Research Article Matrine Attenuates COX-2 and ICAM-1...

Research ArticleMatrine Attenuates COX-2 and ICAM-1 Expressions inHuman Lung Epithelial Cells and Prevents Acute Lung Injury inLPS-Induced Mice

Chian-Jiun Liou12 You-Rong Lai3 Ya-Ling Chen3 Yi-Hsien Chang3

Zih-Ying Li2 and Wen-Chung Huang245

1Department of Nursing Chang Gung University of Science and Technology No 261 Wenhua 1st Road Guishan DistrictTaoyuan City 33303 Taiwan2Chang Gung Memorial Hospital at Linkou Guishan District Taoyuan City 33303 Taiwan3Department of Nutrition and Health Sciences Chang Gung University of Science and Technology No 261 Wenhua 1st RoadGuishan District Taoyuan City 33303 Taiwan4Research Center for Industry of Human Ecology Chang Gung University of Science and Technology No 261 Wenhua 1st RoadGuishan District Taoyuan City 33303 Taiwan5Graduate Institute of Health Industry Technology Chang Gung University of Science Taiwan and TechnologyNo 261 Wenhua 1st Road Guishan District Taoyuan City 33303 Taiwan

Correspondence should be addressed to Wen-Chung Huang wchuanggwcgustedutw

Received 8 September 2015 Revised 8 November 2015 Accepted 10 November 2015

Academic Editor Mirella Giovarelli

Copyright copy 2016 Chian-Jiun Liou et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Matrine is isolated from Sophora flavescens and shows anti-inflammatory effects in macrophages Here we evaluated matrinersquos sup-pressive effects on cyclooxygenase 2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1) expressions in lipopolysaccharide-(LPS-) stimulated human lung epithelial A549 cells Additionally BALBcmice were given variousmatrine doses by intraperitonealinjection and then lung injury was induced via intratracheal instillation of LPS In LPS-stimulated A549 cells matrine inhibited theproductions of interleukin-8 (IL-8)monocyte chemotactic protein-1 and IL-6 and decreasedCOX-2 expressionMatrine treatmentalso decreased ICAM-1 protein expression and suppressed the adhesion of neutrophil-like cells to inflammatory A549 cells In vitroresults demonstrated that matrine significantly inhibited mitogen-activated protein kinase phosphorylation and decreased nucleartranscription factor kappa-B subunit p65 protein translocation into the nucleus In vivo data indicated that matrine significantlyinhibited neutrophil infiltration and suppressed productions of tumor necrosis factor-120572 and IL-6 in mouse bronchoalveolarlavage fluid and serum Analysis of lung tissue showed that matrine decreased the gene expression of proinflammatory cytokineschemokines COX-2 and ICAM-1 Our findings suggest thatmatrine improved lung injury inmice and decreased the inflammatoryresponse in human lung epithelial cells

1 Introduction

Acute lung injury (ALI) is characterized by atelectasis oflung airspaces reducing the total lung capacity [1] In ALIcases the lung tissue releases large amounts of inflammatorycytokines chemokines and inflammatory media such asnitric oxide and cyclooxygenase 2 (COX-2) [2] ALI alsocauses lung cells to secrete more proteases leading to alve-olar cell damage and fluid accumulation in alveoli causing

edema in the lung tissue [3] Alveolar damage results inincreased vascular permeability of blood vessels and neu-trophil infiltration in the lung tissue further exacerbatinglung inflammation and pulmonary edema and potentiallycausing respiratory failure and death [4]

ALI commonly occurs as an early symptom of sepsisor bacterial infection of the respiratory tract [2] Gram-negative bacterial invasion of the lungs can cause bacterialpneumonia and ALI [5] Lipopolysaccharide (LPS) is a cell

Hindawi Publishing CorporationMediators of InflammationVolume 2016 Article ID 3630485 12 pageshttpdxdoiorg10115520163630485

2 Mediators of Inflammation

wall component of Gram-negative bacteria and can inducean innate immunity response activating immune cells tocombat microbial infection [6] Thus LPS is widely used toinduce acute lung inflammation in animal models Activatedmacrophages and lung epithelial cells reportedly releaseproinflammatory cytokines and chemokines aggravatingALI progression Prior studies have used lung epithelialcells to evaluate the anti-inflammatory responses of drugsor natural compounds during LPS-induced inflammatoryresponse [7 8]

In China and Taiwan the root of Sophora flavescens(Leguminosae) is used to treat fever jaundice urinary tractinfections and pyogenic infections [9] Matrine is isolatedfrom S flavescens and can reportedly attenuate cerebralischemic injury in mice and induce apoptosis of chronicmyeloid leukemia cells osteosarcoma cells and cholangio-carcinoma cells [9 10] Zhang et al previously demonstratedthat matrine suppressed proinflammatory cytokine produc-tion in LPS-stimulated mouse macrophages [11] Previouslyour group also found that matrine could improve eosinophilinfiltration airway hyperresponsiveness and Th2-associatedcytokine production in asthmatic mice [12] However themechanisms of matrinersquos anti-inflammatory activity in lungepithelial cells are largely unclear

In the present study we investigated whether matrinereduced inflammatory responses and production of intercel-lular adhesion molecule-1 (ICAM-1) in lung epithelial cellsWe also examined whether matrine protected and preventedacute lung injury in LPS-induced mice

2 Materials and Methods

21 Matrine and Cell Culture Matrine (ge99 by HPLC) waspurchased from Sigma-Aldrich (St Louis MO USA) andwas dissolved in normal saline as previously described [12]The human lung epithelial cell line A549 was purchased fromthe Bioresource Collection and Research Center (BCRCTaiwan) A549 cells were cultured in DMEM medium(Invitrogen-Gibco Paisley Scotland) containing 2mM glu-tamine 100UmL penicillin and streptomycin and 10 fetalbovine serum (Biological Industries Haemek Israel) Cellswere incubated at 37∘C in 5 CO

2humidified air and were

subcultured twice each week

22 Cell Viability Assay Cell viability was assayed using 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT Sigma) as previously described [13] In 96-well cul-ture plates A549 cells were treated with various matrineconcentrations for 24 h The plates were then washed MTTsolution was added and the plates were incubated at 37∘Cfor 4 h Formazan crystals were dissolved in isopropanol andcell viability was spectrophotometrically measured at 570 nmwith a microplate reader (Multiskan FC Thermo WalthamMA USA)

23 Animals Female BALBc mice were purchased fromthe National Laboratory Animal Center in Taiwan Micewere maintained in animal housing with a thermostat and

central air conditioning The care and housing of the micewere approved by the Laboratory Animal Care Committee ofChang Gung University of Science and Technology (IACUCapproval number 2014-007)

24 Acute Lung Injury and Drug Treatment The mice wererandomly divided into four groups of 10 mice each normalcontrol mice (N group) the LPS group 10mgkg matrineplus LPS (M10 group) and 20mgkg matrine plus LPS(M20 group) On days 1ndash7 of the experiment mice wereintraperitoneally injected with matrine On day 8 the micewere anesthetized with isoflurane (Aesica Kent UK) andreceived intratracheal injection of 50120583L LPS (1 120583gmL) ornormal saline Four hours after LPS administration themice were sacrificed and we collected samples of serumbronchoalveolar lavage fluid (BALF) and lung tissue

25 Bronchoalveolar Lavage Fluid and Cell Count BALF wascollected as described previously [14] Briefly the mousetrachea was intubated and the lungs were flushed with1mL normal saline To determine the neutrophil cell countin BALF the cells were stained with Liu stain solution(Polysciences Inc Taipei Taiwan) The supernatants werealso measured for cytokine and chemokine productions

26 Histological Analysis of Lung Tissue Lung tissues werefixed in 10 formalin embedded in paraffin and cut into6 120583m sections The slides were stained with hematoxylin andeosin (HE) The neutrophil infiltration assay was performedas described previously [15]

27 Serum Collection Following anesthetization serum wascollected from the orbital vascular plexus Serum sam-ples were centrifuged at 6000 rpm for 5min as previouslydescribed [16] followed by storage at minus80∘C until use

28 ELISA for Cytokine and Chemokine Production In an invitro experiment A549 cells were pretreated with matrine(100ndash400 120583M) for 1 h in 24-well plates and then they werestimulated with LPS (1 120583gmL) and cultured for 24 hWe thenused specific ELISA kits (RampD Systems Minneapolis MNUSA) to assay IL-8 IL-6 monocyte chemotactic protein-1 (MCP-1) CCL5 and ICAM-1 production Optical density(OD) was spectrophotometrically measured at 450 nm usinga microplate reader (Multiskan FC Thermo) In our invivo study we also used ELISA to measure cytokines andchemokines in serum and BALF

29 RNA Isolation and Real-Time PCR for Gene ExpressionTotal RNA was extracted from lung tissue using TRIzolreagent (Life Technologies Carlsbad CA USA) From thisRNA we synthesized cDNA using a cDNA synthesis kit (LifeTechnologies)We then used a real-time PCR system kit (Bio-Rad Laboratories Hercules CA USA) with a spectrofluo-rometric thermal cycler (iCycler Bio-Rad) to detect cDNAgene expression Table 1 presents the specific gene primersthat were used

Mediators of Inflammation 3

Table 1 Primers used in real-time PCR analyses of mRNA expression

Gene Primers (51015840-31015840 sequence) GenBank accession number Product size (bp)

IL-1120573 ForwardReverse

CACTACAGGCTCCGAGATGACGTTGCTTGGTTCTCCTTGT NM 008361 143

IL-4 ForwardReverse

TCCGTGCTTGAAGAAGAACTCGTGATGTGGACTTGGACTCATT NM 0212832 116

IL-6 ForwardReverse

AGGACCAAGACCATCCAATTCAGCTTAGGCATAACGCACTAGG NM 0311681 97


GCTCCAGCATTGAAGCAGTGCGTGGCAGACAGGAGTGTT NM 0083553 141

TNF-120572 ForwardReverse

GCACCACCATCAAGGACTCAGGCAACCTGACCACTCTC NM 013693 96

MCP-1 ForwardReverse

TTCCACAACCACCTCAAGCATTAAGGCATCACAGTCCGAGTC NM 011333 80

CCL5 ForwardReverse

CGAAGGAACCGCCAAGTGTAGGACTAGAGCAAGCAATGAC NM 0136533 139

CCL11 ForwardReverse

GGCTTCATGTAGTTCCAGATCCATTGTGTTCCTCAATAATCC NM 0113303 145


AGGCAGTGAGAACCAAGTGCGTCAATACCTATGTCCAA NM 0195774 102

iNOS ForwardReverse


COX-2 ForwardReverse

ACCAGCAGTTCCAGTATCAGACAGGAGGATGGAGTTGTTGTAG NM 011198 143

ICAM-1 ForwardReverse

AACAGAATGGTAGACAGCATTCCACCGAGTCCTCTTAG NM 0104932 113

120573-actin ForwardReverse

AAGACCTCTATGCCAACACAGTAGCCAGAGCAGTAATCTCCTTC NM 0073933 92

210 Cell-Cell Adhesion Assay A549 cells were treated withmatrine and incubated with LPS (1120583gmL) for 24 h Thenneutrophil-like HL-60 cells were treated with calcein AMsolution (Sigma) and cocultured with A549 cells for 1 hWe observed the adhesion of HL-60 cells to A549 usingfluorescence microscopy (Olympus Tokyo Japan)

211 Preparation of Total and Nuclear Proteins Followingtreatment with matrine for 1 h A549 cells were stimulatedwith LPS (1120583gmL) for 30min to detect protein phosphoryla-tion or for 24 h to evaluate total protein expressions Proteinswere extracted using protein lysis buffer containing proteaseand phosphatase inhibitors (Sigma) Nuclear proteins wereextracted using NE-PER nuclear and cytoplasmic extractionreagent kits (Pierce Rockford IL USA) All proteins werequantified using the BCA protein assay kit (Pierce)

212 Western Immunoblot Analysis Equal amounts of pro-tein were separated on 10 SDS polyacrylamide gels and thentransferred to polyvinylidene fluoride (PVDF) membranes(Millipore Billerica MA USA) The PVDF membraneswere incubated overnight at 4∘C with primary antibod-ies including COX-2 I120581B-120572 phosphorylated-I120581B-120572 laminB1 p65 phosphorylated-I120581B-120572 (Santa Cruz CA USA)ERK12 p38 JNK phosphorylated-ERK12 phosphorylated-p38 phosphorylated-JNK (Millipore) ICAM-1 and 120573-actin(Sigma) After the overnight incubation the membrane was

washed with TBST buffer (150mM NaCl 10mM Tris pH80 and 01 Tween 20) and incubated with HRP-conjugatedsecondary antibodies for 1 h Finally the membranes werewashed and processed with LuminolEnhancer Solution(Millipore) for detection and quantification of the specificprotein using the BioSpectrum 600 system (UVP UplandCA USA)

213 Transfection and Luciferase Assays Transfected pNF120581B-Luc plasmid evaluatedNF-120581B activity as previously described[13] Briefly A549 cells were transfected with pNF120581B-Lucplasmid (Stratagene California USA) and cells were pre-treated with matrine for 1 h and were stimulated with LPS for4 h The luciferase activity was assayed using a Multi-ModeMicroplate Reader (BioTek SynergyHT BedfordshireUnitedKingdom)

214 Statistical Analysis All data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey-Kramer method for multiple comparisons Values areexpressed as mean plusmn SEM A 119875 value of lt005 was consideredto indicate a statistically significant difference

3 Results

31 Effects of Matrine on Inflammatory Response in ActivatedA549 Cells At doses of le400 120583M matrine did not affect


0

50

100

150

200

250IL

-6 (p

gm

L)

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

lowast

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lowastlowast

LPS (1120583gmL)

(a)

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IL-8

(pg

mL)

+ + + +

100 200 400Matrine (120583M)

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minus minus

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LPS (1120583gmL)

(b)

+ + + +

100 200 400

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CCL5

(pg

mL)

Matrine (120583M)

minus

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LPS (1120583gmL)

(c)

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MCP

-1 (p

gm

L)

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

lowast

lowastlowast

lowastlowast

LPS (1120583gmL)

(d)

Figure 1 Matrine affects cytokine and chemokine productions in LPS-stimulated A549 cells ELISA was used to measure the levels of IL-6(a) IL-8 (b) CCL5 (c) and MCP-1 (d) All data are presented as mean plusmn SEM lowast119875 lt 005 compared with the LPS control group lowastlowast119875 lt 001compared with the LPS control group

the viability of A549 cells (data not shown) Thus matrineconcentrations of 100ndash400120583M were used in the in vitroexperiments First we investigated how matrine modulatedcytokine and chemokine production in LPS-stimulated A549cells (Figure 1)We found thatmatrine significantly decreasedIL-6 production in a concentration-dependent manner IL-6production with LPS alone 1794 plusmn 79 pgmL with 100120583Mmatrine 1854 plusmn 102 pgmL (119875 = 028 versus LPS alone)200120583M matrine 1505 plusmn 112 pgmL (119875 lt 005 versus LPSalone) 400120583Mmatrine 1019 plusmn 115 pgmL (119875 lt 001 versusLPS alone) Matrine also dose-dependently decreased thelevels of IL-8 MCP-1 and CCL5 in LPS-stimulated A549cells

32 Effects of Matrine on LPS-Induced COX-2 and ICAM-1Protein Expressions A549 cells were pretreatedwithmatrinefollowed by stimulation with LPS for 24 h Matrine pre-treatment significantly suppressed COX-2 protein expression

in a concentration-dependent manner compared with LPS-stimulated A549 cells (Figure 2(a)) ELISA (Figure 2(b)) andWestern blot analysis (Figure 2(a)) also showed that matrinesignificantly inhibited ICAM-1 expression LPS alone 2159plusmn243 pgmL matrine 100 120583M 1929 plusmn 103 pgmL (119875 = 012versus LPS alone) matrine 200120583M 1638 plusmn 184 pgmL(119875 lt 005 versus LPS alone) matrine 400 120583M 1306 plusmn175 pgmL (119875 lt 001 versus LPS alone) Inflammatorylung epithelial cells express ICAM-1 to promote neutrophiladhesion [17] Correspondingly we also found that matrinetreatment decreased the adherence of HL-60 cells to LPS-stimulated A549 cells (Figure 2(c))

33 Effects of Matrine on LPS-Induced NF-120581B Activation andPhosphorylation ofMAPKPathways inHumanLungEpithelialCells Upon finding that matrine decreased productionsof chemokines proinflammatory cytokines and COX-2 inLPS-stimulated A549 cells we further investigated whether


+ + + +

100 200 400Matrine (120583M)

minus

minus minus

COX-2

ICAM-1

120573-actin

LPS (1120583gmL)

(a)

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

lowast

lowastlowast

LPS (1120583gmL)0

50

100

150

200

250

300

ICA

M-1

(pg

mL)

(b)N LPS M100

M200 M400

(c)

Figure 2 Matrine affects the LPS-induced productions of COX and ICAM-1 in A549 cells COX-2 and ICAM-1 protein levels were detectedusing Western blots with 120573-actin expression as an internal control (a) and ICAM-1 levels were measured by ELISA (b) (c) Treatment with100ndash400 120583Mmatrine (M) inhibited the adherence of neutrophil-like HL-60 cells to active A549 cells Data are presented as the mean plusmn SEMlowast

119875 lt 005 compared to LPS alone

matrine attenuated activation of the NF-120581B and MAPKpathways in these cells In unstimulated cells I120581B holds theNF-120581B complex (p65 and p50) in the cytoplasm while LPSstimulation induces phosphorylation of I120581B-120572 increasing therelease of p65 into the nucleus [18] Here we found thatmatrine significantly inhibited I120581B-120572 phosphorylation anddecreased I120581B-120572 degradation thus attenuating p65 translo-cation into the nucleus compared with that observed in LPS-stimulated A549 cells (Figures 3(a) and 3(b)) NF-120581B activitywas evaluated using a promoter luciferase assay and it wasfound that matrine could significantly decrease luciferaseactivity (Figure 3(c)) We also observed that matrine sup-pressed phosphorylation of ERK12 JNK and p38 comparedwith LPS-stimulated A549 cells (Figure 4)

34 Matrine Inhibits Neutrophil Infiltration of LPS-InducedLung Injury in Mice After the sacrifice of mice lung tissuewas fixed with formalin and HE stained to analyze thedistribution of neutrophils in lung sections The LPS groupshowed greater neutrophil infiltration compared to the Ngroup (Figure 5) Moreover matrine treatment significantlysuppressed neutrophil infiltration in the lung biopsy sectionsof mice

35Matrine Reduced the Neutrophil Count in BALF from LPS-Challenged Mice Evaluation of inflammatory cells in BALFrevealed that matrine suppressed neutrophil infiltration inacute lung injury mice Intraperitoneal injection of matrinein LPS-induced acute lung injury mice led to a significant


+ + + +

100 200 400Matrine (120583M)

minus

minus minus

120573-actin

Phospho-I120581B-120572

I120581B-120572

LPS (1120583gmL)

(a)

p65

p65

Cyto

sol

Nuc

lear

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

120573-actin

Lamin-B1

LPS (1120583gmL)

(b)

0

20

40

60

80

100

Relat

ive l

ucife

rase

activ

ity (

)

Matrine (120583M)

LPS

lowast

+ + + +

100 200 400minus

minus minus

lowastlowast

lowastlowast

(1120583gmL)

(c)

Figure 3 Matrine inhibits the nuclear translocation of NF-120581B in A549 cells (a) Effects of matrine on the LPS-induced phosphorylation ofI120581B-120572 with total I120581B-120572 levels used as internal controls (b) To assess the nuclear translocation of NF-120581B cells were pretreated with differentmatrine concentrations for 1 h and then incubated with LPS (1 120583gmL) for 1 h The internal controls were lamin B1 in the nucleus and 120573-actinin the cytosol (c) Matrine suppressed luciferase activity by the NF-120581B promoter assay Three independent experiments were analyzed andcompared with the LPS-treated control group Data are presented as the mean plusmn SEM lowast119875 lt 005 compared to LPS alone

decrease of neutrophils compared with the LPS group LPSalone 43 times 105 plusmn 61 times 104 neutrophilsmL M10 27 times 105 plusmn71 times 10

4 neutrophilsmL (119875 = 008 versus LPS alone) M2020 times 10

5

plusmn 61 times 104 neutrophilsmL (119875 lt 005 versus LPS

alone) (Figure 6(a))

36 Effects of Matrine on Cytokine and Chemokine Levels inBALF Acute lung injurymice treatedwithmatrine exhibitedlower IL-6 levels LPS alone 3478plusmn976 pgmLM10 1993plusmn83 pgmL (119875 lt 005 versus LPS alone) M20 1639 plusmn498 pgmL (119875 lt 001 versus LPS alone) (Figure 6(b))Matrine treatment also reduced TNF-120572 levels in these miceLPS alone 5563 plusmn 1119 pgmL M10 4410 plusmn 711 pgmL(119875 = 011 versus LPS alone) M20 2629 plusmn 691 pgmL (119875 lt005 versus LPS alone) (Figure 6(c)) Using real-time PCR toevaluate gene expression we found that matrine significantlydecreased levels of IL-1120573 IL-6 TNF-120572 IL-13 MCP-1 andCCL5 in lung injury tissue (Figure 7) However matrine didnot suppress the gene expressions of IL-4 CCL11 or CCL24

We further found that matrine inhibited the gene expressionsof ICAM-1 iNOS and COX-2 in lung injury tissue

37 Effects of Matrine on Phosphorylation of NF-120581B andMAPK Pathways in Lung Injury Tissue Matrine significantlyinhibited phosphorylation of p65 compared with that inLPS-stimulated lung injury tissue Matrine also suppressedphosphorylation of ERK12 JNK and p38 in the lung injurytissue (Figure 8)

38 Effects of Matrine on Cytokine Production in SerumSerum analysis revealed significantly reduced IL-6 produc-tion and TNF-120572 production in the M20 group compared toin the LPS group (Figure 9)

4 Discussion

Matrine is a major active component isolated from Sophoraflavescens (Kushen) which reportedly shows many biologicaland medical properties including improved inflammatory


ERK

Phospho-ERK

p38

Phospho-p38

Phospho-JNK

JNK

120573-actin

Matrine (120583M)

LPS + + + +

100 200 400minus

minus minus

(1120583gmL)

Figure 4 Matrine affects LPS-induced phosphorylation of MAPK pathway molecules Western blots showed the effects of the indicatedmatrine concentrations on the phosphorylation of ERK (top two rows) p38 (middle two rows) and JNK (bottom two rows)Three independentexperiments were analyzed and compared with the LPS-treated group

N LPS

M20M10

Figure 5 Matrine inhibits neutrophil infiltration Hematoxylin and eosin staining was used for histological examination of airwayinflammation in lung tissue (magnification 400x) N group normal control mice LPS group LPS treatment only M10 group 10mgkgmatrine plus LPS M20 group 20mgkg matrine plus LPS


lowast

0

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6N

eutro

phils

cells

mL

N LPS M10 M20

times105

(a)

lowast

lowastlowast

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IL-6

(pg

mL)

N LPS M10 M20

(b)

lowast

N LPS M10 M200

200

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TNF-120572

(pg

mL)

(c)

Figure 6 Matrine affects neutrophil counts and cytokine and chemokine levels in BALF In the different treatment groups we analyzedneutrophil cell counts (a) and the levels of IL-6 (b) and TNF-120572 (c) were measured by ELISA N group normal control mice LPS groupLPS treatment only M10 group 10mgkg matrine plus LPS M20 group 20mgkg matrine plus LPS All data are presented as mean plusmn SEMlowast

119875 lt 005 compared with the LPS control group lowastlowast119875 lt 001 compared with the LPS control group

responses of the central nervous system antitumor activitiesand immunity-regulating effects [10 19] We previouslydemonstrated that matrine suppresses eosinophil infiltrationin lung tissue and improves airway hyperresponsiveness andsuppresses tracheal goblet cell hyperplasia in asthmatic mice[12] Moreover Zhang et al described lung histopathologicalchanges and inflammatory response at 18 h after LPS injectionin mice reporting that matrine decreased myeloperoxidaseandmalondialdehyde activities and inhibited reactive oxygenspecies and oxidative stress in BALF [20]

In our present study we investigated whether matrineimproved the inflammatory response in acute lung injurymice at 4 h after LPS injection into the trachea and alsoexamined inflammatory responses in LPS-stimulated humanlung epithelial cells Our results showed that matrine sig-nificantly suppressed COX-2 expression and productions ofIL-6 IL-8 MCP-1 and CCL5 in LPS-stimulated A549 cellsMatrine treatment also suppressed ICAM-1 expressions and

correspondingly reduced LPS-stimulated A549 cell adher-ence to neutrophils Finally matrine suppressed componentsof inflammatory signaling pathways including the NF-120581Band MAPK pathways

LPS is an important component of Gram-negative bacte-ria cell walls [21] Upon infection with Gram-negative bacte-ria LPS release leads to inflammation and fever symptomspotentially causing severe acute or chronic organ congestionedema or even septic shock and death [22] LPS stimulatesI120581B phosphorylation emancipating NF-120581B (containing p50and p65) to translocate into the nucleus In the nucleusp65 binds the promoters of inflammatory genes leading tothe expressions of proinflammatory cytokines chemokinesCOX-2 and ICAM-1 in LPS-stimulated A549 cells [21 23]MAPK pathways also modulate NF-120581B activation to alterCOX-2 and ICAM-1 expressions [24] Our present exper-iments demonstrated that matrine significantly inhibitedthe secretion of proinflammatory cytokines and chemokines


N LPS M10 M20

CCL11

02468

10121416

CCL1

1 (fo

ld)

N LPS M10 M20

CCL5

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10CC

L5 (f

old)

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-1 (f

old)

lowast

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CCL24

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4 (fo

ld)

N LPS M10 M20

IL-6

lowast

lowastlowast

010203040506070

IL-6

(fol

d)

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lowastlowast

lowastlowast

IL-1120573

020406080

100120140

IL-1120573

(fold

)

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TNF-120572

(fold

)

TNF-120572

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ld)

Figure 7 Matrine affects the expressions of cytokines chemokines and inflammatory mediators in the lung Gene expressions weredetermined using real-timeRT-PCR Fold expression levels weremeasured relative to120573-actin expression (internal control) Data are presentedas mean plusmn SEM lowast119875 lt 005 compared with LPS control mice lowastlowast119875 lt 001 compared with LPS control mice

from LPS-induced A549 lung epithelial cells This effect mayhave been because matrine inhibited MAPK and NF-120581BactivationThese results suggest thatmatrine treatment couldimprove the inflammatory response in cases of lung bacterialinfections

Previously Zhang et al investigated the administrationof matrine after 30min of LPS stimulation and reportedthat matrine reduces lung inflammation and oxidation byinhibiting inflammatory signal pathways [20] However theirprotocol involved LPS injected intravenously after 60 hours


ERK

Phospho-ERK

p38

Phospho-p38

Phospho-JNK

JNK

120573-actin

N LPS M10 M20

Phospho-NF-120581B

NF-120581B

Figure 8 Matrine affects the LPS-induced nuclear phosphorylation of NF-120581B and phosphorylation of MAPK pathway moleculesPhosphorylation in lung tissue was analyzed by Western blots Three independent experiments were analyzed and results were comparedwith the LPS-treated group

lowast

lowastlowast

N LPS M10 M200

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IL-6

(pg

mL)

(a)

lowast

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TNF-120572

(pg

mL)

(b)

Figure 9 Matrine affects the serum levels of inflammatory cytokines including IL-6 (a) and TNF-120572 (b) All data are presented as means plusmnSEM lowast119875 lt 005 compared with the LPS control group


and the survival rate was only 20 Mice given 20mgkgmatrine did not show an increased survival rate mice given100mgkgmatrine showed a 60 survival rate and decreasedlung damage [20] In contrast our present experimentalmodel of lung injury involved intratracheal LPS injectionto induce local inflammatory lung injury which seems tobe more suitable for assessing the inflammatory responseFurthermore our experiment mainly investigated whethermatrine could provide protection against lung injury inmiceThe mice were treated with intraperitoneal matrine injectionfor continuous 7 days followed by intratracheal LPS injectionon the 8th day and the mice were sacrificed four hours laterThe experimental process did not lead to the death of anymice and our model is more appropriate for observing theearly response of acute lung injury and for evaluating theprotective effects of matrine in early lung injury Our resultsshowed that 20mgkgmatrine could protect and prevent lunginjury symptoms in this experimental model Despite thedifferences between our animal experimental model and thelung injury mouse model used by Zhang et al [20] both setsof experimental results suggest that matrine can reduce lunginjury in mice

In cases of bacterial infection neutrophils in the lungcan ingest invading bacteria and release large amounts ofinflammatorymediators to induce an inflammatory responsein the lung [25] Our experiments showed that treatmentwith 20mgkg of matrine suppressed neutrophil infiltrationin the lung and correspondingly reduced the number ofneutrophils in BALF Chemokinesmdashparticularly IL-8 MCP-1 and CCL5mdashinduce neutrophil migration into inflamma-tory tissue [26] Matrine inhibited the productions of IL-8 MCP-1 and CCL5 in LPS-stimulated A549 cells andsuppressedMCP-1 and CCL5 gene expressions inmouse lungtissue Inflammatory lung epithelial cells also express ICAM-1 adhesion molecules to promote neutrophil adsorption [1827] Our experiments found that matrine suppressed ICAM-1 expression both in vitro and in vivo Overall our resultssuggest that matrine may be useful for reducing neutrophilinfiltration in the inflammatory response of the lungs Thiswould be particularly useful since lung inflammation andlung damage in LPS-induced acute lung injury are mainlycaused by neutrophil infiltration On the other hand matrinedid not effectively reduce the gene expressions of CCL11and CCL24 in the lung and therefore matrine did notimprove eosinophilic infiltration in this LPS-induced lunginjurymousemodelMatrine also did not effectively suppressthe gene expressions of theTh2-associated cytokines IL-4 andIL-13 in lung tissue

5 Conclusion

In conclusion our results demonstrate that matrine signifi-cantly suppressed the inflammatory response in human lungepithelial cells and blocked ICAM-1 expression preventingacute lung injury in LPS-induced mice

Conflict of Interests

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

Authorsrsquo Contribution

Chian-Jiun Liou and You-Rong Lai contributed equally to thepaper

Acknowledgments

This study was supported in part by grants from theChang Gung Memorial Hospital (CMRPF1C0202 andCMRPF3E0051) and from the Chang Gung University ofScience and Technology (EZRPF3E0191)

References

[1] C-L Tsai Y-C Lin H-M Wang and T-C Chou ldquoBaicaleinan active component of Scutellaria baicalensis protects againstlipopolysaccharide-induced acute lung injury in ratsrdquo Journal ofEthnopharmacology vol 153 no 1 pp 197ndash206 2014

[2] Y Aschner R L Zemans C M Yamashita and G PDowney ldquoMatrix metalloproteinases and protein tyrosineKinases potential novel targets in acute lung injury and ARDSrdquoChest vol 146 no 4 pp 1081ndash1091 2014

[3] L Xu J Xu and ZWang ldquoMolecular mechanisms of paraquat-induced acute lung injury a current reviewrdquoDrug andChemicalToxicology vol 37 no 2 pp 130ndash134 2014

[4] X X Liu D D Yu M J Chen et al ldquoHesperidin amelio-rates lipopolysaccharide-induced acute lung injury in mice byinhibiting HMGB1 releaserdquo International Immunopharmacol-ogy vol 25 no 2 pp 370ndash376 2015

[5] M A Taddonio V Dolgachev M Bosmann et al ldquoInfluence oflipopolysaccharide-binding protein on pulmonary inflamma-tion in gram-negative pneumoniardquo Shock vol 43 no 6 pp 612ndash619 2015

[6] T Yokochi ldquoA new experimental murine model forlipopolysaccharide-mediated lethal shock with lung injuryrdquoInnate Immunity vol 18 no 2 pp 364ndash370 2012

[7] H Ma J Shi C Wang et al ldquoBlockade of PDE4B limits lungvascular permeability and lung inflammation in LPS-inducedacute lung injuryrdquo Biochemical and Biophysical Research Com-munications vol 450 no 4 pp 1560ndash1567 2014

[8] B V Patel M R Wilson K P OrsquoDea and M Takata ldquoTNF-induced death signaling triggers alveolar epithelial dysfunctionin acute lung injuryrdquo Journal of Immunology vol 190 no 8 pp4274ndash4282 2013

[9] H Li G Tan X Jiang et al ldquoTherapeutic effects of matrineon primary and metastatic breast cancerrdquo American Journal ofChinese Medicine vol 38 no 6 pp 1115ndash1130 2010

[10] M Sun H Cao L Sun et al ldquoAntitumor activities of kushenliterature reviewrdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2012 Article ID 373219 11 pages 2012

[11] J-P ZhangM Zhang C Jin et al ldquoMatrine inhibits productionand actions of fibrogenic cytokines released by mouse peri-toneal macrophagesrdquo Acta Pharmacologica Sinica vol 22 no8 pp 765ndash768 2001

[12] W-C Huang C-C Chan S-J Wu et al ldquoMatrine attenuatesallergic airway inflammation and eosinophil infiltration bysuppressing eotaxin and Th2 cytokine production in asthmaticmicerdquo Journal of Ethnopharmacology vol 151 no 1 pp 470ndash4772014


[13] W-T Chang W-C Huang and C-J Liou ldquoEvaluationof the anti-inflammatory effects of phloretin and phlorizinin lipopolysaccharide-stimulated mouse macrophagesrdquo FoodChemistry vol 134 no 2 pp 972ndash979 2012

[14] W-C Huang and C-J Liou ldquoDietary acacetin reduces airwayhyperresponsiveness and eosinophil infiltration by modulatingeotaxin-1 and Th2 cytokines in a mouse model of asthmardquoEvidence-Based Complementary and Alternative Medicine vol2012 Article ID 910520 11 pages 2012

[15] Y-L Liu Y-J Liu Y Liu et al ldquoHydroxysafflor yellow Aameliorates lipopolysaccharide-induced acute lung injury inmice via modulating toll-like receptor 4 signaling pathwaysrdquoInternational Immunopharmacology vol 23 no 2 pp 649ndash6572014

[16] C-J Liou and W-C Huang ldquoDehydroepiandrosterone sup-presses eosinophil infiltration and airway hyperresponsivenessviamodulation of chemokines andTh2cytokines in ovalbumin-sensitized micerdquo Journal of Clinical Immunology vol 31 no 4pp 656ndash665 2011

[17] C Lawson and S Wolf ldquoICAM-1 signaling in endothelial cellsrdquoPharmacological Reports vol 61 no 1 pp 22ndash32 2009

[18] W C Huang S J Wu R S Tu Y R Lai and C J LiouldquoPhloretin inhibits interleukin-1120573-induced COX-2 and ICAM-1expression through inhibition of MAPK Akt and NF-120581B sig-naling in human lung epithelial cellsrdquo Food amp Function vol 6no 6 pp 1960ndash1967 2015

[19] Z-J Ma Q Li J-B Wang et al ldquoCombining oxymatrine ormatrine with lamivudine increased its antireplication effectagainst the hepatitis B virus in vitrordquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 1865738 pages 2013

[20] B Zhang Z-Y Liu Y-Y Li et al ldquoAntiinflammatory effects ofmatrine in LPS-induced acute lung injury in micerdquo EuropeanJournal of Pharmaceutical Sciences vol 44 no 5 pp 573ndash5792011

[21] F Peri M Piazza V Calabrese G Damore and R CighettildquoExploring the LPSTLR4 signal pathwaywith smallmoleculesrdquoBiochemical Society Transactions vol 38 no 5 pp 1390ndash13952010

[22] M R Karta M L Gavala C S Curran et al ldquoLPS mod-ulates rhinovirus-induced chemokine secretion in monocytesand macrophagesrdquo American Journal of Respiratory Cell andMolecular Biology vol 51 no 1 pp 125ndash134 2014

[23] K Hostanska J Melzer A Amon and R Saller ldquoSuppression ofinterleukin (IL)-8 and human beta defensin-2 secretion in LPS-andor IL-1120573-stimulated airway epithelial A549 cells by a herbalformulation against respiratory infections (BNO 1030)rdquo Journalof Ethnopharmacology vol 134 no 2 pp 228ndash233 2011

[24] J M Kyriakis and J Avruch ldquoMammalian MAPK signaltransduction pathways activated by stress and inflammation a10-year updaterdquo Physiological Reviews vol 92 no 2 pp 689ndash737 2012

[25] S L Poe M Arora T B Oriss et al ldquoSTAT1-regulated lungMDSC-like cells produce IL-10 and efferocytose apoptotic neu-trophils with relevance in resolution of bacterial pneumoniardquoMucosal Immunology vol 6 no 1 pp 189ndash199 2013

[26] M D Turner B Nedjai T Hurst and D J PenningtonldquoCytokines and chemokines at the crossroads of cell signallingand inflammatory diseaserdquo Biochimica et Biophysica Acta

(BBA)mdashMolecular Cell Research vol 1843 no 11 pp 2563ndash2582 2014

[27] Y Tao Y-C Chen T Lan H Qian YWang and L Jiang ldquoLPS-induced nuclear translocation of RhoA is dependent on NF-120581Bin the human lung cancer cell line A549rdquo Oncology Letters vol3 no 6 pp 1283ndash1287 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


wall component of Gram-negative bacteria and can inducean innate immunity response activating immune cells tocombat microbial infection [6] Thus LPS is widely used toinduce acute lung inflammation in animal models Activatedmacrophages and lung epithelial cells reportedly releaseproinflammatory cytokines and chemokines aggravatingALI progression Prior studies have used lung epithelialcells to evaluate the anti-inflammatory responses of drugsor natural compounds during LPS-induced inflammatoryresponse [7 8]

In China and Taiwan the root of Sophora flavescens(Leguminosae) is used to treat fever jaundice urinary tractinfections and pyogenic infections [9] Matrine is isolatedfrom S flavescens and can reportedly attenuate cerebralischemic injury in mice and induce apoptosis of chronicmyeloid leukemia cells osteosarcoma cells and cholangio-carcinoma cells [9 10] Zhang et al previously demonstratedthat matrine suppressed proinflammatory cytokine produc-tion in LPS-stimulated mouse macrophages [11] Previouslyour group also found that matrine could improve eosinophilinfiltration airway hyperresponsiveness and Th2-associatedcytokine production in asthmatic mice [12] However themechanisms of matrinersquos anti-inflammatory activity in lungepithelial cells are largely unclear

In the present study we investigated whether matrinereduced inflammatory responses and production of intercel-lular adhesion molecule-1 (ICAM-1) in lung epithelial cellsWe also examined whether matrine protected and preventedacute lung injury in LPS-induced mice

2 Materials and Methods

21 Matrine and Cell Culture Matrine (ge99 by HPLC) waspurchased from Sigma-Aldrich (St Louis MO USA) andwas dissolved in normal saline as previously described [12]The human lung epithelial cell line A549 was purchased fromthe Bioresource Collection and Research Center (BCRCTaiwan) A549 cells were cultured in DMEM medium(Invitrogen-Gibco Paisley Scotland) containing 2mM glu-tamine 100UmL penicillin and streptomycin and 10 fetalbovine serum (Biological Industries Haemek Israel) Cellswere incubated at 37∘C in 5 CO

2humidified air and were

subcultured twice each week

22 Cell Viability Assay Cell viability was assayed using 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT Sigma) as previously described [13] In 96-well cul-ture plates A549 cells were treated with various matrineconcentrations for 24 h The plates were then washed MTTsolution was added and the plates were incubated at 37∘Cfor 4 h Formazan crystals were dissolved in isopropanol andcell viability was spectrophotometrically measured at 570 nmwith a microplate reader (Multiskan FC Thermo WalthamMA USA)

23 Animals Female BALBc mice were purchased fromthe National Laboratory Animal Center in Taiwan Micewere maintained in animal housing with a thermostat and

central air conditioning The care and housing of the micewere approved by the Laboratory Animal Care Committee ofChang Gung University of Science and Technology (IACUCapproval number 2014-007)

24 Acute Lung Injury and Drug Treatment The mice wererandomly divided into four groups of 10 mice each normalcontrol mice (N group) the LPS group 10mgkg matrineplus LPS (M10 group) and 20mgkg matrine plus LPS(M20 group) On days 1ndash7 of the experiment mice wereintraperitoneally injected with matrine On day 8 the micewere anesthetized with isoflurane (Aesica Kent UK) andreceived intratracheal injection of 50120583L LPS (1 120583gmL) ornormal saline Four hours after LPS administration themice were sacrificed and we collected samples of serumbronchoalveolar lavage fluid (BALF) and lung tissue

25 Bronchoalveolar Lavage Fluid and Cell Count BALF wascollected as described previously [14] Briefly the mousetrachea was intubated and the lungs were flushed with1mL normal saline To determine the neutrophil cell countin BALF the cells were stained with Liu stain solution(Polysciences Inc Taipei Taiwan) The supernatants werealso measured for cytokine and chemokine productions

26 Histological Analysis of Lung Tissue Lung tissues werefixed in 10 formalin embedded in paraffin and cut into6 120583m sections The slides were stained with hematoxylin andeosin (HE) The neutrophil infiltration assay was performedas described previously [15]

27 Serum Collection Following anesthetization serum wascollected from the orbital vascular plexus Serum sam-ples were centrifuged at 6000 rpm for 5min as previouslydescribed [16] followed by storage at minus80∘C until use

28 ELISA for Cytokine and Chemokine Production In an invitro experiment A549 cells were pretreated with matrine(100ndash400 120583M) for 1 h in 24-well plates and then they werestimulated with LPS (1 120583gmL) and cultured for 24 hWe thenused specific ELISA kits (RampD Systems Minneapolis MNUSA) to assay IL-8 IL-6 monocyte chemotactic protein-1 (MCP-1) CCL5 and ICAM-1 production Optical density(OD) was spectrophotometrically measured at 450 nm usinga microplate reader (Multiskan FC Thermo) In our invivo study we also used ELISA to measure cytokines andchemokines in serum and BALF

29 RNA Isolation and Real-Time PCR for Gene ExpressionTotal RNA was extracted from lung tissue using TRIzolreagent (Life Technologies Carlsbad CA USA) From thisRNA we synthesized cDNA using a cDNA synthesis kit (LifeTechnologies)We then used a real-time PCR system kit (Bio-Rad Laboratories Hercules CA USA) with a spectrofluo-rometric thermal cycler (iCycler Bio-Rad) to detect cDNAgene expression Table 1 presents the specific gene primersthat were used






IL-4 ForwardReverse


IL-6 ForwardReverse








CCL5 ForwardReverse






iNOS ForwardReverse














3 Results



0

50

100

150

200

250IL

-6 (p

gm

L)

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

lowast

lowastlowast

lowastlowast

LPS (1120583gmL)

(a)

0

500

1000

1500

2000

IL-8

(pg

mL)

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

lowastlowast

lowastlowast

lowastlowast

LPS (1120583gmL)

(b)

+ + + +

100 200 400

0

50

100

150

200

250

300

CCL5

(pg

mL)

Matrine (120583M)

minus

minus minus

lowast

lowastlowast

LPS (1120583gmL)

(c)

0

1000

2000

3000

MCP

-1 (p

gm

L)

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

lowast

lowastlowast

lowastlowast

LPS (1120583gmL)

(d)







+ + + +

100 200 400Matrine (120583M)

minus

minus minus

COX-2

ICAM-1

120573-actin

LPS (1120583gmL)

(a)

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

lowast

lowastlowast

LPS (1120583gmL)0

50

100

150

200

250

300

ICA

M-1

(pg

mL)

(b)N LPS M100

M200 M400

(c)







+ + + +

100 200 400Matrine (120583M)

minus

minus minus

120573-actin

Phospho-I120581B-120572

I120581B-120572

LPS (1120583gmL)

(a)

p65

p65

Cyto

sol

Nuc

lear

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

120573-actin

Lamin-B1

LPS (1120583gmL)

(b)

0

20

40

60

80

100

Relat

ive l

ucife

rase

activ

ity (

)

Matrine (120583M)

LPS

lowast

+ + + +

100 200 400minus

minus minus

lowastlowast

lowastlowast

(1120583gmL)

(c)




5







4 Discussion



ERK

Phospho-ERK

p38

Phospho-p38

Phospho-JNK

JNK

120573-actin

Matrine (120583M)

LPS + + + +

100 200 400minus

minus minus

(1120583gmL)


N LPS

M20M10



lowast

0

1

2

3

4

5

6N

eutro

phils

cells

mL

N LPS M10 M20

times105

(a)

lowast

lowastlowast

lowastlowast

0

100

200

300

400

500

IL-6

(pg

mL)

N LPS M10 M20

(b)

lowast

N LPS M10 M200

200

400

600

800

TNF-120572

(pg

mL)

(c)








N LPS M10 M20

CCL11

02468

10121416

CCL1

1 (fo

ld)

N LPS M10 M20

CCL5

0

2

4

6

8

10CC

L5 (f

old)

lowast

N LPS M10 M20

MCP-1

0

20

40

60

80

100

MCP

-1 (f

old)

lowast

N LPS M10 M20

CCL24

0

2

4

6

8

CCL2

4 (fo

ld)

N LPS M10 M20

IL-6

lowast

lowastlowast

010203040506070

IL-6

(fol

d)

N LPS M10 M20

lowastlowast

lowastlowast

IL-1120573

020406080

100120140

IL-1120573

(fold

)

N LPS M10 M20

TNF-120572

(fold

)

TNF-120572

0

10

20

30

40

50

lowast

lowastlowast

N LPS M10 M20

ICAM-1

0

5

10

15

20

25

ICA

M-1

(fol

d)

lowastlowast

lowastlowast

N LPS M10 M20

COX-2

0

10

20

30

40

50

60

COX-

2 (fo

ld)

lowast

lowastlowast

N LPS M10 M20

iNOS

lowast

0

2

4

6

8

10

iNO

S (fo

ld)

N LPS M10 M20

IL-4

0

1

2

3

4

5

IL-4

(fol

d)

N LPS M10 M20

IL-13

lowast

0

2

4

6

8

10

IL-1

3 (fo

ld)





ERK

Phospho-ERK

p38

Phospho-p38

Phospho-JNK

JNK

120573-actin

N LPS M10 M20

Phospho-NF-120581B

NF-120581B


lowast

lowastlowast

N LPS M10 M200

200

400

600

800

1000

IL-6

(pg

mL)

(a)

lowast

lowastlowast

N LPS M10 M200

200

400

600

800

1000

TNF-120572

(pg

mL)

(b)





5 Conclusion






Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















IL-4 ForwardReverse


IL-6 ForwardReverse








CCL5 ForwardReverse






iNOS ForwardReverse














3 Results



0

50

100

150

200

250IL

-6 (p

gm

L)

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

lowast

lowastlowast

lowastlowast

LPS (1120583gmL)

(a)

0

500

1000

1500

2000

IL-8

(pg

mL)

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

lowastlowast

lowastlowast

lowastlowast

LPS (1120583gmL)

(b)

+ + + +

100 200 400

0

50

100

150

200

250

300

CCL5

(pg

mL)

Matrine (120583M)

minus

minus minus

lowast

lowastlowast

LPS (1120583gmL)

(c)

0

1000

2000

3000

MCP

-1 (p

gm

L)

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

lowast

lowastlowast

lowastlowast

LPS (1120583gmL)

(d)







+ + + +

100 200 400Matrine (120583M)

minus

minus minus

COX-2

ICAM-1

120573-actin

LPS (1120583gmL)

(a)

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

lowast

lowastlowast

LPS (1120583gmL)0

50

100

150

200

250

300

ICA

M-1

(pg

mL)

(b)N LPS M100

M200 M400

(c)







+ + + +

100 200 400Matrine (120583M)

minus

minus minus

120573-actin

Phospho-I120581B-120572

I120581B-120572

LPS (1120583gmL)

(a)

p65

p65

Cyto

sol

Nuc

lear

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

120573-actin

Lamin-B1

LPS (1120583gmL)

(b)

0

20

40

60

80

100

Relat

ive l

ucife

rase

activ

ity (

)

Matrine (120583M)

LPS

lowast

+ + + +

100 200 400minus

minus minus

lowastlowast

lowastlowast

(1120583gmL)

(c)




5







4 Discussion



ERK

Phospho-ERK

p38

Phospho-p38

Phospho-JNK

JNK

120573-actin

Matrine (120583M)

LPS + + + +

100 200 400minus

minus minus

(1120583gmL)


N LPS

M20M10



lowast

0

1

2

3

4

5

6N

eutro

phils

cells

mL

N LPS M10 M20

times105

(a)

lowast

lowastlowast

lowastlowast

0

100

200

300

400

500

IL-6

(pg

mL)

N LPS M10 M20

(b)

lowast

N LPS M10 M200

200

400

600

800

TNF-120572

(pg

mL)

(c)








N LPS M10 M20

CCL11

02468

10121416

CCL1

1 (fo

ld)

N LPS M10 M20

CCL5

0

2

4

6

8

10CC

L5 (f

old)

lowast

N LPS M10 M20

MCP-1

0

20

40

60

80

100

MCP

-1 (f

old)

lowast

N LPS M10 M20

CCL24

0

2

4

6

8

CCL2

4 (fo

ld)

N LPS M10 M20

IL-6

lowast

lowastlowast

010203040506070

IL-6

(fol

d)

N LPS M10 M20

lowastlowast

lowastlowast

IL-1120573

020406080

100120140

IL-1120573

(fold

)

N LPS M10 M20

TNF-120572

(fold

)

TNF-120572

0

10

20

30

40

50

lowast

lowastlowast

N LPS M10 M20

ICAM-1

0

5

10

15

20

25

ICA

M-1

(fol

d)

lowastlowast

lowastlowast

N LPS M10 M20

COX-2

0

10

20

30

40

50

60

COX-

2 (fo

ld)

lowast

lowastlowast

N LPS M10 M20

iNOS

lowast

0

2

4

6

8

10

iNO

S (fo

ld)

N LPS M10 M20

IL-4

0

1

2

3

4

5

IL-4

(fol

d)

N LPS M10 M20

IL-13

lowast

0

2

4

6

8

10

IL-1

3 (fo

ld)





ERK

Phospho-ERK

p38

Phospho-p38

Phospho-JNK

JNK

120573-actin

N LPS M10 M20

Phospho-NF-120581B

NF-120581B


lowast

lowastlowast

N LPS M10 M200

200

400

600

800

1000

IL-6

(pg

mL)

(a)

lowast

lowastlowast

N LPS M10 M200

200

400

600

800

1000

TNF-120572

(pg

mL)

(b)





5 Conclusion






Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200

250IL

-6 (p

gm

L)

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

lowast

lowastlowast

lowastlowast

LPS (1120583gmL)

(a)

0

500

1000

1500

2000

IL-8

(pg

mL)

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

lowastlowast

lowastlowast

lowastlowast

LPS (1120583gmL)

(b)

+ + + +

100 200 400

0

50

100

150

200

250

300

CCL5

(pg

mL)

Matrine (120583M)

minus

minus minus

lowast

lowastlowast

LPS (1120583gmL)

(c)

0

1000

2000

3000

MCP

-1 (p

gm

L)

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

lowast

lowastlowast

lowastlowast

LPS (1120583gmL)

(d)







+ + + +

100 200 400Matrine (120583M)

minus

minus minus

COX-2

ICAM-1

120573-actin

LPS (1120583gmL)

(a)

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

lowast

lowastlowast

LPS (1120583gmL)0

50

100

150

200

250

300

ICA

M-1

(pg

mL)

(b)N LPS M100

M200 M400

(c)







+ + + +

100 200 400Matrine (120583M)

minus

minus minus

120573-actin

Phospho-I120581B-120572

I120581B-120572

LPS (1120583gmL)

(a)

p65

p65

Cyto

sol

Nuc

lear

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

120573-actin

Lamin-B1

LPS (1120583gmL)

(b)

0

20

40

60

80

100

Relat

ive l

ucife

rase

activ

ity (

)

Matrine (120583M)

LPS

lowast

+ + + +

100 200 400minus

minus minus

lowastlowast

lowastlowast

(1120583gmL)

(c)




5







4 Discussion



ERK

Phospho-ERK

p38

Phospho-p38

Phospho-JNK

JNK

120573-actin

Matrine (120583M)

LPS + + + +

100 200 400minus

minus minus

(1120583gmL)


N LPS

M20M10



lowast

0

1

2

3

4

5

6N

eutro

phils

cells

mL

N LPS M10 M20

times105

(a)

lowast

lowastlowast

lowastlowast

0

100

200

300

400

500

IL-6

(pg

mL)

N LPS M10 M20

(b)

lowast

N LPS M10 M200

200

400

600

800

TNF-120572

(pg

mL)

(c)








N LPS M10 M20

CCL11

02468

10121416

CCL1

1 (fo

ld)

N LPS M10 M20

CCL5

0

2

4

6

8

10CC

L5 (f

old)

lowast

N LPS M10 M20

MCP-1

0

20

40

60

80

100

MCP

-1 (f

old)

lowast

N LPS M10 M20

CCL24

0

2

4

6

8

CCL2

4 (fo

ld)

N LPS M10 M20

IL-6

lowast

lowastlowast

010203040506070

IL-6

(fol

d)

N LPS M10 M20

lowastlowast

lowastlowast

IL-1120573

020406080

100120140

IL-1120573

(fold

)

N LPS M10 M20

TNF-120572

(fold

)

TNF-120572

0

10

20

30

40

50

lowast

lowastlowast

N LPS M10 M20

ICAM-1

0

5

10

15

20

25

ICA

M-1

(fol

d)

lowastlowast

lowastlowast

N LPS M10 M20

COX-2

0

10

20

30

40

50

60

COX-

2 (fo

ld)

lowast

lowastlowast

N LPS M10 M20

iNOS

lowast

0

2

4

6

8

10

iNO

S (fo

ld)

N LPS M10 M20

IL-4

0

1

2

3

4

5

IL-4

(fol

d)

N LPS M10 M20

IL-13

lowast

0

2

4

6

8

10

IL-1

3 (fo

ld)





ERK

Phospho-ERK

p38

Phospho-p38

Phospho-JNK

JNK

120573-actin

N LPS M10 M20

Phospho-NF-120581B

NF-120581B


lowast

lowastlowast

N LPS M10 M200

200

400

600

800

1000

IL-6

(pg

mL)

(a)

lowast

lowastlowast

N LPS M10 M200

200

400

600

800

1000

TNF-120572

(pg

mL)

(b)





5 Conclusion






Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















+ + + +

100 200 400Matrine (120583M)

minus

minus minus

COX-2

ICAM-1

120573-actin

LPS (1120583gmL)

(a)

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

lowast

lowastlowast

LPS (1120583gmL)0

50

100

150

200

250

300

ICA

M-1

(pg

mL)

(b)N LPS M100

M200 M400

(c)







+ + + +

100 200 400Matrine (120583M)

minus

minus minus

120573-actin

Phospho-I120581B-120572

I120581B-120572

LPS (1120583gmL)

(a)

p65

p65

Cyto

sol

Nuc

lear

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

120573-actin

Lamin-B1

LPS (1120583gmL)

(b)

0

20

40

60

80

100

Relat

ive l

ucife

rase

activ

ity (

)

Matrine (120583M)

LPS

lowast

+ + + +

100 200 400minus

minus minus

lowastlowast

lowastlowast

(1120583gmL)

(c)




5







4 Discussion



ERK

Phospho-ERK

p38

Phospho-p38

Phospho-JNK

JNK

120573-actin

Matrine (120583M)

LPS + + + +

100 200 400minus

minus minus

(1120583gmL)


N LPS

M20M10



lowast

0

1

2

3

4

5

6N

eutro

phils

cells

mL

N LPS M10 M20

times105

(a)

lowast

lowastlowast

lowastlowast

0

100

200

300

400

500

IL-6

(pg

mL)

N LPS M10 M20

(b)

lowast

N LPS M10 M200

200

400

600

800

TNF-120572

(pg

mL)

(c)








N LPS M10 M20

CCL11

02468

10121416

CCL1

1 (fo

ld)

N LPS M10 M20

CCL5

0

2

4

6

8

10CC

L5 (f

old)

lowast

N LPS M10 M20

MCP-1

0

20

40

60

80

100

MCP

-1 (f

old)

lowast

N LPS M10 M20

CCL24

0

2

4

6

8

CCL2

4 (fo

ld)

N LPS M10 M20

IL-6

lowast

lowastlowast

010203040506070

IL-6

(fol

d)

N LPS M10 M20

lowastlowast

lowastlowast

IL-1120573

020406080

100120140

IL-1120573

(fold

)

N LPS M10 M20

TNF-120572

(fold

)

TNF-120572

0

10

20

30

40

50

lowast

lowastlowast

N LPS M10 M20

ICAM-1

0

5

10

15

20

25

ICA

M-1

(fol

d)

lowastlowast

lowastlowast

N LPS M10 M20

COX-2

0

10

20

30

40

50

60

COX-

2 (fo

ld)

lowast

lowastlowast

N LPS M10 M20

iNOS

lowast

0

2

4

6

8

10

iNO

S (fo

ld)

N LPS M10 M20

IL-4

0

1

2

3

4

5

IL-4

(fol

d)

N LPS M10 M20

IL-13

lowast

0

2

4

6

8

10

IL-1

3 (fo

ld)





ERK

Phospho-ERK

p38

Phospho-p38

Phospho-JNK

JNK

120573-actin

N LPS M10 M20

Phospho-NF-120581B

NF-120581B


lowast

lowastlowast

N LPS M10 M200

200

400

600

800

1000

IL-6

(pg

mL)

(a)

lowast

lowastlowast

N LPS M10 M200

200

400

600

800

1000

TNF-120572

(pg

mL)

(b)





5 Conclusion






Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















+ + + +

100 200 400Matrine (120583M)

minus

minus minus

120573-actin

Phospho-I120581B-120572

I120581B-120572

LPS (1120583gmL)

(a)

p65

p65

Cyto

sol

Nuc

lear

+ + + +

100 200 400Matrine (120583M)

minus

minus minus

120573-actin

Lamin-B1

LPS (1120583gmL)

(b)

0

20

40

60

80

100

Relat

ive l

ucife

rase

activ

ity (

)

Matrine (120583M)

LPS

lowast

+ + + +

100 200 400minus

minus minus

lowastlowast

lowastlowast

(1120583gmL)

(c)




5







4 Discussion



ERK

Phospho-ERK

p38

Phospho-p38

Phospho-JNK

JNK

120573-actin

Matrine (120583M)

LPS + + + +

100 200 400minus

minus minus

(1120583gmL)


N LPS

M20M10



lowast

0

1

2

3

4

5

6N

eutro

phils

cells

mL

N LPS M10 M20

times105

(a)

lowast

lowastlowast

lowastlowast

0

100

200

300

400

500

IL-6

(pg

mL)

N LPS M10 M20

(b)

lowast

N LPS M10 M200

200

400

600

800

TNF-120572

(pg

mL)

(c)








N LPS M10 M20

CCL11

02468

10121416

CCL1

1 (fo

ld)

N LPS M10 M20

CCL5

0

2

4

6

8

10CC

L5 (f

old)

lowast

N LPS M10 M20

MCP-1

0

20

40

60

80

100

MCP

-1 (f

old)

lowast

N LPS M10 M20

CCL24

0

2

4

6

8

CCL2

4 (fo

ld)

N LPS M10 M20

IL-6

lowast

lowastlowast

010203040506070

IL-6

(fol

d)

N LPS M10 M20

lowastlowast

lowastlowast

IL-1120573

020406080

100120140

IL-1120573

(fold

)

N LPS M10 M20

TNF-120572

(fold

)

TNF-120572

0

10

20

30

40

50

lowast

lowastlowast

N LPS M10 M20

ICAM-1

0

5

10

15

20

25

ICA

M-1

(fol

d)

lowastlowast

lowastlowast

N LPS M10 M20

COX-2

0

10

20

30

40

50

60

COX-

2 (fo

ld)

lowast

lowastlowast

N LPS M10 M20

iNOS

lowast

0

2

4

6

8

10

iNO

S (fo

ld)

N LPS M10 M20

IL-4

0

1

2

3

4

5

IL-4

(fol

d)

N LPS M10 M20

IL-13

lowast

0

2

4

6

8

10

IL-1

3 (fo

ld)





ERK

Phospho-ERK

p38

Phospho-p38

Phospho-JNK

JNK

120573-actin

N LPS M10 M20

Phospho-NF-120581B

NF-120581B


lowast

lowastlowast

N LPS M10 M200

200

400

600

800

1000

IL-6

(pg

mL)

(a)

lowast

lowastlowast

N LPS M10 M200

200

400

600

800

1000

TNF-120572

(pg

mL)

(b)





5 Conclusion






Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ERK

Phospho-ERK

p38

Phospho-p38

Phospho-JNK

JNK

120573-actin

Matrine (120583M)

LPS + + + +

100 200 400minus

minus minus

(1120583gmL)


N LPS

M20M10



lowast

0

1

2

3

4

5

6N

eutro

phils

cells

mL

N LPS M10 M20

times105

(a)

lowast

lowastlowast

lowastlowast

0

100

200

300

400

500

IL-6

(pg

mL)

N LPS M10 M20

(b)

lowast

N LPS M10 M200

200

400

600

800

TNF-120572

(pg

mL)

(c)








N LPS M10 M20

CCL11

02468

10121416

CCL1

1 (fo

ld)

N LPS M10 M20

CCL5

0

2

4

6

8

10CC

L5 (f

old)

lowast

N LPS M10 M20

MCP-1

0

20

40

60

80

100

MCP

-1 (f

old)

lowast

N LPS M10 M20

CCL24

0

2

4

6

8

CCL2

4 (fo

ld)

N LPS M10 M20

IL-6

lowast

lowastlowast

010203040506070

IL-6

(fol

d)

N LPS M10 M20

lowastlowast

lowastlowast

IL-1120573

020406080

100120140

IL-1120573

(fold

)

N LPS M10 M20

TNF-120572

(fold

)

TNF-120572

0

10

20

30

40

50

lowast

lowastlowast

N LPS M10 M20

ICAM-1

0

5

10

15

20

25

ICA

M-1

(fol

d)

lowastlowast

lowastlowast

N LPS M10 M20

COX-2

0

10

20

30

40

50

60

COX-

2 (fo

ld)

lowast

lowastlowast

N LPS M10 M20

iNOS

lowast

0

2

4

6

8

10

iNO

S (fo

ld)

N LPS M10 M20

IL-4

0

1

2

3

4

5

IL-4

(fol

d)

N LPS M10 M20

IL-13

lowast

0

2

4

6

8

10

IL-1

3 (fo

ld)





ERK

Phospho-ERK

p38

Phospho-p38

Phospho-JNK

JNK

120573-actin

N LPS M10 M20

Phospho-NF-120581B

NF-120581B


lowast

lowastlowast

N LPS M10 M200

200

400

600

800

1000

IL-6

(pg

mL)

(a)

lowast

lowastlowast

N LPS M10 M200

200

400

600

800

1000

TNF-120572

(pg

mL)

(b)





5 Conclusion






Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast

0

1

2

3

4

5

6N

eutro

phils

cells

mL

N LPS M10 M20

times105

(a)

lowast

lowastlowast

lowastlowast

0

100

200

300

400

500

IL-6

(pg

mL)

N LPS M10 M20

(b)

lowast

N LPS M10 M200

200

400

600

800

TNF-120572

(pg

mL)

(c)








N LPS M10 M20

CCL11

02468

10121416

CCL1

1 (fo

ld)

N LPS M10 M20

CCL5

0

2

4

6

8

10CC

L5 (f

old)

lowast

N LPS M10 M20

MCP-1

0

20

40

60

80

100

MCP

-1 (f

old)

lowast

N LPS M10 M20

CCL24

0

2

4

6

8

CCL2

4 (fo

ld)

N LPS M10 M20

IL-6

lowast

lowastlowast

010203040506070

IL-6

(fol

d)

N LPS M10 M20

lowastlowast

lowastlowast

IL-1120573

020406080

100120140

IL-1120573

(fold

)

N LPS M10 M20

TNF-120572

(fold

)

TNF-120572

0

10

20

30

40

50

lowast

lowastlowast

N LPS M10 M20

ICAM-1

0

5

10

15

20

25

ICA

M-1

(fol

d)

lowastlowast

lowastlowast

N LPS M10 M20

COX-2

0

10

20

30

40

50

60

COX-

2 (fo

ld)

lowast

lowastlowast

N LPS M10 M20

iNOS

lowast

0

2

4

6

8

10

iNO

S (fo

ld)

N LPS M10 M20

IL-4

0

1

2

3

4

5

IL-4

(fol

d)

N LPS M10 M20

IL-13

lowast

0

2

4

6

8

10

IL-1

3 (fo

ld)





ERK

Phospho-ERK

p38

Phospho-p38

Phospho-JNK

JNK

120573-actin

N LPS M10 M20

Phospho-NF-120581B

NF-120581B


lowast

lowastlowast

N LPS M10 M200

200

400

600

800

1000

IL-6

(pg

mL)

(a)

lowast

lowastlowast

N LPS M10 M200

200

400

600

800

1000

TNF-120572

(pg

mL)

(b)





5 Conclusion






Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















N LPS M10 M20

CCL11

02468

10121416

CCL1

1 (fo

ld)

N LPS M10 M20

CCL5

0

2

4

6

8

10CC

L5 (f

old)

lowast

N LPS M10 M20

MCP-1

0

20

40

60

80

100

MCP

-1 (f

old)

lowast

N LPS M10 M20

CCL24

0

2

4

6

8

CCL2

4 (fo

ld)

N LPS M10 M20

IL-6

lowast

lowastlowast

010203040506070

IL-6

(fol

d)

N LPS M10 M20

lowastlowast

lowastlowast

IL-1120573

020406080

100120140

IL-1120573

(fold

)

N LPS M10 M20

TNF-120572

(fold

)

TNF-120572

0

10

20

30

40

50

lowast

lowastlowast

N LPS M10 M20

ICAM-1

0

5

10

15

20

25

ICA

M-1

(fol

d)

lowastlowast

lowastlowast

N LPS M10 M20

COX-2

0

10

20

30

40

50

60

COX-

2 (fo

ld)

lowast

lowastlowast

N LPS M10 M20

iNOS

lowast

0

2

4

6

8

10

iNO

S (fo

ld)

N LPS M10 M20

IL-4

0

1

2

3

4

5

IL-4

(fol

d)

N LPS M10 M20

IL-13

lowast

0

2

4

6

8

10

IL-1

3 (fo

ld)





ERK

Phospho-ERK

p38

Phospho-p38

Phospho-JNK

JNK

120573-actin

N LPS M10 M20

Phospho-NF-120581B

NF-120581B


lowast

lowastlowast

N LPS M10 M200

200

400

600

800

1000

IL-6

(pg

mL)

(a)

lowast

lowastlowast

N LPS M10 M200

200

400

600

800

1000

TNF-120572

(pg

mL)

(b)





5 Conclusion






Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ERK

Phospho-ERK

p38

Phospho-p38

Phospho-JNK

JNK

120573-actin

N LPS M10 M20

Phospho-NF-120581B

NF-120581B


lowast

lowastlowast

N LPS M10 M200

200

400

600

800

1000

IL-6

(pg

mL)

(a)

lowast

lowastlowast

N LPS M10 M200

200

400

600

800

1000

TNF-120572

(pg

mL)

(b)





5 Conclusion






Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















5 Conclusion






Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Matrine Attenuates COX-2 and ICAM-1...

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Transcript of Research Article Matrine Attenuates COX-2 and ICAM-1...