Cyclosporine Inhibits Apoptosis in Experimental Murinerophthalamia Conjunctival Epithelium

1、Cyclosporine Inhibits Apoptosis in Experimental Murine rophthalamia Conjunctival EpitheliumJournalofHuazhongUniversityofScienceandTechnologyLMedSciJ华中科技大学医学(英德文)版26(4):469_471,2006469CyclosporineInhibitsApoptosisinExperimentalMurineXe-rophthalamiaConjunctivalEpitheliumSUNJinghua(孙京华),WANGJingxin(王净信

2、)DepartmentofOphthalmology,TongjiHospital,TongfiMedicalCollege,HuazhongUniversityofScienceandTechnology,Wuhan430030,ChinaSummary:ThisstudyexaminedtheinhibitoryefFectoftopicalcyclosporine(CsA)treatmentonconjunctivaepithelialapoptosisinamurinemodelofxerophthalamia.Dryeyewasinducedin3groupsofC57BL6mice

3、bysubcutaneousinjectionofscopolamine(t.i,d1andexposuretoanairdraftandlow.humidityenvironmentforl6heachdavforl2days,Thedryeyecontrolgroupreceivednotopicaltreatment;anothergroupreceivedlLof0.05%CsAtopically(t.i.d,dryeye+CsA);andthethirdgroupreceivedlLofthecastoroilvehicleofCsAtopically(t.i.d,dryeye+ve

4、hicle),Normalmicewereusedasuntreatedcontrols.Twelvedayslater,themicewerekilled.andtheirconjunctivaswereexcised,ThenumberoftheconjunctivalgobletcellswascountedintissuesectionsstainedwithperiodicacidSchiff(PAS)reagent.Theirconjunctivaepitheliumhadbeeninvestigatedbyim-muno.histochemicalstainingtodetect

5、thegobletcellsandtheexpressionofCaspase.3,Baxandbcl一2Ourresultsshowedthatcomparedwithdryeyecontrolanddryeyemice+vehiclegroups.thehum-berofconjunctivalepithelialgobletcellswassignificantlygreaterintheuntreatedcontrolsanddryeyemicereceivingCsAf尸<0.0lforbothgroups).Therewasnosignificantdifierenceint

6、henumberofconjunctivalepithelialgobletcellsbetweenthedryeyecontrolanddryeye+vehiclegroup.Itwasalsotrueofthenumberofconjunctivalepithelialgobletcellswhencomparisonwasmadebetweenthenormalgroupandthedryeye+CsAgroup.ExpressionsofCaspas-3andBaxwereincreasedandex.pressionofbc1.2wasdecreasedinconiunctivale

7、pithelialcellsindryeyecontrolanddryeyemice+vehiclegroups.Therewasasignificantpositivecorrelationbetweengobleteellnumberandthenumberofcellsthatexpressedbcl-2,andanegativecorrelationbetweengobletcellsandCaspase-3andBaxexpression.ItisconcludedthatthetopicaluseofCsAcouldsignificantlyreducecon-iuncti-val

8、epithelialapoptosisandprotectgobleteellagainstthelOSSinexperimenlmurinexero-phathala.mia,InhibitionofapoptosisappearstobeakeymechanismresponsibleforthetherapeuticeffbctofCsAonxerophthalamia.Keywords:xerophthalamia;cyclosporine(CsA);apoptosis;geneD0Il0.1007/sll596.006.0424-8Dvsfunctionofthefunctional

9、unitsofSUl-facelacrimalglandleadstotearnlminstabilityandtheocularsurfaceepithelialdiseasecalledxerophthalamia,whichresultsineyeirritationandvisionimpairment.Thepathologicalmechanismsunderlyingxerophthalamiaremainunknown.butthestudybyYehetalshowedthatacceleratedapoptosisoftheocularsurfaceepitheliamay

10、playaroleintheprocess.CyclosporineA(CsA)isthefirstFDA.approveddrugusedforthetreatmentofxe.rophthalamia.anditspossiblemechanismfortheameliorationofeyesurfaceepitheliumdiseaseliesintheinhibitionofapoptosisJ.ThisstudyistodetermineiftopicalCsAtreatmentforxerophthalamiacaninhibitapoptosisoftheocularsurfa

11、ceepitheliainresponsetoexperimentallyinduceddryeyeinmice.1MATERIALSANDMETHoDS1.1ExperimentalAnimalsFortyC57BL6miceofbothsexes,aged8weeks,SUNJinghua,female,bomin1949,AssociateProfessorweighing(202)g,wereprovidedbyOrganTransplantationCenterofWuhanTongjiMedicalCollege,HuazhongUniversityofScienceandTech

12、nology.Wuhan.China)aged8weeks,weighingofbothsexeswereusedinthestudy.1.2ProcurementandPreparationofSpecimens1.2.1EstablishmentofConjunctivalXerosisModelThirtvC57BL6miceaged8weekswerehypodermicallyinjectedscopolamineatadoseof0.2mL/mg(t.i.d)f0r12days.1.2.2GroupingandTreatmentThe30miceweredividedinto3gr

13、oups.witheachgrouphavingl0animals.Thel0miceindryeyecontrolgroupreceivednoeyedrop;10miceindry-eye+CsAgroupweregivenlL0.05%ciclosporinA(CsA)emulsion(PharmacyofTbngiiHospital,Wuhan,China)3timesadayforatimeof12days.Tenmiceindryeye+bearergroupwereadministeredCsAagentlLcastoroil(TianjinStandardTechnologyC

14、o.,Ltd.,China)3timesadayforatimeperiodofl2days.Anotherl0miceservedasnormalgroupandreceivednotreatment.1.3HarvestingandTreatmentofSpecimenARerabovementionedtreatmentfor12dthemiceofthe4groupsweresacriricedandthetissuesofthe470JournalofHuazhongUniversityofScienceandTechnologyMedSci_2!coniunctivaofbothe

15、yesweretakenandfixedby4%parafo1TII,andsubjectedtoroutineparaffinimbedding.1.4DetectionTechniques1.4.1PASStainingSectionswerestainedwithpen-odicacidSchiff(PAS)reagenttoevaluatetheconjuncti-valgobleteelldensity.Thecaliciformcellsinthe4groupswerecountedunderhighpowermicroscope(400folds)andtheaverageeel

16、lnumberineachhighpowerfieldwascalculated.1.4.2ImmunohistochemistrySectionsadjacenttothoseusedforPASstainingwereusedforimmunohisto-chemicalstainingforactivatedcaspase一3,Baxorbcl-2.SABCkit(WuhanBosterBioengineeringLtd.,China)wasemployed.Thedetailedproceduresweredonebyfollowingtheinstructionsofthekit.T

17、hepolyclonalgoatanti.mousecaspase.3.Baxandbcl一2primaryantibody(PharMingen.USA1usedweredilutedtol:100.Thetom1numberofcellspositiveforimmunohistochemicalstainingin1000conjunctivalepithelialcellswascalcu-lated.1.5CeUCountingandStatisticalAnalysisThetotalnumberofgobletcellsin57hi曲powerfieldswerecounteda

18、ndtheaveragenumberperfieldwascalculated.Thetotalnumberofcellspositivefortheimmunohistochemicalstaininginl000conjunctivalepithelialcellswascalculated.Thedatawereexpressedas士andSPPSl2.0packagewasusedforthestatisticalanalysisfSNKmethod1.2RESULTS2.1FindingsafterTreatmentTwelvedaysafterthetreatment,inthe

19、dryeyecon-trolgroupanddryeye+vehiclegroup,thehairofthemicewascoarse;theircorneawasdryandlackedluster.FluoresceinstainingandroseBengalstainingrevealedscatteredspottyulcer.Rosebengalstainingandbreakuptime(BUT)weremeasuredandtheSchirmerstestwasconducted.Thereweresignificantdifferencesbetweendryeyecontr

20、olgroupanddryeyevehiclegroupandbetweennormalgroupanddryeye+CsAgroup(尸<0.0l,table1).Table1ResultsofRoseBengal,BUTandchirmertest(土)P<0.OOl,ascomparedwithdryeyeanddry+vehiclegroup;,P<0.01,ascomparedwithdryeyeanddryeye+vehiclegroup2.2PASStainingofGobletCellsThereweremainlytwokindsofcellsinnorma

21、lgroupanddryeye+CsAgroup.Onewasglobalcells,whichwerebigandabundant.Thenucleiwereroundandstainedblue.purpleandwaslocatednearoneendofcells.ThecytoplasmwasabundantandcontainedplentyofmucusanditwasstronglyPASpositiveandredpurpleincolor.Theotherkindwasepithelia,whichweresmallandeveninsize.Theywereclosely

22、anddenselyarrangedinpatchesandtheirnucleiwereroundandblueincolorandtendedtolieinthemiddleofthenucleus.Theircytoplasmwasbluishorreddish.Indryeyegroupanddry.eye+vehiclegroup,conjunctivalepithelialcellswereirregularlyarrangedandhadfewgobletcellsfewPAS.positivesubstances.Comparedwithdryeyecontrolgroupan

23、ddryeye+vehiclegroups,numberoIconjunctivalepithelialgobletcellswassignificantlygreaterinthenormalgroupanddryeye+CsAgroup(P<0.01forboth,table2).Table2Gobletcellsandexpressionoftherelatedgenesintheconjunctivaiepitheliumofeachgroupis),P<0.001ascomparedwithdryeyeanddryeye+vehiclegroup.P<0.01asc

24、omparedwithdryeyeanddryeye+vehiclegroup2.3ExpressionsofApoptosisRelativeGeneProteinCaspase-3,Baxandbcl一2Theexpressionsofcaspase一3andBaxcouldbeseenintheconjunctivalepithelialcellsofdryeyegroupanddryeye+vehiclegroup,theexpressionwasobviouslyhilerthanthoseofthenormalgroupandthedry.eye+CsAgroup.Theposit

25、ivereactionproductswerebrownoncolorandmainlyfoundincytoplasma.ThedifferencebetweenthemwasstatisticallysignificantfP<0.0l,table21.Theexpressionofbcl一2couldbeseeninthecon-iunctivalepithelialcellsofnormalgroupanddryeye+CsAgroup,andtheywereobviouslystrongerthanthoseofdryeyegroupandd1eye+vehiclegroupr

26、P<0.0l,.Thepositivereactionproductswerebrownlcolorandmainlyfoundinthecytoplasm(table21.2.4CorrelativeAnalysisofGobletCellPopulationandRelativeGeneExpressionTheexpressionofcaspase.3andBaxinconjunctivalepithelialcellswasnegativelycorrelatedwiththenumberofgobletcellsfnormalgroup:,=一0.79,-0.86;P<O

27、.05;dry.eyecontrolgroup:r=一0.72,一0.83;P<0.05;dry.eye+vehiclegroup:1-=一0.69,一0.77;dry+CsAgroup:y-=.lP<0.05).Theexpressionofbcl一2incon-junctivalepithelialcellshadapositivecorrelationwithJournalofHuazhongUniversityofScienceandTechnologyMedSci26(4):2006471thenumberofgobletcellsrnormalgroup

28、:r0.80;P<0.01:dry-eyecontrolgroup:r0.78;P<0.01,dry-eye+vehiclegroup:r=0.71,P<0.01;dryeye+CsAgroup:r=0.75,P<0.011.3DISCUSS10NAccessorylacrimalglandandconjunctivalgobletcellsmaintainstheocularsurfacemoistfoundationofsecretiongland.Oncetheconjunctivalgobletcellisde-stroyed.evenifthelacrimal

29、glandfunctionisnorma1.theocularsurfacewilldevelopdryness.Ontheotherhand.ifthegobletcellfunctionisnormalevenifthelacrimalglandisremoved,theocularsurfacemayclinicallyshownodamaget1.TheinvestigationsofrecentyearsLJfoundthatMcholinergicreceptorblockerwasoneofthemainfactorswhichimpairsthesecretionoflacri

30、malgland.andscopolaminecouldsuppressthesecretionoftears.Ourpreviousstudyshowedthat12daysafterthehypodermiciniectionofscopolaminetoC57BL6mice.thenumberofgobletcellsofconjunctivalepitheliumwassignificantlylessthanthatofthenormalgroup.indicatingthatapop-tosisisoneoftheimportantcausesforconjunctivalepi-

31、theliuminthepatientswithxerophthalmia.Caspase.3belongstoapoptosiseffectsubgroupCaspaseproleaseintheCaspasefamilyandcutsthestructuralproteinofcells.directlyinducesapoptosisofcells,anditistheterminalexecutingenzymeinapop-tosisBc1.2isextractedfromBcelllymphomaandpossessesevidentapoptosis.inhibitoryeffe

32、ctL)J_Baxpro.teincanpromotecellapoptosis,therebyworkingagainsttheapoptoticefflectofbc1.2protein【.Onthebasisofthefindingsofthisstudy.weareledtoconcludethattheabsenceofbc1.2proteincanleadtoincreasedapoptosisofepitheliumofconjunctivaltissuesinpatientswithxerosisanddamagetheconjunctivalglobetcells.More-

33、over,increas&tpopulationofcellswithpositiveexpres-sionofCaspase.3andBaxproteincanpromoteapop.tosls.ThepresentstudyfurtherconfirmedthattopicalCsAhemicewithexperimentallyinducedxerosis.couldinhibittheapoptosisofconjunctivalepithelialcells.Inthedry.eyegroupanddry.eye+vehiclegroup,thenu

34、mberofconjunctivalgobletcellswassig.nificantlydecreasedbutthegobletcellsincorrespondingregionsofdryeye+CsAgroupremained,suggestingthatthelOSSofgobletcellinxerophthalamiamightbecausedbyapoptosisandthetopicalCsAtreatmentcouldpreventthelOSSofgobletcells.Theexpressionofactivatedcas-pase.3andPASstainingc

35、onfirmedthespeculation.CsAmayinhibitapoptosisbythefollowingmechanisms.Onemechanismmightliesinitsimmuno.modulatoryef_fect.Increasedpro.apoptoticcytokinessuchasTNFandIL.1hadbeendetectedintheconjunctivalepitheliumandtearfluidofhumanpatientswithxerophthalamial/1.Treat.mentofhumanxerophthalamiabytopicalu

36、seofCsAsignificantlyreducedthenumberofactivatedlympho.cytesintheconiunctivaItisspeculatedthatthisre.ductioninthenumberofactivatedTlymphocytesbyCsAmightdecreasethelevelofpro.apoptoticcytokinesontheOcularsurface.Anotherpossiblemechanismfortheanti.apoptoticactionofCsAmightbeitsdirectinhi.bitionofpermea

37、bilizationofmitochondrialmembrane.whichisanearlystepintheapoptosiscascadethatpre.cedesnuclearfragmentation.OpeningoftheCa.stimulatedpermeabilitytransitionporefMPT)dis.ruptstheoutermitochondrialmembrane.therebyleadingtotheswellingofmitochondriaandreleaseofmitochon.drialproteins.suchascytochromeC.into

38、thecyto-plasm.1lThisresultsinactivationofcysteineproteases(caspases).whicharetheeffectorsofDNAfragmenta.tioninapoptosis.CsAinhibitsMTPopening.cytoplas-mictranslocationofcytochromeCandcaspaseactiva.tion9-1o1.Tosumup,ourexperimentdemonstratedthatCsAcanprotectgobletcellsandinhibitapoptosis,therebymaint

39、ainingtheessentialsecretionoflacrimalgland.REFERENCESlYehS.SongXJ.FarleyWa1.Apoptosisofocularsur-facecellsinexperimentallyinduceddryeye.InvestOph-thalmolVisSci.2003:44:l24-l292SallK.StevensonOD.MundorfTKeta1.Twomulticen.ter.randomizedstudiesoftheefficacyandsaf.etvofcyc.1osporine.ophthalmicemulsioninmoderatetoseveredryeyedisease.CsAPhase3StudyGroup.Ophtha