淺論外源性一氧化碳釋放分子抑制膿毒癥炎癥反應(yīng)的實(shí)驗(yàn)研究【摘要】目的：探討外源性一氧化碳釋放分子對膿毒癥炎癥反應(yīng)的抑制作用及可能的機(jī)制。方法：應(yīng)用盲腸結(jié)扎及穿孔膿毒癥小鼠模型，使用外源性一氧化碳釋放分子進(jìn)行干預(yù)。檢測肝、肺臟髓過氧化物酶(MPO)活性。應(yīng)用內(nèi)毒素(LPS，10g/ml)刺激的人臍靜脈內(nèi)皮細(xì)胞炎癥模型，使用外源性一氧化碳釋放分子進(jìn)行干預(yù)。檢測核因子κB活性,內(nèi)皮細(xì)胞黏附分子的表達(dá)，氧化產(chǎn)物、NO產(chǎn)物以及多形核白細(xì)胞對內(nèi)皮細(xì)胞的黏附作用。結(jié)果：盲腸結(jié)扎及穿孔膿毒癥小鼠模型使用外源性一氧化碳釋放分子干預(yù)后肝、肺組織MPO活性明顯下降。CORM2抑制了LPS刺激導(dǎo)致的NFκB活性上調(diào)。同時，NO產(chǎn)物下降，內(nèi)皮細(xì)胞ICAM1的表達(dá)抑制，白細(xì)胞對內(nèi)皮細(xì)胞的黏附作用明顯抑制。結(jié)論：外源性一氧化碳釋放分子通過抑制NFκB活性，抑制ICAM1蛋白和NO的表達(dá)，抑制白細(xì)胞對內(nèi)皮細(xì)胞的黏附作用，進(jìn)而有效抑制膿毒癥炎癥反應(yīng)。

【關(guān)鍵詞】一氧化碳；盲腸結(jié)扎及穿孔；炎癥反應(yīng)；核因子κB

CLP(cecalligationandpuncture)mayinducetheactivationofaninflammatorycascade,causedamagetomultipleorgansdistantfromtheoriginalburnwoundandmayleadtosepsisandmultipleorganfailure［1］.Therehavebeenseveralreportsindicatingthattheinflammatoryresponsesyndrome,whichcontributestooxidativecell/tissuedamage,mightfrequentlybeaccompaniedbyleukocytesequestrationinmanyimportantorgansystemsinthebody［2］.Theincreaseofproductionofproinflammatorymediatorssuchasinterleukin(IL)1βandtumornecrosisfactor(TNF)αiscloselyassociatedwithactivationofleukocytesandmacrophageswhichweresequestratedinthetissue［3,4］.

Leukocytessequestrationandtheirsubsequentinfiltrationinorgantissuecancauseleukocyteactivationandcontributetovasculardamageandthedevelopmentofsystemicinflammatorytheprerequisite,activationofleukocytesandendothelialcellsresultsinaggregationofleukocytes,plateletsanderythrocytesinmayfavordisseminatedintravasalcoagulationandfurthermultipleorganfailure.

Carbonmonoxide(CO)haslongbeenknowninbiologyandmedicineasatoxiccompound,duetoitsabilitytobindhemoglobinwithamuchhigheraffinitythanoxygen［5］.Evidenceaccumulatedtodatesuggeststhatendogenouscarbonmonoxide(CO),abiproductofinduciblehemeoxygenase(HO1)canmodulateinflammation,inhibitslipopolysaccharide(LPS)inducedproductionofcytokinesbothinvivoandinvitro,andconsequentlyexhibitsimportantcytoprotectivefunctionandantiinflammatorypropertiesthatarebeneficialfortheresolutionofacuteinflammation［6-8］.InhaledCOatconcentrationsof250～500partspermillion(ppm)hasalsobeenshowntobebeneficialinanumberoflunginjurymodels,includinghyperoxicinjury［9,10］allergeninducedinflammation［11］.

Recently,transitionalmetalcarbonylshavebeenidentifiedaspotentialCOreleasingmolecules(CORMs)withthepotentialtofacilitatethepharmaceuticaluseofCObydeliveringittotissuesandorgans［12］.CORMshavebeenshowntoactpharmacologicallyinrataorticandcardiactissuewhereliberationofCOproducedvasorelaxanteffects［13-16］anddecreasedmyocardialischemiareperfusiondamage［17,18］intheabsenceofdramaticchangesinbloodcarboxyhemoglobin(COHb)levels.

Onthebasisofthesedata,thepresentstudywas,therefore,designedasaprospectivelaboratoryexperimenttoinvestigatetheeffectsoftricarbonyldichlororuthenium(Ⅱ)dimer(CORM2),oneofthenovelgroupofCORMs,onattenuationofleukocytesequestrationanddecreaseofinflammatoryresponsesandoxidativestressintheorgansofCLPinducedmiceandLPSinducedHUVEC(humanumbilicalveinendothelialcell),anddiscussedthepossiblemolecularmechanisms.

1Materialandmethods

Materials

Medium199(M199),fetalcalfserum(FCS),penicillin,andstreptomycinwerepurchasedfromGIBCOBRL(GlandIsland,NY).Tricarbonyldichlororuthenium(II)dimer(CORM2)wasobtainedfromSigmaAldrichandsolubilizedindimethylsulfoxide(DMSO)toobtaina10mmol/L(Escherichiacoliserotype055:B5)waspurchasedfromICAM1polyclonalantibodywaspurchasedfromTransductionLaboratories(Lexington,KY).AntimouseIgGconjugatedtohorseradishperoxidasewaspurchasedfromKirkegaardandPerryLaboratories(Gaithersburg,MD).

Animals

TheC57BL/6mice［male,N=21;bw(20±2)g］werefedastandardlaboratorydietandwateradwereassignedtothreegroupsinthreerespectiveeachexperiment,miceinshamgroup(n=7)wereunderwentshamprocedure,whereasmiceinCLPgroup(n=7)receivedcecalligationandpunctureandmiceinCORM2group(n=7)underwentthesameinjurywithimmediateadministrationofCORM2(8mg/kg,).TheconcentrationofCORM2usedinthepresentstudywasbasedonapreviousreportinoftheuseofthiscompoundinmice［19］andthepreliminaryexperimentsinourlabbymeasuringdynamicCOHblevelsandpeaklevelswhichdidnotaveraged15%±5%abovenormalexperimentalprotocolwasapprovedbyTheCouncilonAnimalCareatJiangsuUniversityontheprotectionandthewelfareofanimalsandmetNationalInstitutesofHealthguidelinesforthecareanduseofexperimentalanimals.

CLPMicewereanesthetizedwith2%isofluraneinoxygenviaa1to2cmmidlineincisionwasmadethroughtheabdominalwall;thececumwasidentifiedandligatedwitha3-0silktie1cmfromthewastakennottocausebowelsinglepunctureofthececalwallwasperformedwitha20gaugececumwaslightlysqueezedtoexpressasmallamountofstoolfromthepuncturesitetoassureafullthicknesscarewastakentopreservecontinuityofflowbetweenthesmallandlargeofmiceatvariousintervalsafterCLPdidnotrevealevidenceofbowelcecumwasreturnedtotheabdominalcavity,andtheincisionwasclosedwithmiceunderwentanesthesiaandmidlinelaparotomy;thececumwasexteriorizedandreturnedtotheabdomen,andthewoundwasclosedwithreceivedinjectionofCORM2(mg/kg,)immediatelyaftermicereceived160μl%DMSOnormalsalineinthesameofMPOintheliverwasperformedat24hafterCLP.

MPOactivityMPOactivityasanassessmentofneutrophilinfluxwasmeasuredaccordingtoestablishedprotocols［20］.Inbrief,tissuewashomogenizedinmlof50mmol/Lpotassiumphosphatebuffer(pH)andcentrifugedat10,000r/minat4℃for30remainingpelletwasresuspendedinmlof50mmol/LpotassiumbufferpHwith%hexadecyltrimethylammoniumbromide,sonicatedonice,andthencentrifugedat12,000r/minat4℃for10werethenassayedata1∶20dilutioninreactionbuffercontaining50mmol/LPB,530mmol/Lodianisidine,and20mmol/LH2O2unitofenzymeactivitywasdefinedastheamountofMPOpresentthatcausedachangeinabsorbancemeasuredat460nmfor3activitywasexpressedasU/gtissue.

Cells

Isolationandcultureofhumanumbilicalveinendothelialcells(HUVEC)Humanumbilicalveinendothelialcells(HUVEC)wereharvestedfromthefreshhumanumbilicalveinofnewbornsbycollagenasetreatment(WorthingtonBiochem,Freehold,NJ)aspreviouslydescribed［21］.Thecellsweregrowninmedium199(M199;GIBCO,Burlington,Canada)supplementedwith10%heatinactivatedFCS(Intergen,Purchase,NY),mg/Lthymidine(SigmaChemical,Oakville,Canada),10IU/mlheparinsodium,antibiotics(100U/mlpenicillinand100μg/mlstreptomycin;GIBCO),μg/mlfungizone(GIBCO),and80μg/mlendothelialmitogen(Biomedicalchnologies,Stoughten,MA).Thecellcultureswereincubatedinroomairwith5%CO2at37℃and95%humidityandwereexpandedbybrieftrypsinizationwith%trypsininPBScontaining%experimentswereconductedonpassage318h,themediumwaschangedto500μloffreshcompletewerestimulatedwithLPS(10g/ml).Aftertreatmentfor4h,thecellsandmediumwereharvestedseparately.

PMNadhesionassaysHumanneutrophilicPMNwereisolatedfromthevenousbloodofhealthyadultsusingstandarddextransedimentationandgradientseparationonHistopaqueprocedureyieldsaPMNpopulationthatis95%～98%viable(trypanblueexclusion)and98%pure(aceticacidcrystalvioletstaining).

Forthestaticadhesionassay,isolatedneutrophilsweresuspendedinPBSbufferandradiolabeledbyincubatingthecellsat5×107cells/mlwith50μCiNa51CrO4/mlPMNsuspensionat37℃for60cellswerethenwashedwithcoldPBStoremoveunincorporatedPMN(5×105/well)wereaddedtoHUVECmonolayersgrownin48wellplates(Costar),and30minlaterthepercentageofaddedPMNthatremainedadherentafterawashprocedurewasquantitatedasfollows:%PMNadherence=lysate(cpm)/［supernatant(cpm)+wash(cpm)+lysate(cpm)］,wherecpmiscountsperminute.

OxidantproductionOxidantproductionwithinHUVECwasassessedbymeasuringtheoxidationofintracellulardihydrorhodamine123(DHR123;MolecularProbes,Inc.),anoxidantsensitivefluorochrome,asdescribedpreviously［22］.Briefly,thecellsweretreatedwithDHR123(5mmol/L)for1hbeforebeingsubjectedtoLPSLPSstimulationthecellswerewashedwithPBS,lysed,andDHR123oxidationwasassessedspectrofluorometricallyatexcitationandemissionwavelengthsof502and523nm,respectively.

NitricoxideproductionNOproductionbyHUVECwasassessedbymeasuringthefluorescenceof4amino5methylamino2′,7′difluorofluoresceindiacetate(DAFFMdiacetate),aspecificNOprobe(MolecularProbes,Inc.)［23］.Briefly,DAFFMdiacetate(10mmol/L)inM199wasaddedtotheHUVEC1hbeforetheLPSLPSstimulation,theHUVECandsupernatantswerecollectedandanalyzedspectrofluorometricallyatexcitationandemissionwavelengthsof495nmand515nm,respectively.

SDSpolyacrylamidegelelectrophoresisandWesternblottingSDSpolyacrylamidegelelectrophoresisandWesternblottingwereperformedasdescribedpreviously［24］.Samples(10μgofprotein)weresubjectedtoelectrophoresison7%(forICAM1)SDSpolyacrylamidegels,withtheuseofthediscontinuoussystemandtransferredontonitrocellulosemembraneswereprobedwithantiICAM1monoclonalantibody(1∶2500).AntimouseIgGconjugatedtohorseradishperoxidase(1∶2500)wasusedasasecondarybandswerevisualizedbytheuseofECLreagentandHyperfilmECL(Amersham,ArlingtonHeights,IL)asdescribedbythewerescannedusingaflatbedscannerandthebandswerequantifiedusingBasicQuantifiersoftware(BioImage,AnnArbor,MI),animageanalysisprogram,oncomputer.

HUVECnuclearproteinextractionandElectrophoreticmobilityshiftassay(EMSA)NuclearproteinwasextractedfromHUVECaspreviouslydescribed［25］.CellsweregrowntoconfluenceinPetridish,scraped,washedwithcoldPBS,andincubatedin150μlofbufferE(+)(%NonidetP40,10mmol/LTris(pH),60mmol/LNaCl,1mmol/LEDTA,mmol/Ldithiothreitol(DTT),1μg/mlaprotinin,1μg/mlleupeptin,and1mmol/Lphenylmethylsulfonylfluoride)for5minonwerecentrifugedat4℃for5minat500r/supernatantwasthenremoved,andthepellets(nuclei)wereresuspendedin150μlofbufferE(10mmol/LTris(pH),60mmol/LNaCl,1mmol/LEDTA,andmmol/LDTT)andcentrifugedat500r/minfor5minat4℃.Thenucleiwerethenextractedin30～50μlofbufferE(c)(20mmol/LHEPES,mmol/Lspermidine,mmol/Lspermine,mmol/LEDTA,2mmol/LEGTA,2mmol/LDTT,20%glycerol,and1mmol/LPMSF(4℃)inthepresenceofmol/LNaCl)andwereincubatedonicefor20,thesampleswerecentrifugedfor10minat500r/min(4℃),andthesupernatantswerecollectedandsavedasthenuclearproteinwerestoredat-80℃.

Thedoublestrandedoligonucleotidecontainingconsensus(58AGGGACTTCCGCTGGGGACTTTCC38)bindingsitesforNFκB(synthesizedonsite;BeckmanOligo1000mol/LDNAsynthesizer)wereendlabeledwith［γ-32P］ATP(Amersham)byusingT4polynucleotidekinase(MBIFermentas,Flamborough,ON),asdescribedpreviously［25］.Onepicomoleofthelabeledoligonucleotidewasincubatedwith5μgofnuclearextractproteininthepresenceorabsenceof50×excessofcoldwereincubatedfor30minatroomtemperatureandthenrunthrougha4%nondenaturingpolyacrylamidegelat280Vfor45～60gelwasdriedandthenexposedtoXrayfilm(Kodak)incassettesfor2～4hat-80℃withintensifyingscreens.

CellELISAForassessmentofICAM1surfaceexpressionlevel,anELISAwasperformed［26］onHUVECgrownin96wellcellcultureplates(Corning).HUVECwerefixedin4%paraformaldehydeat4℃for30cellswerethenwashedtwotimeswithcoldPBSandwereincubatedwiththemouseprimarymonoclonalantibody(MAb)againsthumanICAM1(Dako)ataconcentrationof10μg/mlfor1hatroomthistreatment,immunocytochemicalstainingofHUVECmonolayerswasperformedusinganavidinbiotinconjugatedperoxidasemouseIgGstainingkit(Vectastain),andMAbbindingwassubsequentlyquantifiedwithamicroplatereader(model3550UV;BioRad)at450nmwavelength.

StatisticalAnalysis

Allofthevaluesarepresentedasmeans±analysiswasperformedwiththeuseofANOVAandStudent′sttestforthevalueofwasconsideredtobestatisticallysignificant.

2Results

EffectofCORM2onMPOactivityinlungandliverofCLPchallengedmice

TodeterminewhetherCLPinducedincreaseinPMNaccumulationinthelungandliverwaseffectivelypreventedbyCORM2,theactivityofMPO,anenzymeinazurophilicgranulesofneutrophils,wasoftheorganssampleswereexaminedforcontentofMPOat24hafterCLPactivityinorgansobtainedfromCLPinducedmicewasmarkedlyincreasedcomparedtosham(P),whileitsignificantlydecreasedbytreatmentwithCORM2().

Fig1EffectsofCORM2onMPOactivityinthe

lungandliverofCLPchallengedmice

Micewereinjected()withCORM2(8mg/kg)immediatelyafterCLPmicereceived160μlbolusinjectionof%DMSO/activitiesinthelungandliverwereassessed24hfollowingaremean±SEofthreeexperiments(threemicepergroup),*comparedtoshammice.﹟PcomparedtoCLPmice.

EffectofCORM2onexpressionofICAM1inthelungandliverofCLPchallengedmice

At24hafterCLPinduction,theexpressionofICAM1inlungandlivertissuessignificantlyincreasedcomparedtotheshamvivoadministrationofCORM2(8mg/kg,),expressionofICAM1wassignificantlydecreased()

Fig2EffectsofCORM2onproteinexpressionofICAM1

intheliverandlungofCLPinducedmice

MicewerechallengedwithCLPandtreatedwithCORM2asdescribedinexpressionofICAM1wasperformedbywesternblottingat24hafteraremean±SE.*comparedtoshammice.﹟PcomparedtoCLPmice.

EffectofCORM2onactivitiesofNFκBinlungandlivertissueofCLPchallengedmice

BindingactivitiesofnuclearproteintotheradiolabeledconsensusbindingsequencesofNFκBwasassessedby24hafterCLPinduction,theNFκBactivationinlungandliverwasmarkedlyincreasedandthisactivitywasinhibitedbyinvivoadministrationofCORM2(8mg/kg,)().

Fig3EffectsofCORM2onNFκBactivationinthelung

andliverofCLPinducedmice(EMSA)

MicewerechallengedwithCLPandtreatedwithCORM2asdescribedinofNFκBactivitywasperformedbymobilityshiftassay(EMSA)with32PlabeledNFκBprobeand5μgofnuclearextractfromliverofsham,CLPandCLP+CORM2miceat24hafterCLP.

EffectofCORM2onintracellularproductionofROSandNOinHUVECstimulatedbyLPS

Asshownin,theLPSstimulationresultedinasignificantincreaseinHUVECoxidantHUVECwithdifferentconcentrationofCORM2inducedlessDHRoxidationcomparedtotheLPSwithaconcentrationdependentshownin,HUVECproducedsignificantlymoreNOduringtheLPSstimulationascomparedtosignificantlydecreasedtheirNOproductionaftertreatmentwithCORM2asaconcentrationdependentmanner.

Fig4EffectsofCORM2onintracellularproductionof

ROSandNOinLPSstimulatedHUVEC

HUVECweregrowntoconfluencein48wellcellcultureplatesandloadedwithdihydrorhodamine123(DHR123)ordiaminofluoresceinFM(DAFFM)for1,HUVECwerestimulatedwithLPS(10μg/ml)for4hinthepresenceorabsenceofCORM2(10,50,100μmol/L).Oxidativestress(DHR123oxidation)(a)andNOproduction(DAFFMnitration)(b)werevaluesareexpressedasmean±SE(n=4).*ascomparedtocontrol,#ascomparedtothatLPSinducedoxidativestressandNOproductionwereinhibitedbyCORM2inadosedependentmanner.

EffectofCORM2onICAM1expressioninLPSstimulatedHUVEC(cellELISAandWesternblotting)

At24hafterLPSstimulation,theexpressionofICAM1inHUVECsignificantlyincreasedcomparedtothethepresentofCORM2(10,50and100μmol/L),expressionofICAM1(,ELISAand5b,Westernblotting)wassignificantlydecreased.

Allvaluesareexpressedasmean±SE(n=4).*ascomparedtocontrol,#ascomparedtothatLPSinducedexpressionofICAM1wasdownregulatedbyCORM2inadosedependentmanner.

Fig5EffectsofCORM2onICAM1expressioninLPS

stimulatedHUVEC(cellELISAandWesternblotting)

EffectofCORM2onactivitiesofNFκBinLPSchallengedHUVEC

BindingactivitiesofnuclearproteintotheradiolabeledconsensusbindingsequencesofNFκBwasassessedby4hafterLPSstimulation,theNFκBactivationinHUVECwasmarkedlyincreasedandthisactivitywasinhibitedbyadministrationofCORM2withaconcentrationdependentmanner().

Fig6EffectsofCORM2onNFκBactivationinLPS

stimulatedHUVEC(EMSA)

At4hafterLPSstimulation,theNFκBactivationinHUVECwasmarkedlyincreasedandthisactivitywasinhibitedbyadministrationofCORM2withaconcentrationdependentmanner.

EffectofCORM2onPMNadhesiontoHUVECstimulatedbyLPS

Asshownin,adhesionofPMNtoHUVECislowinmonolayerofendothelialcellswerestimulatedbyLPSfor4h,adhesionofPMNtoHUVECsignificantlyincreased(comparedtocontrol).However,coincubationwithLPSandCORM2(10,50and100μmol/L)markedlydecreasedleukocyteendothelialcellsadhesion(comparedtoLPSgroup).

ConfluentmonolayersofHUVECwereincubatedwithLPS(10μg/ml)inthepresenceorabsenceofCORM2(10,50and100μmol/L)for4hfollowedbyPMNadditionintheadherencewasvaluesareexpressedasmeans±SE(n=3).*Pcomparedtocontrol;﹟PcomparedtoLPS.

Fig7EffectofCORM2onPMNadhesionto

LPSstimulatedHUVEC

3Discussion

Sepsisisacommonandseriousmedicalconditioncausedbyaseveresystemicinfectionleadingtoasystemicinflammatoryresponse,whichfrequentlyoccursafterhemorrhage,trauma,burn,orabdominalisaleadingcauseofmorbidityandmortalityinseverelyillpatients［27］.AlthoughsomeinformationhasbeengeneratedfromtheLPSinjectionstudies,LPSinjectionisanadequatemodelofendotoxemiaandcannotpreciselymimicthechangesobservedduringtheotherhand,cecalligationandpuncture(CLP)modelseemstoresemblequalitativelyaswellasquantitativelytheclinicalobservationsofvascularreactivityandinflammationduringpolymicrobialperitonitis,bacteremia,andsystemicsepsis［28］.Therefore,theaimofthisstudyistoevaluatethepossibleroleofCORMderivedCOinCLPinducedsepsis.

ManyexperimentalstudieshavehighlightedthespecificandindependentroleofexogenousCOinthemodulationofinflammation［29,30］.Asanewmetalcarbonylbasedcompounds,COreleasingmoleculars(CORMs)havetheabilitytoreleaseCOinbiologicalvasoactive,antihypertensiveandantirejectioneffectsofCORMshavebeendemonstratedtobeduetotheCOliberatedbythe2,aDMSOsolubleCORM,alsohasexhibitedantiinflammatoryactionsinaninvitromodelofLPSstimulatedmacrophages［31］.Therefore,theaimofthisstudywastoinvestigatetheeffectsofCORM2onthedynamicsofleukocytessequestrationinthelungandliver,andonthedecreaseofROSandNOproductionintheLPSstimulatedHUVEC.

Leukocytessequestrationandtheirsubsequentinfiltrationinlungandlivertissuescancauseleukocyteactivationandcontributetovasculardamageandthedevelopmentofsystemicinflammatory(MPO)isanenzymethatisfoundpredominantlyintheazurophilicgranulesofpolymorphonuclearleukocytes(PMN).TissueMPOactivityisfrequentlyutilizedtoestimatetissuePMNaccumulationininflamedtissuesandcorrelatessignificantlywiththenumberofPMNdeterminedhistochemicallyintissues［32］.Inthepresentstudy,wefoundthattissueMPOactivitywasmarkedlyelevatedafterCLPandinvivoadministrationofCORM2ledtothesignificantlydownregulationofMPOindicatedthatCORM2effectivelypreventsPMNchemotaxisandinfiltrationinthetissueafterCLP,consequentlydecreasedtheproductionofoxidants,reducedtissueoxidativeinjury.

ThedirectcauseofleukocytessequestrationafterCLPisconsideredtobethemoreexpressionofadhesionmolecule(ICAM1).ICAM1activatesleukocytesandendothelialcells(ECs),whichinturnpromptthereleaseofvariousinflammatorymediators,resultinginsystemicinflammatoryresponsesyndrome(SIRS),acuterespiratorydistresssyndrome(ARDS)andmultiorgandysfunctionsyndrome(MODS)［33-35］.Thepresentresultsshowedthatat24hpostCLP,theexpressionlevelsofICAM1inlungandlivertissueweremarkedlyvivoadministrationofCORM2wasabletoinhibittheupregulationofICAM1inducedbyaddition,CORM2alsoinhibitstheincreaseofICAM1expressioninLPSstimulatedparallel,theresultsofinvitroexperimentsshowedthatLPSstimulationcausedsignificantincreaseofPMNsadhesiontoHUVEC,CORM2treatmenteffectivelypreventedthisincrease.

ThereisnodoubtthatthenuclearfactorκB(NFκB)isaubiquitous,rapidlyactingtranscriptionfactorinvolvedinimmuneandinflammatoryreactions,itexertsitsimmuneandinflammatoryresponsebyregulatingexpressionofcytokines,chemokines,celladhesionmolecules,andgrowthfactors［36,37］.Inthisstudy,NFκBactivitiesinlungandlivertissueandinLPSstimulatedHUVECwereelevatedbyCLPwhileitwasmarkedlyinhibitedbyadministrationofCORMdatashowedthatCORM2playsapivotalroleininhibitionofNFκBactivity,subsequentlydecreasedtheexpressionofcellularadhesionmolecules(ICAM1)andCLPinducedproinflammatory,aneffectivetherapeuticstrategythatinhibitthistranscriptionfactorwouldbeexpectedtoimproveorganfunctionsafterCLP.

Insummary,thepresentstudiesservetoclarifytheroleofCORM2,oneofthenovelCOreleasingmolecules,onthemechanismsofattenuationofleukocyteofCORM2onCLPmiceattenuatedPMNsaccumulation,preventedactivationofNFκB，andsubsequentlydecreasedtheproductionofinflammatorymediatorsinthelungandwasaccompaniedbyadecreaseoftheexpressionofICAMparallel,expressionofICAM1,PMNadhesiontoHUVECstimulatedbyLPSweremarkedlydecreasedafterCORM2togetherthesefindingsindicatethatCORMreleasedCOattenuatesleukocytessequestrationandinflammatoryresponsesbyinterferingwithNFκBactivation,proteinexpressionofICAM1andthereforesuppressingendothelialcellsproadhesivephenotype.

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