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    EFFECT

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    ;32?JournalofShanghaiSecondMedicalUniversity2008Vo1.20No.J ;EFFECToFPREoPERATIVEGLUT?DEADM[[NISTRArrIoN

    ;ONICAM1EXPRESSIONINRATLUNGINDUCEDBY

    ;DIESTDAIISCHEMA.REPERFUSIoN

    ;GENGGuiqi(耿桂启),JIANGHong(姜虹),ZHUYesen(朱也森)

    ;DepartmentofAnesthesiology,theNinthPeople’sHospital,SchoolofMedicine,ShanghaiJiaotongUniversity,Shanghai

    ;200011,China

    ;ObjectiveToevaluatetheeffectofpreoperativeglutamineadministrationonintracellular

    ;adhesionmolecule,(1CAM

    ,)expressioninratlunginducedbYintestinalischemiareperfusion(I/R).M

    ethods

    ;SpragueDawleyrats(n=25)wererandomlydividedinto5groups:shamgroup(shamsu~ery),glutaminegroups

    ;(threedifferentdoses)andcontrolgroup.Allgroupsexceptshamweresubjectedtointestinal1/Ri~ury.and

    ;superiormesentericartery(SMA)occludedfor60minfollowedbY90minofr

eperfusion.Lunginjurywasevaluated

    ;withEvansblueayeconcentrationandhistopathologicexamination.TheimmunohistochemicalexpressionandmRNA

    ;expressionof1CAM

    ,weremeasuredwithimmunohistochemicalstainingandRT-PCRmethodrespectively.Thelevel

    ;ofmyeloperoxidase(MPO)wasalsomeasuredwithbiochemistrymethod.ResultsIntestinal1/Rresultedin

    ;lunginjurycharacterizedbyanincreaseinEvansblueayeconcentration,neutrophilsequestration,andobvious

    ;stainingforexpressionofpulmonary1CAM-1,comparedwithshamgroup.Theexpressionof1CAM-1andthelevel

    ;ofMPOinratlungwereloweringlutaminegroupscomparedwithcontrolgroup.Conclusion1-Rin2ury

    ;increasestheexpressionof1CAM

    ,withinthelung.Thismaycontributetothemigration.accumulationand ;activationofpolymorphonuclearneutrophils(PMNs)aftersuchinjury.Preoperativeglutamineadministration

    ;attenuatesratlunginjuryinducedbyintestinal1-R.andinhibiting1CAM,expressionmaybeoneofthepotential

    ;mechanisms.

    ;1unginjuryintracellularadhesionmoleculeglutamineintestinalischemia

    ;reperfusion

    cell ;Adhesionmoleculesplayakeyroleincell

    ;interactionsandcellextracellularmatrixinteractions. ;Intracellularadhesionmolecule1(ICAM1)and

    ;vascularcelladhesionmolecule1(VCAM1)are

    ;membersoftheimmunoglobinsuperfamilyofcellad

    ;hesionmolecules(CAMs).ICAM1isanadhesion

    ;moleculeexpressedbyneutrophilsandlymphocytes ;whichisinvolvedincel1.cellandcel1.matrixbind

    ;ing.CeUadhesionmoleculesareimportantinthead

    ;hesionofmonocytes,lymphocytesandneutrophilsto ;activateendothelium….

    ;Giutamine(Gin),formerlyclassifiedasanon

    ;essentialaminoacid,isconsideredtobeessential ;duringcertaincatabolicconditions.Astudyhas ;shownthatglutaminecanattenuatecytokinerelease, ;reduceorgandamage,andimprovesurvivalinarat ;modelofendotoxemia.Uptonow.thereisno

    ;studyinvestigatingtheeffectofGinontheexpression ;ofICAM??1inratlungafterintestinalischemia??reper- ;fusion(I/R).Thehypothesisforthisratmodelstudy

;wasthatpretreatmentwithglutaminereducestheex

    ;pressionofICAM1inducedbyintestinalI/R.We ;haveattemptedtoclarifythe

    ;Ginthroughtheinterrelation

    ;sionandICAM1.

    ;Correspondingauthor:ZHUYeIsen(朱也

    ),E-mail:zhuyesenjh@yahoo.corn

    ;cytoprotectiveeffectsof

    ;betweenHSP-70expres

    ;MATERLSANDMETHoDS

    ;Allim~sTwentyfivemaleSDrats(300350g

    ;

    ;JournalofShanghaiSecondMedicalUniversity2008Vd.20No.J?33?

    ;wereallowed1weektoacclimatizebeforesurgery. ;Theratswereexposedtoadaily12-hlight-dark

    ;cycle,andfedwaterandratchowadlibitum.This ;studywasapprovedbytheAnimalEthicsCommittee, ;ShanghaiJiaotongUniversity.

    ;proceduresandGinadministration

    ;Beforeintroductionofanesthesiafortheexperi

    ;ment,eachratwasfastedfor12hbeforeintestinal ;I/Rinjurybutallowedfreeaccesstowater.Therats

    ;wereanaesthetizedbyanintraperitonealinjectionof ;chloralhydrate300mg/kg,thenplacedonaheating ;pad.Gin(Sigma,USA)waspreparedassolution ;dissolvedinlactatedRinger(LR)immediatelybefore ;uso.Ginsolutionswerefilteredwitha0.22mfilter

    ;beforeadministration.GInsolutionorLRcontrolwas ;administered30minbeforeoperationviathelateral ;tailveinofanesthetizedratsbyusingasyringepump ;atarateof0.5mL/min.ThetotalvolumeofGInor ;LRwas5mL.Glndoseswere0.15,0.45and0.75 ;g/kginrespectivegroup.Theanimalswererandomly ;dividedintoshamgroup(shamsurgery),glutamine ;groupandcontrolgroup[superiormesentericartery ;(SMA)occludedfor60minfollowedby90minof ;reperfusion].TomakeallintestinalI/Rinjurymod

    ;el,lapamtomywasperformedusingthesteriletech

    ;niques,andSMAwascarefullydissectedandocclu

    ;dedatitsproximalportionwithamicrovascular ;clampfor60min.Afterconfirmationofblanchingof ;thesmallintestine,thentheabdomenwasclosed ;witII3-0nylonsilktominimizeheatandfluidlosses. ;Forresuscitation,allanimalswereadministeredwith

    ;25mL/kgof0.9%salinesubcutaneouslyafterSMA ;occlusion.After60minofSMAocclusion,theabdo

    ;menwasreopened,andtheclipwascarefullyre

    ;movedtoallowreperfusionofthebloodflow.After ;confirmingbowelreperfusionbyreturnofbowel ;color.theabdomenwasclosedagain.Atthetimeof ;initiallaparotomy,immediatelybeforeSMAocclu

    ;sion,Evansblue30mg/kgwasinjectedintothetail ;vein.Systemicmeanarterialbloodpressure(MAP) ;andrectaltemperaturewererecordedevery5min ;throughouttheexperiment.

    ;Ratsweresacrificedattheendofreperfusion. ;Afterthethoracoabdominalcavitywasopened,The ;hilusoftherightlungwasclampedandresectedfor ;immunohistochemicalstaining.biochemistryexami

    ;nationandRTPCR.Toeliminateresidualbloodand ;Evansbluedyefromthepulmonarybed,theleftlung ;wasperfusedviathepulmonaryarteryfor2minwith ;saline(37oC)at0.04mL?g?minusinganin

    ;fusionpump,finally,theleftlungwasexcised, ;rinsedexternallywithsalineandblotteddry.The ;lungwasweighted,placedinanovenandheatedat

    ;90?f0r16h.thedriedtissuewereincubatedin ;2mLofformamideat37oCfor24h.Thedyecon

     ;centrationoftheeluentwasthenmeasuredbyspec

    ;trophotometryat620nm.TheconcentrationofEvans ;bluedyeextractedfromthelungswasexpressedas ;microgramsofdyepermilligramwetlungweight. ;Real-timePCRanalysisAftergenerating ;cDNA.RTPCRwasperformedforratICAM1.andrat

    ;glyceraldehyde-3phosphatedehydrogenase(GAPDH) ;genesfromthesamecDNAsamplesbyusingRTPCR

    ;detectionsystem(RotorGene3000,CorbettRe

    ;search).Reactionmixture(25L)forPCRcon

    ;tained10molofprimer,2.5U/LTaqpolymerase ;(Promega),2.5mmol/LdNTP,and1.5mmol/L ;MgC12.PCRwasrunfor40cyclesafteraninitial ;denaturationstepat95oCfor5minwithanamplifi

    ;cationprofileofeachcycleconsistingofdenaturation ;f0r10sat95oC.primerannealingfor15sat60oC. ;andelongationfor20sat72oC.Afterthelastcycle ;ofamplification,thesampleswerethenincubated ;f0r2minat72oC.Oligonucleotideprimersforrat ;GAPDHcDNAwere5’AAGAAGGTGGTGAAGCAG

;GC3’.and5’-TCCACCACCCTGTrGCTGTA-3’.Oli—

    ;gonucleotideprimersforratICAM1cDNAwere5’

    ;CCCGTGAAATrATGGTCAATCC-3’and5’GCCTAC

    ;CCTCCCACAACAGC-3’.Expressionofthehouse—

    ;keepinggeneGAPDHservedascontro1.

    ;hnmunohistochemicalanalysisParaffinsec

    ;tionswereprocessedforstandardimmunohistologic ;stainingbythelabeledstreptavidinbiotinmethod

    -1antibodydi’- ;usingpolyclonalrabbitanti?-ratICAM

    ;luted1:100(BosterBiotechnologiesCo,Wuhan, ;China).Thesecondaryantibodyconsistedofbiotiny

    ;

    ;?

    ;34?JournalofShanghaiSecondM

    ;edicalUniversity2008Vo1.20No.

    ;latedsheepantirabbitimmunoglobulins(SantaCruz ;Biotechnology,SantaCruz,USA)containing10per ;centnormalratserum.Thetertiaryantibodywasa ;treptavidinhorseradishperoxidaseconjugate(Santa ;CruzBiotechnology,SantaCruz,USA).Sections ;werecounterstainedwithhematoxylinandperoxidase

    ;positivecellswereidentifiedmorphometricallvbv

    ;brownstainingaroundthecytoplasm.Positivecells ;wereexpressedasthenumberofpositivecellsper ;highpowerfield(HPF).

    ;Assessmentofpulmonaryneutrophilseques. ;trationTheupperhalfofeachrightlungspecimen ;wasremovedandfixedin10percentformalinsolu

    ;tionf0ratleast24h.Paraffinembedded4-ixmsec- ;tionswerestainedwithhematoxylinandeosinandex

    ;aminedunderalightmicroscope.Asinglepatholo

    ;gist,whowasblindedtoallgroups.examinedthe ;pathologicalspecimens.Atleasttwodifferentsec

    ;tionsofeachspecimenwereexaminedtodetermine ;thedegreeofinjury.Lungpolymorphonuclearneutro.. ;phil(PMN)sequestrationwasquantifiedbycounting ;alveolarseptalwallPMNs.Onlyperipherallungpa

    ;renchymawasexamined.Microscopicfieldscontai

    ;ningotherstructuressuchasairways,largevessels ;andpleurawereexcluded.Whitebloodcellentrap

    ;mentwasexpressedasthemeannumberofPMNsper ;GlnO75

    ;Sham

    ;HPF.

    ;Thelevelofmyeloperoxidase(MPO)Wasa1so ;measuredwithbiochemistrymethodusingreagentkit

    Company,Nanjing,China)accord ;(JianChengBio

    ;ingtothedirection.

    ;StatisticalanalysisDatawereexpressedas ;X?s.ComparisonsweremadebyusingANOVAand ;theStudent’St-test.AP<O.05wasconsideredtobe ;statisticallysignificant.

    ;RESULTS

    ;ICAM1expressioninthelungwassignificantlv ;higherinthecontrolgroupcomparedwithshamgroup ;(Tab1,Fig1).IntestinalI/Rresultedintheup- ;regulationofpulmonaryendothelialadhesionmoIe

    ;cules.TherewasasignificantdecreaseinICAM.1 ;expressioninthelungsharvestedfromanimalspre- ;treatedwithGlnthanafterintestinalI/Ralone. ;Tab1ICAM-1positiveceHsandgeneexpression(~?.n:5) ;Comparedwithcontrol:P<0.O1:P<0.05 ;Gln045GlnOl5

    ;Fig1EffectsofGinOilICAM?1expressioninratlungafterintestinalI/Rx200

    ;

    ;JmdlofShanghaiSecondMedicalUniversity2008Vo1.20No.j?35?

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