Research Article Hydrogen-Rich Saline Inhibits NLRP3...

Research ArticleHydrogen-Rich Saline Inhibits NLRP3 Inflammasome Activationand Attenuates Experimental Acute Pancreatitis in Mice

Jian-Dong Ren Jie Ma Jun Hou Wen-Jin Xiao Wei-Hua Jin Juan Wu and Kai-Hua Fan

Department of Pharmacy Chengdu Military General Hospital No 270 Rongdu Avenue Jinniu DistrictChengdu Sichuan 610083 China

Correspondence should be addressed to Jian-Dong Ren keanrjd126com and Kai-Hua Fan fan kai hua163com

Received 6 June 2014 Revised 5 August 2014 Accepted 5 August 2014 Published 20 August 2014

Academic Editor Yung-Hsiang Chen

Copyright copy 2014 Jian-Dong Ren et alThis 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

Increasing evidence has demonstrated that reactive oxygen species (ROS) induces oxidative stress and plays a crucial role in thepathogenesis of acute pancreatitis (AP) Hydrogen-rich saline (HRS) a well-known ROS scavenger has been shown to possesstherapeutic benefit on AP in many animal experiments Recent findings have indicated that the NOD-like receptor family pyrindomain-containing 3 (NLRP3) inflammasome an intracellular multiprotein complex required for the maturation of interleukin-(IL-) 1120573 may probably be a potential target of HRS in the treatment of AP Therefore in this study we evaluated the activation ofNLRP3 inflammasome andmeanwhile assessed the degree of oxidative stress and inflammatory cascades as well as the histologicalalterations in mice suffering from cerulein-induced AP after the treatment of HRS The results showed that the activation ofNLRP3 inflammasome in AP mice was substantially inhibited following the administration of HRS which was paralleled with thedecreasedNF-120581B activity and cytokines production attenuated oxidative stress and the amelioration of pancreatic tissue damage Inconclusion our study has for the first time revealed that inhibition of the activation ofNLRP3 inflammasome probably contributedto the therapeutic potential of HRS in AP

1 Introduction

Acute pancreatitis (AP) a debilitating inflammation of thepancreas is usually triggered by inappropriate intra-acinaractivation of the trypsinogen Although most cases of AParemild or self-limiting sometimes uncontrolled subsequentevents including activation of immune cells and furthersystemic progression of inflammatory response are capableof converting AP to a severe form [1 2] Vigorous releaseof inflammatory mediators such as cytokines chemokinesand reactive oxygen species (ROS) during systemic inflam-matory responses results in a life-threatening condition withmultiple-organ dysfunction which carries substantial mor-bidity and mortality

Currently much research attention has been focused onthe involvement of ROS and resultant oxidative stress in theinflammatory processes during the development of pancre-atitis [3ndash5] At the early stage of AP several pathogeneticmechanisms such as proteolytic damage tissue ischaemiaand activation of polymorphonuclear leucocytes stimulatethe production of ROS that act as the molecular trigger of

various inflammatory processes [6] In general the detrimen-tal ROS can be detoxified either by endogenous antioxidantslike glutathione (GSH) and superoxide dismutase (SOD)or when needed by exogenous dietary antioxidants likecarotenoids flavonoids and vitamin E However once theproduction of ROS is excessive and overwhelms endogenousantioxidants oxidative stress can occur with increasing cellu-lar injury Therefore the theory that attack of oxidative stressis responsible for not only the amplification of pancreaticdamage but also the progression of the disease from a localdamage to a systemic organ failure is generally accepted asfact [3] Therefore supplementation with exogenous antioxi-dants is regarded as a beneficial therapeutic strategy at least auseful adjunctive option in the treatment of AP as evidencedin some animal models [7ndash9]

Among numerous antioxidants molecular hydrogen(H2) is emerging as a promising candidate in the therapeutic

approaches of oxidative stress-implicated inflammatory dis-eases because this simple molecule is a selective scavenger ofdetrimental ROS such as hydroxyl radical and peroxynitrite[10 11] In some animal experiments hydrogen gas can be

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 930894 9 pageshttpdxdoiorg1011552014930894

2 Mediators of Inflammation

given by inhalation or more conveniently administered byinjection when it dissolves in normal saline to achieve thesaturation level (called hydrogen-rich saline HRS) Recentstudies including our previous work have provided evidencethat treatment of HRS relieved the severity of pancreatitisin rats describing the potential of HRS in the managementof AP caused by different pathogenetic mechanisms [12 13]Although the molecular mechanism underlying the protec-tive effect of HRS against AP has not been fully elucidatedit is well considered that the direct ROS elimination activityof HRS is mostly responsible for its therapeutic benefitsdue to the specific scavenging activity of cytotoxic ROSInterestingly emerging evidence has been presented that thepotential of HRS to suppress the proinflammatory cascadealso contributes to the reduction of the severity of the dis-ease Recently the finding that activation of an intracellularmultiprotein complex the NOD-like receptor family pyrindomain-containing 3 (NLRP3) inflammasome is involvedin the proinflammatory process in pancreas provides valu-able insights into the pathogenesis of pancreatitis [14]As the most well-characterized inflammasome the NLRP3inflammasome has been shown to possess the function ofregulating the maturation and release of proinflammatorycytokine interleukin- (IL-) 1120573 In previous work we foundthat augmented secretion of IL-1120573 in rats suffering fromtrauma-induced pancreatitis was substantially depressed bythe treatment of HRS which implies a possibility that HRSis able to inhibit the activation of NLRP3 inflammasomethereby contributing to the suppression of IL-1120573 production[13]Thus the herein study extended previous studies to eval-uate the potential of HRS to inhibit NLRP3 inflammasomeactivation which probablymediated the protective activity ofHRS against experimental AP

2 Materials and Methods

21 Materials Cerulein was purchased from Sigma (St Lou-is MO USA) Antibodies against the p65 subunit of nuclearfactor-120581B (NF-120581B p65) I-120581B120572 phosphorylated-I-120581B120572 (p-I-120581B120572) thioredoxin-interacting protein (TXNIP) NLRP3p20 subunit of caspase-1 (casp-1-p20) 120573-actin and horse-radish peroxidase conjugated goat anti-rabbit antibody wereobtained from Santa Cruz Biotechnology (Santa Cruz CAUSA) Enzyme-linked immunosorbent assay (ELISA) kits forIL-1120573 and tumor necrosis factor-120572 (TNF-120572) were obtainedfrom RampD system (Minneapolis MN USA)

HRS was prepared as described previously [15] In briefH2gas (04MPa) was dissolved in normal saline (NS) for 6

hours to achieve a concentration of 06mmolL and the con-centration of H

2in NS was detected using a dissolved hydro-

gen analyzer (DH-35A DKKTOA Tokyo Japan) The HRSwas freshly prepared every week and then stored under at-mospheric pressure at 4∘C in an aluminum bag with no deadvolume and sterilized by gamma radiation before use

22 Experimental Mouse AP Model and Treatments MaleBalbc mice (20ndash25 g) were obtained from the ExperimentalAnimal Center of Sichuan University (Chengdu Sichuan

China) Experimental AP model was induced by hourlyintraperitoneal injections of cerulein (50 120583gkg) to mice for6 hours One hour after the final injection animals receivedan intraperitoneal injection of HRS at various doses (2 4and 8mLkg every 20min for 3 times) or control saline (airdissolved in NS at a pressure of 04MPa for 6 h) respectivelyMice in sham group were treated with only NS Tail veinblood sampleswere collected 2 h after the final administrationof cerulein and 24 h after the initiation of AP all the animalswere sacrificed by carbon dioxide asphyxiation followed bythe collection of pancreatic tissue samples All proceduresinvolving the mice were carried out in accordance withthe guidelines of the Institutional Animal Care and UseCommittee of Sichuan University

23 Analysis of NLRP3 Inflammasome Activation in Pan-creas Pancreas tissues were collected and homogenized in20mM phosphate buffer (pH 74) containing 05mM buty-lated hydroxytoluene followed by a centrifugation at 4∘Cat 1500timesg for 15 minutes Then cytoplasmic proteins intissue homogenate were extracted using cytoplasmic extrac-tion reagents according to the manufacturerrsquos instructions(Pierce Biotechnology Rockford IL USA)The BCA proteinassay kit was employed to determine protein concentrationSubsequently the extracted proteins were separated by SDS-polyacrylamide gels and then transferred to a polyvinylidenedifluoride membrane (Millipore Billerica MA USA) Themembrane was incubated with blocking solution containing5 skim milk for more than 1 h at room temperature andprobed by 1 1000 dilutions of rabbit polyclonal antibodiesagainst TXNIP NLRP3 and casp-1-p20 respectively Afterwashing membrane was incubated with secondary goat anti-rabbit antibody conjugated to horseradish peroxidase for 1 hat room temperature Immunoreactive proteins were visual-ized by chemiluminescence using theWestern blotting detec-tion system (Amersham Biosciences Piscataway NJ USA)

24 Analysis of NF-120581B Activation in Pancreas The nuclearand cytoplasmic proteins in pancreatic tissue homogenatewere extracted using a protein extraction kit (Pierce Biotech-nology Rockford ILUSA)TheDNA-binding activity ofNF-120581B p65 in nuclear extracts was detected using a sandwichELISA kit (TransAM NF-120581B p65 Active Motif CarlsbadCA USA) Briefly oligonucleotide containing the NF-120581Bconsensus-binding site (51015840-GGGACTTTCC-31015840) was preim-mobilized onto 96-well plates The nuclear extract (1120583g)diluted in complete lysis buffer was added to each wellcontaining 30 120583L of complete binding buffer and incubatedat room temperature for 1 h with mild agitation Afterwashing three times with wash buffer the antibody againstNF-120581B p65 subunit was added (1 1000) followed by 1 hincubation at room temperature After washing the wellswere incubated with the secondary antibody conjugated tohorseradish peroxidase (1 1000) for 1 h at room temperatureThen the DNA-binding activity of NF-120581B p65 was quantifiedby measuring the absorbance at 450 nm with a referencewavelength of 655 nm after the wells were incubated withdeveloping solution for 5min at room temperature

Mediators of Inflammation 3

Moreover the degradation of I-120581B caused by NF-120581Bactivation was evaluated by determining the phosphorylationof I-120581B120572 in pancreatic extracts using Western blotting assayas described above

25 Analysis of SOD Activities in Pancreas A colorimetricassay kit purchased from Cayman Chemical Company (AnnArbor MI USA) was used to measure the activity of SODin pancreas which uses a tetrazolium salt for the detectionof superoxide radicals generated by xanthine oxidase andhypoxanthine Briefly the pancreatic tissuewas homogenizedat 4∘C in 20mM HEPES buffer (containing 1mM EGTA210mM mannitol and 70mM sucrose pH 72) and thencentrifuged at 1500timesg for 5min at 4∘C Then the radicaldetector (tetrazolium salt) and xanthine oxidase were addedAfter mixed the sample was incubated at room temperaturefor 20min and the absorbance was read at 450 nm One unit(U) of SOD was defined as the amount of enzyme neededto produce 50 dismutation of superoxide radical The totaltissue protein concentrationwas determined by a commercialkit (Jiancheng Corp Nanjing China) and the activity ofSOD was expressed as Umg of protein

26 Analysis of Malondialdehyde (MDA) and GSH Levels inPancreas Tissue MDA level a marker of lipid peroxidationwas detected using a commercial MDA-586 assay kit (Oxis-Research Portland OR USA) In brief pancreatic tissueswere homogenized in 20mM phosphate buffer (pH 74)containing 05mM butylated hydroxytoluene followed by acentrifugation at 4∘C at 1500timesg for 15 minutes After proteinconcentration measurement equal amounts of proteins wereused in triplicate to react with a chromogenic reagent N-methyl-2-phenylindole to form a stable carbocyanine dyewith a maximum absorption at 586 nm The level of MDA insample was then calculated with the standard curve obtainedfrom the kit according to the manufacturerrsquos instructions andexpressed as 120583molg of total protein

The level of GSH in pancreas tissue wasmeasured accord-ing to the method reported by Tietze [16] In brief pancreatichomogenate was centrifuged at 1500timesg for 20min and 1mLof the supernatant was mixed with 1mL of 5 trichloroaceticacid for 30min Subsequently 05mL of Ellmanrsquos reagent(55-dithiobis-2-nitrobenzoic acid) and 3mL of phosphatebuffer (pH 80) were added After mixed thoroughly theabsorbance was recorded at 412 nm The GSH level in tissuewas normalized against total protein (120583molg)

27 Pancreatic Enzymes Activity and Cytokines Concentrationsin Plasma Plasma samples were obtained from harvestedblood specimens by centrifugation (1800timesg for 15min at4∘C) and the amylase or lipase activity was analyzed throughcolorimetric method using a commercial kit for amylaseor lipase (Jiancheng Co Nanjing China) respectively Theactivity of amylase or lipase was expressed as units per liter(UL) To determine the concentrations of proinflammatorycytokines in plasma quantitative ELISA kits for IL-1120573 andTNF-120572 were used according to the manufacturerrsquos instruc-tions

120573-Actin

Casp-1-p20

TXNIP

NLRP3

Sham

AP

HRS (M)HRS (L) HRS (H) CSNotreatment

Figure 1 Evaluation of pancreatic NLRP3 inflammasome activationin mice by Western blot assay The HRS at various doses or controlsaline (CS) was injected intraperitoneally to AP mice 1 h after thefinal injection of cerulein respectively Twenty-four hours lateranimals in each group (119899 = 6) were killed and the pancreatictissue samples were immediately collected and then homogenizedon ice After the measurement of protein concentration the NLRP3inflammasome activation was evaluated by detecting the proteinlevels of TXNIP NLRP3 and casp-1-p20 in pancreatic homogenatesL low dose (2mLkg times 3) M middle dose (4mLkg times 3) H highdose (8mLkg times 3)

28 Histological Examination Pancreatic tissues were fixedin formaldehyde and embedded in paraffin Then sampleswere sectioned and stained with hematoxylin and eosinusing a standard staining procedure Histopathological eval-uation was performed under light microscope by an experi-enced laboratory pathologist who was blinded to the groupidentity for the samples Edema inflammatory infiltrationhemorrhage and necrosis were evaluated in accordance withthe scoring scale reported by Rongione et al [17]

29 Statistical Comparisons and Presentation of Data Allexperimental values are expressed as mean plusmn SE Statisticalcomparisons among the groups were assessed by one-wayanalysis of variance (ANOVA) using SPSS 130 softwareDifferences in values were considered significant if119875 values lt005

3 Results

31 HRS Inhibited NLRP3 Inflammasome Activation in Pan-creas during AP TheNLRP3 inflammasome is an intracellu-lar multiprotein complex triggering inflammatory responsesThe protein expression of NLRP3 a major component of thisinflammasome was remarkably increased in the progressionof AP Moreover the development of cerulein-induced pan-creatitis conspicuously stimulated the expression of TXNIPthe critical regulator of NLRP3 activity Similar elevationwas also found in the protein expression of p20 subunit ofactivated caspase-1 used as one of themarkers for the NLRP3inflammasome activation However treatment of HRS dose-dependently diminished expression levels of NLRP3 TXNIPand casp-1-p20 exhibiting the significant inhibitory activityon the NLRP3 inflammasome activation (Figure 1)


0

03

06

09

Sham Notreatment

HRS (L) HRS (M) HRS (H) CS

NF-120581

B p6

5 D

NA

bin

ding

activ

ity (O

D 4

50 nm

)

AP model

lowast

lowast

lowast

lowast

(a)

I-120581B120572

p-I-120581B120572

Sham

AP

HRS (M)HRS (L) HRS (H) CSNo

120573-Actin

treatment

(b)

Figure 2 Evaluation of pancreatic NF-120581B activation in mice The HRS at various doses or control saline (CS) was injected intraperitoneallyto AP mice 1 h after the final injection of cerulein respectively The pancreatic tissue samples were collected from sacrificed animals in eachgroup (119899 = 6) 24 h after the initiation of AP and then homogenized on ice After the measurement of protein concentration NF-120581B activationwas evaluated by measuring the DNA-binding activity of NF-120581B p65 in nuclear extracts using an ELISA kit (a) and by detecting the proteinlevels of I-120581B120572 and p-I-120581B120572 in pancreatic homogenates (b) L low dose (2mLkg times 3) M middle dose (4mLkg times 3) H high dose (8mLkgtimes 3)

AP model

Sham Notreatment

HRS (L) HRS (M) HRS (H) CS0

3000

6000

9000

12000

15000

18000

Seru

m am

ylas

e act

ivity

(UL

)

(a)

0

500

1000

1500

2000

2500

3000

3500

4000

Sham Notreatment

HRS (L) HRS (M) HRS (H)

AP model

CS

Seru

m li

pase

activ

ity (U

L)

(b)

Figure 3 Determination of plasma amylase and lipase activities in mice Experimental AP model was induced by hourly intraperitonealinjections of cerulein (50120583gkg times 6) to mice Then the HRS at various doses or control saline (CS) was injected intraperitoneally to APmice 1 h after the final injection of cerulein respectively Animals in each group (119899 = 6) were sacrificed by CO

2asphyxiation 1 h after the

final administration of HRS and immediately the blood and tissue samples were collected The amylase and lipase activities in plasma weredetermined with a colorimetric method using the commercial kits for amylase and lipase respectively L low dose (2mLkg times 3) M middledose (4mLkg times 3) H high dose (8mLkg times 3)

32 HRS Inhibited NF-120581B Activation in Pancreas during APIt was known that NF-120581B is ubiquitous transcription factorthat controls proinflammatory gene expression during thedevelopment of inflammation related diseases including APIn present study the DNA-binding activity of NF-120581B p65

in pancreas measured by the ELISA increased significantlyin response to the induction of pancreatitis indicating theactivation of NF-120581B whereas it was substantially depressedby the administration of HRS in a dose-response manner(Figure 2(a)) In line with the ELISA data protein expression


(I) (II)

(III) (IV)

(V) (VI)

(a)

0123456789

1011

Hist

opat

holo

gica

l sco

re

Total histopathological scores

0

1

2

3

Hist

opat

holo

gica

l sco

re

Edema

0

1

2

3

Hist

opat

holo

gica

l sco

re

Necrosis

0

1

2

3

Hist

opat

holo

gica

l sco

re

Hemorrhage

0

APAP + HRS (L)AP + HRS (M)

AP + HRS (H)AP + CS

1

2

3

Hist

opat

holo

gica

l sco

reInflammatory infiltration

lowastlowast

lowastlowast

lowastlowast

lowast

(b)

Figure 4 Histopathological examination of pancreatic tissue from mice The HRS at various doses or control saline (CS) was injectedintraperitoneally to AP mice 1 h after the final injection of cerulein respectively One hour later animals in each group (119899 = 6) werekilled and the pancreatic tissue samples were immediately collected After fixed in formaldehyde and embedded in paraffin the sampleswere sectioned and stained with hematoxylin and eosin using a standard staining procedure Histopathological evaluation was performedunder light microscope and the scores were calculated according to histopathological changes including edema inflammatory infiltrationhemorrhage and necrosis (a) Images of histological sections I sham II AP model group III AP + CS IV AP + HRS (L) V AP + HRS(M) VI AP + HRS (H) scale bars = 50120583m Significant histopathological changes like cytoplasmic vacuole generation inflammatory cellsinfiltration and parenchyma necrosis were indicated by blue arrows (b) Histopathological scores lowast119875 lt 005 compared with AP group

of cytoplasmic p-I-120581B120572 in pancreatic homogenates from APmice rose markedly demonstrating the degradation of I-120581B Likewise HRS showed a dose-dependent inhibition onthe degradation of I-120581B which was in agreement with theinhibitory effect on NLRP3 activity (Figure 2(b))

33 HRS Ameliorated Pancreatic Damage in AP Mice Asshown in Figures 3 and 4 experimental AP was inducedby cerulein administration in mice with significant ele-vated plasma pancreatic enzymes activities and pathologicaldamage in pancreas There were no significant changes of

plasma amylase and lipase activities in AP mice after thetreatment of HRS at various doses (Figure 3) Moreover onlyat the higher doses (4 and 8mLkg times 3) HRS conferredfavorable histopathological changes in pancreatic tissuesmainly characterized by reduced cytoplasmic vacuole gener-ation alleviated inflammatory infiltration and parenchymanecrosis which was paralleled with lower histopathologicalscores (Figures 4(a) and 4(b))

34 HRS Decreased Plasma Cytokines Concentration in APMice The proinflammatory cytokines TNF-120572 and IL-1120573 are


0

200

400

600

800

1000

Sham Notreatment


TNF-120572

(pg

mL)

lowast

lowast lowast

lowast

AP model

(a)

Sham Notreatment


50

100

150

200

250

IL-1120573

(pg

mL)

AP model

lowast

lowast

lowast

lowast

(b)

Figure 5 Measurement of plasma cytokines concentration in mice The HRS at various doses or control saline (CS) was injectedintraperitoneally to AP mice 1 h after the final injection of cerulein respectively One hour later animals in each group (119899 = 6) were killedand the blood samples were immediately collected The plasma concentrations of TNF-120572 and IL-1120573 were measured using quantitative ELISAkits according to the manufacturerrsquos instructions lowast119875 lt 005 compared with the group of AP mice with no treatment 119875 lt 005 comparedin groups of AP mice treated with HRS at different doses L low dose (2mLkg times 3) M middle dose (4mLkg times 3) H high dose (8mLkg times3)

known to mediate amplification of inflammatory processduring pancreatitis Here it was shown that plasma concen-trations of TNF-120572 and IL-1120573 markedly increased in micesuffering from cerulein-inducedAP which were substantiallyabolished by the treatment of HRS (Figure 5) In additiona significant dose-dependent effect of HRS to decline theplasma IL-1120573 level was observed however middle dose ofHRS (4mLkg times 3) failed to demonstrate superiority inreducing TNF-120572 release in APmice when compared with thelow dose (2mLkg times 3)

35 HRS Inhibited Oxidative Stress in Pancreas during APIn order to verify the potential of HRS to reduce oxidativestress during AP we measured the changes of pancreaticMDA level an indicator of lipid peroxidation in oxidativestress process as well as the endogenous antioxidants GSHand SOD As expected enhanced oxidative stress in pancreaswas observed in mice after the initiation of AP which wascharacterized by increased amount of MDA and decreasedSOD activity and GSH level In contrast administration ofHRS dose-dependently diminished the MDA productionaccompanied with the elevated levels of SOD and GSH(Figure 6) Moreover the capacity of HRS to help recruitendogenous antioxidants was observed only at the middle(4mLkg times 3) or high dose (8mLkg times 3) in our experiment

4 Discussion

Over the past years there has been continuing controversyabout the benefits of supplementation with exogenous anti-oxidants for the management of oxidative stress relatedinflammatory diseases largely because once it was consideredthat these antioxidants only served as ROS scavengers and

thereby helped correcting the pro- or antioxidative imbalance[18] However recent findings that elimination of ROS byantioxidants suppressed activation of NLRP3 inflammasomeand the resultant maturation of IL-1120573 have shed new lighton understanding the roles of antioxidants in pathogenesisof inflammation related diseases [19] Here we found thatalthough HRS failed to suppress plasma pancreatic enzymesactivity it significantly attenuated the pancreatic damage inmice in response to the induction of pancreatitis togetherwith the impaired NLRP3 inflammasome activity suggestingthat the potential of HRS to block the NLRP3 inflammasomeactivation was probably involved in the amelioration ofpancreatic injury Moreover the data from this study havedemonstrated the possible mechanism by which HRS inhib-ited the activation of NLRP3 inflammasome Firstly becausetheNF-120581B activation is shown previously to be the traditionalpriming signal to induce the NLRP3 expression blockade ofNF-120581B activity by HRS with the following downregulation ofexpression of NLRP3 probably conferred the suppression ofNLRP3 inflammasome activation Furthermore treatment ofHRS promoted the elimination of ROS and thereby reducedthe expression of TXNIP the ROS-sensing protein requiredforNLRP3 activation also resulting in the blockade ofNLRP3inflammasome activation

As an important component of innate immune systemthe NLRP3 inflammasome was initially found to be activatedby pathogens [20ndash23] In recent years some endogenousmolecules known as damage-associated molecular patterns(DAMPs) such as extracellular glucose uric acid amyloid-120573and hyaluronan have been demonstrated to activate NLRP3inflammasome playing important roles in the metabolicdisorders and sterile inflammatory responses including typeII diabetes gout Alzheimerrsquos disease and tissue injury aftertrauma [24ndash27] As for AP some known extracellular NLRP3


0

5

10

15

20

25

30

35Pa

ncre

atic

GSH

leve

l (120583

mol

g)

lowast

lowast

lowast

Sham Notreatment


AP model

(a)

0

20

40

60

80

100

Panc

reat

ic M

DA

leve

l (120583

mol

g)

lowast

lowastlowast

lowast

Sham Notreatment


AP model

(b)

AP model

0

10

20

30

40

50

Sham Notreatment


lowast

lowast

lowast

Panc

reas

SO

D ac

tivity

(Um

g)

(c)

Figure 6 Determination of pancreatic tissue levels of GSH MDA and SOD in mice The HRS at various doses or control saline (CS) wasinjected intraperitoneally to AP mice 1 h after the final injection of cerulein respectively One hour later animals in each group (119899 = 6)were killed and the pancreatic tissue samples were immediately collected and then homogenized on ice After the measurement of proteinconcentration the levels of GSH (a) and MDA (b) and the activity of SOD (c) in pancreatic homogenates were determined according to themethods reported previously lowast119875 lt 005 compared with the group of AP mice with no treatment 119875 lt 005 compared in groups of AP micetreated with HRS at different doses L low dose (2mLkg times 3) M middle dose (4mLkg times 3) H high dose (8mLkg times 3)

activators like DNA adenosine triphosphate (ATP) highmobility group box 1 (HMGB1) and nucleotide binding oli-gomerization domain 2 (NOD2) are all associated with theinflammation during the development of pancreatitis More-over some researches including our previous work haverevealed that heparan sulfate (HS) a degradation productof extracellular matrix known as an important member ofDAMPs triggered intracellular proinflammatory responsesresulting in the aggravation of pancreatitis [28ndash30] There-fore the above-mentioned findings hint at a possible factthat HS might act as a previously unknown agonist ofNLRP3 inflammasome and thereby promote the progressionof AP In this context there arises an interesting questionrequired to be addressed by future work that is whetherHRSpossesses the ability to inhibit the presumedHS-induced

activation of NLRP3 during the development of pancreatitisObviously the elucidation of this question is of considerablesignificance to further understand the beneficial roles ofhydrogen molecule in AP

In addition although the mechanism underlying thetherapeutic potential of HRS is probably shared by otherantioxidants due to their ROS scavenging capacity HRS stillexhibits significant advantages in the treatment of oxidativestress-implicated diseases At first it was known that mam-malian species are lacking endogenous detoxification systemsfor the hydroxyl radical which is considered as one of thestrongest oxidant species having harmful reactions to nucleicacids lipids and proteins further causing irreversible cellulardamage Compared with other known exogenous antioxi-dants hydrogen molecule can selectively quench hydroxyl


radical forming water due to the unique molecular struc-ture and chemical property meanwhile without excessivelyinterfering the physiological roles of the beneficial ROS likesuperoxide anions [10] Moreover HRS can be easily injectedor administered orally and even used as the dissolventfor other therapeutic agents Therefore the advantages ofHRS mentioned above make it a superior candidate for thedetoxification of oxidative stress in clinical application

In summary our work has for the first time revealedthat HRS can act as an antagonist of NLRP3 inflammasomeresulting in attenuation of cellular inflammatory processesand consequently relieve the pancreatic injury during APThis finding allows for a further understanding of themecha-nism underlying the protective effects of HRS against AP andfurthermore marks the NLRP3 inflammasome as a potentialtarget for future therapeutic approaches of this dangerousdisease

Conflict of Interests

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

Acknowledgments

This work was supported by both the Medical ScientificResearch Foundation of Sichuan Provincial Health Depart-ment (no 110477) and the Research Fund of ChengduMilitary General Hospital (no 2011YG-A18) granted to Jian-Dong Ren

References

[1] M Bhatia M Brady S Shokuhi S Christmas J P Neoptole-mos and J Slavin ldquoInflammatorymediators in acute pancreati-tisrdquoThe Journal of Pathology vol 190 no 2 pp 117ndash125 2000

[2] M Bhatia L W Fei Y Cao et al ldquoPathophysiology of acutepancreatitisrdquo Pancreatology vol 5 no 2-3 pp 132ndash144 2005

[3] K Tsai S-S Wang T-S Chen et al ldquoOxidative stress animportant phenomenon with pathogenetic significance in theprogression of acute pancreatitisrdquo Gut vol 42 no 6 pp 850ndash855 1998

[4] A Dabrowski S J Konturek J W Konturek and AGabryelewicz ldquoRole of oxidative stress in the pathogenesisof caerulein-induced acute pancreatitisrdquo European Journal ofPharmacology vol 377 no 1 pp 1ndash11 1999

[5] J Escobar J Pereda A Arduini et al ldquoCross-talk betweenoxidative stress and pro-inflammatory cytokines in acute pan-creatitis a key role for protein phosphatasesrdquo Current Pharma-ceutical Design vol 15 no 26 pp 3027ndash3042 2009

[6] B Poch F Gansauge B Rau et al ldquoThe role of polymorphonu-clear leukocytes and oxygen-derived free radicals in experi-mental acute pancreatitis mediators of local destruction andactivators of inflammationrdquoTheFEBS Letters vol 461 no 3 pp268ndash272 1999

[7] A Demols J Van Laethem E Quertinmont et al ldquoN-acetyl-cysteine decreases severity of acute pancreatitis in micerdquo Pan-creas vol 20 no 2 pp 161ndash169 2000

[8] H Shapiro P Singer Z Halpern and R Bruck ldquoPolyphenolsin the treatment of inflammatory bowel disease and acutepancreatitisrdquo Gut vol 56 no 3 pp 426ndash435 2007

[9] C Niederau M Niederau F Borchard et al ldquoEffects of antiox-idants and free radical scavengers in three different models ofacute pancreatitisrdquo Pancreas vol 7 no 4 pp 486ndash496 1992

[10] I Ohsawa M Ishikawa K Takahashi et al ldquoHydrogen actsas a therapeutic antioxidant by selectively reducing cytotoxicoxygen radicalsrdquo Nature Medicine vol 13 no 6 pp 688ndash6942007

[11] S Ohta ldquoMolecular hydrogen as a preventive and therapeuticmedical gas initiation development and potential of hydrogenmedicinerdquo Pharmacology ampTherapeutics 2014

[12] H Chen Y P Sun Y Li et al ldquoHydrogen-rich saline amelioratesthe severity of l-arginine-induced acute pancreatitis in ratsrdquoBiochemical and Biophysical Research Communications vol 393no 2 pp 308ndash313 2010

[13] J Ren Z Luo F Tian QWang K Li andCWang ldquoHydrogen-rich saline reduces the oxidative stress and relieves the severityof trauma-induced acute pancreatitis in ratsrdquo Journal of Traumaand Acute Care Surgery vol 72 no 6 pp 1555ndash1561 2012

[14] R Hoque M Sohail A Malik et al ldquoTLR9 and the NLRP3inflammasome link acinar cell deathwith inflammation in acutepancreatitisrdquo Gastroenterology vol 141 no 1 pp 358ndash369 2011

[15] X Zheng Y Mao J Cai et al ldquoHydrogen-rich saline protectsagainst intestinal ischemiareperfusion injury in ratsrdquo FreeRadical Research vol 43 no 5 pp 478ndash484 2009

[16] F Tietze ldquoEnzymic method for quantitative determination ofnanogram amounts of total and oxidized glutathione appli-cations to mammalian blood and other tissuesrdquo AnalyticalBiochemistry vol 27 no 3 pp 502ndash522 1969

[17] A J RongioneAMKusske KKwan SWAshleyHA Reberand D W McFadden ldquoInterleukin 10 reduces the severity ofacute pancreatitis in ratsrdquo Gastroenterology vol 112 no 3 pp960ndash967 1997

[18] K M Oldham and P E Bowen ldquoOxidative stress in criticalcare Is antioxidant supplementation beneficialrdquo Journal of theAmericanDietetic Association vol 98 no 9 pp 1001ndash1008 1998

[19] A Rubartelli ldquoRedox control of NLRP3 inflammasome activa-tion in health and diseaserdquo Journal of Leukocyte Biology vol 92no 5 pp 951ndash958 2012

[20] V A K Rathinam S K Vanaja L Waggoner et al ldquoTRIFlicenses caspase-11-dependent NLRP3 inflammasome activa-tion by gram-negative bacteriardquoCell vol 150 no 3 pp 606ndash6192012

[21] L Mao L Zhang H Li et al ldquoPathogenic fungusMicrosporumcanis activates the NLRP3 inflammasomerdquo Infection and Immu-nity vol 82 no 2 pp 882ndash892 2014

[22] J LMcAuleyM D Tate C JMacKenzie-Kludas et al ldquoActiva-tion of theNLRP3 inflammasome by IAVvirulence protein PB1-F2 contributes to severe pathophysiology and diseaserdquo PLoSPathogens vol 9 no 5 Article ID e1003392 2013

[23] T M Lawrence A W Hudacek M R de Zoete R A Flavelland M J Schnell ldquoRabies virus is recognized by the NLRP3inflammasome and activates interleukin-1120573 release in murinedendritic cellsrdquo Journal of Virology vol 87 no 10 pp 5848ndash58572013

[24] R Zhou A Tardivel B Thorens I Choi and J TschoppldquoThioredoxin-interacting protein links oxidative stress to in-flammasome activationrdquo Nature Immunology vol 11 no 2 pp136ndash140 2010


[25] F Martinon V Petrilli A Mayor A Tardivel and J TschoppldquoGout-associated uric acid crystals activate the NALP3 inflam-masomerdquo Nature vol 440 no 7081 pp 237ndash241 2006

[26] A Halle V Hornung G C Petzold et al ldquoThe NALP3inflammasome is involved in the innate immune response toamyloid-120573rdquoNature Immunology vol 9 no 8 pp 857ndash865 2008

[27] K Yamasaki J Muto K R Taylor et al ldquoNLRP3cryopyrinis necessary for interleukin-1120573 (IL-1120573) release in response tohyaluronan an endogenous trigger of inflammation in responseto injuryrdquo The Journal of Biological Chemistry vol 284 no 19pp 12762ndash12771 2009

[28] J D Ren L Fan F Z Tian et al ldquoInvolvement of a membranepotassium channel in heparan sulfate induced activation ofmacrophagesrdquo Immunology vol 141 no 3 pp 345ndash352 2014

[29] H Liu Y Li L Wang H Chen J Guan and Z Zhou ldquoAg-gravation of acute pancreatitis by heparan sulfate in micerdquoScandinavian Journal of Gastroenterology vol 44 no 5 pp 626ndash632 2009

[30] G B Johnson G J Brunn and J L Platt ldquoCutting edgean endogenous pathway to systemic inflammatory responsesyndrome (SIRS)-like reactions through Toll-like receptor-4rdquoJournal of Immunology vol 172 no 1 pp 20ndash24 2004

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


given by inhalation or more conveniently administered byinjection when it dissolves in normal saline to achieve thesaturation level (called hydrogen-rich saline HRS) Recentstudies including our previous work have provided evidencethat treatment of HRS relieved the severity of pancreatitisin rats describing the potential of HRS in the managementof AP caused by different pathogenetic mechanisms [12 13]Although the molecular mechanism underlying the protec-tive effect of HRS against AP has not been fully elucidatedit is well considered that the direct ROS elimination activityof HRS is mostly responsible for its therapeutic benefitsdue to the specific scavenging activity of cytotoxic ROSInterestingly emerging evidence has been presented that thepotential of HRS to suppress the proinflammatory cascadealso contributes to the reduction of the severity of the dis-ease Recently the finding that activation of an intracellularmultiprotein complex the NOD-like receptor family pyrindomain-containing 3 (NLRP3) inflammasome is involvedin the proinflammatory process in pancreas provides valu-able insights into the pathogenesis of pancreatitis [14]As the most well-characterized inflammasome the NLRP3inflammasome has been shown to possess the function ofregulating the maturation and release of proinflammatorycytokine interleukin- (IL-) 1120573 In previous work we foundthat augmented secretion of IL-1120573 in rats suffering fromtrauma-induced pancreatitis was substantially depressed bythe treatment of HRS which implies a possibility that HRSis able to inhibit the activation of NLRP3 inflammasomethereby contributing to the suppression of IL-1120573 production[13]Thus the herein study extended previous studies to eval-uate the potential of HRS to inhibit NLRP3 inflammasomeactivation which probablymediated the protective activity ofHRS against experimental AP

2 Materials and Methods

21 Materials Cerulein was purchased from Sigma (St Lou-is MO USA) Antibodies against the p65 subunit of nuclearfactor-120581B (NF-120581B p65) I-120581B120572 phosphorylated-I-120581B120572 (p-I-120581B120572) thioredoxin-interacting protein (TXNIP) NLRP3p20 subunit of caspase-1 (casp-1-p20) 120573-actin and horse-radish peroxidase conjugated goat anti-rabbit antibody wereobtained from Santa Cruz Biotechnology (Santa Cruz CAUSA) Enzyme-linked immunosorbent assay (ELISA) kits forIL-1120573 and tumor necrosis factor-120572 (TNF-120572) were obtainedfrom RampD system (Minneapolis MN USA)

HRS was prepared as described previously [15] In briefH2gas (04MPa) was dissolved in normal saline (NS) for 6

hours to achieve a concentration of 06mmolL and the con-centration of H

2in NS was detected using a dissolved hydro-

gen analyzer (DH-35A DKKTOA Tokyo Japan) The HRSwas freshly prepared every week and then stored under at-mospheric pressure at 4∘C in an aluminum bag with no deadvolume and sterilized by gamma radiation before use

22 Experimental Mouse AP Model and Treatments MaleBalbc mice (20ndash25 g) were obtained from the ExperimentalAnimal Center of Sichuan University (Chengdu Sichuan

China) Experimental AP model was induced by hourlyintraperitoneal injections of cerulein (50 120583gkg) to mice for6 hours One hour after the final injection animals receivedan intraperitoneal injection of HRS at various doses (2 4and 8mLkg every 20min for 3 times) or control saline (airdissolved in NS at a pressure of 04MPa for 6 h) respectivelyMice in sham group were treated with only NS Tail veinblood sampleswere collected 2 h after the final administrationof cerulein and 24 h after the initiation of AP all the animalswere sacrificed by carbon dioxide asphyxiation followed bythe collection of pancreatic tissue samples All proceduresinvolving the mice were carried out in accordance withthe guidelines of the Institutional Animal Care and UseCommittee of Sichuan University

23 Analysis of NLRP3 Inflammasome Activation in Pan-creas Pancreas tissues were collected and homogenized in20mM phosphate buffer (pH 74) containing 05mM buty-lated hydroxytoluene followed by a centrifugation at 4∘Cat 1500timesg for 15 minutes Then cytoplasmic proteins intissue homogenate were extracted using cytoplasmic extrac-tion reagents according to the manufacturerrsquos instructions(Pierce Biotechnology Rockford IL USA)The BCA proteinassay kit was employed to determine protein concentrationSubsequently the extracted proteins were separated by SDS-polyacrylamide gels and then transferred to a polyvinylidenedifluoride membrane (Millipore Billerica MA USA) Themembrane was incubated with blocking solution containing5 skim milk for more than 1 h at room temperature andprobed by 1 1000 dilutions of rabbit polyclonal antibodiesagainst TXNIP NLRP3 and casp-1-p20 respectively Afterwashing membrane was incubated with secondary goat anti-rabbit antibody conjugated to horseradish peroxidase for 1 hat room temperature Immunoreactive proteins were visual-ized by chemiluminescence using theWestern blotting detec-tion system (Amersham Biosciences Piscataway NJ USA)

24 Analysis of NF-120581B Activation in Pancreas The nuclearand cytoplasmic proteins in pancreatic tissue homogenatewere extracted using a protein extraction kit (Pierce Biotech-nology Rockford ILUSA)TheDNA-binding activity ofNF-120581B p65 in nuclear extracts was detected using a sandwichELISA kit (TransAM NF-120581B p65 Active Motif CarlsbadCA USA) Briefly oligonucleotide containing the NF-120581Bconsensus-binding site (51015840-GGGACTTTCC-31015840) was preim-mobilized onto 96-well plates The nuclear extract (1120583g)diluted in complete lysis buffer was added to each wellcontaining 30 120583L of complete binding buffer and incubatedat room temperature for 1 h with mild agitation Afterwashing three times with wash buffer the antibody againstNF-120581B p65 subunit was added (1 1000) followed by 1 hincubation at room temperature After washing the wellswere incubated with the secondary antibody conjugated tohorseradish peroxidase (1 1000) for 1 h at room temperatureThen the DNA-binding activity of NF-120581B p65 was quantifiedby measuring the absorbance at 450 nm with a referencewavelength of 655 nm after the wells were incubated withdeveloping solution for 5min at room temperature







120573-Actin

Casp-1-p20

TXNIP

NLRP3

Sham

AP





3 Results



0

03

06

09

Sham Notreatment


NF-120581

B p6

5 D

NA

bin

ding

activ

ity (O

D 4

50 nm

)

AP model

lowast

lowast

lowast

lowast

(a)

I-120581B120572

p-I-120581B120572

Sham

AP


120573-Actin

treatment

(b)


AP model

Sham Notreatment


3000

6000

9000

12000

15000

18000

Seru

m am

ylas

e act

ivity

(UL

)

(a)

0

500

1000

1500

2000

2500

3000

3500

4000

Sham Notreatment


AP model

CS

Seru

m li

pase

activ

ity (U

L)

(b)







(I) (II)

(III) (IV)

(V) (VI)

(a)

0123456789

1011

Hist

opat

holo

gica

l sco

re


0

1

2

3

Hist

opat

holo

gica

l sco

re

Edema

0

1

2

3

Hist

opat

holo

gica

l sco

re

Necrosis

0

1

2

3

Hist

opat

holo

gica

l sco

re

Hemorrhage

0


AP + HRS (H)AP + CS

1

2

3

Hist

opat

holo

gica

l sco


lowastlowast

lowastlowast

lowastlowast

lowast

(b)







0

200

400

600

800

1000

Sham Notreatment


TNF-120572

(pg

mL)

lowast

lowast lowast

lowast

AP model

(a)

Sham Notreatment


50

100

150

200

250

IL-1120573

(pg

mL)

AP model

lowast

lowast

lowast

lowast

(b)




4 Discussion





0

5

10

15

20

25

30

35Pa

ncre

atic

GSH

leve

l (120583

mol

g)

lowast

lowast

lowast

Sham Notreatment


AP model

(a)

0

20

40

60

80

100

Panc

reat

ic M

DA

leve

l (120583

mol

g)

lowast

lowastlowast

lowast

Sham Notreatment


AP model

(b)

AP model

0

10

20

30

40

50

Sham Notreatment


lowast

lowast

lowast

Panc

reas

SO

D ac

tivity

(Um

g)

(c)










Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















120573-Actin

Casp-1-p20

TXNIP

NLRP3

Sham

AP





3 Results



0

03

06

09

Sham Notreatment


NF-120581

B p6

5 D

NA

bin

ding

activ

ity (O

D 4

50 nm

)

AP model

lowast

lowast

lowast

lowast

(a)

I-120581B120572

p-I-120581B120572

Sham

AP


120573-Actin

treatment

(b)


AP model

Sham Notreatment


3000

6000

9000

12000

15000

18000

Seru

m am

ylas

e act

ivity

(UL

)

(a)

0

500

1000

1500

2000

2500

3000

3500

4000

Sham Notreatment


AP model

CS

Seru

m li

pase

activ

ity (U

L)

(b)







(I) (II)

(III) (IV)

(V) (VI)

(a)

0123456789

1011

Hist

opat

holo

gica

l sco

re


0

1

2

3

Hist

opat

holo

gica

l sco

re

Edema

0

1

2

3

Hist

opat

holo

gica

l sco

re

Necrosis

0

1

2

3

Hist

opat

holo

gica

l sco

re

Hemorrhage

0


AP + HRS (H)AP + CS

1

2

3

Hist

opat

holo

gica

l sco


lowastlowast

lowastlowast

lowastlowast

lowast

(b)







0

200

400

600

800

1000

Sham Notreatment


TNF-120572

(pg

mL)

lowast

lowast lowast

lowast

AP model

(a)

Sham Notreatment


50

100

150

200

250

IL-1120573

(pg

mL)

AP model

lowast

lowast

lowast

lowast

(b)




4 Discussion





0

5

10

15

20

25

30

35Pa

ncre

atic

GSH

leve

l (120583

mol

g)

lowast

lowast

lowast

Sham Notreatment


AP model

(a)

0

20

40

60

80

100

Panc

reat

ic M

DA

leve

l (120583

mol

g)

lowast

lowastlowast

lowast

Sham Notreatment


AP model

(b)

AP model

0

10

20

30

40

50

Sham Notreatment


lowast

lowast

lowast

Panc

reas

SO

D ac

tivity

(Um

g)

(c)










Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

03

06

09

Sham Notreatment


NF-120581

B p6

5 D

NA

bin

ding

activ

ity (O

D 4

50 nm

)

AP model

lowast

lowast

lowast

lowast

(a)

I-120581B120572

p-I-120581B120572

Sham

AP


120573-Actin

treatment

(b)


AP model

Sham Notreatment


3000

6000

9000

12000

15000

18000

Seru

m am

ylas

e act

ivity

(UL

)

(a)

0

500

1000

1500

2000

2500

3000

3500

4000

Sham Notreatment


AP model

CS

Seru

m li

pase

activ

ity (U

L)

(b)







(I) (II)

(III) (IV)

(V) (VI)

(a)

0123456789

1011

Hist

opat

holo

gica

l sco

re


0

1

2

3

Hist

opat

holo

gica

l sco

re

Edema

0

1

2

3

Hist

opat

holo

gica

l sco

re

Necrosis

0

1

2

3

Hist

opat

holo

gica

l sco

re

Hemorrhage

0


AP + HRS (H)AP + CS

1

2

3

Hist

opat

holo

gica

l sco


lowastlowast

lowastlowast

lowastlowast

lowast

(b)







0

200

400

600

800

1000

Sham Notreatment


TNF-120572

(pg

mL)

lowast

lowast lowast

lowast

AP model

(a)

Sham Notreatment


50

100

150

200

250

IL-1120573

(pg

mL)

AP model

lowast

lowast

lowast

lowast

(b)




4 Discussion





0

5

10

15

20

25

30

35Pa

ncre

atic

GSH

leve

l (120583

mol

g)

lowast

lowast

lowast

Sham Notreatment


AP model

(a)

0

20

40

60

80

100

Panc

reat

ic M

DA

leve

l (120583

mol

g)

lowast

lowastlowast

lowast

Sham Notreatment


AP model

(b)

AP model

0

10

20

30

40

50

Sham Notreatment


lowast

lowast

lowast

Panc

reas

SO

D ac

tivity

(Um

g)

(c)










Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(I) (II)

(III) (IV)

(V) (VI)

(a)

0123456789

1011

Hist

opat

holo

gica

l sco

re


0

1

2

3

Hist

opat

holo

gica

l sco

re

Edema

0

1

2

3

Hist

opat

holo

gica

l sco

re

Necrosis

0

1

2

3

Hist

opat

holo

gica

l sco

re

Hemorrhage

0


AP + HRS (H)AP + CS

1

2

3

Hist

opat

holo

gica

l sco


lowastlowast

lowastlowast

lowastlowast

lowast

(b)







0

200

400

600

800

1000

Sham Notreatment


TNF-120572

(pg

mL)

lowast

lowast lowast

lowast

AP model

(a)

Sham Notreatment


50

100

150

200

250

IL-1120573

(pg

mL)

AP model

lowast

lowast

lowast

lowast

(b)




4 Discussion





0

5

10

15

20

25

30

35Pa

ncre

atic

GSH

leve

l (120583

mol

g)

lowast

lowast

lowast

Sham Notreatment


AP model

(a)

0

20

40

60

80

100

Panc

reat

ic M

DA

leve

l (120583

mol

g)

lowast

lowastlowast

lowast

Sham Notreatment


AP model

(b)

AP model

0

10

20

30

40

50

Sham Notreatment


lowast

lowast

lowast

Panc

reas

SO

D ac

tivity

(Um

g)

(c)










Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

200

400

600

800

1000

Sham Notreatment


TNF-120572

(pg

mL)

lowast

lowast lowast

lowast

AP model

(a)

Sham Notreatment


50

100

150

200

250

IL-1120573

(pg

mL)

AP model

lowast

lowast

lowast

lowast

(b)




4 Discussion





0

5

10

15

20

25

30

35Pa

ncre

atic

GSH

leve

l (120583

mol

g)

lowast

lowast

lowast

Sham Notreatment


AP model

(a)

0

20

40

60

80

100

Panc

reat

ic M

DA

leve

l (120583

mol

g)

lowast

lowastlowast

lowast

Sham Notreatment


AP model

(b)

AP model

0

10

20

30

40

50

Sham Notreatment


lowast

lowast

lowast

Panc

reas

SO

D ac

tivity

(Um

g)

(c)










Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

15

20

25

30

35Pa

ncre

atic

GSH

leve

l (120583

mol

g)

lowast

lowast

lowast

Sham Notreatment


AP model

(a)

0

20

40

60

80

100

Panc

reat

ic M

DA

leve

l (120583

mol

g)

lowast

lowastlowast

lowast

Sham Notreatment


AP model

(b)

AP model

0

10

20

30

40

50

Sham Notreatment


lowast

lowast

lowast

Panc

reas

SO

D ac

tivity

(Um

g)

(c)










Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















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 Hydrogen-Rich Saline Inhibits NLRP3...

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Transcript of Research Article Hydrogen-Rich Saline Inhibits NLRP3...