoneal macrophages The effects of NJ fractions on cell viability were measured byMTT assay Peritoneal macrophages were incubated with or without NJ fractions at indicated doses (120583gmL) for 24 h The values are mean plusmnSE of three independent experiments

119875 lt 0001 versus saline

a nitrocellulose membraneThemembrane was then blockedwith 5 skim milk in PBS-Tween-20 for 2 h at room temper-ature and incubated overnight with antibodies against phos-phorylated ERK 12 phosphorylated JNK phosphorylatedp38 and I120581-B120572 After washing three times each blot wasincubated with a secondary antibody for 1 h and immunore-active bands were visualized using an enhanced chemilumi-nescence detection system (Amersham Piscataway NJ USA)according to the manufacturerrsquos recommended protocol

212 Immunostaining Peritoneal macrophages were platedin a chamber slide and incubated with 500 ngmL of LPSfor 1 h at 37∘C The cells were fixed in 4 paraformaldehydefor 30min at RT and washed three times with PBS Thecells were treated with 01 Triton X-100 for 15min at roomtemperature After washing the cells were incubated with ablocking serum for 1 h and incubated overnight with a 1 100dilution of primaryNF-120581Bp65 antibody (SantaCruzBiotech-nology Santa Cruz CA USA) The cells were then washedand incubated with a 1 2000 dilution of Alexafluor 568-conjugated goat anti-mouse IgG (Invitrogen USA) for 2 h ina dark room For nuclear staining the cells were incubatedwith a 1 1000 dilution of DAPI for 5minThe slide was finally

washed and mounted for microscopic examination Stainingwith NF-120581B p65 antibody exhibited red fluorescence whichwas detected by fluorescence microscopy

213 Animal Model of LPS-Induced Endotoxin Shock Endo-toxin shock was induced in 6- to 8-week-old female C57BL6mice by intraperitoneal (ip) injection of bacterial endotoxin(LPS from E coli serotype O55B5 375mgkg) At 1 h afterNJ fraction administration LPS (375mgsdotkgminus1) was injectedintraperitoneally Survival was monitored for 120 h Animaluse and relevant experimental procedures were conductedin accordance with the National Institutes of Health (NIH)Guidelines for the Care and Use of Laboratory Animals Allexperiments were approved by the Animal Care Committeeof Wonkwang University

214 Statistical Analysis The results are expressed as themean plusmn SE of independent experiments Two-way ANOVAwas used to analyze the statistical significance of resultsamong groups If results were found to be statistically sig-nificant post hoc analysis was performed using the Dun-can method as a multiple comparison among groups All

Evidence-Based Complementary and Alternative Medicine 5

0 0

5

10

15

20

25

10 20 50 100

5

10

15

20

25

0

5

10

15

20

25

0

5

10

15

20

25

0

5

10

15

20

25

0

5

10

15

20

25

LPS LPS LPS

LPS LPS LPS

NJ-1 minus minus 10 20 50 100NJ-2 minus minus 10 20 50 100NJ-3 minus minus

10 20 50 100NJ-6 minus minus10 20 50 100NJ-5 minus minus10 20 50 100NJ-4 minus minus

Nitr

ite (120583

M)

Nitr

ite (120583

M)

Nitr

ite (120583

M)

Nitr

ite (120583

M)

Nitr

ite (120583

M)

Nitr

ite (120583

M)

lowast lowast lowast lowast lowast lowast

lowastdagger lowastdagger

lowastdaggerlowastdagger

lowast

lowastdagger

lowastlowast lowastlowast lowast lowast

lowastdagger

lowast

lowastdaggerlowastdaggerlowastdaggerlowastdagger

lowastdagger

lowastdagger lowastdagger

lowastdaggerlowastdagger

lowastdagger

Figure 2 Effects of NJ fractions on LPS-induced NO production Peritoneal macrophages were pretreated with NJ fractions at indicateddoses (120583gmL) for 1 h and then stimulated with LPS (500 ngmL) for 24 h The effects of NJ fractions on LPS-induced NO production weremeasured by Griess assay The values are mean plusmn SE of three independent experiments lowast119875 lt 005 versus saline +119875 lt 005 versus LPS

statistical analyses were performed using SPSS version 100statistical analysis software

3 Results

31 Cytotoxicity of NJ Fractions in Peritoneal MacrophagesThe first approach to study the biological activity of anycompound or plant extract is to ensure lack of effect oncellular metabolism To determine whether fraction of NJaffects cell viability peritoneal macrophages were incubatedfor 24 h with varying concentrations of extract (120583gmL) andcell viability was evaluated by MTT assay NJ-1 NJ-2 NJ-3 and NJ-6 did not demonstrate any significant cytotoxiceffects on peritoneal macrophages However at a relativelyhigher dose NJ-4 and NJ-5 showed significant cytotoxicity( 119875 lt 0001) (Figure 1) Therefore because higher levels

of NJ-4 (ge100 120583gmL) and NJ-5 (ge50120583gmL) could affectcellular metabolism we excluded data of NJ-4 and NJ-5 atthese concentrations

32 Effects of NJ Fractions on LPS-Induced NO Produc-tion Next to examine the anti-inflammatory effect of NJfractions we measured NO production Murine peritonealmacrophages were pretreated with the indicated concentra-tions of NJ fractions for 1 h and then stimulated with LPS(500 ngmL) for 24 h As shown in Figure 2 LPS stimulationincreased NO production However pretreatment with NJ-1 NJ-3 NJ-4 and NJ-6 significantly inhibited LPS-inducedproduction of NO but not with NJ-2 and NJ-5 (Figure 2)

33 Effects of NJ Fractions on LPS-Induced ProinflammatoryCytokine Production To examine the effects of NJ fractions

6 Evidence-Based Complementary and Alternative Medicine

0

30

60

90

120

150

180

210

0

30

60

90

120

150

180

210

0

30

60

90

120

150

180

210

0

30

60

90

120

150

180

210

10 20 50 100NJ-1 minus minus 10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus

IL-1120573

(pg

mL)

IL-1120573

(pg

mL)

IL-1120573

(pg

mL)

IL-1120573

(pg

mL)

lowastlowastlowastlowastlowastlowast lowast lowastlowast lowast lowast lowast lowast

lowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdagger

(a)

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

IL-6

(ng

mL)

IL-6

(ng

mL)

IL-6

(ng

mL)

IL-6

(ng

mL)

lowast lowast lowast lowast lowastlowast

lowast

lowastlowastlowast lowast lowast lowast lowast

lowastdagger

lowastdaggerlowastdagger

lowastdaggerlowastdagger

lowastdagger

(b)

0

1

2

3

4

0

1

2

3

4

0

1

2

3

4

0

1

2

3

4

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

LPSLPSLPS LPS

TNF-120572

(ng

mL)

TNF-120572

(ng

mL)

TNF-120572

(ng

mL)

TNF-120572

(ng

mL)

lowastlowastlowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastdagger

lowastdagger

lowastdagger lowastdagger

lowastdagger

(c)

Figure 3 Effects of NJ fractions on LPS-induced cytokine productions Peritoneal macrophages were treated with NJ fractions (120583gmL) for1 h and then stimulated with LPS at indicated time pointsThe supernatants were harvested and the levels of (a) IL-1120573 (b) IL-6 and (c) TNF-120572were measured by ELISA The values are mean plusmn SE of three independent experiments lowast119875 lt 005 versus saline +119875 lt 005 versus LPS

on LPS-induced proinflammatory cytokine production peri-toneal macrophages were pretreated with NJ fractions at theindicated concentrations for 1 h and then stimulated with500 ngmL of LPS for 24 h In particular we treated cells withNJ-1 NJ-3 NJ-4 and NJ-6 fractions which demonstratedinhibition of NO production As shown in Figures 3 and 4protein and mRNA levels of IL-1120573 IL-6 and TNF-120572 wereincreased by LPS stimulation However pretreatment withNJ-3 NJ-4 and NJ-6 inhibited the production of IL-1120573 IL-6and TNF-120572 significantly but not with NJ-1 fraction (Figures 3and 4)

34 Effects of NJ Fractions on the Activation of MAPKs andNF-120581B To examine inhibitory mechanism(s) of NJ fractionson production of NO and cytokines MAPK and NF-120581B were

measured The cells were pretreated with NJ-1 (100 120583gmL)NJ-3 (100 120583gmL) NJ-4 (50 120583gmL) and NJ-6 (100 120583gmL)for 1 h and then stimulatedwith 500 ngmLof LPS for 0 15 30or 60min Pretreatment withNJ-1 NJ-3 or NJ-4 significantlyinhibited the LPS-induced activation of JNK and p38 but notERK 12 (Figures 5(a)ndash5(c)) In addition pretreatment withNJ-6 significantly inhibited the LPS-induced activation ofERK 12 JNK and p38 (Figure 5(d)) To determine whetherNJ fractions inhibit LPS-inducedNF-120581B activation we exam-ined degradation of I120581-B120572 and NF-120581B p65 translocationTheNJ-1 NJ-3 andNJ-4 fractions did not inhibit the degradationof I120581-B120572 (Figures 6(a)ndash6(c)) However NJ-6 inhibited thedegradation of I120581-B120572 and translocation of NF-120581B p65 intonucleus suggesting that NJ-6 may influence LPS-inducedNF-120581B activation (Figures 6(d) and 6(e))

Evidence-Based Complementary and Alternative Medicine 7

0

50

100

150

200

0

50

100

150

200

0

50

100

150

200

0

50

100

150

200

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

Rela

tive I

L-1120573

Rela

tive I

L-1120573

Rela

tive I

L-1120573

Rela

tive I

L-1120573 lowast

lowast

lowast

lowast

lowast

lowastlowast lowast lowast lowast lowast

lowastdagger

lowastdaggerlowastdaggerlowastdagger

lowastdagger

lowastdagger

lowastdaggerlowastdagger

lowastdagger

(a)

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

Rela

tive I

L-6

Rela

tive I

L-6

Rela

tive I

L-6

Rela

tive I

L-6

lowast lowast lowast lowast lowast lowastlowastlowast lowast

lowast

lowast

lowastdagger

lowastdaggerlowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdaggerlowastdagger

lowastdagger

(b)

0

5

10

15

20

0

5

10

15

20

0

5

10

15

20

0

5

10

15

20

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

LPSLPSLPSLPS

lowast lowast lowast lowast

lowast

lowastlowast

lowast

lowastlowastlowastlowastlowastlowast

lowastdagger

lowastdaggerlowastdagger

lowastdagger

lowastdaggerlowastdagger

Rela

tive T

NF-120572

Rela

tive T

NF-120572

Rela

tive T

NF-120572

Rela

tive T

NF-120572

(c)

Figure 4 Effects of NJ fractions onmRNA levels of LPS-induced cytokine expression Peritoneal macrophages were treated with NJ fractions(120583gmL) for 1 h and then stimulated with LPS at indicated time points Cells were collected for real-time RT-PCR Total RNA (1 120583g) wasprepared for the real-time RT-PCR of (a) IL-1120573 (b) IL-6 and (c) TNF-120572 The values are mean plusmn SE of three independent experimentslowast

119875 lt 005 versus saline +119875 lt 005 versus LPS

35 Effects of NJ Fractions on LPS-Induced Endotoxin ShockTo verify effects of NJ fractions in vivo we used a model LPS-induced endotoxin shock NJ fractions (NJ-1 NJ-3 NJ-4 orNJ-6) were injected at the indicated doses After 3 h a lethaldose of LPS (375mgkg) was administered intraperitoneallyThe survival rate was recorded every 12 h after LPS treat-ment Generally LPS-injected septic mice died within 48 hConsistent with in vitro experiments NJ fractions exhibitedstrong anti-inflammatory activities in vivo as shown bysignificant inhibition of LPS-induced death in the treatedmice (Figure 7) In particular NJ-4 and NJ-6 showed themost dramatic protection against LPS-induced endotoxinshock these fractions prevented nearly all harmful effects ofLPS (Figures 7(c) and 7(d))

4 Discussion

Recently our laboratory reported the beneficial effects of NJon various diseases such as endotoxin shock pancreatitisand diabetes [6ndash10] Particularly in endotoxin shock NJshowed a marked preventive effect and strong therapeuticpotential against LPS challenge [7] Although many of ourstudies have indicated the benefits of NJ on inflamma-tory diseases which compounds of NJ could exhibit anti-inflammatory properties are unknown Thus in this paperbringing more bright insight to identify the constituent com-pounds from NJ we selected and assorted effective fractionsthat would possess anti-inflammatory potential Overall weobtained six fractions from NJ Using the fourth fraction

8 Evidence-Based Complementary and Alternative Medicine

Time (min)LPS

60301506030150NJ-1 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(a)

LPSTime (min) 60301506030150

NJ-3 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(b)

LPSTime (min) 60301506030150

NJ-4 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(c)

LPSTime (min) 60301506030150

NJ-6 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(d)

Figure 5 Effects of NJ fractions on MAPKs activation ((a)ndash(d)) Peritoneal macrophages were treated with NJ fractions (100120583gmL) for 1 hand then stimulated with LPS (500 ngmL) at indicated time points Cells were harvested for western blot Total cellular proteins (20120583g) wereresolved by SDS-PAGE transferred to PVDF membrane and detected with phospho-specific ERK 12 (4244 kDa) JNK (4654 kDa) andp38 (43 kDa) antibody ERK JNK and p38 were used as a loading control A representative western blot of three experiments is shown

we evaluated the protective effects on cerulein-induced acutepancreatitis (AP) However the sixth fraction remained to beexamined for potential to prevent an inflammatory responseto stimuli such as LPS Therefore in this study we used sixfractions isolated from NJ to examine the anti-inflammatoryactivity against LPS on murine macrophages

Generally sepsis is a life threatening condition caused bythe bodyrsquos response to bacterial products such as endotoxinwhich is often present with severe fever shock and respira-tory damage [12] Partially due to this phenomenon sepsisis often regarded as an uncontrolled inflammatory response[12] In the context of sepsis TNF-120572 IL-1120573 IL-6 and IL-8 arethemost frequently altered cytokines [13]When injected intoanimals these cytokines recapitulate many clinical and labo-ratory features of sepsis supporting the concept that sepsisrepresents a ldquocytokine stormrdquo Thus regulation of cytokinesmay prove valuable for the prevention and treatment ofsepsis In this study just as the total extract of NJ inhibitedthe production of cytokines [7] fractions 3 4 and 6 also

inhibited the production of cytokines (Figures 3 and 4)These results suggest that the ability of NJ to inhibit cytokineproduction may originate from fractions 3 4 andor 6

Using the fractions that were found to be effectiveagainst LPS we examined activation of MAPKs and NF-120581Bas possible regulatory mechanisms Generally it has beenreported that the production of proinflammatory mediatorsis modulated by the MAPKs and NF-120581B pathways Threemajor groups of MAPKs include the ERK JNK and p38which are all activated by phosphorylation [14ndash16] NF-120581Bactivation occurs mainly through the degradation of I120581Bthereby leading to subsequent release of NF-120581B dimers [17]Subsequently NF-120581B dimers translocate from the cytoplasmto the nucleus and activate the transcription of multiple 120581B-dependent target genes [17] In this study we examine thephosphorylation of MAPKs and degradation of I120581-B120572 NJ-1NJ-3 and NJ-4 inhibited the phosphorylation and activationof JNK and p38 but not ERK12 However NJ-6 inhibitednot only the activation of ERK12 JNK and p38 but also

Evidence-Based Complementary and Alternative Medicine 9

Time (min)LPS

60301506030150NJ-1 + LPS

120573-Actin

I120581-B120572

(a)

Time (min)LPS

60301506030150NJ-4 + LPS

120573-Actin

I120581-B120572

(b)

Time (min)LPS

60301506030150NJ-3 + LPS

120573-Actin

I120581-B120572

(c)

Time (min)LPS

60301506030150NJ-6 + LPS

120573-Actin

I120581-B120572

(d)

DAPI Merge

None

LPS

NJ-6

NF-120581B p65

(e)

Figure 6 Effects of NJ fractions onNF-120581B activation ((a)ndash(d)) Peritonealmacrophages were treatedwithNJ fractions (100120583gmL) for 1 h andthen stimulated with LPS (500 ngmL) at indicated time points The cells were harvested for western blot Total cellular proteins (20 120583g) wereresolved by SDS-PAGE transferred to PVDF membrane and detected with I120581-B120572 (40 kDa) antibody 120573-Actin was used as loading controlA representative western blot of three experiments is shown (e) Peritoneal macrophages were treated with NJ fractions (100120583gmL) for 1 hand then stimulated with LPS (500 ngmL) for 1 h Then nuclear translocation of NF-120581B p65 was examined using immunostaining NF-120581Bp65 was shown as red and the nucleus was shown as blue (DAPI) The values are means plusmn SE of three independent experiments lowast119875 lt 005versus saline +119875 lt 005 versus LPS

10 Evidence-Based Complementary and Alternative Medicine

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-1 (01mgkg) + LPS

NJ-1 (1mgkg) + LPSNJ-1 (10mgkg) + LPS

(a)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-3 (01mgkg) + LPS

NJ-3 (1mgkg) + LPSNJ-3 (10mgkg) + LPS

(b)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-4 (01mgkg) + LPS

NJ-4 (1mgkg) + LPSNJ-4 (10mgkg) + LPS

(c)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-6 (01mgkg) + LPS

NJ-6 (1mgkg) + LPSNJ-6 (10mgkg) + LPS

(d)

Figure 7 Effect of NJ fractions on LPS-induced endotoxin shock ((a)ndash(d)) Endotoxin shock was induced in 6ndash8-week-old female C57BL6mice as described in Section 2 NJ fractions were administered intraperitoneally to mice at 01 1 or 10mgkg 1 h before LPS injection ThenLPS (375mgkg) was injected to induce endotoxin shock The survival rates of endotoxin shock mice were monitored for survival for 120 hThis figure shows data for 10 mice per group

degradation of I120581-B120572 These results suggest that inhibitionof NO and cytokine production by NJ fractions is primarilythrough MAPKs or NF-120581B pathways

In this study we isolated two beneficial fractions of NJThe first one was NJ-4 which could show similar effectsto NJ total extract NJ-4 inhibited cytokine production andLPS-induced endotoxin shock similar to NJ total extract [7]Furthermore the regulatory mechanisms were similar NJtotal extract inhibited only MAPKs but NJ-4 inhibited theJNK and p38 Secondly NJ-6 showed dramatic inhibitionof LPS-induced inflammatory response Indeed NJ-6 inhib-ited the cytokine production and LPS-induced endotoxinshock dramatically However NJ-6 differed from total extractmechanistically NJ-6 inhibited the degradation of I120581-B120572and translocation of NF-120581B p65 which was not regulatedby the NJ total extract Further isolation of the NJ-4 andNJ-6 fractions could potentially yield a single compounddemonstrating inhibitory activity resembling that of NJ totalextract

In conclusion this study demonstrated which fractionsof NJ exhibit inhibitory activity against LPS-induced inflam-mation Among the 6 fractions isolated NJ-1 NJ-3 NJ-4 andNJ-6 showed the inhibition of NO and NJ-3 NJ-4 and NJ-6 showed the inhibition of cytokines In addition NJ-4 andNJ-6 exhibited dramatic prevention against LPS-endotoxinshock These results suggest that NJ-4 and NJ-6 may beeffective and beneficial candidates to ameliorate LPS-inducedinflammation

Abbreviations

ELISA Enzyme-linked immunosorbent assayERK Extracellular signal-regulated kinaseFBS Fetal bovine serumIL InterleukinJNK c-jun NH

2-terminal kinase

LPS LipopolysaccharideMAPKs Mitogen-activated protein kinases

Evidence-Based Complementary and Alternative Medicine 11

MTT 3-(45 Dimethylthiazol)-25-diphenyltetrazolium bromide

NF Nuclear factorNJ Nardostachys jatamansiNO Nitric oxidePCR Polymerase chain reactionTG ThioglycollateTLR Toll-like receptorTNF Tumor necrosis factor

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Gi-Sang Bae and Kwang-Ho Heo equally contributed to thiswork

Acknowledgments

This research was supported by a Basic Science ResearchProgram through theNational Research Foundation of Korea(NRF) fundedby theMinistryofEducation ScienceandTech-nology (NRF-2012R1A1A2040916 2012R1A5A2A35671257and 2012M2B2B1055246)

References

[1] C-S Sung and C-S Wong ldquoCellular mechanisms of neuroin-flammatory pain the role of interleukin-1120573rdquo Acta Anaesthesio-logica Taiwanica vol 45 no 2 pp 103ndash109 2007

[2] F O Martinez L Helming and S Gordon ldquoAlternative activa-tion of macrophages an immunologic functional perspectiverdquoAnnual Review of Immunology vol 27 pp 451ndash483 2009

[3] J D Schilling H M Machkovech L He A Diwan and J ESchaffer ldquoTLR4 activation under lipotoxic conditions leads tosynergistic macrophage cell death through a TRIF-dependentpathwayrdquo Journal of Immunology vol 190 no 3 pp 1285ndash12962013

[4] Y Ben-Neriah andM Karin ldquoInflammation meets cancer withNF-120581B as the matchmakerrdquo Nature Immunology vol 12 no 8pp 715ndash723 2011

[5] B C Bose Y Vijayvargiya and J N Bhatnagar ldquoNardostachysjatamansiDC a phyto-chemical study of its active constituentsrdquoIndian Journal of Medical Sciences vol 11 no 10 pp 799ndash8021957

[6] G-S Bae H-J Park D-Y Kim et al ldquoNardostachys jatamansiprotects against cerulein-induced acute pancreatitisrdquo Pancreasvol 39 no 4 pp 520ndash529 2010

[7] G-S Bae S-W Seo M-S Kim et al ldquoThe roots of Nar-dostachys jatamansi inhibits lipopolysaccharide-induced endo-toxin shockrdquo Journal of Natural Medicines vol 65 no 1 pp 63ndash72 2011

[8] G S Bae K C Park B S Koo et al ldquoThe inhibitory effectsof Nardostachys jatamansi on alcoholic chronic pancreatitisrdquoBMB Reports vol 45 no 7 pp 402ndash407 2012

[9] G S Bae K C Park B S Koo et al ldquoThe beneficial effectsof Nardostachys jatamansi extract on diet-induced severe acutepancreatitisrdquo Pancreas vol 42 no 2 pp 362ndash363 2013

[10] M-Y Song U-J Bae B-H Lee et al ldquoNardostachys jatamansiextract protects against cytokineinduced 120573-cell damage andstreptozotocin-induced diabetesrdquo World Journal of Gastroen-terology vol 16 no 26 pp 3249ndash3257 2010

[11] G S Bae M S Kim K C Park et al ldquoEffect of biologicallyactive fraction of Nardostachys jatamansi on cerulein-inducedacute pancreatitisrdquo World Journal of Gastroenterology vol 18no 25 pp 3223ndash3234 2012

[12] R S Hotchkiss and I E Karl ldquoThe pathophysiology andtreatment of sepsisrdquoThe New England Journal of Medicine vol348 no 2 pp 138ndash150 2003

[13] T S Blackwell and J W Christman ldquoSepsis and cytokinescurrent statusrdquo British Journal of Anaesthesia vol 77 no 1 pp110ndash117 1996

[14] H B Jijon W J Panenka K L Madsen and H G Par-sons ldquoMAP kinases contribute to IL-8 secretion by intestinalepithelial cells via a posttranscriptional mechanismrdquo AmericanJournal of PhysiologymdashCell Physiology vol 283 no 1 pp C31ndashC41 2002

[15] M Sanchez-Hidalgo A R Martın I Villegas and C Alarconde la Lastra ldquoRosiglitazone a PPAR120574 ligand modulates signaltransduction pathways during the development of acute TNBS-induced colitis in ratsrdquo European Journal of Pharmacology vol562 no 3 pp 247ndash258 2007

[16] F Scaldaferri M Sans S Vetrano et al ldquoThe role of MAPKin governing lymphocyte adhesion to and migration acrossthemicrovasculature in inflammatory bowel diseaserdquo EuropeanJournal of Immunology vol 39 no 1 pp 290ndash300 2009

[17] S Chae L Eckmann Y Miyamoto C Pothoulakis M KarinandM FKagnoff ldquoEpithelial cell I120581B-kinase120573has an importantprotective role in Clostridiumdifficile toxinA-inducedmucosalinjuryrdquo Journal of Immunology vol 177 no 2 pp 1214ndash12202006

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

0

20

40

60

80

100

10 20 50 1000

20

40

60

80

100

10 20 50 1000

20

40

60

80

100

10 20 50 100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

Cel

l via

bilit

y (

)

NJ-2NJ-1 NJ-3minus minus

10 20 50 100 10 20 50 100NJ-5 NJ-6minus minus

minus

10 20 50 100NJ-4 minus

Figure 1 Cytotoxic effects of NJ fractions on murine peritoneal macrophages The effects of NJ fractions on cell viability were measured byMTT assay Peritoneal macrophages were incubated with or without NJ fractions at indicated doses (120583gmL) for 24 h The values are mean plusmnSE of three independent experiments

119875 lt 0001 versus saline

a nitrocellulose membraneThemembrane was then blockedwith 5 skim milk in PBS-Tween-20 for 2 h at room temper-ature and incubated overnight with antibodies against phos-phorylated ERK 12 phosphorylated JNK phosphorylatedp38 and I120581-B120572 After washing three times each blot wasincubated with a secondary antibody for 1 h and immunore-active bands were visualized using an enhanced chemilumi-nescence detection system (Amersham Piscataway NJ USA)according to the manufacturerrsquos recommended protocol

212 Immunostaining Peritoneal macrophages were platedin a chamber slide and incubated with 500 ngmL of LPSfor 1 h at 37∘C The cells were fixed in 4 paraformaldehydefor 30min at RT and washed three times with PBS Thecells were treated with 01 Triton X-100 for 15min at roomtemperature After washing the cells were incubated with ablocking serum for 1 h and incubated overnight with a 1 100dilution of primaryNF-120581Bp65 antibody (SantaCruzBiotech-nology Santa Cruz CA USA) The cells were then washedand incubated with a 1 2000 dilution of Alexafluor 568-conjugated goat anti-mouse IgG (Invitrogen USA) for 2 h ina dark room For nuclear staining the cells were incubatedwith a 1 1000 dilution of DAPI for 5minThe slide was finally

washed and mounted for microscopic examination Stainingwith NF-120581B p65 antibody exhibited red fluorescence whichwas detected by fluorescence microscopy

213 Animal Model of LPS-Induced Endotoxin Shock Endo-toxin shock was induced in 6- to 8-week-old female C57BL6mice by intraperitoneal (ip) injection of bacterial endotoxin(LPS from E coli serotype O55B5 375mgkg) At 1 h afterNJ fraction administration LPS (375mgsdotkgminus1) was injectedintraperitoneally Survival was monitored for 120 h Animaluse and relevant experimental procedures were conductedin accordance with the National Institutes of Health (NIH)Guidelines for the Care and Use of Laboratory Animals Allexperiments were approved by the Animal Care Committeeof Wonkwang University

214 Statistical Analysis The results are expressed as themean plusmn SE of independent experiments Two-way ANOVAwas used to analyze the statistical significance of resultsamong groups If results were found to be statistically sig-nificant post hoc analysis was performed using the Dun-can method as a multiple comparison among groups All

Evidence-Based Complementary and Alternative Medicine 5

0 0

5

10

15

20

25

10 20 50 100

5

10

15

20

25

0

5

10

15

20

25

0

5

10

15

20

25

0

5

10

15

20

25

0

5

10

15

20

25

LPS LPS LPS

LPS LPS LPS

NJ-1 minus minus 10 20 50 100NJ-2 minus minus 10 20 50 100NJ-3 minus minus

10 20 50 100NJ-6 minus minus10 20 50 100NJ-5 minus minus10 20 50 100NJ-4 minus minus

Nitr

ite (120583

M)

Nitr

ite (120583

M)

Nitr

ite (120583

M)

Nitr

ite (120583

M)

Nitr

ite (120583

M)

Nitr

ite (120583

M)

lowast lowast lowast lowast lowast lowast

lowastdagger lowastdagger

lowastdaggerlowastdagger

lowast

lowastdagger

lowastlowast lowastlowast lowast lowast

lowastdagger

lowast

lowastdaggerlowastdaggerlowastdaggerlowastdagger

lowastdagger

lowastdagger lowastdagger

lowastdaggerlowastdagger

lowastdagger

Figure 2 Effects of NJ fractions on LPS-induced NO production Peritoneal macrophages were pretreated with NJ fractions at indicateddoses (120583gmL) for 1 h and then stimulated with LPS (500 ngmL) for 24 h The effects of NJ fractions on LPS-induced NO production weremeasured by Griess assay The values are mean plusmn SE of three independent experiments lowast119875 lt 005 versus saline +119875 lt 005 versus LPS

statistical analyses were performed using SPSS version 100statistical analysis software

3 Results

31 Cytotoxicity of NJ Fractions in Peritoneal MacrophagesThe first approach to study the biological activity of anycompound or plant extract is to ensure lack of effect oncellular metabolism To determine whether fraction of NJaffects cell viability peritoneal macrophages were incubatedfor 24 h with varying concentrations of extract (120583gmL) andcell viability was evaluated by MTT assay NJ-1 NJ-2 NJ-3 and NJ-6 did not demonstrate any significant cytotoxiceffects on peritoneal macrophages However at a relativelyhigher dose NJ-4 and NJ-5 showed significant cytotoxicity( 119875 lt 0001) (Figure 1) Therefore because higher levels

of NJ-4 (ge100 120583gmL) and NJ-5 (ge50120583gmL) could affectcellular metabolism we excluded data of NJ-4 and NJ-5 atthese concentrations

32 Effects of NJ Fractions on LPS-Induced NO Produc-tion Next to examine the anti-inflammatory effect of NJfractions we measured NO production Murine peritonealmacrophages were pretreated with the indicated concentra-tions of NJ fractions for 1 h and then stimulated with LPS(500 ngmL) for 24 h As shown in Figure 2 LPS stimulationincreased NO production However pretreatment with NJ-1 NJ-3 NJ-4 and NJ-6 significantly inhibited LPS-inducedproduction of NO but not with NJ-2 and NJ-5 (Figure 2)

33 Effects of NJ Fractions on LPS-Induced ProinflammatoryCytokine Production To examine the effects of NJ fractions

6 Evidence-Based Complementary and Alternative Medicine

0

30

60

90

120

150

180

210

0

30

60

90

120

150

180

210

0

30

60

90

120

150

180

210

0

30

60

90

120

150

180

210

10 20 50 100NJ-1 minus minus 10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus

IL-1120573

(pg

mL)

IL-1120573

(pg

mL)

IL-1120573

(pg

mL)

IL-1120573

(pg

mL)

lowastlowastlowastlowastlowastlowast lowast lowastlowast lowast lowast lowast lowast

lowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdagger

(a)

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

IL-6

(ng

mL)

IL-6

(ng

mL)

IL-6

(ng

mL)

IL-6

(ng

mL)

lowast lowast lowast lowast lowastlowast

lowast

lowastlowastlowast lowast lowast lowast lowast

lowastdagger

lowastdaggerlowastdagger

lowastdaggerlowastdagger

lowastdagger

(b)

0

1

2

3

4

0

1

2

3

4

0

1

2

3

4

0

1

2

3

4

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

LPSLPSLPS LPS

TNF-120572

(ng

mL)

TNF-120572

(ng

mL)

TNF-120572

(ng

mL)

TNF-120572

(ng

mL)

lowastlowastlowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastdagger

lowastdagger

lowastdagger lowastdagger

lowastdagger

(c)

Figure 3 Effects of NJ fractions on LPS-induced cytokine productions Peritoneal macrophages were treated with NJ fractions (120583gmL) for1 h and then stimulated with LPS at indicated time pointsThe supernatants were harvested and the levels of (a) IL-1120573 (b) IL-6 and (c) TNF-120572were measured by ELISA The values are mean plusmn SE of three independent experiments lowast119875 lt 005 versus saline +119875 lt 005 versus LPS

on LPS-induced proinflammatory cytokine production peri-toneal macrophages were pretreated with NJ fractions at theindicated concentrations for 1 h and then stimulated with500 ngmL of LPS for 24 h In particular we treated cells withNJ-1 NJ-3 NJ-4 and NJ-6 fractions which demonstratedinhibition of NO production As shown in Figures 3 and 4protein and mRNA levels of IL-1120573 IL-6 and TNF-120572 wereincreased by LPS stimulation However pretreatment withNJ-3 NJ-4 and NJ-6 inhibited the production of IL-1120573 IL-6and TNF-120572 significantly but not with NJ-1 fraction (Figures 3and 4)

34 Effects of NJ Fractions on the Activation of MAPKs andNF-120581B To examine inhibitory mechanism(s) of NJ fractionson production of NO and cytokines MAPK and NF-120581B were

measured The cells were pretreated with NJ-1 (100 120583gmL)NJ-3 (100 120583gmL) NJ-4 (50 120583gmL) and NJ-6 (100 120583gmL)for 1 h and then stimulatedwith 500 ngmLof LPS for 0 15 30or 60min Pretreatment withNJ-1 NJ-3 or NJ-4 significantlyinhibited the LPS-induced activation of JNK and p38 but notERK 12 (Figures 5(a)ndash5(c)) In addition pretreatment withNJ-6 significantly inhibited the LPS-induced activation ofERK 12 JNK and p38 (Figure 5(d)) To determine whetherNJ fractions inhibit LPS-inducedNF-120581B activation we exam-ined degradation of I120581-B120572 and NF-120581B p65 translocationTheNJ-1 NJ-3 andNJ-4 fractions did not inhibit the degradationof I120581-B120572 (Figures 6(a)ndash6(c)) However NJ-6 inhibited thedegradation of I120581-B120572 and translocation of NF-120581B p65 intonucleus suggesting that NJ-6 may influence LPS-inducedNF-120581B activation (Figures 6(d) and 6(e))

Evidence-Based Complementary and Alternative Medicine 7

0

50

100

150

200

0

50

100

150

200

0

50

100

150

200

0

50

100

150

200

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

Rela

tive I

L-1120573

Rela

tive I

L-1120573

Rela

tive I

L-1120573

Rela

tive I

L-1120573 lowast

lowast

lowast

lowast

lowast

lowastlowast lowast lowast lowast lowast

lowastdagger

lowastdaggerlowastdaggerlowastdagger

lowastdagger

lowastdagger

lowastdaggerlowastdagger

lowastdagger

(a)

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

Rela

tive I

L-6

Rela

tive I

L-6

Rela

tive I

L-6

Rela

tive I

L-6

lowast lowast lowast lowast lowast lowastlowastlowast lowast

lowast

lowast

lowastdagger

lowastdaggerlowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdaggerlowastdagger

lowastdagger

(b)

0

5

10

15

20

0

5

10

15

20

0

5

10

15

20

0

5

10

15

20

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

LPSLPSLPSLPS

lowast lowast lowast lowast

lowast

lowastlowast

lowast

lowastlowastlowastlowastlowastlowast

lowastdagger

lowastdaggerlowastdagger

lowastdagger

lowastdaggerlowastdagger

Rela

tive T

NF-120572

Rela

tive T

NF-120572

Rela

tive T

NF-120572

Rela

tive T

NF-120572

(c)

Figure 4 Effects of NJ fractions onmRNA levels of LPS-induced cytokine expression Peritoneal macrophages were treated with NJ fractions(120583gmL) for 1 h and then stimulated with LPS at indicated time points Cells were collected for real-time RT-PCR Total RNA (1 120583g) wasprepared for the real-time RT-PCR of (a) IL-1120573 (b) IL-6 and (c) TNF-120572 The values are mean plusmn SE of three independent experimentslowast

119875 lt 005 versus saline +119875 lt 005 versus LPS

35 Effects of NJ Fractions on LPS-Induced Endotoxin ShockTo verify effects of NJ fractions in vivo we used a model LPS-induced endotoxin shock NJ fractions (NJ-1 NJ-3 NJ-4 orNJ-6) were injected at the indicated doses After 3 h a lethaldose of LPS (375mgkg) was administered intraperitoneallyThe survival rate was recorded every 12 h after LPS treat-ment Generally LPS-injected septic mice died within 48 hConsistent with in vitro experiments NJ fractions exhibitedstrong anti-inflammatory activities in vivo as shown bysignificant inhibition of LPS-induced death in the treatedmice (Figure 7) In particular NJ-4 and NJ-6 showed themost dramatic protection against LPS-induced endotoxinshock these fractions prevented nearly all harmful effects ofLPS (Figures 7(c) and 7(d))

4 Discussion

Recently our laboratory reported the beneficial effects of NJon various diseases such as endotoxin shock pancreatitisand diabetes [6ndash10] Particularly in endotoxin shock NJshowed a marked preventive effect and strong therapeuticpotential against LPS challenge [7] Although many of ourstudies have indicated the benefits of NJ on inflamma-tory diseases which compounds of NJ could exhibit anti-inflammatory properties are unknown Thus in this paperbringing more bright insight to identify the constituent com-pounds from NJ we selected and assorted effective fractionsthat would possess anti-inflammatory potential Overall weobtained six fractions from NJ Using the fourth fraction

8 Evidence-Based Complementary and Alternative Medicine

Time (min)LPS

60301506030150NJ-1 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(a)

LPSTime (min) 60301506030150

NJ-3 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(b)

LPSTime (min) 60301506030150

NJ-4 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(c)

LPSTime (min) 60301506030150

NJ-6 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(d)

Figure 5 Effects of NJ fractions on MAPKs activation ((a)ndash(d)) Peritoneal macrophages were treated with NJ fractions (100120583gmL) for 1 hand then stimulated with LPS (500 ngmL) at indicated time points Cells were harvested for western blot Total cellular proteins (20120583g) wereresolved by SDS-PAGE transferred to PVDF membrane and detected with phospho-specific ERK 12 (4244 kDa) JNK (4654 kDa) andp38 (43 kDa) antibody ERK JNK and p38 were used as a loading control A representative western blot of three experiments is shown

we evaluated the protective effects on cerulein-induced acutepancreatitis (AP) However the sixth fraction remained to beexamined for potential to prevent an inflammatory responseto stimuli such as LPS Therefore in this study we used sixfractions isolated from NJ to examine the anti-inflammatoryactivity against LPS on murine macrophages

Generally sepsis is a life threatening condition caused bythe bodyrsquos response to bacterial products such as endotoxinwhich is often present with severe fever shock and respira-tory damage [12] Partially due to this phenomenon sepsisis often regarded as an uncontrolled inflammatory response[12] In the context of sepsis TNF-120572 IL-1120573 IL-6 and IL-8 arethemost frequently altered cytokines [13]When injected intoanimals these cytokines recapitulate many clinical and labo-ratory features of sepsis supporting the concept that sepsisrepresents a ldquocytokine stormrdquo Thus regulation of cytokinesmay prove valuable for the prevention and treatment ofsepsis In this study just as the total extract of NJ inhibitedthe production of cytokines [7] fractions 3 4 and 6 also

inhibited the production of cytokines (Figures 3 and 4)These results suggest that the ability of NJ to inhibit cytokineproduction may originate from fractions 3 4 andor 6

Using the fractions that were found to be effectiveagainst LPS we examined activation of MAPKs and NF-120581Bas possible regulatory mechanisms Generally it has beenreported that the production of proinflammatory mediatorsis modulated by the MAPKs and NF-120581B pathways Threemajor groups of MAPKs include the ERK JNK and p38which are all activated by phosphorylation [14ndash16] NF-120581Bactivation occurs mainly through the degradation of I120581Bthereby leading to subsequent release of NF-120581B dimers [17]Subsequently NF-120581B dimers translocate from the cytoplasmto the nucleus and activate the transcription of multiple 120581B-dependent target genes [17] In this study we examine thephosphorylation of MAPKs and degradation of I120581-B120572 NJ-1NJ-3 and NJ-4 inhibited the phosphorylation and activationof JNK and p38 but not ERK12 However NJ-6 inhibitednot only the activation of ERK12 JNK and p38 but also

Evidence-Based Complementary and Alternative Medicine 9

Time (min)LPS

60301506030150NJ-1 + LPS

120573-Actin

I120581-B120572

(a)

Time (min)LPS

60301506030150NJ-4 + LPS

120573-Actin

I120581-B120572

(b)

Time (min)LPS

60301506030150NJ-3 + LPS

120573-Actin

I120581-B120572

(c)

Time (min)LPS

60301506030150NJ-6 + LPS

120573-Actin

I120581-B120572

(d)

DAPI Merge

None

LPS

NJ-6

NF-120581B p65

(e)

Figure 6 Effects of NJ fractions onNF-120581B activation ((a)ndash(d)) Peritonealmacrophages were treatedwithNJ fractions (100120583gmL) for 1 h andthen stimulated with LPS (500 ngmL) at indicated time points The cells were harvested for western blot Total cellular proteins (20 120583g) wereresolved by SDS-PAGE transferred to PVDF membrane and detected with I120581-B120572 (40 kDa) antibody 120573-Actin was used as loading controlA representative western blot of three experiments is shown (e) Peritoneal macrophages were treated with NJ fractions (100120583gmL) for 1 hand then stimulated with LPS (500 ngmL) for 1 h Then nuclear translocation of NF-120581B p65 was examined using immunostaining NF-120581Bp65 was shown as red and the nucleus was shown as blue (DAPI) The values are means plusmn SE of three independent experiments lowast119875 lt 005versus saline +119875 lt 005 versus LPS

10 Evidence-Based Complementary and Alternative Medicine

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-1 (01mgkg) + LPS

NJ-1 (1mgkg) + LPSNJ-1 (10mgkg) + LPS

(a)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-3 (01mgkg) + LPS

NJ-3 (1mgkg) + LPSNJ-3 (10mgkg) + LPS

(b)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-4 (01mgkg) + LPS

NJ-4 (1mgkg) + LPSNJ-4 (10mgkg) + LPS

(c)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-6 (01mgkg) + LPS

NJ-6 (1mgkg) + LPSNJ-6 (10mgkg) + LPS

(d)

Figure 7 Effect of NJ fractions on LPS-induced endotoxin shock ((a)ndash(d)) Endotoxin shock was induced in 6ndash8-week-old female C57BL6mice as described in Section 2 NJ fractions were administered intraperitoneally to mice at 01 1 or 10mgkg 1 h before LPS injection ThenLPS (375mgkg) was injected to induce endotoxin shock The survival rates of endotoxin shock mice were monitored for survival for 120 hThis figure shows data for 10 mice per group

degradation of I120581-B120572 These results suggest that inhibitionof NO and cytokine production by NJ fractions is primarilythrough MAPKs or NF-120581B pathways

In this study we isolated two beneficial fractions of NJThe first one was NJ-4 which could show similar effectsto NJ total extract NJ-4 inhibited cytokine production andLPS-induced endotoxin shock similar to NJ total extract [7]Furthermore the regulatory mechanisms were similar NJtotal extract inhibited only MAPKs but NJ-4 inhibited theJNK and p38 Secondly NJ-6 showed dramatic inhibitionof LPS-induced inflammatory response Indeed NJ-6 inhib-ited the cytokine production and LPS-induced endotoxinshock dramatically However NJ-6 differed from total extractmechanistically NJ-6 inhibited the degradation of I120581-B120572and translocation of NF-120581B p65 which was not regulatedby the NJ total extract Further isolation of the NJ-4 andNJ-6 fractions could potentially yield a single compounddemonstrating inhibitory activity resembling that of NJ totalextract

In conclusion this study demonstrated which fractionsof NJ exhibit inhibitory activity against LPS-induced inflam-mation Among the 6 fractions isolated NJ-1 NJ-3 NJ-4 andNJ-6 showed the inhibition of NO and NJ-3 NJ-4 and NJ-6 showed the inhibition of cytokines In addition NJ-4 andNJ-6 exhibited dramatic prevention against LPS-endotoxinshock These results suggest that NJ-4 and NJ-6 may beeffective and beneficial candidates to ameliorate LPS-inducedinflammation

Abbreviations

ELISA Enzyme-linked immunosorbent assayERK Extracellular signal-regulated kinaseFBS Fetal bovine serumIL InterleukinJNK c-jun NH

2-terminal kinase

LPS LipopolysaccharideMAPKs Mitogen-activated protein kinases

Evidence-Based Complementary and Alternative Medicine 11

MTT 3-(45 Dimethylthiazol)-25-diphenyltetrazolium bromide

NF Nuclear factorNJ Nardostachys jatamansiNO Nitric oxidePCR Polymerase chain reactionTG ThioglycollateTLR Toll-like receptorTNF Tumor necrosis factor

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Gi-Sang Bae and Kwang-Ho Heo equally contributed to thiswork

Acknowledgments

This research was supported by a Basic Science ResearchProgram through theNational Research Foundation of Korea(NRF) fundedby theMinistryofEducation ScienceandTech-nology (NRF-2012R1A1A2040916 2012R1A5A2A35671257and 2012M2B2B1055246)

References

[1] C-S Sung and C-S Wong ldquoCellular mechanisms of neuroin-flammatory pain the role of interleukin-1120573rdquo Acta Anaesthesio-logica Taiwanica vol 45 no 2 pp 103ndash109 2007

[2] F O Martinez L Helming and S Gordon ldquoAlternative activa-tion of macrophages an immunologic functional perspectiverdquoAnnual Review of Immunology vol 27 pp 451ndash483 2009

[3] J D Schilling H M Machkovech L He A Diwan and J ESchaffer ldquoTLR4 activation under lipotoxic conditions leads tosynergistic macrophage cell death through a TRIF-dependentpathwayrdquo Journal of Immunology vol 190 no 3 pp 1285ndash12962013

[4] Y Ben-Neriah andM Karin ldquoInflammation meets cancer withNF-120581B as the matchmakerrdquo Nature Immunology vol 12 no 8pp 715ndash723 2011

[5] B C Bose Y Vijayvargiya and J N Bhatnagar ldquoNardostachysjatamansiDC a phyto-chemical study of its active constituentsrdquoIndian Journal of Medical Sciences vol 11 no 10 pp 799ndash8021957

[6] G-S Bae H-J Park D-Y Kim et al ldquoNardostachys jatamansiprotects against cerulein-induced acute pancreatitisrdquo Pancreasvol 39 no 4 pp 520ndash529 2010

[7] G-S Bae S-W Seo M-S Kim et al ldquoThe roots of Nar-dostachys jatamansi inhibits lipopolysaccharide-induced endo-toxin shockrdquo Journal of Natural Medicines vol 65 no 1 pp 63ndash72 2011

[8] G S Bae K C Park B S Koo et al ldquoThe inhibitory effectsof Nardostachys jatamansi on alcoholic chronic pancreatitisrdquoBMB Reports vol 45 no 7 pp 402ndash407 2012

[9] G S Bae K C Park B S Koo et al ldquoThe beneficial effectsof Nardostachys jatamansi extract on diet-induced severe acutepancreatitisrdquo Pancreas vol 42 no 2 pp 362ndash363 2013

[10] M-Y Song U-J Bae B-H Lee et al ldquoNardostachys jatamansiextract protects against cytokineinduced 120573-cell damage andstreptozotocin-induced diabetesrdquo World Journal of Gastroen-terology vol 16 no 26 pp 3249ndash3257 2010

[11] G S Bae M S Kim K C Park et al ldquoEffect of biologicallyactive fraction of Nardostachys jatamansi on cerulein-inducedacute pancreatitisrdquo World Journal of Gastroenterology vol 18no 25 pp 3223ndash3234 2012

[12] R S Hotchkiss and I E Karl ldquoThe pathophysiology andtreatment of sepsisrdquoThe New England Journal of Medicine vol348 no 2 pp 138ndash150 2003

[13] T S Blackwell and J W Christman ldquoSepsis and cytokinescurrent statusrdquo British Journal of Anaesthesia vol 77 no 1 pp110ndash117 1996

[14] H B Jijon W J Panenka K L Madsen and H G Par-sons ldquoMAP kinases contribute to IL-8 secretion by intestinalepithelial cells via a posttranscriptional mechanismrdquo AmericanJournal of PhysiologymdashCell Physiology vol 283 no 1 pp C31ndashC41 2002

[15] M Sanchez-Hidalgo A R Martın I Villegas and C Alarconde la Lastra ldquoRosiglitazone a PPAR120574 ligand modulates signaltransduction pathways during the development of acute TNBS-induced colitis in ratsrdquo European Journal of Pharmacology vol562 no 3 pp 247ndash258 2007

[16] F Scaldaferri M Sans S Vetrano et al ldquoThe role of MAPKin governing lymphocyte adhesion to and migration acrossthemicrovasculature in inflammatory bowel diseaserdquo EuropeanJournal of Immunology vol 39 no 1 pp 290ndash300 2009

[17] S Chae L Eckmann Y Miyamoto C Pothoulakis M KarinandM FKagnoff ldquoEpithelial cell I120581B-kinase120573has an importantprotective role in Clostridiumdifficile toxinA-inducedmucosalinjuryrdquo Journal of Immunology vol 177 no 2 pp 1214ndash12202006

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

0 0

5

10

15

20

25

10 20 50 100

5

10

15

20

25

0

5

10

15

20

25

0

5

10

15

20

25

0

5

10

15

20

25

0

5

10

15

20

25

LPS LPS LPS

LPS LPS LPS

NJ-1 minus minus 10 20 50 100NJ-2 minus minus 10 20 50 100NJ-3 minus minus

10 20 50 100NJ-6 minus minus10 20 50 100NJ-5 minus minus10 20 50 100NJ-4 minus minus

Nitr

ite (120583

M)

Nitr

ite (120583

M)

Nitr

ite (120583

M)

Nitr

ite (120583

M)

Nitr

ite (120583

M)

Nitr

ite (120583

M)

lowast lowast lowast lowast lowast lowast

lowastdagger lowastdagger

lowastdaggerlowastdagger

lowast

lowastdagger

lowastlowast lowastlowast lowast lowast

lowastdagger

lowast

lowastdaggerlowastdaggerlowastdaggerlowastdagger

lowastdagger

lowastdagger lowastdagger

lowastdaggerlowastdagger

lowastdagger

Figure 2 Effects of NJ fractions on LPS-induced NO production Peritoneal macrophages were pretreated with NJ fractions at indicateddoses (120583gmL) for 1 h and then stimulated with LPS (500 ngmL) for 24 h The effects of NJ fractions on LPS-induced NO production weremeasured by Griess assay The values are mean plusmn SE of three independent experiments lowast119875 lt 005 versus saline +119875 lt 005 versus LPS

statistical analyses were performed using SPSS version 100statistical analysis software

3 Results

31 Cytotoxicity of NJ Fractions in Peritoneal MacrophagesThe first approach to study the biological activity of anycompound or plant extract is to ensure lack of effect oncellular metabolism To determine whether fraction of NJaffects cell viability peritoneal macrophages were incubatedfor 24 h with varying concentrations of extract (120583gmL) andcell viability was evaluated by MTT assay NJ-1 NJ-2 NJ-3 and NJ-6 did not demonstrate any significant cytotoxiceffects on peritoneal macrophages However at a relativelyhigher dose NJ-4 and NJ-5 showed significant cytotoxicity( 119875 lt 0001) (Figure 1) Therefore because higher levels

of NJ-4 (ge100 120583gmL) and NJ-5 (ge50120583gmL) could affectcellular metabolism we excluded data of NJ-4 and NJ-5 atthese concentrations

32 Effects of NJ Fractions on LPS-Induced NO Produc-tion Next to examine the anti-inflammatory effect of NJfractions we measured NO production Murine peritonealmacrophages were pretreated with the indicated concentra-tions of NJ fractions for 1 h and then stimulated with LPS(500 ngmL) for 24 h As shown in Figure 2 LPS stimulationincreased NO production However pretreatment with NJ-1 NJ-3 NJ-4 and NJ-6 significantly inhibited LPS-inducedproduction of NO but not with NJ-2 and NJ-5 (Figure 2)

33 Effects of NJ Fractions on LPS-Induced ProinflammatoryCytokine Production To examine the effects of NJ fractions

6 Evidence-Based Complementary and Alternative Medicine

0

30

60

90

120

150

180

210

0

30

60

90

120

150

180

210

0

30

60

90

120

150

180

210

0

30

60

90

120

150

180

210

10 20 50 100NJ-1 minus minus 10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus

IL-1120573

(pg

mL)

IL-1120573

(pg

mL)

IL-1120573

(pg

mL)

IL-1120573

(pg

mL)

lowastlowastlowastlowastlowastlowast lowast lowastlowast lowast lowast lowast lowast

lowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdagger

(a)

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

IL-6

(ng

mL)

IL-6

(ng

mL)

IL-6

(ng

mL)

IL-6

(ng

mL)

lowast lowast lowast lowast lowastlowast

lowast

lowastlowastlowast lowast lowast lowast lowast

lowastdagger

lowastdaggerlowastdagger

lowastdaggerlowastdagger

lowastdagger

(b)

0

1

2

3

4

0

1

2

3

4

0

1

2

3

4

0

1

2

3

4

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

LPSLPSLPS LPS

TNF-120572

(ng

mL)

TNF-120572

(ng

mL)

TNF-120572

(ng

mL)

TNF-120572

(ng

mL)

lowastlowastlowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastdagger

lowastdagger

lowastdagger lowastdagger

lowastdagger

(c)

Figure 3 Effects of NJ fractions on LPS-induced cytokine productions Peritoneal macrophages were treated with NJ fractions (120583gmL) for1 h and then stimulated with LPS at indicated time pointsThe supernatants were harvested and the levels of (a) IL-1120573 (b) IL-6 and (c) TNF-120572were measured by ELISA The values are mean plusmn SE of three independent experiments lowast119875 lt 005 versus saline +119875 lt 005 versus LPS

on LPS-induced proinflammatory cytokine production peri-toneal macrophages were pretreated with NJ fractions at theindicated concentrations for 1 h and then stimulated with500 ngmL of LPS for 24 h In particular we treated cells withNJ-1 NJ-3 NJ-4 and NJ-6 fractions which demonstratedinhibition of NO production As shown in Figures 3 and 4protein and mRNA levels of IL-1120573 IL-6 and TNF-120572 wereincreased by LPS stimulation However pretreatment withNJ-3 NJ-4 and NJ-6 inhibited the production of IL-1120573 IL-6and TNF-120572 significantly but not with NJ-1 fraction (Figures 3and 4)

34 Effects of NJ Fractions on the Activation of MAPKs andNF-120581B To examine inhibitory mechanism(s) of NJ fractionson production of NO and cytokines MAPK and NF-120581B were

measured The cells were pretreated with NJ-1 (100 120583gmL)NJ-3 (100 120583gmL) NJ-4 (50 120583gmL) and NJ-6 (100 120583gmL)for 1 h and then stimulatedwith 500 ngmLof LPS for 0 15 30or 60min Pretreatment withNJ-1 NJ-3 or NJ-4 significantlyinhibited the LPS-induced activation of JNK and p38 but notERK 12 (Figures 5(a)ndash5(c)) In addition pretreatment withNJ-6 significantly inhibited the LPS-induced activation ofERK 12 JNK and p38 (Figure 5(d)) To determine whetherNJ fractions inhibit LPS-inducedNF-120581B activation we exam-ined degradation of I120581-B120572 and NF-120581B p65 translocationTheNJ-1 NJ-3 andNJ-4 fractions did not inhibit the degradationof I120581-B120572 (Figures 6(a)ndash6(c)) However NJ-6 inhibited thedegradation of I120581-B120572 and translocation of NF-120581B p65 intonucleus suggesting that NJ-6 may influence LPS-inducedNF-120581B activation (Figures 6(d) and 6(e))

Evidence-Based Complementary and Alternative Medicine 7

0

50

100

150

200

0

50

100

150

200

0

50

100

150

200

0

50

100

150

200

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

Rela

tive I

L-1120573

Rela

tive I

L-1120573

Rela

tive I

L-1120573

Rela

tive I

L-1120573 lowast

lowast

lowast

lowast

lowast

lowastlowast lowast lowast lowast lowast

lowastdagger

lowastdaggerlowastdaggerlowastdagger

lowastdagger

lowastdagger

lowastdaggerlowastdagger

lowastdagger

(a)

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

Rela

tive I

L-6

Rela

tive I

L-6

Rela

tive I

L-6

Rela

tive I

L-6

lowast lowast lowast lowast lowast lowastlowastlowast lowast

lowast

lowast

lowastdagger

lowastdaggerlowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdaggerlowastdagger

lowastdagger

(b)

0

5

10

15

20

0

5

10

15

20

0

5

10

15

20

0

5

10

15

20

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

LPSLPSLPSLPS

lowast lowast lowast lowast

lowast

lowastlowast

lowast

lowastlowastlowastlowastlowastlowast

lowastdagger

lowastdaggerlowastdagger

lowastdagger

lowastdaggerlowastdagger

Rela

tive T

NF-120572

Rela

tive T

NF-120572

Rela

tive T

NF-120572

Rela

tive T

NF-120572

(c)

Figure 4 Effects of NJ fractions onmRNA levels of LPS-induced cytokine expression Peritoneal macrophages were treated with NJ fractions(120583gmL) for 1 h and then stimulated with LPS at indicated time points Cells were collected for real-time RT-PCR Total RNA (1 120583g) wasprepared for the real-time RT-PCR of (a) IL-1120573 (b) IL-6 and (c) TNF-120572 The values are mean plusmn SE of three independent experimentslowast

119875 lt 005 versus saline +119875 lt 005 versus LPS

35 Effects of NJ Fractions on LPS-Induced Endotoxin ShockTo verify effects of NJ fractions in vivo we used a model LPS-induced endotoxin shock NJ fractions (NJ-1 NJ-3 NJ-4 orNJ-6) were injected at the indicated doses After 3 h a lethaldose of LPS (375mgkg) was administered intraperitoneallyThe survival rate was recorded every 12 h after LPS treat-ment Generally LPS-injected septic mice died within 48 hConsistent with in vitro experiments NJ fractions exhibitedstrong anti-inflammatory activities in vivo as shown bysignificant inhibition of LPS-induced death in the treatedmice (Figure 7) In particular NJ-4 and NJ-6 showed themost dramatic protection against LPS-induced endotoxinshock these fractions prevented nearly all harmful effects ofLPS (Figures 7(c) and 7(d))

4 Discussion

Recently our laboratory reported the beneficial effects of NJon various diseases such as endotoxin shock pancreatitisand diabetes [6ndash10] Particularly in endotoxin shock NJshowed a marked preventive effect and strong therapeuticpotential against LPS challenge [7] Although many of ourstudies have indicated the benefits of NJ on inflamma-tory diseases which compounds of NJ could exhibit anti-inflammatory properties are unknown Thus in this paperbringing more bright insight to identify the constituent com-pounds from NJ we selected and assorted effective fractionsthat would possess anti-inflammatory potential Overall weobtained six fractions from NJ Using the fourth fraction

8 Evidence-Based Complementary and Alternative Medicine

Time (min)LPS

60301506030150NJ-1 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(a)

LPSTime (min) 60301506030150

NJ-3 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(b)

LPSTime (min) 60301506030150

NJ-4 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(c)

LPSTime (min) 60301506030150

NJ-6 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(d)

Figure 5 Effects of NJ fractions on MAPKs activation ((a)ndash(d)) Peritoneal macrophages were treated with NJ fractions (100120583gmL) for 1 hand then stimulated with LPS (500 ngmL) at indicated time points Cells were harvested for western blot Total cellular proteins (20120583g) wereresolved by SDS-PAGE transferred to PVDF membrane and detected with phospho-specific ERK 12 (4244 kDa) JNK (4654 kDa) andp38 (43 kDa) antibody ERK JNK and p38 were used as a loading control A representative western blot of three experiments is shown

we evaluated the protective effects on cerulein-induced acutepancreatitis (AP) However the sixth fraction remained to beexamined for potential to prevent an inflammatory responseto stimuli such as LPS Therefore in this study we used sixfractions isolated from NJ to examine the anti-inflammatoryactivity against LPS on murine macrophages

Generally sepsis is a life threatening condition caused bythe bodyrsquos response to bacterial products such as endotoxinwhich is often present with severe fever shock and respira-tory damage [12] Partially due to this phenomenon sepsisis often regarded as an uncontrolled inflammatory response[12] In the context of sepsis TNF-120572 IL-1120573 IL-6 and IL-8 arethemost frequently altered cytokines [13]When injected intoanimals these cytokines recapitulate many clinical and labo-ratory features of sepsis supporting the concept that sepsisrepresents a ldquocytokine stormrdquo Thus regulation of cytokinesmay prove valuable for the prevention and treatment ofsepsis In this study just as the total extract of NJ inhibitedthe production of cytokines [7] fractions 3 4 and 6 also

inhibited the production of cytokines (Figures 3 and 4)These results suggest that the ability of NJ to inhibit cytokineproduction may originate from fractions 3 4 andor 6

Using the fractions that were found to be effectiveagainst LPS we examined activation of MAPKs and NF-120581Bas possible regulatory mechanisms Generally it has beenreported that the production of proinflammatory mediatorsis modulated by the MAPKs and NF-120581B pathways Threemajor groups of MAPKs include the ERK JNK and p38which are all activated by phosphorylation [14ndash16] NF-120581Bactivation occurs mainly through the degradation of I120581Bthereby leading to subsequent release of NF-120581B dimers [17]Subsequently NF-120581B dimers translocate from the cytoplasmto the nucleus and activate the transcription of multiple 120581B-dependent target genes [17] In this study we examine thephosphorylation of MAPKs and degradation of I120581-B120572 NJ-1NJ-3 and NJ-4 inhibited the phosphorylation and activationof JNK and p38 but not ERK12 However NJ-6 inhibitednot only the activation of ERK12 JNK and p38 but also

Evidence-Based Complementary and Alternative Medicine 9

Time (min)LPS

60301506030150NJ-1 + LPS

120573-Actin

I120581-B120572

(a)

Time (min)LPS

60301506030150NJ-4 + LPS

120573-Actin

I120581-B120572

(b)

Time (min)LPS

60301506030150NJ-3 + LPS

120573-Actin

I120581-B120572

(c)

Time (min)LPS

60301506030150NJ-6 + LPS

120573-Actin

I120581-B120572

(d)

DAPI Merge

None

LPS

NJ-6

NF-120581B p65

(e)

Figure 6 Effects of NJ fractions onNF-120581B activation ((a)ndash(d)) Peritonealmacrophages were treatedwithNJ fractions (100120583gmL) for 1 h andthen stimulated with LPS (500 ngmL) at indicated time points The cells were harvested for western blot Total cellular proteins (20 120583g) wereresolved by SDS-PAGE transferred to PVDF membrane and detected with I120581-B120572 (40 kDa) antibody 120573-Actin was used as loading controlA representative western blot of three experiments is shown (e) Peritoneal macrophages were treated with NJ fractions (100120583gmL) for 1 hand then stimulated with LPS (500 ngmL) for 1 h Then nuclear translocation of NF-120581B p65 was examined using immunostaining NF-120581Bp65 was shown as red and the nucleus was shown as blue (DAPI) The values are means plusmn SE of three independent experiments lowast119875 lt 005versus saline +119875 lt 005 versus LPS

10 Evidence-Based Complementary and Alternative Medicine

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-1 (01mgkg) + LPS

NJ-1 (1mgkg) + LPSNJ-1 (10mgkg) + LPS

(a)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-3 (01mgkg) + LPS

NJ-3 (1mgkg) + LPSNJ-3 (10mgkg) + LPS

(b)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-4 (01mgkg) + LPS

NJ-4 (1mgkg) + LPSNJ-4 (10mgkg) + LPS

(c)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-6 (01mgkg) + LPS

NJ-6 (1mgkg) + LPSNJ-6 (10mgkg) + LPS

(d)

Figure 7 Effect of NJ fractions on LPS-induced endotoxin shock ((a)ndash(d)) Endotoxin shock was induced in 6ndash8-week-old female C57BL6mice as described in Section 2 NJ fractions were administered intraperitoneally to mice at 01 1 or 10mgkg 1 h before LPS injection ThenLPS (375mgkg) was injected to induce endotoxin shock The survival rates of endotoxin shock mice were monitored for survival for 120 hThis figure shows data for 10 mice per group

degradation of I120581-B120572 These results suggest that inhibitionof NO and cytokine production by NJ fractions is primarilythrough MAPKs or NF-120581B pathways

In this study we isolated two beneficial fractions of NJThe first one was NJ-4 which could show similar effectsto NJ total extract NJ-4 inhibited cytokine production andLPS-induced endotoxin shock similar to NJ total extract [7]Furthermore the regulatory mechanisms were similar NJtotal extract inhibited only MAPKs but NJ-4 inhibited theJNK and p38 Secondly NJ-6 showed dramatic inhibitionof LPS-induced inflammatory response Indeed NJ-6 inhib-ited the cytokine production and LPS-induced endotoxinshock dramatically However NJ-6 differed from total extractmechanistically NJ-6 inhibited the degradation of I120581-B120572and translocation of NF-120581B p65 which was not regulatedby the NJ total extract Further isolation of the NJ-4 andNJ-6 fractions could potentially yield a single compounddemonstrating inhibitory activity resembling that of NJ totalextract

In conclusion this study demonstrated which fractionsof NJ exhibit inhibitory activity against LPS-induced inflam-mation Among the 6 fractions isolated NJ-1 NJ-3 NJ-4 andNJ-6 showed the inhibition of NO and NJ-3 NJ-4 and NJ-6 showed the inhibition of cytokines In addition NJ-4 andNJ-6 exhibited dramatic prevention against LPS-endotoxinshock These results suggest that NJ-4 and NJ-6 may beeffective and beneficial candidates to ameliorate LPS-inducedinflammation

Abbreviations

ELISA Enzyme-linked immunosorbent assayERK Extracellular signal-regulated kinaseFBS Fetal bovine serumIL InterleukinJNK c-jun NH

2-terminal kinase

LPS LipopolysaccharideMAPKs Mitogen-activated protein kinases

Evidence-Based Complementary and Alternative Medicine 11

MTT 3-(45 Dimethylthiazol)-25-diphenyltetrazolium bromide

NF Nuclear factorNJ Nardostachys jatamansiNO Nitric oxidePCR Polymerase chain reactionTG ThioglycollateTLR Toll-like receptorTNF Tumor necrosis factor

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Gi-Sang Bae and Kwang-Ho Heo equally contributed to thiswork

Acknowledgments

This research was supported by a Basic Science ResearchProgram through theNational Research Foundation of Korea(NRF) fundedby theMinistryofEducation ScienceandTech-nology (NRF-2012R1A1A2040916 2012R1A5A2A35671257and 2012M2B2B1055246)

References

[1] C-S Sung and C-S Wong ldquoCellular mechanisms of neuroin-flammatory pain the role of interleukin-1120573rdquo Acta Anaesthesio-logica Taiwanica vol 45 no 2 pp 103ndash109 2007

[2] F O Martinez L Helming and S Gordon ldquoAlternative activa-tion of macrophages an immunologic functional perspectiverdquoAnnual Review of Immunology vol 27 pp 451ndash483 2009

[3] J D Schilling H M Machkovech L He A Diwan and J ESchaffer ldquoTLR4 activation under lipotoxic conditions leads tosynergistic macrophage cell death through a TRIF-dependentpathwayrdquo Journal of Immunology vol 190 no 3 pp 1285ndash12962013

[4] Y Ben-Neriah andM Karin ldquoInflammation meets cancer withNF-120581B as the matchmakerrdquo Nature Immunology vol 12 no 8pp 715ndash723 2011

[5] B C Bose Y Vijayvargiya and J N Bhatnagar ldquoNardostachysjatamansiDC a phyto-chemical study of its active constituentsrdquoIndian Journal of Medical Sciences vol 11 no 10 pp 799ndash8021957

[6] G-S Bae H-J Park D-Y Kim et al ldquoNardostachys jatamansiprotects against cerulein-induced acute pancreatitisrdquo Pancreasvol 39 no 4 pp 520ndash529 2010

[7] G-S Bae S-W Seo M-S Kim et al ldquoThe roots of Nar-dostachys jatamansi inhibits lipopolysaccharide-induced endo-toxin shockrdquo Journal of Natural Medicines vol 65 no 1 pp 63ndash72 2011

[8] G S Bae K C Park B S Koo et al ldquoThe inhibitory effectsof Nardostachys jatamansi on alcoholic chronic pancreatitisrdquoBMB Reports vol 45 no 7 pp 402ndash407 2012

[9] G S Bae K C Park B S Koo et al ldquoThe beneficial effectsof Nardostachys jatamansi extract on diet-induced severe acutepancreatitisrdquo Pancreas vol 42 no 2 pp 362ndash363 2013

[10] M-Y Song U-J Bae B-H Lee et al ldquoNardostachys jatamansiextract protects against cytokineinduced 120573-cell damage andstreptozotocin-induced diabetesrdquo World Journal of Gastroen-terology vol 16 no 26 pp 3249ndash3257 2010

[11] G S Bae M S Kim K C Park et al ldquoEffect of biologicallyactive fraction of Nardostachys jatamansi on cerulein-inducedacute pancreatitisrdquo World Journal of Gastroenterology vol 18no 25 pp 3223ndash3234 2012

[12] R S Hotchkiss and I E Karl ldquoThe pathophysiology andtreatment of sepsisrdquoThe New England Journal of Medicine vol348 no 2 pp 138ndash150 2003

[13] T S Blackwell and J W Christman ldquoSepsis and cytokinescurrent statusrdquo British Journal of Anaesthesia vol 77 no 1 pp110ndash117 1996

[14] H B Jijon W J Panenka K L Madsen and H G Par-sons ldquoMAP kinases contribute to IL-8 secretion by intestinalepithelial cells via a posttranscriptional mechanismrdquo AmericanJournal of PhysiologymdashCell Physiology vol 283 no 1 pp C31ndashC41 2002

[15] M Sanchez-Hidalgo A R Martın I Villegas and C Alarconde la Lastra ldquoRosiglitazone a PPAR120574 ligand modulates signaltransduction pathways during the development of acute TNBS-induced colitis in ratsrdquo European Journal of Pharmacology vol562 no 3 pp 247ndash258 2007

[16] F Scaldaferri M Sans S Vetrano et al ldquoThe role of MAPKin governing lymphocyte adhesion to and migration acrossthemicrovasculature in inflammatory bowel diseaserdquo EuropeanJournal of Immunology vol 39 no 1 pp 290ndash300 2009

[17] S Chae L Eckmann Y Miyamoto C Pothoulakis M KarinandM FKagnoff ldquoEpithelial cell I120581B-kinase120573has an importantprotective role in Clostridiumdifficile toxinA-inducedmucosalinjuryrdquo Journal of Immunology vol 177 no 2 pp 1214ndash12202006

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

0

30

60

90

120

150

180

210

0

30

60

90

120

150

180

210

0

30

60

90

120

150

180

210

0

30

60

90

120

150

180

210

10 20 50 100NJ-1 minus minus 10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus

IL-1120573

(pg

mL)

IL-1120573

(pg

mL)

IL-1120573

(pg

mL)

IL-1120573

(pg

mL)

lowastlowastlowastlowastlowastlowast lowast lowastlowast lowast lowast lowast lowast

lowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdagger

(a)

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

IL-6

(ng

mL)

IL-6

(ng

mL)

IL-6

(ng

mL)

IL-6

(ng

mL)

lowast lowast lowast lowast lowastlowast

lowast

lowastlowastlowast lowast lowast lowast lowast

lowastdagger

lowastdaggerlowastdagger

lowastdaggerlowastdagger

lowastdagger

(b)

0

1

2

3

4

0

1

2

3

4

0

1

2

3

4

0

1

2

3

4

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

LPSLPSLPS LPS

TNF-120572

(ng

mL)

TNF-120572

(ng

mL)

TNF-120572

(ng

mL)

TNF-120572

(ng

mL)

lowastlowastlowastlowastlowast

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastdagger

lowastdagger

lowastdagger lowastdagger

lowastdagger

(c)

Figure 3 Effects of NJ fractions on LPS-induced cytokine productions Peritoneal macrophages were treated with NJ fractions (120583gmL) for1 h and then stimulated with LPS at indicated time pointsThe supernatants were harvested and the levels of (a) IL-1120573 (b) IL-6 and (c) TNF-120572were measured by ELISA The values are mean plusmn SE of three independent experiments lowast119875 lt 005 versus saline +119875 lt 005 versus LPS

on LPS-induced proinflammatory cytokine production peri-toneal macrophages were pretreated with NJ fractions at theindicated concentrations for 1 h and then stimulated with500 ngmL of LPS for 24 h In particular we treated cells withNJ-1 NJ-3 NJ-4 and NJ-6 fractions which demonstratedinhibition of NO production As shown in Figures 3 and 4protein and mRNA levels of IL-1120573 IL-6 and TNF-120572 wereincreased by LPS stimulation However pretreatment withNJ-3 NJ-4 and NJ-6 inhibited the production of IL-1120573 IL-6and TNF-120572 significantly but not with NJ-1 fraction (Figures 3and 4)

34 Effects of NJ Fractions on the Activation of MAPKs andNF-120581B To examine inhibitory mechanism(s) of NJ fractionson production of NO and cytokines MAPK and NF-120581B were

measured The cells were pretreated with NJ-1 (100 120583gmL)NJ-3 (100 120583gmL) NJ-4 (50 120583gmL) and NJ-6 (100 120583gmL)for 1 h and then stimulatedwith 500 ngmLof LPS for 0 15 30or 60min Pretreatment withNJ-1 NJ-3 or NJ-4 significantlyinhibited the LPS-induced activation of JNK and p38 but notERK 12 (Figures 5(a)ndash5(c)) In addition pretreatment withNJ-6 significantly inhibited the LPS-induced activation ofERK 12 JNK and p38 (Figure 5(d)) To determine whetherNJ fractions inhibit LPS-inducedNF-120581B activation we exam-ined degradation of I120581-B120572 and NF-120581B p65 translocationTheNJ-1 NJ-3 andNJ-4 fractions did not inhibit the degradationof I120581-B120572 (Figures 6(a)ndash6(c)) However NJ-6 inhibited thedegradation of I120581-B120572 and translocation of NF-120581B p65 intonucleus suggesting that NJ-6 may influence LPS-inducedNF-120581B activation (Figures 6(d) and 6(e))

Evidence-Based Complementary and Alternative Medicine 7

0

50

100

150

200

0

50

100

150

200

0

50

100

150

200

0

50

100

150

200

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

Rela

tive I

L-1120573

Rela

tive I

L-1120573

Rela

tive I

L-1120573

Rela

tive I

L-1120573 lowast

lowast

lowast

lowast

lowast

lowastlowast lowast lowast lowast lowast

lowastdagger

lowastdaggerlowastdaggerlowastdagger

lowastdagger

lowastdagger

lowastdaggerlowastdagger

lowastdagger

(a)

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

Rela

tive I

L-6

Rela

tive I

L-6

Rela

tive I

L-6

Rela

tive I

L-6

lowast lowast lowast lowast lowast lowastlowastlowast lowast

lowast

lowast

lowastdagger

lowastdaggerlowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdaggerlowastdagger

lowastdagger

(b)

0

5

10

15

20

0

5

10

15

20

0

5

10

15

20

0

5

10

15

20

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

LPSLPSLPSLPS

lowast lowast lowast lowast

lowast

lowastlowast

lowast

lowastlowastlowastlowastlowastlowast

lowastdagger

lowastdaggerlowastdagger

lowastdagger

lowastdaggerlowastdagger

Rela

tive T

NF-120572

Rela

tive T

NF-120572

Rela

tive T

NF-120572

Rela

tive T

NF-120572

(c)

Figure 4 Effects of NJ fractions onmRNA levels of LPS-induced cytokine expression Peritoneal macrophages were treated with NJ fractions(120583gmL) for 1 h and then stimulated with LPS at indicated time points Cells were collected for real-time RT-PCR Total RNA (1 120583g) wasprepared for the real-time RT-PCR of (a) IL-1120573 (b) IL-6 and (c) TNF-120572 The values are mean plusmn SE of three independent experimentslowast

119875 lt 005 versus saline +119875 lt 005 versus LPS

35 Effects of NJ Fractions on LPS-Induced Endotoxin ShockTo verify effects of NJ fractions in vivo we used a model LPS-induced endotoxin shock NJ fractions (NJ-1 NJ-3 NJ-4 orNJ-6) were injected at the indicated doses After 3 h a lethaldose of LPS (375mgkg) was administered intraperitoneallyThe survival rate was recorded every 12 h after LPS treat-ment Generally LPS-injected septic mice died within 48 hConsistent with in vitro experiments NJ fractions exhibitedstrong anti-inflammatory activities in vivo as shown bysignificant inhibition of LPS-induced death in the treatedmice (Figure 7) In particular NJ-4 and NJ-6 showed themost dramatic protection against LPS-induced endotoxinshock these fractions prevented nearly all harmful effects ofLPS (Figures 7(c) and 7(d))

4 Discussion

Recently our laboratory reported the beneficial effects of NJon various diseases such as endotoxin shock pancreatitisand diabetes [6ndash10] Particularly in endotoxin shock NJshowed a marked preventive effect and strong therapeuticpotential against LPS challenge [7] Although many of ourstudies have indicated the benefits of NJ on inflamma-tory diseases which compounds of NJ could exhibit anti-inflammatory properties are unknown Thus in this paperbringing more bright insight to identify the constituent com-pounds from NJ we selected and assorted effective fractionsthat would possess anti-inflammatory potential Overall weobtained six fractions from NJ Using the fourth fraction

8 Evidence-Based Complementary and Alternative Medicine

Time (min)LPS

60301506030150NJ-1 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(a)

LPSTime (min) 60301506030150

NJ-3 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(b)

LPSTime (min) 60301506030150

NJ-4 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(c)

LPSTime (min) 60301506030150

NJ-6 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(d)

Figure 5 Effects of NJ fractions on MAPKs activation ((a)ndash(d)) Peritoneal macrophages were treated with NJ fractions (100120583gmL) for 1 hand then stimulated with LPS (500 ngmL) at indicated time points Cells were harvested for western blot Total cellular proteins (20120583g) wereresolved by SDS-PAGE transferred to PVDF membrane and detected with phospho-specific ERK 12 (4244 kDa) JNK (4654 kDa) andp38 (43 kDa) antibody ERK JNK and p38 were used as a loading control A representative western blot of three experiments is shown

we evaluated the protective effects on cerulein-induced acutepancreatitis (AP) However the sixth fraction remained to beexamined for potential to prevent an inflammatory responseto stimuli such as LPS Therefore in this study we used sixfractions isolated from NJ to examine the anti-inflammatoryactivity against LPS on murine macrophages

Generally sepsis is a life threatening condition caused bythe bodyrsquos response to bacterial products such as endotoxinwhich is often present with severe fever shock and respira-tory damage [12] Partially due to this phenomenon sepsisis often regarded as an uncontrolled inflammatory response[12] In the context of sepsis TNF-120572 IL-1120573 IL-6 and IL-8 arethemost frequently altered cytokines [13]When injected intoanimals these cytokines recapitulate many clinical and labo-ratory features of sepsis supporting the concept that sepsisrepresents a ldquocytokine stormrdquo Thus regulation of cytokinesmay prove valuable for the prevention and treatment ofsepsis In this study just as the total extract of NJ inhibitedthe production of cytokines [7] fractions 3 4 and 6 also

inhibited the production of cytokines (Figures 3 and 4)These results suggest that the ability of NJ to inhibit cytokineproduction may originate from fractions 3 4 andor 6

Using the fractions that were found to be effectiveagainst LPS we examined activation of MAPKs and NF-120581Bas possible regulatory mechanisms Generally it has beenreported that the production of proinflammatory mediatorsis modulated by the MAPKs and NF-120581B pathways Threemajor groups of MAPKs include the ERK JNK and p38which are all activated by phosphorylation [14ndash16] NF-120581Bactivation occurs mainly through the degradation of I120581Bthereby leading to subsequent release of NF-120581B dimers [17]Subsequently NF-120581B dimers translocate from the cytoplasmto the nucleus and activate the transcription of multiple 120581B-dependent target genes [17] In this study we examine thephosphorylation of MAPKs and degradation of I120581-B120572 NJ-1NJ-3 and NJ-4 inhibited the phosphorylation and activationof JNK and p38 but not ERK12 However NJ-6 inhibitednot only the activation of ERK12 JNK and p38 but also

Evidence-Based Complementary and Alternative Medicine 9

Time (min)LPS

60301506030150NJ-1 + LPS

120573-Actin

I120581-B120572

(a)

Time (min)LPS

60301506030150NJ-4 + LPS

120573-Actin

I120581-B120572

(b)

Time (min)LPS

60301506030150NJ-3 + LPS

120573-Actin

I120581-B120572

(c)

Time (min)LPS

60301506030150NJ-6 + LPS

120573-Actin

I120581-B120572

(d)

DAPI Merge

None

LPS

NJ-6

NF-120581B p65

(e)

Figure 6 Effects of NJ fractions onNF-120581B activation ((a)ndash(d)) Peritonealmacrophages were treatedwithNJ fractions (100120583gmL) for 1 h andthen stimulated with LPS (500 ngmL) at indicated time points The cells were harvested for western blot Total cellular proteins (20 120583g) wereresolved by SDS-PAGE transferred to PVDF membrane and detected with I120581-B120572 (40 kDa) antibody 120573-Actin was used as loading controlA representative western blot of three experiments is shown (e) Peritoneal macrophages were treated with NJ fractions (100120583gmL) for 1 hand then stimulated with LPS (500 ngmL) for 1 h Then nuclear translocation of NF-120581B p65 was examined using immunostaining NF-120581Bp65 was shown as red and the nucleus was shown as blue (DAPI) The values are means plusmn SE of three independent experiments lowast119875 lt 005versus saline +119875 lt 005 versus LPS

10 Evidence-Based Complementary and Alternative Medicine

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-1 (01mgkg) + LPS

NJ-1 (1mgkg) + LPSNJ-1 (10mgkg) + LPS

(a)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-3 (01mgkg) + LPS

NJ-3 (1mgkg) + LPSNJ-3 (10mgkg) + LPS

(b)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-4 (01mgkg) + LPS

NJ-4 (1mgkg) + LPSNJ-4 (10mgkg) + LPS

(c)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-6 (01mgkg) + LPS

NJ-6 (1mgkg) + LPSNJ-6 (10mgkg) + LPS

(d)

Figure 7 Effect of NJ fractions on LPS-induced endotoxin shock ((a)ndash(d)) Endotoxin shock was induced in 6ndash8-week-old female C57BL6mice as described in Section 2 NJ fractions were administered intraperitoneally to mice at 01 1 or 10mgkg 1 h before LPS injection ThenLPS (375mgkg) was injected to induce endotoxin shock The survival rates of endotoxin shock mice were monitored for survival for 120 hThis figure shows data for 10 mice per group

degradation of I120581-B120572 These results suggest that inhibitionof NO and cytokine production by NJ fractions is primarilythrough MAPKs or NF-120581B pathways

In this study we isolated two beneficial fractions of NJThe first one was NJ-4 which could show similar effectsto NJ total extract NJ-4 inhibited cytokine production andLPS-induced endotoxin shock similar to NJ total extract [7]Furthermore the regulatory mechanisms were similar NJtotal extract inhibited only MAPKs but NJ-4 inhibited theJNK and p38 Secondly NJ-6 showed dramatic inhibitionof LPS-induced inflammatory response Indeed NJ-6 inhib-ited the cytokine production and LPS-induced endotoxinshock dramatically However NJ-6 differed from total extractmechanistically NJ-6 inhibited the degradation of I120581-B120572and translocation of NF-120581B p65 which was not regulatedby the NJ total extract Further isolation of the NJ-4 andNJ-6 fractions could potentially yield a single compounddemonstrating inhibitory activity resembling that of NJ totalextract

In conclusion this study demonstrated which fractionsof NJ exhibit inhibitory activity against LPS-induced inflam-mation Among the 6 fractions isolated NJ-1 NJ-3 NJ-4 andNJ-6 showed the inhibition of NO and NJ-3 NJ-4 and NJ-6 showed the inhibition of cytokines In addition NJ-4 andNJ-6 exhibited dramatic prevention against LPS-endotoxinshock These results suggest that NJ-4 and NJ-6 may beeffective and beneficial candidates to ameliorate LPS-inducedinflammation

Abbreviations

ELISA Enzyme-linked immunosorbent assayERK Extracellular signal-regulated kinaseFBS Fetal bovine serumIL InterleukinJNK c-jun NH

2-terminal kinase

LPS LipopolysaccharideMAPKs Mitogen-activated protein kinases

Evidence-Based Complementary and Alternative Medicine 11

MTT 3-(45 Dimethylthiazol)-25-diphenyltetrazolium bromide

NF Nuclear factorNJ Nardostachys jatamansiNO Nitric oxidePCR Polymerase chain reactionTG ThioglycollateTLR Toll-like receptorTNF Tumor necrosis factor

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Gi-Sang Bae and Kwang-Ho Heo equally contributed to thiswork

Acknowledgments

This research was supported by a Basic Science ResearchProgram through theNational Research Foundation of Korea(NRF) fundedby theMinistryofEducation ScienceandTech-nology (NRF-2012R1A1A2040916 2012R1A5A2A35671257and 2012M2B2B1055246)

References

[1] C-S Sung and C-S Wong ldquoCellular mechanisms of neuroin-flammatory pain the role of interleukin-1120573rdquo Acta Anaesthesio-logica Taiwanica vol 45 no 2 pp 103ndash109 2007

[2] F O Martinez L Helming and S Gordon ldquoAlternative activa-tion of macrophages an immunologic functional perspectiverdquoAnnual Review of Immunology vol 27 pp 451ndash483 2009

[3] J D Schilling H M Machkovech L He A Diwan and J ESchaffer ldquoTLR4 activation under lipotoxic conditions leads tosynergistic macrophage cell death through a TRIF-dependentpathwayrdquo Journal of Immunology vol 190 no 3 pp 1285ndash12962013

[4] Y Ben-Neriah andM Karin ldquoInflammation meets cancer withNF-120581B as the matchmakerrdquo Nature Immunology vol 12 no 8pp 715ndash723 2011

[5] B C Bose Y Vijayvargiya and J N Bhatnagar ldquoNardostachysjatamansiDC a phyto-chemical study of its active constituentsrdquoIndian Journal of Medical Sciences vol 11 no 10 pp 799ndash8021957

[6] G-S Bae H-J Park D-Y Kim et al ldquoNardostachys jatamansiprotects against cerulein-induced acute pancreatitisrdquo Pancreasvol 39 no 4 pp 520ndash529 2010

[7] G-S Bae S-W Seo M-S Kim et al ldquoThe roots of Nar-dostachys jatamansi inhibits lipopolysaccharide-induced endo-toxin shockrdquo Journal of Natural Medicines vol 65 no 1 pp 63ndash72 2011

[8] G S Bae K C Park B S Koo et al ldquoThe inhibitory effectsof Nardostachys jatamansi on alcoholic chronic pancreatitisrdquoBMB Reports vol 45 no 7 pp 402ndash407 2012

[9] G S Bae K C Park B S Koo et al ldquoThe beneficial effectsof Nardostachys jatamansi extract on diet-induced severe acutepancreatitisrdquo Pancreas vol 42 no 2 pp 362ndash363 2013

[10] M-Y Song U-J Bae B-H Lee et al ldquoNardostachys jatamansiextract protects against cytokineinduced 120573-cell damage andstreptozotocin-induced diabetesrdquo World Journal of Gastroen-terology vol 16 no 26 pp 3249ndash3257 2010

[11] G S Bae M S Kim K C Park et al ldquoEffect of biologicallyactive fraction of Nardostachys jatamansi on cerulein-inducedacute pancreatitisrdquo World Journal of Gastroenterology vol 18no 25 pp 3223ndash3234 2012

[12] R S Hotchkiss and I E Karl ldquoThe pathophysiology andtreatment of sepsisrdquoThe New England Journal of Medicine vol348 no 2 pp 138ndash150 2003

[13] T S Blackwell and J W Christman ldquoSepsis and cytokinescurrent statusrdquo British Journal of Anaesthesia vol 77 no 1 pp110ndash117 1996

[14] H B Jijon W J Panenka K L Madsen and H G Par-sons ldquoMAP kinases contribute to IL-8 secretion by intestinalepithelial cells via a posttranscriptional mechanismrdquo AmericanJournal of PhysiologymdashCell Physiology vol 283 no 1 pp C31ndashC41 2002

[15] M Sanchez-Hidalgo A R Martın I Villegas and C Alarconde la Lastra ldquoRosiglitazone a PPAR120574 ligand modulates signaltransduction pathways during the development of acute TNBS-induced colitis in ratsrdquo European Journal of Pharmacology vol562 no 3 pp 247ndash258 2007

[16] F Scaldaferri M Sans S Vetrano et al ldquoThe role of MAPKin governing lymphocyte adhesion to and migration acrossthemicrovasculature in inflammatory bowel diseaserdquo EuropeanJournal of Immunology vol 39 no 1 pp 290ndash300 2009

[17] S Chae L Eckmann Y Miyamoto C Pothoulakis M KarinandM FKagnoff ldquoEpithelial cell I120581B-kinase120573has an importantprotective role in Clostridiumdifficile toxinA-inducedmucosalinjuryrdquo Journal of Immunology vol 177 no 2 pp 1214ndash12202006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

0

50

100

150

200

0

50

100

150

200

0

50

100

150

200

0

50

100

150

200

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

Rela

tive I

L-1120573

Rela

tive I

L-1120573

Rela

tive I

L-1120573

Rela

tive I

L-1120573 lowast

lowast

lowast

lowast

lowast

lowastlowast lowast lowast lowast lowast

lowastdagger

lowastdaggerlowastdaggerlowastdagger

lowastdagger

lowastdagger

lowastdaggerlowastdagger

lowastdagger

(a)

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

Rela

tive I

L-6

Rela

tive I

L-6

Rela

tive I

L-6

Rela

tive I

L-6

lowast lowast lowast lowast lowast lowastlowastlowast lowast

lowast

lowast

lowastdagger

lowastdaggerlowastdagger

lowastdagger

lowastdagger

lowastdagger

lowastdaggerlowastdagger

lowastdagger

(b)

0

5

10

15

20

0

5

10

15

20

0

5

10

15

20

0

5

10

15

20

10 20 50 100NJ-3 minus minus 10 20 50 100NJ-4 minus minus 10 20 50 100NJ-6 minus minus10 20 50 100NJ-1 minus minus

LPSLPSLPSLPS

lowast lowast lowast lowast

lowast

lowastlowast

lowast

lowastlowastlowastlowastlowastlowast

lowastdagger

lowastdaggerlowastdagger

lowastdagger

lowastdaggerlowastdagger

Rela

tive T

NF-120572

Rela

tive T

NF-120572

Rela

tive T

NF-120572

Rela

tive T

NF-120572

(c)

Figure 4 Effects of NJ fractions onmRNA levels of LPS-induced cytokine expression Peritoneal macrophages were treated with NJ fractions(120583gmL) for 1 h and then stimulated with LPS at indicated time points Cells were collected for real-time RT-PCR Total RNA (1 120583g) wasprepared for the real-time RT-PCR of (a) IL-1120573 (b) IL-6 and (c) TNF-120572 The values are mean plusmn SE of three independent experimentslowast

119875 lt 005 versus saline +119875 lt 005 versus LPS

35 Effects of NJ Fractions on LPS-Induced Endotoxin ShockTo verify effects of NJ fractions in vivo we used a model LPS-induced endotoxin shock NJ fractions (NJ-1 NJ-3 NJ-4 orNJ-6) were injected at the indicated doses After 3 h a lethaldose of LPS (375mgkg) was administered intraperitoneallyThe survival rate was recorded every 12 h after LPS treat-ment Generally LPS-injected septic mice died within 48 hConsistent with in vitro experiments NJ fractions exhibitedstrong anti-inflammatory activities in vivo as shown bysignificant inhibition of LPS-induced death in the treatedmice (Figure 7) In particular NJ-4 and NJ-6 showed themost dramatic protection against LPS-induced endotoxinshock these fractions prevented nearly all harmful effects ofLPS (Figures 7(c) and 7(d))

4 Discussion

Recently our laboratory reported the beneficial effects of NJon various diseases such as endotoxin shock pancreatitisand diabetes [6ndash10] Particularly in endotoxin shock NJshowed a marked preventive effect and strong therapeuticpotential against LPS challenge [7] Although many of ourstudies have indicated the benefits of NJ on inflamma-tory diseases which compounds of NJ could exhibit anti-inflammatory properties are unknown Thus in this paperbringing more bright insight to identify the constituent com-pounds from NJ we selected and assorted effective fractionsthat would possess anti-inflammatory potential Overall weobtained six fractions from NJ Using the fourth fraction

8 Evidence-Based Complementary and Alternative Medicine

Time (min)LPS

60301506030150NJ-1 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(a)

LPSTime (min) 60301506030150

NJ-3 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(b)

LPSTime (min) 60301506030150

NJ-4 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(c)

LPSTime (min) 60301506030150

NJ-6 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(d)

Figure 5 Effects of NJ fractions on MAPKs activation ((a)ndash(d)) Peritoneal macrophages were treated with NJ fractions (100120583gmL) for 1 hand then stimulated with LPS (500 ngmL) at indicated time points Cells were harvested for western blot Total cellular proteins (20120583g) wereresolved by SDS-PAGE transferred to PVDF membrane and detected with phospho-specific ERK 12 (4244 kDa) JNK (4654 kDa) andp38 (43 kDa) antibody ERK JNK and p38 were used as a loading control A representative western blot of three experiments is shown

we evaluated the protective effects on cerulein-induced acutepancreatitis (AP) However the sixth fraction remained to beexamined for potential to prevent an inflammatory responseto stimuli such as LPS Therefore in this study we used sixfractions isolated from NJ to examine the anti-inflammatoryactivity against LPS on murine macrophages

Generally sepsis is a life threatening condition caused bythe bodyrsquos response to bacterial products such as endotoxinwhich is often present with severe fever shock and respira-tory damage [12] Partially due to this phenomenon sepsisis often regarded as an uncontrolled inflammatory response[12] In the context of sepsis TNF-120572 IL-1120573 IL-6 and IL-8 arethemost frequently altered cytokines [13]When injected intoanimals these cytokines recapitulate many clinical and labo-ratory features of sepsis supporting the concept that sepsisrepresents a ldquocytokine stormrdquo Thus regulation of cytokinesmay prove valuable for the prevention and treatment ofsepsis In this study just as the total extract of NJ inhibitedthe production of cytokines [7] fractions 3 4 and 6 also

inhibited the production of cytokines (Figures 3 and 4)These results suggest that the ability of NJ to inhibit cytokineproduction may originate from fractions 3 4 andor 6

Using the fractions that were found to be effectiveagainst LPS we examined activation of MAPKs and NF-120581Bas possible regulatory mechanisms Generally it has beenreported that the production of proinflammatory mediatorsis modulated by the MAPKs and NF-120581B pathways Threemajor groups of MAPKs include the ERK JNK and p38which are all activated by phosphorylation [14ndash16] NF-120581Bactivation occurs mainly through the degradation of I120581Bthereby leading to subsequent release of NF-120581B dimers [17]Subsequently NF-120581B dimers translocate from the cytoplasmto the nucleus and activate the transcription of multiple 120581B-dependent target genes [17] In this study we examine thephosphorylation of MAPKs and degradation of I120581-B120572 NJ-1NJ-3 and NJ-4 inhibited the phosphorylation and activationof JNK and p38 but not ERK12 However NJ-6 inhibitednot only the activation of ERK12 JNK and p38 but also

Evidence-Based Complementary and Alternative Medicine 9

Time (min)LPS

60301506030150NJ-1 + LPS

120573-Actin

I120581-B120572

(a)

Time (min)LPS

60301506030150NJ-4 + LPS

120573-Actin

I120581-B120572

(b)

Time (min)LPS

60301506030150NJ-3 + LPS

120573-Actin

I120581-B120572

(c)

Time (min)LPS

60301506030150NJ-6 + LPS

120573-Actin

I120581-B120572

(d)

DAPI Merge

None

LPS

NJ-6

NF-120581B p65

(e)

Figure 6 Effects of NJ fractions onNF-120581B activation ((a)ndash(d)) Peritonealmacrophages were treatedwithNJ fractions (100120583gmL) for 1 h andthen stimulated with LPS (500 ngmL) at indicated time points The cells were harvested for western blot Total cellular proteins (20 120583g) wereresolved by SDS-PAGE transferred to PVDF membrane and detected with I120581-B120572 (40 kDa) antibody 120573-Actin was used as loading controlA representative western blot of three experiments is shown (e) Peritoneal macrophages were treated with NJ fractions (100120583gmL) for 1 hand then stimulated with LPS (500 ngmL) for 1 h Then nuclear translocation of NF-120581B p65 was examined using immunostaining NF-120581Bp65 was shown as red and the nucleus was shown as blue (DAPI) The values are means plusmn SE of three independent experiments lowast119875 lt 005versus saline +119875 lt 005 versus LPS

10 Evidence-Based Complementary and Alternative Medicine

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-1 (01mgkg) + LPS

NJ-1 (1mgkg) + LPSNJ-1 (10mgkg) + LPS

(a)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-3 (01mgkg) + LPS

NJ-3 (1mgkg) + LPSNJ-3 (10mgkg) + LPS

(b)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-4 (01mgkg) + LPS

NJ-4 (1mgkg) + LPSNJ-4 (10mgkg) + LPS

(c)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-6 (01mgkg) + LPS

NJ-6 (1mgkg) + LPSNJ-6 (10mgkg) + LPS

(d)

Figure 7 Effect of NJ fractions on LPS-induced endotoxin shock ((a)ndash(d)) Endotoxin shock was induced in 6ndash8-week-old female C57BL6mice as described in Section 2 NJ fractions were administered intraperitoneally to mice at 01 1 or 10mgkg 1 h before LPS injection ThenLPS (375mgkg) was injected to induce endotoxin shock The survival rates of endotoxin shock mice were monitored for survival for 120 hThis figure shows data for 10 mice per group

degradation of I120581-B120572 These results suggest that inhibitionof NO and cytokine production by NJ fractions is primarilythrough MAPKs or NF-120581B pathways

In this study we isolated two beneficial fractions of NJThe first one was NJ-4 which could show similar effectsto NJ total extract NJ-4 inhibited cytokine production andLPS-induced endotoxin shock similar to NJ total extract [7]Furthermore the regulatory mechanisms were similar NJtotal extract inhibited only MAPKs but NJ-4 inhibited theJNK and p38 Secondly NJ-6 showed dramatic inhibitionof LPS-induced inflammatory response Indeed NJ-6 inhib-ited the cytokine production and LPS-induced endotoxinshock dramatically However NJ-6 differed from total extractmechanistically NJ-6 inhibited the degradation of I120581-B120572and translocation of NF-120581B p65 which was not regulatedby the NJ total extract Further isolation of the NJ-4 andNJ-6 fractions could potentially yield a single compounddemonstrating inhibitory activity resembling that of NJ totalextract

In conclusion this study demonstrated which fractionsof NJ exhibit inhibitory activity against LPS-induced inflam-mation Among the 6 fractions isolated NJ-1 NJ-3 NJ-4 andNJ-6 showed the inhibition of NO and NJ-3 NJ-4 and NJ-6 showed the inhibition of cytokines In addition NJ-4 andNJ-6 exhibited dramatic prevention against LPS-endotoxinshock These results suggest that NJ-4 and NJ-6 may beeffective and beneficial candidates to ameliorate LPS-inducedinflammation

Abbreviations

ELISA Enzyme-linked immunosorbent assayERK Extracellular signal-regulated kinaseFBS Fetal bovine serumIL InterleukinJNK c-jun NH

2-terminal kinase

LPS LipopolysaccharideMAPKs Mitogen-activated protein kinases

Evidence-Based Complementary and Alternative Medicine 11

MTT 3-(45 Dimethylthiazol)-25-diphenyltetrazolium bromide

NF Nuclear factorNJ Nardostachys jatamansiNO Nitric oxidePCR Polymerase chain reactionTG ThioglycollateTLR Toll-like receptorTNF Tumor necrosis factor

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Gi-Sang Bae and Kwang-Ho Heo equally contributed to thiswork

Acknowledgments

This research was supported by a Basic Science ResearchProgram through theNational Research Foundation of Korea(NRF) fundedby theMinistryofEducation ScienceandTech-nology (NRF-2012R1A1A2040916 2012R1A5A2A35671257and 2012M2B2B1055246)

References

[1] C-S Sung and C-S Wong ldquoCellular mechanisms of neuroin-flammatory pain the role of interleukin-1120573rdquo Acta Anaesthesio-logica Taiwanica vol 45 no 2 pp 103ndash109 2007

[2] F O Martinez L Helming and S Gordon ldquoAlternative activa-tion of macrophages an immunologic functional perspectiverdquoAnnual Review of Immunology vol 27 pp 451ndash483 2009

[3] J D Schilling H M Machkovech L He A Diwan and J ESchaffer ldquoTLR4 activation under lipotoxic conditions leads tosynergistic macrophage cell death through a TRIF-dependentpathwayrdquo Journal of Immunology vol 190 no 3 pp 1285ndash12962013

[4] Y Ben-Neriah andM Karin ldquoInflammation meets cancer withNF-120581B as the matchmakerrdquo Nature Immunology vol 12 no 8pp 715ndash723 2011

[5] B C Bose Y Vijayvargiya and J N Bhatnagar ldquoNardostachysjatamansiDC a phyto-chemical study of its active constituentsrdquoIndian Journal of Medical Sciences vol 11 no 10 pp 799ndash8021957

[6] G-S Bae H-J Park D-Y Kim et al ldquoNardostachys jatamansiprotects against cerulein-induced acute pancreatitisrdquo Pancreasvol 39 no 4 pp 520ndash529 2010

[7] G-S Bae S-W Seo M-S Kim et al ldquoThe roots of Nar-dostachys jatamansi inhibits lipopolysaccharide-induced endo-toxin shockrdquo Journal of Natural Medicines vol 65 no 1 pp 63ndash72 2011

[8] G S Bae K C Park B S Koo et al ldquoThe inhibitory effectsof Nardostachys jatamansi on alcoholic chronic pancreatitisrdquoBMB Reports vol 45 no 7 pp 402ndash407 2012

[9] G S Bae K C Park B S Koo et al ldquoThe beneficial effectsof Nardostachys jatamansi extract on diet-induced severe acutepancreatitisrdquo Pancreas vol 42 no 2 pp 362ndash363 2013

[10] M-Y Song U-J Bae B-H Lee et al ldquoNardostachys jatamansiextract protects against cytokineinduced 120573-cell damage andstreptozotocin-induced diabetesrdquo World Journal of Gastroen-terology vol 16 no 26 pp 3249ndash3257 2010

[11] G S Bae M S Kim K C Park et al ldquoEffect of biologicallyactive fraction of Nardostachys jatamansi on cerulein-inducedacute pancreatitisrdquo World Journal of Gastroenterology vol 18no 25 pp 3223ndash3234 2012

[12] R S Hotchkiss and I E Karl ldquoThe pathophysiology andtreatment of sepsisrdquoThe New England Journal of Medicine vol348 no 2 pp 138ndash150 2003

[13] T S Blackwell and J W Christman ldquoSepsis and cytokinescurrent statusrdquo British Journal of Anaesthesia vol 77 no 1 pp110ndash117 1996

[14] H B Jijon W J Panenka K L Madsen and H G Par-sons ldquoMAP kinases contribute to IL-8 secretion by intestinalepithelial cells via a posttranscriptional mechanismrdquo AmericanJournal of PhysiologymdashCell Physiology vol 283 no 1 pp C31ndashC41 2002

[15] M Sanchez-Hidalgo A R Martın I Villegas and C Alarconde la Lastra ldquoRosiglitazone a PPAR120574 ligand modulates signaltransduction pathways during the development of acute TNBS-induced colitis in ratsrdquo European Journal of Pharmacology vol562 no 3 pp 247ndash258 2007

[16] F Scaldaferri M Sans S Vetrano et al ldquoThe role of MAPKin governing lymphocyte adhesion to and migration acrossthemicrovasculature in inflammatory bowel diseaserdquo EuropeanJournal of Immunology vol 39 no 1 pp 290ndash300 2009

[17] S Chae L Eckmann Y Miyamoto C Pothoulakis M KarinandM FKagnoff ldquoEpithelial cell I120581B-kinase120573has an importantprotective role in Clostridiumdifficile toxinA-inducedmucosalinjuryrdquo Journal of Immunology vol 177 no 2 pp 1214ndash12202006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

Time (min)LPS

60301506030150NJ-1 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(a)

LPSTime (min) 60301506030150

NJ-3 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(b)

LPSTime (min) 60301506030150

NJ-4 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(c)

LPSTime (min) 60301506030150

NJ-6 + LPS

P-ERK12

ERK12

P-JNK

JNK

P-p38

p38

(d)

Figure 5 Effects of NJ fractions on MAPKs activation ((a)ndash(d)) Peritoneal macrophages were treated with NJ fractions (100120583gmL) for 1 hand then stimulated with LPS (500 ngmL) at indicated time points Cells were harvested for western blot Total cellular proteins (20120583g) wereresolved by SDS-PAGE transferred to PVDF membrane and detected with phospho-specific ERK 12 (4244 kDa) JNK (4654 kDa) andp38 (43 kDa) antibody ERK JNK and p38 were used as a loading control A representative western blot of three experiments is shown

we evaluated the protective effects on cerulein-induced acutepancreatitis (AP) However the sixth fraction remained to beexamined for potential to prevent an inflammatory responseto stimuli such as LPS Therefore in this study we used sixfractions isolated from NJ to examine the anti-inflammatoryactivity against LPS on murine macrophages

Generally sepsis is a life threatening condition caused bythe bodyrsquos response to bacterial products such as endotoxinwhich is often present with severe fever shock and respira-tory damage [12] Partially due to this phenomenon sepsisis often regarded as an uncontrolled inflammatory response[12] In the context of sepsis TNF-120572 IL-1120573 IL-6 and IL-8 arethemost frequently altered cytokines [13]When injected intoanimals these cytokines recapitulate many clinical and labo-ratory features of sepsis supporting the concept that sepsisrepresents a ldquocytokine stormrdquo Thus regulation of cytokinesmay prove valuable for the prevention and treatment ofsepsis In this study just as the total extract of NJ inhibitedthe production of cytokines [7] fractions 3 4 and 6 also

inhibited the production of cytokines (Figures 3 and 4)These results suggest that the ability of NJ to inhibit cytokineproduction may originate from fractions 3 4 andor 6

Using the fractions that were found to be effectiveagainst LPS we examined activation of MAPKs and NF-120581Bas possible regulatory mechanisms Generally it has beenreported that the production of proinflammatory mediatorsis modulated by the MAPKs and NF-120581B pathways Threemajor groups of MAPKs include the ERK JNK and p38which are all activated by phosphorylation [14ndash16] NF-120581Bactivation occurs mainly through the degradation of I120581Bthereby leading to subsequent release of NF-120581B dimers [17]Subsequently NF-120581B dimers translocate from the cytoplasmto the nucleus and activate the transcription of multiple 120581B-dependent target genes [17] In this study we examine thephosphorylation of MAPKs and degradation of I120581-B120572 NJ-1NJ-3 and NJ-4 inhibited the phosphorylation and activationof JNK and p38 but not ERK12 However NJ-6 inhibitednot only the activation of ERK12 JNK and p38 but also

Evidence-Based Complementary and Alternative Medicine 9

Time (min)LPS

60301506030150NJ-1 + LPS

120573-Actin

I120581-B120572

(a)

Time (min)LPS

60301506030150NJ-4 + LPS

120573-Actin

I120581-B120572

(b)

Time (min)LPS

60301506030150NJ-3 + LPS

120573-Actin

I120581-B120572

(c)

Time (min)LPS

60301506030150NJ-6 + LPS

120573-Actin

I120581-B120572

(d)

DAPI Merge

None

LPS

NJ-6

NF-120581B p65

(e)

Figure 6 Effects of NJ fractions onNF-120581B activation ((a)ndash(d)) Peritonealmacrophages were treatedwithNJ fractions (100120583gmL) for 1 h andthen stimulated with LPS (500 ngmL) at indicated time points The cells were harvested for western blot Total cellular proteins (20 120583g) wereresolved by SDS-PAGE transferred to PVDF membrane and detected with I120581-B120572 (40 kDa) antibody 120573-Actin was used as loading controlA representative western blot of three experiments is shown (e) Peritoneal macrophages were treated with NJ fractions (100120583gmL) for 1 hand then stimulated with LPS (500 ngmL) for 1 h Then nuclear translocation of NF-120581B p65 was examined using immunostaining NF-120581Bp65 was shown as red and the nucleus was shown as blue (DAPI) The values are means plusmn SE of three independent experiments lowast119875 lt 005versus saline +119875 lt 005 versus LPS

10 Evidence-Based Complementary and Alternative Medicine

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-1 (01mgkg) + LPS

NJ-1 (1mgkg) + LPSNJ-1 (10mgkg) + LPS

(a)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-3 (01mgkg) + LPS

NJ-3 (1mgkg) + LPSNJ-3 (10mgkg) + LPS

(b)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-4 (01mgkg) + LPS

NJ-4 (1mgkg) + LPSNJ-4 (10mgkg) + LPS

(c)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-6 (01mgkg) + LPS

NJ-6 (1mgkg) + LPSNJ-6 (10mgkg) + LPS

(d)

Figure 7 Effect of NJ fractions on LPS-induced endotoxin shock ((a)ndash(d)) Endotoxin shock was induced in 6ndash8-week-old female C57BL6mice as described in Section 2 NJ fractions were administered intraperitoneally to mice at 01 1 or 10mgkg 1 h before LPS injection ThenLPS (375mgkg) was injected to induce endotoxin shock The survival rates of endotoxin shock mice were monitored for survival for 120 hThis figure shows data for 10 mice per group

degradation of I120581-B120572 These results suggest that inhibitionof NO and cytokine production by NJ fractions is primarilythrough MAPKs or NF-120581B pathways

In this study we isolated two beneficial fractions of NJThe first one was NJ-4 which could show similar effectsto NJ total extract NJ-4 inhibited cytokine production andLPS-induced endotoxin shock similar to NJ total extract [7]Furthermore the regulatory mechanisms were similar NJtotal extract inhibited only MAPKs but NJ-4 inhibited theJNK and p38 Secondly NJ-6 showed dramatic inhibitionof LPS-induced inflammatory response Indeed NJ-6 inhib-ited the cytokine production and LPS-induced endotoxinshock dramatically However NJ-6 differed from total extractmechanistically NJ-6 inhibited the degradation of I120581-B120572and translocation of NF-120581B p65 which was not regulatedby the NJ total extract Further isolation of the NJ-4 andNJ-6 fractions could potentially yield a single compounddemonstrating inhibitory activity resembling that of NJ totalextract

In conclusion this study demonstrated which fractionsof NJ exhibit inhibitory activity against LPS-induced inflam-mation Among the 6 fractions isolated NJ-1 NJ-3 NJ-4 andNJ-6 showed the inhibition of NO and NJ-3 NJ-4 and NJ-6 showed the inhibition of cytokines In addition NJ-4 andNJ-6 exhibited dramatic prevention against LPS-endotoxinshock These results suggest that NJ-4 and NJ-6 may beeffective and beneficial candidates to ameliorate LPS-inducedinflammation

Abbreviations

ELISA Enzyme-linked immunosorbent assayERK Extracellular signal-regulated kinaseFBS Fetal bovine serumIL InterleukinJNK c-jun NH

2-terminal kinase

LPS LipopolysaccharideMAPKs Mitogen-activated protein kinases

Evidence-Based Complementary and Alternative Medicine 11

MTT 3-(45 Dimethylthiazol)-25-diphenyltetrazolium bromide

NF Nuclear factorNJ Nardostachys jatamansiNO Nitric oxidePCR Polymerase chain reactionTG ThioglycollateTLR Toll-like receptorTNF Tumor necrosis factor

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Gi-Sang Bae and Kwang-Ho Heo equally contributed to thiswork

Acknowledgments

This research was supported by a Basic Science ResearchProgram through theNational Research Foundation of Korea(NRF) fundedby theMinistryofEducation ScienceandTech-nology (NRF-2012R1A1A2040916 2012R1A5A2A35671257and 2012M2B2B1055246)

References

[1] C-S Sung and C-S Wong ldquoCellular mechanisms of neuroin-flammatory pain the role of interleukin-1120573rdquo Acta Anaesthesio-logica Taiwanica vol 45 no 2 pp 103ndash109 2007

[2] F O Martinez L Helming and S Gordon ldquoAlternative activa-tion of macrophages an immunologic functional perspectiverdquoAnnual Review of Immunology vol 27 pp 451ndash483 2009

[3] J D Schilling H M Machkovech L He A Diwan and J ESchaffer ldquoTLR4 activation under lipotoxic conditions leads tosynergistic macrophage cell death through a TRIF-dependentpathwayrdquo Journal of Immunology vol 190 no 3 pp 1285ndash12962013

[4] Y Ben-Neriah andM Karin ldquoInflammation meets cancer withNF-120581B as the matchmakerrdquo Nature Immunology vol 12 no 8pp 715ndash723 2011

[5] B C Bose Y Vijayvargiya and J N Bhatnagar ldquoNardostachysjatamansiDC a phyto-chemical study of its active constituentsrdquoIndian Journal of Medical Sciences vol 11 no 10 pp 799ndash8021957

[6] G-S Bae H-J Park D-Y Kim et al ldquoNardostachys jatamansiprotects against cerulein-induced acute pancreatitisrdquo Pancreasvol 39 no 4 pp 520ndash529 2010

[7] G-S Bae S-W Seo M-S Kim et al ldquoThe roots of Nar-dostachys jatamansi inhibits lipopolysaccharide-induced endo-toxin shockrdquo Journal of Natural Medicines vol 65 no 1 pp 63ndash72 2011

[8] G S Bae K C Park B S Koo et al ldquoThe inhibitory effectsof Nardostachys jatamansi on alcoholic chronic pancreatitisrdquoBMB Reports vol 45 no 7 pp 402ndash407 2012

[9] G S Bae K C Park B S Koo et al ldquoThe beneficial effectsof Nardostachys jatamansi extract on diet-induced severe acutepancreatitisrdquo Pancreas vol 42 no 2 pp 362ndash363 2013

[10] M-Y Song U-J Bae B-H Lee et al ldquoNardostachys jatamansiextract protects against cytokineinduced 120573-cell damage andstreptozotocin-induced diabetesrdquo World Journal of Gastroen-terology vol 16 no 26 pp 3249ndash3257 2010

[11] G S Bae M S Kim K C Park et al ldquoEffect of biologicallyactive fraction of Nardostachys jatamansi on cerulein-inducedacute pancreatitisrdquo World Journal of Gastroenterology vol 18no 25 pp 3223ndash3234 2012

[12] R S Hotchkiss and I E Karl ldquoThe pathophysiology andtreatment of sepsisrdquoThe New England Journal of Medicine vol348 no 2 pp 138ndash150 2003

[13] T S Blackwell and J W Christman ldquoSepsis and cytokinescurrent statusrdquo British Journal of Anaesthesia vol 77 no 1 pp110ndash117 1996

[14] H B Jijon W J Panenka K L Madsen and H G Par-sons ldquoMAP kinases contribute to IL-8 secretion by intestinalepithelial cells via a posttranscriptional mechanismrdquo AmericanJournal of PhysiologymdashCell Physiology vol 283 no 1 pp C31ndashC41 2002

[15] M Sanchez-Hidalgo A R Martın I Villegas and C Alarconde la Lastra ldquoRosiglitazone a PPAR120574 ligand modulates signaltransduction pathways during the development of acute TNBS-induced colitis in ratsrdquo European Journal of Pharmacology vol562 no 3 pp 247ndash258 2007

[16] F Scaldaferri M Sans S Vetrano et al ldquoThe role of MAPKin governing lymphocyte adhesion to and migration acrossthemicrovasculature in inflammatory bowel diseaserdquo EuropeanJournal of Immunology vol 39 no 1 pp 290ndash300 2009

[17] S Chae L Eckmann Y Miyamoto C Pothoulakis M KarinandM FKagnoff ldquoEpithelial cell I120581B-kinase120573has an importantprotective role in Clostridiumdifficile toxinA-inducedmucosalinjuryrdquo Journal of Immunology vol 177 no 2 pp 1214ndash12202006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 9

Time (min)LPS

60301506030150NJ-1 + LPS

120573-Actin

I120581-B120572

(a)

Time (min)LPS

60301506030150NJ-4 + LPS

120573-Actin

I120581-B120572

(b)

Time (min)LPS

60301506030150NJ-3 + LPS

120573-Actin

I120581-B120572

(c)

Time (min)LPS

60301506030150NJ-6 + LPS

120573-Actin

I120581-B120572

(d)

DAPI Merge

None

LPS

NJ-6

NF-120581B p65

(e)

Figure 6 Effects of NJ fractions onNF-120581B activation ((a)ndash(d)) Peritonealmacrophages were treatedwithNJ fractions (100120583gmL) for 1 h andthen stimulated with LPS (500 ngmL) at indicated time points The cells were harvested for western blot Total cellular proteins (20 120583g) wereresolved by SDS-PAGE transferred to PVDF membrane and detected with I120581-B120572 (40 kDa) antibody 120573-Actin was used as loading controlA representative western blot of three experiments is shown (e) Peritoneal macrophages were treated with NJ fractions (100120583gmL) for 1 hand then stimulated with LPS (500 ngmL) for 1 h Then nuclear translocation of NF-120581B p65 was examined using immunostaining NF-120581Bp65 was shown as red and the nucleus was shown as blue (DAPI) The values are means plusmn SE of three independent experiments lowast119875 lt 005versus saline +119875 lt 005 versus LPS

10 Evidence-Based Complementary and Alternative Medicine

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-1 (01mgkg) + LPS

NJ-1 (1mgkg) + LPSNJ-1 (10mgkg) + LPS

(a)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-3 (01mgkg) + LPS

NJ-3 (1mgkg) + LPSNJ-3 (10mgkg) + LPS

(b)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-4 (01mgkg) + LPS

NJ-4 (1mgkg) + LPSNJ-4 (10mgkg) + LPS

(c)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-6 (01mgkg) + LPS

NJ-6 (1mgkg) + LPSNJ-6 (10mgkg) + LPS

(d)

Figure 7 Effect of NJ fractions on LPS-induced endotoxin shock ((a)ndash(d)) Endotoxin shock was induced in 6ndash8-week-old female C57BL6mice as described in Section 2 NJ fractions were administered intraperitoneally to mice at 01 1 or 10mgkg 1 h before LPS injection ThenLPS (375mgkg) was injected to induce endotoxin shock The survival rates of endotoxin shock mice were monitored for survival for 120 hThis figure shows data for 10 mice per group

degradation of I120581-B120572 These results suggest that inhibitionof NO and cytokine production by NJ fractions is primarilythrough MAPKs or NF-120581B pathways

In this study we isolated two beneficial fractions of NJThe first one was NJ-4 which could show similar effectsto NJ total extract NJ-4 inhibited cytokine production andLPS-induced endotoxin shock similar to NJ total extract [7]Furthermore the regulatory mechanisms were similar NJtotal extract inhibited only MAPKs but NJ-4 inhibited theJNK and p38 Secondly NJ-6 showed dramatic inhibitionof LPS-induced inflammatory response Indeed NJ-6 inhib-ited the cytokine production and LPS-induced endotoxinshock dramatically However NJ-6 differed from total extractmechanistically NJ-6 inhibited the degradation of I120581-B120572and translocation of NF-120581B p65 which was not regulatedby the NJ total extract Further isolation of the NJ-4 andNJ-6 fractions could potentially yield a single compounddemonstrating inhibitory activity resembling that of NJ totalextract

In conclusion this study demonstrated which fractionsof NJ exhibit inhibitory activity against LPS-induced inflam-mation Among the 6 fractions isolated NJ-1 NJ-3 NJ-4 andNJ-6 showed the inhibition of NO and NJ-3 NJ-4 and NJ-6 showed the inhibition of cytokines In addition NJ-4 andNJ-6 exhibited dramatic prevention against LPS-endotoxinshock These results suggest that NJ-4 and NJ-6 may beeffective and beneficial candidates to ameliorate LPS-inducedinflammation

Abbreviations

ELISA Enzyme-linked immunosorbent assayERK Extracellular signal-regulated kinaseFBS Fetal bovine serumIL InterleukinJNK c-jun NH

2-terminal kinase

LPS LipopolysaccharideMAPKs Mitogen-activated protein kinases

Evidence-Based Complementary and Alternative Medicine 11

MTT 3-(45 Dimethylthiazol)-25-diphenyltetrazolium bromide

NF Nuclear factorNJ Nardostachys jatamansiNO Nitric oxidePCR Polymerase chain reactionTG ThioglycollateTLR Toll-like receptorTNF Tumor necrosis factor

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Gi-Sang Bae and Kwang-Ho Heo equally contributed to thiswork

Acknowledgments

This research was supported by a Basic Science ResearchProgram through theNational Research Foundation of Korea(NRF) fundedby theMinistryofEducation ScienceandTech-nology (NRF-2012R1A1A2040916 2012R1A5A2A35671257and 2012M2B2B1055246)

References

[1] C-S Sung and C-S Wong ldquoCellular mechanisms of neuroin-flammatory pain the role of interleukin-1120573rdquo Acta Anaesthesio-logica Taiwanica vol 45 no 2 pp 103ndash109 2007

[2] F O Martinez L Helming and S Gordon ldquoAlternative activa-tion of macrophages an immunologic functional perspectiverdquoAnnual Review of Immunology vol 27 pp 451ndash483 2009

[3] J D Schilling H M Machkovech L He A Diwan and J ESchaffer ldquoTLR4 activation under lipotoxic conditions leads tosynergistic macrophage cell death through a TRIF-dependentpathwayrdquo Journal of Immunology vol 190 no 3 pp 1285ndash12962013

[4] Y Ben-Neriah andM Karin ldquoInflammation meets cancer withNF-120581B as the matchmakerrdquo Nature Immunology vol 12 no 8pp 715ndash723 2011

[5] B C Bose Y Vijayvargiya and J N Bhatnagar ldquoNardostachysjatamansiDC a phyto-chemical study of its active constituentsrdquoIndian Journal of Medical Sciences vol 11 no 10 pp 799ndash8021957

[6] G-S Bae H-J Park D-Y Kim et al ldquoNardostachys jatamansiprotects against cerulein-induced acute pancreatitisrdquo Pancreasvol 39 no 4 pp 520ndash529 2010

[7] G-S Bae S-W Seo M-S Kim et al ldquoThe roots of Nar-dostachys jatamansi inhibits lipopolysaccharide-induced endo-toxin shockrdquo Journal of Natural Medicines vol 65 no 1 pp 63ndash72 2011

[8] G S Bae K C Park B S Koo et al ldquoThe inhibitory effectsof Nardostachys jatamansi on alcoholic chronic pancreatitisrdquoBMB Reports vol 45 no 7 pp 402ndash407 2012

[9] G S Bae K C Park B S Koo et al ldquoThe beneficial effectsof Nardostachys jatamansi extract on diet-induced severe acutepancreatitisrdquo Pancreas vol 42 no 2 pp 362ndash363 2013

[10] M-Y Song U-J Bae B-H Lee et al ldquoNardostachys jatamansiextract protects against cytokineinduced 120573-cell damage andstreptozotocin-induced diabetesrdquo World Journal of Gastroen-terology vol 16 no 26 pp 3249ndash3257 2010

[11] G S Bae M S Kim K C Park et al ldquoEffect of biologicallyactive fraction of Nardostachys jatamansi on cerulein-inducedacute pancreatitisrdquo World Journal of Gastroenterology vol 18no 25 pp 3223ndash3234 2012

[12] R S Hotchkiss and I E Karl ldquoThe pathophysiology andtreatment of sepsisrdquoThe New England Journal of Medicine vol348 no 2 pp 138ndash150 2003

[13] T S Blackwell and J W Christman ldquoSepsis and cytokinescurrent statusrdquo British Journal of Anaesthesia vol 77 no 1 pp110ndash117 1996

[14] H B Jijon W J Panenka K L Madsen and H G Par-sons ldquoMAP kinases contribute to IL-8 secretion by intestinalepithelial cells via a posttranscriptional mechanismrdquo AmericanJournal of PhysiologymdashCell Physiology vol 283 no 1 pp C31ndashC41 2002

[15] M Sanchez-Hidalgo A R Martın I Villegas and C Alarconde la Lastra ldquoRosiglitazone a PPAR120574 ligand modulates signaltransduction pathways during the development of acute TNBS-induced colitis in ratsrdquo European Journal of Pharmacology vol562 no 3 pp 247ndash258 2007

[16] F Scaldaferri M Sans S Vetrano et al ldquoThe role of MAPKin governing lymphocyte adhesion to and migration acrossthemicrovasculature in inflammatory bowel diseaserdquo EuropeanJournal of Immunology vol 39 no 1 pp 290ndash300 2009

[17] S Chae L Eckmann Y Miyamoto C Pothoulakis M KarinandM FKagnoff ldquoEpithelial cell I120581B-kinase120573has an importantprotective role in Clostridiumdifficile toxinA-inducedmucosalinjuryrdquo Journal of Immunology vol 177 no 2 pp 1214ndash12202006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Evidence-Based Complementary and Alternative Medicine

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-1 (01mgkg) + LPS

NJ-1 (1mgkg) + LPSNJ-1 (10mgkg) + LPS

(a)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-3 (01mgkg) + LPS

NJ-3 (1mgkg) + LPSNJ-3 (10mgkg) + LPS

(b)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-4 (01mgkg) + LPS

NJ-4 (1mgkg) + LPSNJ-4 (10mgkg) + LPS

(c)

Time after LPS injection (h)

Surv

ival

rate

()

0 12 24 36 48 60 72 84 96 108 120

0

20

40

60

80

100

LPSNJ-6 (01mgkg) + LPS

NJ-6 (1mgkg) + LPSNJ-6 (10mgkg) + LPS

(d)

Figure 7 Effect of NJ fractions on LPS-induced endotoxin shock ((a)ndash(d)) Endotoxin shock was induced in 6ndash8-week-old female C57BL6mice as described in Section 2 NJ fractions were administered intraperitoneally to mice at 01 1 or 10mgkg 1 h before LPS injection ThenLPS (375mgkg) was injected to induce endotoxin shock The survival rates of endotoxin shock mice were monitored for survival for 120 hThis figure shows data for 10 mice per group

degradation of I120581-B120572 These results suggest that inhibitionof NO and cytokine production by NJ fractions is primarilythrough MAPKs or NF-120581B pathways

In this study we isolated two beneficial fractions of NJThe first one was NJ-4 which could show similar effectsto NJ total extract NJ-4 inhibited cytokine production andLPS-induced endotoxin shock similar to NJ total extract [7]Furthermore the regulatory mechanisms were similar NJtotal extract inhibited only MAPKs but NJ-4 inhibited theJNK and p38 Secondly NJ-6 showed dramatic inhibitionof LPS-induced inflammatory response Indeed NJ-6 inhib-ited the cytokine production and LPS-induced endotoxinshock dramatically However NJ-6 differed from total extractmechanistically NJ-6 inhibited the degradation of I120581-B120572and translocation of NF-120581B p65 which was not regulatedby the NJ total extract Further isolation of the NJ-4 andNJ-6 fractions could potentially yield a single compounddemonstrating inhibitory activity resembling that of NJ totalextract

In conclusion this study demonstrated which fractionsof NJ exhibit inhibitory activity against LPS-induced inflam-mation Among the 6 fractions isolated NJ-1 NJ-3 NJ-4 andNJ-6 showed the inhibition of NO and NJ-3 NJ-4 and NJ-6 showed the inhibition of cytokines In addition NJ-4 andNJ-6 exhibited dramatic prevention against LPS-endotoxinshock These results suggest that NJ-4 and NJ-6 may beeffective and beneficial candidates to ameliorate LPS-inducedinflammation

Abbreviations

ELISA Enzyme-linked immunosorbent assayERK Extracellular signal-regulated kinaseFBS Fetal bovine serumIL InterleukinJNK c-jun NH

2-terminal kinase

LPS LipopolysaccharideMAPKs Mitogen-activated protein kinases

Evidence-Based Complementary and Alternative Medicine 11

MTT 3-(45 Dimethylthiazol)-25-diphenyltetrazolium bromide

NF Nuclear factorNJ Nardostachys jatamansiNO Nitric oxidePCR Polymerase chain reactionTG ThioglycollateTLR Toll-like receptorTNF Tumor necrosis factor

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Gi-Sang Bae and Kwang-Ho Heo equally contributed to thiswork

Acknowledgments

This research was supported by a Basic Science ResearchProgram through theNational Research Foundation of Korea(NRF) fundedby theMinistryofEducation ScienceandTech-nology (NRF-2012R1A1A2040916 2012R1A5A2A35671257and 2012M2B2B1055246)

References

[1] C-S Sung and C-S Wong ldquoCellular mechanisms of neuroin-flammatory pain the role of interleukin-1120573rdquo Acta Anaesthesio-logica Taiwanica vol 45 no 2 pp 103ndash109 2007

[2] F O Martinez L Helming and S Gordon ldquoAlternative activa-tion of macrophages an immunologic functional perspectiverdquoAnnual Review of Immunology vol 27 pp 451ndash483 2009

[3] J D Schilling H M Machkovech L He A Diwan and J ESchaffer ldquoTLR4 activation under lipotoxic conditions leads tosynergistic macrophage cell death through a TRIF-dependentpathwayrdquo Journal of Immunology vol 190 no 3 pp 1285ndash12962013

[4] Y Ben-Neriah andM Karin ldquoInflammation meets cancer withNF-120581B as the matchmakerrdquo Nature Immunology vol 12 no 8pp 715ndash723 2011

[5] B C Bose Y Vijayvargiya and J N Bhatnagar ldquoNardostachysjatamansiDC a phyto-chemical study of its active constituentsrdquoIndian Journal of Medical Sciences vol 11 no 10 pp 799ndash8021957

[6] G-S Bae H-J Park D-Y Kim et al ldquoNardostachys jatamansiprotects against cerulein-induced acute pancreatitisrdquo Pancreasvol 39 no 4 pp 520ndash529 2010

[7] G-S Bae S-W Seo M-S Kim et al ldquoThe roots of Nar-dostachys jatamansi inhibits lipopolysaccharide-induced endo-toxin shockrdquo Journal of Natural Medicines vol 65 no 1 pp 63ndash72 2011

[8] G S Bae K C Park B S Koo et al ldquoThe inhibitory effectsof Nardostachys jatamansi on alcoholic chronic pancreatitisrdquoBMB Reports vol 45 no 7 pp 402ndash407 2012

[9] G S Bae K C Park B S Koo et al ldquoThe beneficial effectsof Nardostachys jatamansi extract on diet-induced severe acutepancreatitisrdquo Pancreas vol 42 no 2 pp 362ndash363 2013

[10] M-Y Song U-J Bae B-H Lee et al ldquoNardostachys jatamansiextract protects against cytokineinduced 120573-cell damage andstreptozotocin-induced diabetesrdquo World Journal of Gastroen-terology vol 16 no 26 pp 3249ndash3257 2010

[11] G S Bae M S Kim K C Park et al ldquoEffect of biologicallyactive fraction of Nardostachys jatamansi on cerulein-inducedacute pancreatitisrdquo World Journal of Gastroenterology vol 18no 25 pp 3223ndash3234 2012

[12] R S Hotchkiss and I E Karl ldquoThe pathophysiology andtreatment of sepsisrdquoThe New England Journal of Medicine vol348 no 2 pp 138ndash150 2003

[13] T S Blackwell and J W Christman ldquoSepsis and cytokinescurrent statusrdquo British Journal of Anaesthesia vol 77 no 1 pp110ndash117 1996

[14] H B Jijon W J Panenka K L Madsen and H G Par-sons ldquoMAP kinases contribute to IL-8 secretion by intestinalepithelial cells via a posttranscriptional mechanismrdquo AmericanJournal of PhysiologymdashCell Physiology vol 283 no 1 pp C31ndashC41 2002

[15] M Sanchez-Hidalgo A R Martın I Villegas and C Alarconde la Lastra ldquoRosiglitazone a PPAR120574 ligand modulates signaltransduction pathways during the development of acute TNBS-induced colitis in ratsrdquo European Journal of Pharmacology vol562 no 3 pp 247ndash258 2007

[16] F Scaldaferri M Sans S Vetrano et al ldquoThe role of MAPKin governing lymphocyte adhesion to and migration acrossthemicrovasculature in inflammatory bowel diseaserdquo EuropeanJournal of Immunology vol 39 no 1 pp 290ndash300 2009

[17] S Chae L Eckmann Y Miyamoto C Pothoulakis M KarinandM FKagnoff ldquoEpithelial cell I120581B-kinase120573has an importantprotective role in Clostridiumdifficile toxinA-inducedmucosalinjuryrdquo Journal of Immunology vol 177 no 2 pp 1214ndash12202006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 11

MTT 3-(45 Dimethylthiazol)-25-diphenyltetrazolium bromide

NF Nuclear factorNJ Nardostachys jatamansiNO Nitric oxidePCR Polymerase chain reactionTG ThioglycollateTLR Toll-like receptorTNF Tumor necrosis factor

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Gi-Sang Bae and Kwang-Ho Heo equally contributed to thiswork

Acknowledgments

This research was supported by a Basic Science ResearchProgram through theNational Research Foundation of Korea(NRF) fundedby theMinistryofEducation ScienceandTech-nology (NRF-2012R1A1A2040916 2012R1A5A2A35671257and 2012M2B2B1055246)

References

[1] C-S Sung and C-S Wong ldquoCellular mechanisms of neuroin-flammatory pain the role of interleukin-1120573rdquo Acta Anaesthesio-logica Taiwanica vol 45 no 2 pp 103ndash109 2007

[2] F O Martinez L Helming and S Gordon ldquoAlternative activa-tion of macrophages an immunologic functional perspectiverdquoAnnual Review of Immunology vol 27 pp 451ndash483 2009

[3] J D Schilling H M Machkovech L He A Diwan and J ESchaffer ldquoTLR4 activation under lipotoxic conditions leads tosynergistic macrophage cell death through a TRIF-dependentpathwayrdquo Journal of Immunology vol 190 no 3 pp 1285ndash12962013

[4] Y Ben-Neriah andM Karin ldquoInflammation meets cancer withNF-120581B as the matchmakerrdquo Nature Immunology vol 12 no 8pp 715ndash723 2011

[5] B C Bose Y Vijayvargiya and J N Bhatnagar ldquoNardostachysjatamansiDC a phyto-chemical study of its active constituentsrdquoIndian Journal of Medical Sciences vol 11 no 10 pp 799ndash8021957

[6] G-S Bae H-J Park D-Y Kim et al ldquoNardostachys jatamansiprotects against cerulein-induced acute pancreatitisrdquo Pancreasvol 39 no 4 pp 520ndash529 2010

[7] G-S Bae S-W Seo M-S Kim et al ldquoThe roots of Nar-dostachys jatamansi inhibits lipopolysaccharide-induced endo-toxin shockrdquo Journal of Natural Medicines vol 65 no 1 pp 63ndash72 2011

[8] G S Bae K C Park B S Koo et al ldquoThe inhibitory effectsof Nardostachys jatamansi on alcoholic chronic pancreatitisrdquoBMB Reports vol 45 no 7 pp 402ndash407 2012

[9] G S Bae K C Park B S Koo et al ldquoThe beneficial effectsof Nardostachys jatamansi extract on diet-induced severe acutepancreatitisrdquo Pancreas vol 42 no 2 pp 362ndash363 2013

[10] M-Y Song U-J Bae B-H Lee et al ldquoNardostachys jatamansiextract protects against cytokineinduced 120573-cell damage andstreptozotocin-induced diabetesrdquo World Journal of Gastroen-terology vol 16 no 26 pp 3249ndash3257 2010

[11] G S Bae M S Kim K C Park et al ldquoEffect of biologicallyactive fraction of Nardostachys jatamansi on cerulein-inducedacute pancreatitisrdquo World Journal of Gastroenterology vol 18no 25 pp 3223ndash3234 2012

[12] R S Hotchkiss and I E Karl ldquoThe pathophysiology andtreatment of sepsisrdquoThe New England Journal of Medicine vol348 no 2 pp 138ndash150 2003

[13] T S Blackwell and J W Christman ldquoSepsis and cytokinescurrent statusrdquo British Journal of Anaesthesia vol 77 no 1 pp110ndash117 1996

[14] H B Jijon W J Panenka K L Madsen and H G Par-sons ldquoMAP kinases contribute to IL-8 secretion by intestinalepithelial cells via a posttranscriptional mechanismrdquo AmericanJournal of PhysiologymdashCell Physiology vol 283 no 1 pp C31ndashC41 2002

[15] M Sanchez-Hidalgo A R Martın I Villegas and C Alarconde la Lastra ldquoRosiglitazone a PPAR120574 ligand modulates signaltransduction pathways during the development of acute TNBS-induced colitis in ratsrdquo European Journal of Pharmacology vol562 no 3 pp 247ndash258 2007

[16] F Scaldaferri M Sans S Vetrano et al ldquoThe role of MAPKin governing lymphocyte adhesion to and migration acrossthemicrovasculature in inflammatory bowel diseaserdquo EuropeanJournal of Immunology vol 39 no 1 pp 290ndash300 2009

[17] S Chae L Eckmann Y Miyamoto C Pothoulakis M KarinandM FKagnoff ldquoEpithelial cell I120581B-kinase120573has an importantprotective role in Clostridiumdifficile toxinA-inducedmucosalinjuryrdquo Journal of Immunology vol 177 no 2 pp 1214ndash12202006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom