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1、CellStressandChaperones(2011)16:287296DOI10.1007/s12192-010-0242-6ORIGINALPAPERLipopolysaccharidepretreatmentprotectsagainstischemia/reperfusioninjuryviaincreaseofHSP70andinhibitionofNF-BYong-weiYao&Guo-huiZhang&Ying-yuZhang&Wei-dongLi&Cheng-huaWang&Chun-yangYin&Fu-minZhangRe
2、ceived:4September2010/Revised:25October2010/Accepted:27October2010/Publishedonline:16November2010#CellStressSocietyInternational2010AbstractIthasbeenreportedthatpretreatmentofratswithlipopolysaccharide(LPS)increasesmyocardialfunctionalrecoveryinischemia/reperfusion(I/R)hearts.However,themechanismsby
3、whichLPSinducescardioprotectionagainstI/Rinjuryhavenotbeenfullyelucidated.Inthisstudy,wepretreatedratswithLPS(1.0mg/kg)24hbeforetheyweresubjectedtoI/Rinjury,andthenexaminedtherolesofheatshockprotein-70(HSP70)andnucleusfactor-B(NF-B)inLPS-inducedcardioprotection.Weobservedthatpre-treatmentwithlow-dos
4、eLPSresultedinsignificantlyincreasedlevelsofHSP70inthemyocardium,whichcoulddramaticallyinhibitNF-Btranslocationandreducedegra-dationofinhibitoryB.InhibitionofNF-B,inturn,attenuatedreleaseofinflammatorycytokines(tumornecro-sisfactor-,interleukin(IL)-1,andIL-6)andreducedapoptosisofmyocardiumandinfarct
5、areafollowingI/Rinjury.Moreover,HSP70couldameliorateoxidativestressfollowingI/Rinjury.TofurtherinvestigatewhetherincreaseofHSP70mightberesponsibleforprotectionofthemyocardiumagainstI/Rinjury,weco-administeredtheY.-w.YaoG.-h.Zhang(*)Y.-y.ZhangW.-d.LiC.-h.Wang:C.-y.YinDepartmentofCardiology,Affiliated
6、PeoplesHospitalofJiangsuUniversity,No.8,DianLiRoad,Zhenjiang,JiangsuProvince212000,PeoplesRepublicofChinae-mail:laoyao_1978Y.-w.Yaoe-mail:ywyao78F.-m.ZhangDepartmentofCardiology,FirstAffiliatedHospitalofNanjingMedicalUniversity,300GuangzhouRoad,Nanjing,JiangsuProvince210029,PeoplesRepublicofChinaHSP
7、70inhibitor,quercetin,withLPSbeforeI/Rinjury.WefoundthatLPS-inducedcardioprotectionwasattenuatedbyco-administrationwithquercetin.Herein,weconcludedthatincreasedlevelsofHSP70throughLPSpretreatmentledtoinhibitionofNF-BactivityinthemyocardiumafterI/Rinjury.OurresultsindicatedthatLPS-inducedcardiopro-te
8、ctionwasmediatedpartlythroughinhibitionofNF-BviaincreaseofHSP70,andLPSpretreatmentcouldprovideameansofreducingmyocardialI/Rinjury.KeywordsLipopolysaccharide.Heatshockprotein70.NF-B.Ischemia/reperfusioninjuryIntroductionReperfusionafteraperiodofischemiahasdeleteriouseffectsonthemyocardium,rangingfrom
9、contractileimpairmenttoactualnecrosis.Asubstantialamountofevidencesupportstheideathatischemia/reperfusion(I/R)-inducedinjurytotheheartisduetothereleaseofreactiveoxygenspecies(ROS;Pchejetskietal.2007;Oshimaetal.2005).AsanintracellulartargetofROS,nucleusfactor-B(NF-B)issequesteredinthecytoplasminanina
10、ctivestateduetoitsassociationwithaclassofinhibitoryproteinstermedinhibitoryB(IB).Ischemia/reperfusioninjurycausesarapiddegradationofIB.Then,NF-Btrans-locatesintothenucleusandactivatesBcontaininggenessuchastumornecrosisfactor-(TNF-),interleukin-1(IL-1),andinterleukin-6(IL-6;Cepinskasetal.2002;Lietal.
11、1999).Theselocallyoverexpressedmyocardialcytokinesmayplayacriticalroleintheprogressionofmyocardialdysfunction,includingmyocardialremodeling,cardiachypertrophy,andheartfailure(Detenetal.2002).288Herein,NF-BplaysapivotalroleinI/Rinjury,andinhibitionofNF-BcanprotectmyocardiumfromI/Rinjury.Lipopolysacch
12、aride(LPS),theantigeniccomponentofthegram-negativebacterialcellwall,isknownastheexogenousligandofToll-likereceptor-4(Chowetal.1999).CombinationofLPSanditsreceptorleadstotheactivationofMyD88-dependentsignaltransductionpathwayandnucleartranslo-cationofNF-B.ThedysregulationofNF-Bmayleadtotheexcessivepr
13、oductionofpro-inflammatorymediators,resultinginmyocardiumdamage,heartfailure,andevendeath(Nemotoetal.2002).ExcessivestimulationofcardiaccellsbyLPSresultsinnecrosisandapoptosisofmyocardiumingram-negativesepticshock.Interestingly,ithasbeenreportedthatpretreatmentofratswithlow-doseLPSincreasesmyocardia
14、lfunctionalrecoveryinischemia/reper-fusionhearts(Brownetal.1989;Songetal.1996;Haetal.2008).OurpreviousstudyalsohasshownthatLPScouldprotectmesenchymalstemcells(MSCs)againstoxidativestress-inducedapoptosis,andLPSpretreatmentenhancestheefficacyofMSCstransplantationinaratmodelofacutemyocardialinfarction
15、(Wangetal.2009a,b;Yaoetal.2009).However,themechanismsbywhichLPSinducecardiopro-tectionagainstI/Rinjuryhavenotbeenfullyelucidated.Heatshockproteins(HSPs)arehighlyconservedcellularstressproteinswhicharepresentineveryorganismfrombacteriatomammaliananimals.ManystudieshaveshowntheimportanceofHSPsforthesu
16、rvivalofcellsunderstressconditions(BaoandLiu2008;Shinoharaetal.2007).HSP70,asmolecularchaperon,couldrespondtoawidevarietyofstress,suchasheatshock,ischemia,andinflammation(Zhangetal.2009).OverexpressionofHSP70couldinhibitthetranslocationofNF-B,attenuatethereleaseofinflammatoryfactors,andreducetheapop
17、tosisofmyocardium(Dokladnyetal.2010).Inthepresentstudy,weexaminedtheroleofHSP70andNF-BinLPS-inducedcardioprotection.Weobservedthatpretreatmentwithlow-doseLPSresultedinsignificantlyincreasedlevelsofHSP70inthemyocardium,whichcoulddramaticallyinhibitNF-Btranslocationandreducereleaseofinflammatorycytoki
18、nesinthefollowingI/Rinjury.LPS-inducedcardioprotectionwasattenuatedbyco-administrationwithapharmacologicalinhibitorofHSP70.OurresultsindicatedthatLPS-inducedcardioprotectionwasmediatedpartlythroughinhibitionofNF-BviaincreaseofHSP70.MaterialsandmethodsAnimalpreparationAdultmaleWistarrats(wildtype,210
19、250g)wereprovidedbySlacCompany(Shanghai,China).TheY.-w.Ycedurewasperformedinaccordancewiththe“GuidefortheCareandUseofLaboratoryAnimals”(NIHPublicationNo.85-23,NationalAcademyPress,Washington,DC,revised1996).ThestudyprotocolwasapprovedbytheAnimalCareandUseCommitteeofJiangsuUniversity.Experi
20、mentalprotocolsRatsintheLPSpretreatmentgroup(LPS+I/Rgroup,n=12)werepretreatedwithLPS(1.0mg/kg)byintraper-itonealinjection.ToexaminetheroleofHSP70intheLPS-inducedcardioprotection,anothergroupofrats(Q-LPS+I/Rgroup,n=12)wasintraperitoneallyinjectedwithquerce-tin(100mg/kg),inhibitorofHSP70,2hbeforeLPSad
21、ministration.Thethirdgroupofrats(I/Rgroup,n=12)wasgiventhesamevolumeofnormalsaline.Twenty-fourhourslater,threegroupsofratswereanesthetizedwithketamine(100mg/kg)byintraperitonealinjectionandmechanicallyventilated.Underasterilecondition,theheartwasexposedthroughaleftthoracotomy,andtheleftanteriordesce
22、ndingcoronaryarterywasligatedwithsilksuture(7.0)2mmtoitsoriginwithaslipknot.Aftercompletionofthe30minofocclusion,thecoronaryarterywasreperfusedbyreleasingtheknotfor3h.Controlgroup(n=12)wasintraperitoneallyinjectedwiththesamevolumeofnormalsalineandgivenashamsurgicaloperation.Afterthat,ratsweresacrifi
23、ced,andbloodandheartswerecollectedforthefollowingexperiments.TUNELassayTodetectapoptoticcardiomyocytesintheheart,theLVmyocardium(n=5,each)wasfixedin4%paraformalde-hyde,cuttransversely,andembeddedinparaffin.Apoptoticcardiomyocyteswereevaluatedbyterminaldeoxynucleo-tidyltransferasedUTPnickendlabeling(
24、TUNEL)assayusinganinsituCellDeathDetectionKit(Roche,Germany)accordingtothemanufacturersinstructions.Apoptoticcellswereidentifiedbybrowncolorintheirnuclei.Tissuesectionswereexaminedmicroscopically,andthepercent-ageofapoptoticcellspertotalcellswasdeterminedineightrandomlychosenfields.Determinationofmy
25、ocardialinfarctsizeToassesstheischemicareaatrisk,1%Evansbluewasinfusedintotheaortaandcoronaryarteriesinretrogradefashion.Heartswereexcisedandslicedintofive1-mmcrosssections.Theheartsectionswereincubatedwith1%triphenyltetrazoliumchloride(TTC)solution(Sigma-Aldrich)at37°Cfor15min.Ischemicmyocardi
26、um,whichwasstillviable,wasstainedredwithTTC,whereasthenecroticmyocardiumwasnotstainedandappearedpaleLipopolysaccharidepretreatmentprotectsagainstI/Rinjurywhite.Theinfarctarea(white)andtheareaatrisk(redandwhite)fromeachsectionweremeasuredusingNIHImageandSpotsoftware.Ratiosofriskareavs.leftventricle(R
27、A/LV)andinfarctareavs.riskarea(IA/RA)werecalculatedandexpressedasapercentage.SerumconcentrationsofTNF-,IL-1,andIL-6Bloodwascollectedbeforeandafterischemia/reperfusioninjury,andserumwasprepared.ConcentrationofTNF-,IL-1,andIL-6inserumwasdeterminedbyELISAkits(R&DSystems)accordingtotheinstructionoft
28、hemanufacturer.Experimentswererepeatedthreetimesforverificationofresults.ElectrophoreticmobilityshiftassayHeartnuclearextractsforelectrophoreticmobilityshiftassaywerepreparedusingnuclear-cytosolextractionkit(ApplygenTechnologiesInc.).Double-strandedNF-Bconsensusoligonucleotideprobe(5-AGTTGAGGGGACTTT
29、CCCAGGC-3)wasend-labeledwithbiotin.Electrophoreticmobilityshiftassaykit(Pierce)wasusedtoperformthereaction.Bindingreactions(20lintotal)consistedof35fmolofbiotin-labeledDNA,10gofnuclearprotein,2.5%glycerol,5mMMgCl2,and50ng/lpoly(dIdC)andincubatedatroomtemperaturefor20min.DNAproteincomplexesweresubjec
30、tedtonon-denaturing4%polyacrylamidegelinlowionicstrengthbuffer(45mMTris-borate,1mMEDTA)at75mV/8mAforapproxi-mately3h.Gelswerevacuum-driedandexposedtoX-rayfilmat70°C.Thequantificationoftheradioactiveintensityofautoradiographicsignalswasperformedbyusinganenhancedchemiluminescence(Amersham,USA).We
31、sternblotanalysisofIB,caspase-3,andHSPsCytoplasmicproteinwaspreparedfromhearttissue,andimmunoblotwasperformedasdescribedpreviously(Huetal.2008).Proteinwasanalyzedusing10%sodiumdodecylsulfate-polyacrylamidegelelectrophoresisandtransferredtonitrocellulosemembranes(Bio-Rad).Mem-braneswerethenincubatedw
32、ithprimaryantibodiesincludingIB(1:1,000,CellSignaling);cleavedcaspase-3(1:1,000,CellSignaling);HSP27,HSP60,HSP70,andHSP90(1:1,000,CellSignaling);and-actin(1:5,000,Sigma)at4°Covernight,respectively.Themembraneswerethenincubatedwithperoxidase-labeledsecondaryantibody(1:1,000,SantaCruz,USA)at37
33、76;Cfor2h.Signalsweredetectedbyenhancedchemilumi-nescence(Amersham,USA).DensitometricanalysisfortheblotswasperformedwithNIHimagesoftware.289Experimentswererepeatedsixtimesforverificationofresults.OxidativestressassayAfterI/R,theheartswerecollectedtodetecthomogenatelipidperoxidesbymeasuringmalondiald
34、ehyde(MDA)concentrationwithMDAELISAkit(UscnLifeScienceInc.).AntioxidantenzymesweremeasuredbySuperoxideDismutase(SOD)AssayKit(sigma)accordingtotheillustrationofthemanufacturer.StatisticalanalysisDataareexpressedasmean±SEMandanalyzedwithSPSS11.0statisticalsoftware.Comparisonsofdatabetweengroupswe
35、remadeusingone-wayanalysisofvariancewithBonferronispost-test.Statisticalsignifi-cancewassetat<0.05level.ResultLPSpretreatmentattenuatesmyocardialapoptosisfollowingischemia/reperfusioninjuryInordertostudymyocardialapoptosisfollowingI/Rinjuryandanti-apoptoticeffectofLPSpretreatment,TUNELassaywasper
36、formed.TUNEL-positivenucleistaining(browncolorintheirnuclei)wasdetectableinbothcontrolandexperimentalgroups(Fig.1a).AsshowninFig.1b,cardiacmyocyteapoptosiswasgreatlyincreasedinI/Rgroupcomparedwiththatincontrolgroup(28.6±2.2%vs.2.3±0.4%,P<0.01).LPSpretreatmentsignificantlyde-creasedthenu
37、mberofTUNEL-positivecellsinLPS+I/RgroupcomparedwiththatinI/Rgroup(20.0±2.6%vs.28.6±2.2%,P<0.01).ToinvestigatetheroleofHSP70intheLPSpretreatment,quercetin,theinhibitorofHSP70,wasusedintheexperiment.Wecouldseethatquercetinpartlycanceledtheanti-apoptoticeffectofLPSinQ-LPS+I/Rgroup(25.5
38、7;1.5%).Tofurtherexploretheanti-apoptoticeffectofLPSpretreatment,caspase-3cleavageandactivityweremea-suredbyWesternblot(Fig.1c).Theproteinlevelsforeachsampleweredeterminedasaratiototheircorresponding-actinlevels(Fig.1d).Comparedwithcontrolgroup,I/Rinjuryinducedgreatactivationofpro-caspase-3inI/Rgrou
39、p(0.23±0.02vs.2.10±0.08,P<0.01).LPSpretreat-mentdramaticallyinhibitedthepro-caspase-3cleavageinLPS+I/RgroupcomparedwiththatinI/Rgroup(0.81±0.04vs.2.10±0.08,P<0.01).However,inhibitionofpro-caspase-3cleavagewasattenuatedbyquercetinadminis-trationinQ-LPS+I/Rgroup(1.24±0.0
40、3).290Y.-w.Yaoetal.Fig.1LPSpretreatmentattenuatesmyocardialapoptosisfollowingI/Rinjury.aRepresentativephotomicrographsofTUNEL-positivenucleistaining(brown)inischemiccardiacmusclecells(×400).bCardiacmyocyteapoptosiswasincreasedafterischemic/reperfusioninjury.LPSpretreatmentsignificantlydecreased
41、thenumberofTUNEL-positivecellsintheheartfollowingI/Rinjury.Quercetinpartlycanceledtheanti-apoptoticeffectofLPS(n=5ineachgroupandeightrandomlychosenfieldsfromeachtissuesectionwerecalculated).cCaspase-3activitybyWesternblotanalysiswithspecificanti-cleavedcaspase-3antibody(n=6ineachgroup).Corresponding
42、-actinblotsareshownasacontrolforsampleloading.dTheproteinlevelsforeachsampleweredeterminedasaratiototheircorresponding-actinlevels.LPSpretreatmentsignificantlyde-creasedthelevelofcleavedcaspase-3intheheartfollowingI/Rinjury.However,quercetinattenuatedtheanti-apoptoticeffectofLPSpretreatment.Dataarem
43、ean±SEM.*P<0.01comparedwithindicatedgroupsLPSpretreatmentreducesmyocardialinfarctsizefollowingischemia/reperfusioninjuryAsshowninFig.2,therewasnosignificantdifferenceintheriskareaamongthethreeexperimentalgroups(RA/LV),averaging36.3±1.0%(I/Rgroup),34.5±1.8%(LPS+I/Rgroup),and36.7
44、7;0.6%(Q-LPS+I/Rgroup;P>0.05).I/Rinjuryresultedinasignificantmyocardialinjurywhichwasindexedasinfarctarea/riskarea(IA/RA,48.5±1.8%).However,infarctareainLPS+I/RgroupwassignificantlyreducedcomparedwiththatinI/Rgroup(15.8±0.7vs.48.5±1.8%,P<0.01).ToinvestigatewhetherincreaseofHSP70
45、mayberesponsibleformyocardialprotectionfromI/Rinjury,weadministeredtheHSP70inhibitor,quercetin,beforeI/R.HeartsectionsfromQ-LPS+I/RgroupshowedalargernecroticinfarctareathanthatinLPS+I/Rgroup(IA/RA31.7±0.9vs.15.8±0.7%,P<0.01).LPSpretreatmentincreasesHSP70levelsinthemyocardiumbeforeandaft
46、erischemia/reperfusioninjuryBecauseHSPsarereportedtoprotectheartagainstischemia/reperfusioninjury(Williamsonetal.2008),inductionofHSP70mayberesponsibleforLPS-inducedmyocardialprotection.Toconfirmoursupposition,weinvestigatedthelevelsofHSP70inthreeexperimentalgroupsbeforeandafterI/Rinjury(Fig.3a).Ass
47、howninFig.3b,beforeI/Rinjury,immunoblottinganalysisofHSP70showedthatLPSLipopolysaccharidepretreatmentprotectsagainstI/RinjuryFig.2LPSpretreatmentreducesmyocardialinfarctsizefollowingischemia/reperfusioninjury.aTheheartsinthreeexperimentalgroupswereharvestedandinfarctsizewasdeterminedbytriphenyltetra
48、zo-liumchloride(TTC)staining(n=8ineachgroup).ViablemyocardiumstainsbluewithTTC.Ischemicmyocardium,whichisstillviable,stainsredwithTTC.Necroticordeadmyocardiumdoesnotstainandappearspalewhite.bBargraphofinfarctsizeandriskareainthethreegroups.Theinfarctarea(white)andtheareaatrisk(redandwhiteRatio)offro
49、minfarcteachareasectionvs.wereriskmeasuredarea(IA/RAusing)andanriskimagineareaanalyzer.vs.leftventriclearea(RA/LV)werecalculatedandarepresentedinthegraph.I/Rgroupheartsdevelopedlargeinfarctsthatrepresented48.5±1.8%ofriskzonesize.AscomparedwithI/Rgroup,LPS+I/RgroupandQ-LPS+I/Rgrouphadsignificant
50、lysmallerinfarcts,representing15.8±0.7%and31.7±0.9%ofriskzone,respectively.Theriskareawassimilaramongthethreegroups,averaging36.3±1.0%(I/Rgroup),34.5±1.8%(LPS+I/Rgroup),and36.7±0.6%(Q-LPS+I/Rgroup).Dataaremean±SEM.*P<0.01comparedwithindicatedgroupspretreatmentresulte
51、dinahighercontentofHSP70inLPS+I/RgroupthanthatinI/Rgroup(72.3±2.6%vs.12.3±1.9%,P<0.01).However,levelofHSP70wasinhibitedsignifi-cantlybyitsinhibitor,quercetin,inQ-LPS+I/Rgroup(24.6±2.9%).AfterI/Rinjury,levelsofHSP70were291increasedineveryexperimentalgroups.Interestingly,elevatedHSP7
52、0inLPS+I/RgroupwasstillmorethanthatinI/Rgroup(91.4±2.8%vs.25.3±3.1%,P<0.01).Theproteinlevelsforeachsampleweredeterminedasapercentagetotheircorresponding-actinlevels.ToinvestigatethepossibleeffectsofotherHSPmolecules,weperformedWesternblotanalysisofotherHSPs,includingHSP27,HSP60,andHSP90
53、.IncontrasttoHSP70,HSP27,HSP60,andHSP90,showedaminimalincreaseafterLPSpretreatmentinheart(Fig.3c).LPSpretreatmentreducesexpressionofinflammatoryfactorsfollowingischemia/reperfusioninjuryI/Rinjurycouldinducereleaseofinflammatoryfactors,suchasTNF-,IL-1,andIL-6,whichplayanimportantroleinthefibrosisofmy
54、ocardiumandheartfailure(Detenetal.2002).ReductionofinflammatoryfactorscouldprotectheartsfromI/Rinjury.SerumconcentrationsofTNF-,IL-1,andIL-6weremeasuredbeforeandafterI/Rinjuryinthreeexperimentalgroups.BeforeI/Rinjury,concentrationsofinflammatoryfactors(TNF-,IL-1,IL-6)fromLPS+I/Rgrouphadmoderatelyinc
55、reasedcomparedwiththatinI/Rgroup(TNF-2,556±30vs.698±12pg/ml,IL-11,077±15vs.458±8pg/ml,IL-6872±13vs.384±6pg/ml,P<0.01,respectively).However,afterischemiafor30minandreperfusionfor3h,serumfromLPS+I/Rgroupratshaddecreasedconcentrationsofinflammatoryfactorscomparedwithser
56、umfromI/Rgrouprats(TNF-3,643±46vs.11,785±146pg/ml,IL-11,643±124vs.7,663pg/ml,IL-61,038±13vs.4,720±53pg/ml,P<0.01,respectively).LPSpretreatmentcouldinducethereleaseofinflammatoryfactorsmoderatelybeforeI/RbutreducethereleaseofinflammatoryfactorssignificantlyinthefollowingI/
57、Rinjury.AsaninhibitorofHSP70,quercetinincurredhigherlevelofinflammatoryfactorsinQ-LPS+I/RgroupthanthatinLPS+I/Rgroup(TNF-7,267±46vs.3,643±46pg/ml,IL-14,274±67vs.1,643±124pg/ml,IL-63,380±35vs.1,038±13pg/ml,P<0.01,respectively).TheseresultsindicatedthatHSP70inducedbyLP
58、SpretreatmentwasresponsibleforthereductionofinflammatoryfactorsfollowingI/Rinjury(Fig.4).LPSpretreatmentattenuatesactivationofNF-BanddegradationofIBfollowingischemia/reperfusioninjuryNF-Bactivationinthenuclearextractsofmyocardiumwasdeterminedbyelectrophoreticmobilityshiftassay(EMSA).AsshowninFig.5a,
59、NF-Bbindingactivitywaspresentatverylowlevelsincontrolgroupbutsignificantlyincreasedafter292Y.-w.Yaoetal.Fig.3LevelsofHSP70inthreeexperimentalgroupsbeforeandafterischemia/reperfusioninjury.aRepresentativeimmunoblotsforHSP70proteininheartsbeforeandafterI/Rinjury.-actinwasalsoexaminedasaninternalcontro
60、l(n=6ineachgroup).bSummaryofdensitometricanalysesoftheimmunoblotsofHSP70proteininhearts.Theproteinlevelsforeachsampleweredeterminedasapercentagetotheircorresponding-actinlevels.BeforeI/Rinjury,LPSpretreat-mentcouldinducetheexpressionofHSP70inmyocardium,andquercetinadministrationdecreasedthelevelofHSP70inQ-LPS+I/Rgroup.AfterI/Rinjury,levelsofHSP70wereincreasedineveryexperimentalgroups.How
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