生长激素加重内毒素腹腔感染大鼠肺毛细血管损伤：循环中性粒细胞核因子—κB活性增加

生长激素加重内毒素腹腔感染大鼠肺毛细血管损伤：循环中性粒细胞核因子—κB活性增加 生长激素加重内毒素腹腔感染大鼠肺毛细 血管损伤:循环中性粒细胞核因子—κB活 性增加 @2002.ActaPharmacologicaSinica ISSN167卜4083 ShanghaiInstituteofMateriaMedica ChineseAcademyofSciences LiuZHPf?f/ActaPharmacolSin2002Oct;23(1O):887—892887 _'?11?1?1??t3rowthhormonenacreaseslunKmicrovascularnalurV ''''''' peritoni.rats:possiblei】 [,_voM.,mentinllpOpOIVsaccharlcteperltonltlSrats:ossiDleolvement?W ofNF一1activationincirculatingneutrophils LIUZhi—Hal,YUYing—Qun,LIWei—Qin,LIJie—Shou ResearchInstituteofGeneralSurgery,NanjingUniversitySchoolofMedicine;2Department ofAnesthesiology;3Departmentof GeneralSurgery,JinlingHospital,NanjingUniversitySchoolofMedicine,Nanjing210002, China KEYWORDSsomatotropin;neutrophilinfiltration;NF— KB:Iqd3;lipopolysaccharides;lung;capillarypermeability A玎:ToinvestigatetheeffectsofgrowthhormonefGHonNF— KBactivityinneutrophilsandneutrophils—mediated organinjuryinducedbylipopolysaccharide(LPS)inrats.METHODS:MaleWistarratschall engedwithorwithout LPS(5mg~g)weretreatedwithvarieddosesofGH(0.5,1.0,and2.0mg/kg)for2or4h.NF— KBactivitiesin circulatingneutrophilsweremeasuredwithelectrophoreticmobilityshiftassays(EMSA).andI—ld3levelsincircu— latingneutrophilsweredetectedbyWesternblot.Lungneutrophilssequestrationandlungmicrovascularperme— abilityweremeasuredat4hafterLPSchallenge.RESUITS:CirculatingneutrophilsinLPSchallengedratshad increasedNF—ld3activityanddecreasedI— ld3levelascomparedwithcontrols.GHdramaticallyincreasedNF.1d3 activityandI— ld3degradationinducedbVLPSchallengeinneutrophils.Also,subsequently,GHtreatmentincreased lungneutrophilssequestrationandlungmicrovascularinjuryinducedbyLPS.CONCLUSION:Theseresults suggestthattreatmentofGHisharmful,insteadofbeneficial,toLPS— inducedorganinjury.Increasedneutrophils' NF— KBactivityandlungneutrophilssequestrationarecriticalvivomechanismsmediafingGHactiononLPS— inducedorganinjury. Althoughmanytranscriptionalregulatoryproteins havebeenpurifiedanddescribed,nuclearfactorkappa B(NF—KB1hasparticularimportanceinmodulatingex— pressionofimmunoregulatorygenesrelevanttocritical illness.Inparticular.NF—KBplaysacentralrolein regulatingthetranscriptionofcytokines,adhesion SupportedbytheKeyProjectofTenth-FivePlanFoundation OfPLA.No01Z011. CorrespondencetoProfLIJie—Shou.Phn86-25.482.4804. Fax86-25?480-3956.E-mailLijiesou@public1.ptt.js.ca Received2001--11?26Accepted2002-06?18 molecules,andothermediatorsinvolvedintheacute respiratorydistresssyndrome,sepsis,ormultipleor— gansystemfailuret~】.ExcessiveactivationofNF—KB resultsinanoverlyexuberantinflammatoryresponse thatthenleadstoacuteinflammatoryinjurytothe1ungs andotherorgans,andthedevelopmentofmultipleor— gandysfunction,acommonproblemincriticallyill patients【,引.NF—KBisincreasedinneutrophilsin endotoxemia.andelevationofNF—ld3predictspoorsur— vivalinsepticpatientsandinthemousemodelof endotoxemia【引.I-KBisacytosolicproteinwhichfunc. tionstoinhibitactivationofthetranscriptionfactorNF. KB.Stimuliknowntoinducecytokineproduction usuallyleadtocleavageofI-1~BfromNF.1d3withsub. 888LiuZHetal/ActaPharmacolSin2002Oct;23(1O):887?892 sequentdegradationofI-riBandnucleartranslocation ofNF—nB[. Growthhormone(GH)hasbeenshowntoenhance immunefunctionbyprimingphagocytesforthepro— ductionofsuperoxideanionsandcytokines【.'.Also GHhasbeenidentifiedasafactorinvolvedintheregu— lationofneutrophilsfunctionbypriming,ortheresponse ofneutrophilstoanactivatingstimulusispoten— tiatedt8,9]. TheGHreceptorbelongstothecytokinere— ceptorfamily.whichiscoupledwiththeNF一1cBsignal— ingpathway.ItseemspossiblethatGHcouldregulate theexpressionofcytokines,chemokines,andadhesion moleculesthroughNF—n13signalingpathway. Growthhormonestimulatesproteinsynthesisand attenuatesthenitrogenlossafterinjuryandhasbeen administratedtoimprovenitrogenbalanceincriticalill— nessforabouttwodecades.However.theoutcomeis ambiguoust0?".Recentdatasuggestthatadministra— tionofrecombinanthumangrowthhormone(rhGH1 maynotbebeneficialincriticalillpatients.Ina prospective,placebo—controlled,multicenterresearch studiedbvTakalaetalt0,highdosesofgrowthhot— monewereassociatedwithincreasedmorbidityand mortalityinpatientswithprolongedcriticalillness.In— creasedincidenceofsepsis,septicshock,uncontrolled infection.andmultipleorganfailureintherhGHtreated groupsuggeststheimmunesystemmaybeaffected. Thisfindingpromotesaninvestigationatourlabora— torytodeterminewhetherGHadministrationincreases NF—KBactivityandlungneutrophilssequestration.and subsequentlyenhancesmicrovascularinjuryduring sepsis.Thepurposeofthisstudyistodeterminethe effectsofGHonNF一1cBactivityandI一1cBlevelin neutrophils,lungneutrophilssequestration,andlung microvascularinjuryinlipopolysaccharide(LPS)peri— tonitisrats. AnimalprotocolsMaleWistarrats(GradeII, Certificate?97001.ExperimentalAnimalCenter. JinlingHospita1)weighingbetween250gand300g werefedstandardratchowwithfreeaccesstotap waterandkeptintemperatureandhumiditycontrolled animalquartersundera12一hlightanddarkcycle.The ratsweredividedintosixgroups(n=7pergroup)in randomizedmanner:control,LPS(5mg&g,ip;Escheri— chiacolf055:B5;SigmaChemicalCo,StLouis,MO), LPSplusGH(0.5,1.0,or2.0mg/l【g,sc,atthetimeof LPSchallenge;fromLaboratoriesSeronoSASwitzer— land),andGHalone(2.0mg/kg,sc,foranequivalent periodoftime).Allprocedureswereapprovedbythe InstitutionalAnimalCareCommittee. AnimalsweresacriricedbyexsanguinatiOns,and bloodsampleswerecollectedat2hpost..LPScha1.. 1engeforelectrophoreticmobilityshiftassays(EMSA) andWesternblotanalysis.Lungsampleswerecol— lected4hpost—LPSchallenge,frozeninliquidnitrogen, andstoredat一80?untilanalysistodetermine myeloperoxidaseactivity.Inanothersetofexperiments animalsunderwentthesametreatments.andlungmi— crovascularendothelialpermeabilitywasdeterminedat 4hafterLPSchallenge. Isolationofratbloodneutrophils乃oleblood fromratswasdrawnintosyringescontainingheparin astheanticoagulant.Neutrophilswereisolatedusing Ficoll—PaquegradientcentrifugatiOnanddextran sedimentation.Afterhypotonic1ysisofresidualred bloodcells,neutrophilswerereadyforproteinextrac— tionfollowedbvEMSAandWestemblotanalysis. NuclearproteinextractandEMSANuclearex— tractsoftheneutrophilswerepreparedbyhypotonic lysisfollowedbyhighsaltextraction.Inbrief,thesepa— ratedcellswerelysedin0.5mLbufferAcomposedof HEPES10mmol/L,pH7.9,KCl10mmol/L,MgC1,2 mmol/L,dithiothreitol(DTT)1.0mmol/L,edeticacid 0.1mmol/L,andphenylmethylsulfonylfluoride(PMSF) 0.5mmol/Lonicefor15min.then50LLLOf10% NonidetP一40solutionwasadded,andthemixturewas vortexedvigorouslyfor15sandcentrifugedfor30sat 12000xg.Thecrudenuclearpelletwasresuspendedin 50uLofbufferBcontainingHEPES50mmo1/L,PH 7.9,MgC1,1.5mmo1/L,NaCl300mmol/L,DTT0.5 mmo1/L,edeticacid0.1mmol/L,PMSF0.5mmol/L, 1O%glycerol,andleupeptidin4~aol/L(SigmaChemi— calCo,StLouis,MO)andincubatedonicefor20min withintermittentmixing.Thesuspensionwascentri— fugedat12000xgat4?for5min.Thesupematant containingnuclearproteinswascollectedandkeptat 一 70?foruse.Proteinconcentrationwasdetermined usingbicinchOninicacidassaykitwithbovineserum albuminasstandard(Pierce,Rockford,IL). Electrophoreticmobilityshiftassaywasperformed usingcommercialkit(GelShiftAssaySystem;Promega, Madison,WI).NF—nBconsensusoligonucleotideprobe f5'.AGTrGAGGGGACTITCCCAGGC.31wasend—la— beledwith32p】ATP(YahuiBiotech,Beijing,China) withT4polynucleotidekinase.Nuclearprotein(10g) LiuZHetal/ActaPharmacolSin2002Oct;23f10):887—892 waspreincubatedin9gLofabindingbufferconsisted ofTris—C110mmol/L,pH7.5,MgC121mmol/L,NaC1 50mmol/L.DTT0.5iIlmo1/L.edeticacid0.5inino儿, 4%glycerol,and2ggofpoly(deoxyinosinic?deoxy— cytidylicacid)for10minatroomtemperature.After additionoftheP—labeledOligOnucleOtideprobe,the incubationwascontinuedfor20minatroom temperature.ThespecificityoftheDNA/proteinbind— ingwasdeterminedbycompetitionreactionsinwhich 50一f0ldmolarexcessofunlabeledNF—rJ3oligonucle— otidewasaddedtothebindingreaction10minbefore theadditionofradiolabeledprobe.Apositivecontrol wasrunusingHelanuclearextract.Reactionwas stoppedbyadding1Lofgelloadingbufferandsub— jectedtonondenaturing4%polyacrylamidegelelectro— phoresisin0.25xTBEbufferfTris—borate—edeticacid). Gelwasvacuum—driedandexposedtoX.rayfilm(Fuji hyperfilm,at一70?withanintensifyingscreen. Proteinextractionand,vesternblotanalysis Theseparatedneutrophilsweresuspendedin0.5mL ofice.coldproteinextractingbuffercontainingTris—HCl 25mmol/L,edeticacid0.5mmol/L,egtazicacid0.5 mmol/L,PMSF0.1g/L,leupeptin10mg/L,and pepstatin1~rnol/L(SigmaChemicalCo,StLouis,MO). Thehomogenatewascentrifugedat16000xgat4? for15min,andtheresultingsupematantwascollected ascytosolicfraction.Proteinconcentrationwasdeter- minedusingbicinchOninicacidassaykitwithbovine semmalbuminasstandard. Equalamountsofproteins(30tag/lane)wereloaded andseparatedon12.5%SDS—polyacrylamideslabgel underdenaturingconditions.Lowmolecularprotein molecularweightmarker(PharmaciaBiotech,Piscata— way,NJ)wasusedasstandard.Proteinswereelectro— blottedtonitrocellulosemembrane(Bio—Rad,Hercules, CA).Afterincubationinblockingsolution『5%dry milkinTBST(sbufferedsalinewithTWeen20)1at roomtemperaturefor1h.themembranewas immunoblottedtotherabbitpolyclonalanti—I—rd3anti— body(SantaCruzBiotechnology,SantaCruz,CA)over- nightat4?.Thesecondaryantibodieswerehorse— radishperoxidase—conjugatedgoatanti—rabbitantibodies. Peroxidaselabelingwasdetectedwithenhancedchemi— luminescenceWestemblottingdetectionsystemfPierce. Rockford,IL)accordingtothemanufacturer'srecom— mendations. Assessmentoflungneutrophilsaccumulation Lungmyeloperoxidase(MPO)activitywasdetermined asanindexoftissueneutrophilsaccumulation.Tomea一 889 suretissueMPOactivity,frozenlungswerethawedand extractedforMPO,followingthehomogenizationand sonicationprocedureasdescribedpreviouslyt13J.MPO activityinsupematantwasmeasuredandcalculatedfrom theabsorbance(at460nm)changesresultingfromde— compositionofH,O,inthepresenceofo.dianisidine. Measurementofpulmonaryvascularinjury Pulmonaryvascularinjurywasassessedbyquantitat— ingextravasationofEvan'sbluedyeintolungparen— chyma.Briefly,Evans'sbluedye(SigmaChemicalCo, StLouis,MO)wasgivenintravenously30minpriorto harvest.Fiveminuteslater1mLofbloodwasobtained andcentrifugedat400xgfor15minandtheplasma wassaved.Twenty—fiveminutesfollowingtheEvan's blueadministrationtheratswereeuthanizedanda bronchoalveolarlavage(BAL)wasperformedwith5 mLofnormalsalinerepeatedthreetimes.TheBAL fluidwasthencomparedtoserialdilutionsoftheplasma collected5minfollowingdyeadministrationfBAL/ Plasma). StatisticalanalysisA11dataweregivenas mean_+SD.Statisticalsignificancewasdeterminedby onewayanalysisofvariancefollowedbyNewman— Keulstest.P<0.05wasconsideredassignificant. RESI『I GHincreasedLPS-inducedNF-1activation invivoNF—r33activityinneutrophilswasincreasedin LPS—treatedratscomparedwithcontrols.Therats treatedwithLPSplusdifferentdosesofGHhadhigher levelsofNF—KBactivitythanLPS—challengedrats (Fig1.ThespecificityoftheshiftbandsinEMSA wasverifiedbycompetitionassay.A11theshiftbands weresuppressedbyincubationwith50一f0ldexcessof unlabeledNF—w.Bprobeandunchangedbycompetition withasimilaramountofanotherirrelevantAP2oligo— nucleotidefFig2). GHincreasedLPS-inducedI.'c】Bdegradation invivoLPSreducedtheneutrophilsI—rd3proteincon— tentdramatically,whereasthiswasenhancedbytreat— mentwichGH0.5,1.0.and2.0mg/kg.Controlrats andratstreatedwithGHalonehadsimilarI—rd3levelsin neutrophilsfFig3). GHincreasedLPS-inducedlungneutrophils sequestrationMyeloperoxidase(MPO)levelswere significantlyincreasedfrom(1.71?0.13)U/gincontrol animalsto(3.14_+0.18)U/gintheLPSgroupofrats, andto(4.67+0.21)U/g,(4.9-+0.3)U/g,and(4.82-+ 890LiuZHelal/ActaPharmacolSin2002Oct;23f1O:887—892 23456 NF一B—+室鸶 Fig1.Aut0radi0graph0felectr0ph0reticmobilityshiftas. sayshowingenhancementbygrowthhormoneofLPS..in.. ducedNF-1cBactivationintheneutrophilsofratschallenged withLPSfor2h.Lane1,control;lane2,LPSalone(5m2, kg);lane3,LPSplusGH0.5mg/kg;lane4,LPSplusGH 1.0mg/kg;lane5,LPSplusGH2.0mg/kg;lane6,GHalone (2.0mg/kg).Thisautoradiographisarepresentativeofthree separateexperimentsusingthreeanimalsineachgroup. Supershift NF—KB_+ Non-specific- Freeprobe 234 Fig2.Resultsofcompetitiveelectr0ph0reticmobilityshift assayforNF-'activity.Lane1,negativecontrol,noHeLa nuclearextract:lane2,positivecontrol,HeLanuclear extract;lane3,HeLanuclearextractplus50-f0Idmolar excessofunlabeledNF一1cBconsensusoligo(specific competitor);lane4.HeLanuclearextractplus50.f0Idmo. 1arexcessofunlabeledAP2consensusoligo(nonspecific competitor).Thisautoradiographisarepresentativeoftwo independentexperiments. 0.15)U/gintheratstreatedwithLPSplus0.5,1.0,or 2.0mg/kgGH.ControlratsandratstreatedwithGH alonehadsimilarlungMPOactivities(Fig4). GHenhancedLPS?inducedincreaseinlung I-.KB 23456 Fig3.WesternblotphotographshowingincreaseofI-1cB degradationbyGHintheneutrophilsofratschallenged withLPSf0r2h.Lane1,control;lane2,LPSalone(5mg, kg);lane3,LPSplusGH0.5mg/kg;lane4,LPSplusGH 1.0mg/kg;lane5,LPSplusGH2.0mg/kg;lane6,GHalone (2.0mg/kg).Thisisarepresentativeofthreeseparateex. perimentsfromthreeanimalsineachgroup. '; = 矗 0 芒 = LPS5mg/kg— GH/mg?kg-一 + - + 0.52.02.0 Fig4.GHincreasedLPS-inducedincreaseinlungneutro- philsaccumulationinratschallengedwithLPSfor4h. n=7.Mean+SD.bp<o.05VSLPSalonegroup. microvascularpermeabilityChallengewithLPS causeda2.4一foldincreaseintheEvan'sbluedyeleakin lungs.TreatmentoftheLPSchallengedanimalswith GH0.5,1.0,and2.0mg/kgincreasedtheLPS—induced elevationinpermeability(P<0.05,Fig5). DISCUSSION ItiswellestablishedthatGHisaphysiological mediatorofimmunecellfunctions,andmanyofthe actionsofthisstimuliarelikelytobetransducedthrough theJanuskinase2(Jak21pathway_I4l.引.JeaySetal demonstratedthatGHexertedantiapoptoticandproli~ erativeeffectsthroughtwodifferentpathways, involvingNF一1d3andphosphatidylinositol3一kinase(PI 3-kinase).AsNF—w.Bplaysacentralroleinregulat— ingthetranscriptionofcytokines,adhesionmolecules, andothermediatorsinvolvedinthemultipleorgansys— ternfailure,itisimportanttodeterminetheroleofGH wh3activationandthesubsequentorganinjury. inNF— Inthepresentstudy.alowbase—lineactivityofNF—w.B wasobservedincontrols.whilechallengewitl1LPSfor 65432lO LiuZHetal/ActaPharmacolSin2002Oct;23(1O):887-892 LPS5mg/kg? GH/mg?kg'1? + ? + 0.5 + 2.0 Fig5.GHincreasedLPS-inducedincreaseinmicrovascu- larpermeabilityinthelungsofratschallengedwithLPS for4h.,l=7.Mean~SD.P<0.05vsLPSalonegroup. 2hcouldinduceNF.rd3activationmarkedly.Treat. mentofLPS.challengedratswithdifferentdosesof GHvariablyincreasedNF.rd3activities.whereasthis effectwasnotobservedincontrolstreatedwithsaline plusGH. ThebindingofGHtoitsreceptorcausesdimeri. zationoftwogrowthhormonereceptor,whichinturn initiatesthesignaltransductioninthecel1.Themecha. nismsoftheactionofGHremainobscure舱found thatGHtreatmentinsepticratsincreasedsepsis..in.. ducedincreaseofCD1lbexpressionandoxidativeburst activityinneutrophils(datanotshown).Reactiveoxy— genintermediates(RO