Dexamethasone rapidly suppresses IL-33-stimulated mast cell function-博泰典藏网
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Dexamethasone rapidly suppresses IL-33-stimulated mast cell function
导读：EpubaheadofprintJuly21,2016-doi:10.1189/jlb.3ARArticleDexamethasonerapidlysuppressesIL-33-stimulatedmastcellfunctionbyblockingtranscriptionfactoractivityAnuyaParanjape,*,1OEpub ahead of print July 21, 2016 - doi:10.1189/jlb.3AR ArticleDexamethasonerapidlysuppressesIL-33-stimulatedmastcellfunctionbyblockingtranscriptionfactoractivityAnuyaParanjape,*,1OksanaChernushevich,*,1AminaAbdulQayum,*AndrewJ.Spence,*MarcelaT.Taruselli,*DanielAbebayehu,*BrianO.Barnstein,*JamieJosephineAvilaMcLeod,*BiancaBaker,*GurjasS.Bajaj,*AlenaP.Chumanevich,?CaroleA.Oskeritzian,?andJohnJ.Ryan*,2*DepartmentofBiology,VirginiaCommonwealthUniversity,Richmond,Virginia,USA;and,?DepartmentofPathology,MicrobiologyandImmunology,UniversityofSouthCarolinaSchoolofMedicine,Columbia,SouthCarolina,USARECEIVEDMARCH9,2016;REVISEDJUNE7,2016;ACCEPTEDJULY1,2016.DOI:10.1189/jlb.3ARABSTRACTMastcellsarecriticaleffectorsofallergicdiseaseandcanbeactivatedbyIL-33,aproinflammatorymemberoftheIL-1cytokinefamily.IL-33worsensthepathologyofmastcellCmediateddiseases,buttherapiestoantagonizeIL-33arestillforthcoming.Becausesteroidsarethemainstayofallergicdiseasetreatmentandarewellknowntosuppressmastcellactivationbyotherstimuli,weexaminedtheeffectsofthesteroiddexamethasoneonIL-33-mediatedmastcellfunction.Wefoundthatdexametha-sonepotentlyandrapidlysuppressedcytokineproductionelicitedbyIL-33frommurinebonemarrowCderivedandperitonealmastcells.IL-33enhancesIgE-mediatedmastcellcytokineproduction,anactivitythatwasalsoantag-onizedbydexamethasone.Theseeffectswereconsistentinhumanmastcells.WeadditionallyobservedthatIL-33augmentedmigrationofIgE-sensitizedmastcellstowardantigen.Thisenhancingeffectwassimilarlyreversedbydexamethasone.Simultaneousadditionofdexametha-sonewithIL-33hadnoeffectonthephosphorylationofMAPkinasesorNFkBp65however,dexameth-asoneantagonizedAP-1-andNFkB-mediatedtranscrip-tionalactivity.IntraperitonealadministrationofdexamethasonecompletelyabrogatedIL-33-mediatedperitonealneutrophilrecruitmentandpreventedplasmaIL-6elevation.ThesedatademonstratethatsteroidtherapymaybeaneffectivemeansofantagonizingtheeffectsofIL-33onmastcellsinvitroandinvivo,actingpartlybysuppressingIL-33-inducedNFkBandAP-1activity.J.Leukoc.Biol.100:000C000;2016.IntroductionIL-33isnowappreciatedasanimportantearlyinducerofbothprotectiveandpathologicin?ammation.AnuclearcytokineAbbreviations:AP-1=activatorprotein-1,BMMC=bonemarrow-derivedmastcell,Dex=dexamethasone,DNP-HAS=dinitrophenyl-coupledhumanserumalbumin,gMFI=geometricmeanfluorescenceintensity,GR=glucocorticoidreceptor,MC=mastcellbelongingtotheIL-1family,IL-33isunusual,inthatitfunctionsasbothaproin?ammatorycytokineandaregulatoroftranscription,becauseitcanbindtochromatin[1].IL-33isconstitutivelyexpressedinepithelialbarriertissues,keratino-cytes,endothelialcells,?broblasticreticularcells,andlymphoidorgans[2].Althoughexpressioncanbeinducedinmacrophagesanddendriticcells,tissue-derivedIL-33ratherthanimmunecell-derivedIL-33hasprovedtobecrucialforin?ammation[3].Biologicallyactivefull-lengthIL-33isreleasedasan“alarmin”aftercellstressorinjury.SecretedIL-33bindsacomplexofST2(IL-1RL1)andIL-1receptoraccessoryprotein(IL-1RAcP),activatinggroup2innatelymphoidcells,basophils,eosinophils,Th2cells,NKcells,andMCs[4,5].IL-33isimportantforprotectivetype2responsestopathogens,suchasparasites[2,6],anditisalsoelevatedinlungin?ammation,atopicdermatitis,psoriasis,andmanyrheumatologicaldiseases.Inthesein?am-matorydisorders,antagonizingIL-33maybeclinicallybene?cial.MCsarepresentinmucosalandconnectivetissues,wheretheyserveasmasterregulatorsofallergicresponses[7].IL-33,asurvivalfactorforMCs[8],stimulatesMCstoproducepro-in?ammatorycytokinesandchemokines,independentofIgE-mediatedstimulation.IL-33alsoampli?esIgE-inducedMCfunction,exacerbatingallergicdisease[9C11].Collectively,theseeffectshavebeenshowntobefunctionallyimportant.Forexample,recentstudieshaveindicatedthatIL-33-inducedMCactivationworsensairwayhyperreactivityduringasthma[12,13].TheIL-33-MCaxisalsoexacerbatescollagen-inducedarthritisandin?ammatoryarthritisinmurinemodels[10,14].Neutral-izingIL-33hasbeenshowntoreducelate-phaseresponsesduringantigen-inducedpassivecutaneousanaphylaxis[15],supportingtherationalefortargetingIL-33inMCmediateddiseases.Asanoetal.[16]recentlyshowedthatdexamethasonesuppressesIL-33-mediatedacutelungin?ammation.Firstmade1.Theseauthorscontributedequallytothiswork.2.Correspondence:VCUBiologyDepartment,Box842012,Richmond,VA.E-mail:jjryan@vcu.edu/?SocietyforLeukocyteBiologyVolume100,October2016JournalofLeukocyteBiology1Copyright 2016 by The Society for Leukocyte Biology. in1957,Dexisasyntheticglucocorticoid.Ananalogofcortisone,Dexisthemostpotentofthecorticosteroids.Ithasbeenwidelyandeffectivelyusedforthetreatmentofrheumatoidarthritis,rheumaticfever,bronchialasthma,allergy,lupus,andulcerativecolitis[17C19].DexamethasonesuppressesIgE-mediatedMCfunctionsincludingdegranulation,lipidmediatorrelease,andcytokineproductioninvitroandpassivecutaneousanaphylaxisandwheal-and-?arereactionsinvivo[20C24].ThesestudiespromptedustoinvestigatetheeffectsofDexonIL-33-inducedMCfunction.DexwasapotentsuppressorofIL-33effectsinvitroandinvivoinmouseandhumanMCs.Theseeffectswererapidandselective,actingtoinhibittheproin?ammatorytranscriptionfactorsAP-1andNFkB.Ourdatasuggestthatthisandothercorticosteroidswouldbeareliablemeansofantagonizingpathologicin?ammatoryresponsesinvolvingtheIL-33-MCaxis.MATERIALSANDMETHODSReagentsRecombinantmouseIL-3,stemcellfactor,mature,cleavedhumanandmouseIL-33,andFITC-conjugatedanti-mouseCD11b[(cat.no.)101206],PE-conjugatedanti-mouseGr-1(108408),APC-conjugatedanti-mouseTNF(506308),APC-conjugatedanti-mouseIL-6(504508)werepurchasedfromBioLegend(SanDiego,CA,USA).FITC-conjugatedanti-mouseT1/ST2(101001F)waspurchasedfromMDBiosciences(St.Paul,MN,USA).Anti-mouseCD16/32Fcblock(clone2.4G2),andpuri?edmouseIgE(cloneC38-2,kisotype)werepurchasedfromBDBiosciences(SanDiego,CA,USA).DNP-HSAwaspurchasedfromSigma-Aldrich(St.Louis,MO,USA).DexamethasoneandRU-486werepurchasedfromTocrisBioscience(Bristol,UK).AllWesternblotantibodieswerepurchasedfromCellSignalingTechnology(Danvers,MA,USA).Phospho-p38MAPK(T180/Y182)(D3F9)rabbit4511,p38MAPKrabbit(9212),phospho-p44/42MAPK(T202/Y204)rabbit(9101),p-44/42MAPK(Erk1/2)(L34F12)mouse(4696),phospho-NFkBp65(Ser536)(93H1)rabbit(3033),NFkBp65(L8F6)mouse(6956),phospho-SAPK/JNK(T183/Y185)rabbit(9251),andSAPK/JNKrabbit(9252)wereusedasprimaryantibodies.Anti-rabbitIgG(H+L)(DyLight8004XPEGConjugate)(5151)andanti-mouseIgG(H+L)(DyLight6804XPEGconjugate)(5470)wereusedassecondaryantibodies.LuciferasereporterassayreagentswerepurchasedfromPromega(Madison,WI,USA).AnimalsC57BL/6Jand129/SvJmicewerepurchasedfromTheJacksonLaboratory(BarHarbor,ME,USA)andusedataminimumof10wkold,withapprovalfromtheVirginiaCommonwealthUniversityInstitutionalAnimalCareandUseCommittee.MouseMCculturesMousebonemarrow-derivedMCs(BMMCs)wereculturedaspublished[25].PeritoneallavagecellswereculturedincompleteRPMIcontaining10?S,IL-3(10ng/ml),andstemcellfactor(10ng/ml)for10dbeforeuse.HumanMCcultureProtocolsinvolvinghumantissueswereapprovedbythehumanstudiesInstitutionalReviewBoardattheUniversityofSouthCarolina.SurgicalskinsampleswereobtainedfromtheCooperativeHumanTissueNetworkoftheNationalCancerInstitute.SkinMCswereculturedasdescribedelsewhere[26]andwereusedafter8C16wk.MCpuritywasdeterminedtobe100%purebytoluidinebluestaining.IgE-mediatedactivationHumanMCsandmouseBMMCsweresensitizedovernightwithDNP-speci?cmouseIgE(1.0mg/mlforhumanMC;0.5mg/mlforBMMCs).Cellswere2JournalofLeukocyteBiologyVolume100,October2016washed,resuspended13106cells/mlincompletemediumwithcytokines,andstimulatedfor6(mousecells)or16(humancells)hourswithDNP-HSA(50ng/ml)forassessmentofcytokinesinsupernatant.CytokinemeasurementbyELISAMurineIL-13ELISAkitwaspurchasedfromPeprotech(RockyHill,NJ,USA).IL-6,MCP-1,andTNFELISAkitswerepurchasedfromBioLegend.ELISAswereperformedwithculturesupernatantsaccordingtothemanufacturer’sprotocols.ELISAsforhumancytokinesweredevelopedusingBDOptEIAreagentsfromBDBiosciences(FranklinLakes,NJ,).CytokinemRNAanalysisBMMCswereactivatedwithIL-33for2h.Cellswereharvested,andtotalRNAwasextractedwithTRIzolreagent(ThermoFisherScienti?c,GrandIsland,NY,USA).RNAwasquanti?edwiththeNanoDrop1000UVCvisspectropho-tometer(ThermoFisherScienti?c,Waltham,MA,USA),accordingtothemanufacturer’srecommendedprotocol.ForcytokinemRNAdetection,cDNAwassynthesizedbyusingqScriptcDNASynthesisfromQuantaBiosciences(Gaithersburg,MD,USA).TheCFX96TouchReal-TimePCRDetectionSystem(Bio-Rad,Hercules,CA,USA)wasusedtoamplifymessagewithPerfeCTaSYBRGreenSuperMix(QuantaBiosciences).PrimersforIL-6(forward:59-TCCAGTTGCCTTCTTGGGAC-39,reverse:59-TCCAGTTGCC-TTCTTGGGAC-39),TNF(forward:59-AGCACAGAAAGCATCATCCGC-39,reverse:59-TGCCACAAGCAGGAATGAGAAG-39),b-actin(forward:59-GATGACGATATCGCTGCGC-39,reverse:59-CTCGTCACCCACATAGGAGTC-39),werepurchasedfromEuro?nsMWGOperon(Huntsville,AL,USA).Ampli?cationconditionsforqPCRconsistedofaheat-activationstepat95°Cfor2minfollowedby40cyclesof95°Cfor15s,55°Cfor30s,and60°Cfor1min.Allmeltingcurveanalyseswereperformedbetween50°Cand95°C.ResultswerenormalizedtohousekeepinggenesbyusingtherelativeLivakMethod.WesternblotanalysisWesternblotanalysiswasperformedwith25mgtotalcellularprotein,asdescribedpreviously[25].Blotswerevisualizedandquanti?edwithanOdysseyCLxinfraredimagingsystem(LiCor,Lincoln,NE,USA).LuciferaseassayBMMCs(33106/condition)weretransfectedwith1.2mgofpGL4.74[hRluc/TK]vectorencodingluciferasegenefromRenillareniformisundertheHSV-TKpromoterand6mgofeitherpGL4.44[luc2p/AP1RE/Hygro]orpGL4.32[luc2p/NFkBRE/Hygro]vectorencodingtheluciferasegenefromPhotinuspyralis(?re?y)undertheAP-1andNFkBresponseelements,respectively.AlltransfectionexperimentswereperformedwithAmaxaNucleofector(LAllendale,NJ,USA)withprogramT-5in20?Sand50mMHEPES(pH7.5)[27].Cellswereused48haftertransfection.LuciferaseactivityamongthelysateswasmeasuredwithaDual-LuciferaseReporterAssaySystem,bytheGloMax20/20luminometer,programDLR-2-INJ(Promega).Flowcytometry.Forsurfacestaining,cellswerewashedinPBSaftertheappropriatetreatment.Fordirectlylabeledantibodystaining,cellpelletswereincubatedinFcblock+stainingorisotypecontrolantibodiesfor30minat4°C,washedwithPBSandresuspendedinFACSbuffer(PBS,3?S,and0.1%sodiumazide),andanalyzedby?owcytometryonaFACSCalibur(BDBiosciences)afterpropidiumiodideexclusionstaining.In-cellstainingforcytokines.BMMCswereactivatedwithIL-33(50ng/ml)6Dex(1mM)for90min,thentreatedwith5mMmonensinfor5h,?xed20minin4%paraformaldehyde,washedtwiceinPBSandstoredovernightat4°C.Cellswerethenpelletedandresuspendedinsaponinbuffer(PBS,0.1%BSA,0.01MHEPES,and0.5%saponin)for20minatroomtemperature.WashedcellpelletswereincubatedinFcblock+stainingorisotypecontrolantibodiesfor30minat4°C.Migrationassay.IgE-sensitizedBMMCswerewashedandresuspendedat23106cells/mlinmigrationmedium[cRPMIinwhichFBSisreplacedby10mg/mlBSA)+IL-3(1ng/ml)]6Dex(1mM)orvehicle,1-hbeforeuse.Polycarbonate(8mm)24-wellTranswellinserts(Corning,CorningNY,USA)werecoatedinwww.jleukbio.orgParanjapeetal.migrationmediumfor1hat37°Cbeforeuse.Migrationwellscontained900mlmigrationmedium6antigen(50ng/ml)6IL-33(50ng/ml)6Dex(1mM)orvehicle.Coatedinsertswereplacedinthemigrationwells,and200mloftheresuspendedcellswasplacedinthetopchamber.Cellswereincubatedfor16hat37°C,afterwhichcellsfromquadruplicatealiquotsfromthemigrationwellwerecountedvia?owcytometrywithpropidium-iodideexclusionstaining.DexamethasoneinhibitsIL-33effectsonmastcellseuthanizedbyCO2asphyxiation,andperitoneallavagewasperformed.CellsintheperitoneallavagewereanalyzedforsurfaceexpressionofGr-1andCD11bby?owcytometry.Bloodwascollectedviacardiacpuncture.PlasmaisolatedfrombloodsampleswasanalyzedforcytokinesbyELISA.StatisticalanalysisDatashownineach?gurearetheSEMoftheindicatednumberofsamples,unlessspeci?edotherwise.P-valueswerecalculatedbypairedorunpairedStudent’sttestunlessmentionedotherwise.P,0.05indicatedstatisticalsigni?cance(PGraphPad,SanDiego,CA,USA).NeutrophilrecruitmentassayAge-andgender-matchedgroupsofC57BL/6mice(10C16wkold)receivedintraperitonealinjectionsofDex(2mg/kg)orvehicleand1mgrecombinantIL-33in200mlofsterilePBS.Fourhlater,micewereFigure1.DexsuppressesIL-33-inducedcytokineproductioninmouseBMMCs.BMMCsweretreatedwith2mM(A)ortheindicatedconcentrations(B)ofDexattheindicatedtimesin(A)orsimultaneouslywithIL-33(50ng/ml)in(B).CellswerestimulatedwithIL-33for6h,andsupernatantswereanalyzedbyELISA.Dottedline:backgroundcytokineproductionwithoutactivation.(C)BMMCsweretreatedwith1mMDexasin(B)andassessedforTNFproductionbyintracellularstainingand?owcytometry.Dotplotsarerepresentativeof3BMMCpopulations.NumbersindicatepercentageofcellspositiveforTNF.Quanti?cationofvehicle-orDex-treatedcells(bottom).(D)RT-qPCRofcytokinemRNAsisolatedfromBMMCs2haftertreatmentwith1mMDexasin(B).Foldinductionwascalculatedbynormalizingtreatmentgroupstothevehicle-treated,unactivatedgroup.Datashownarerepresentativeof3(ACC)oranaverageof2(D)independentexperimentsperformedintriplicate.UnpairedStudentttestswereperformedtocomparethevehicle-treatedcellstothedrug-treatedcellsateachtimepointin(A).Dunnett’smultiple-comparisontestwasperformedtocompareeachgrouptreatedwithaparticulardoseofthedrugwiththecontrolgroupthatwastreatedonlywiththevehiclein(B).Tukey’smultiple-comparisontestwasusedtocalculatetheP-valuesin(D).*P,0.05;***P,0.001;****P,0.0001.www.jleukbio.orgVolume100,October2016JournalofLeukocyteBiology3RESULTSDexrapidlysuppressesIL-33-inducedcytokineproductionPreviousstudiesindicatedthatthelengthofpretreatmentiscriticalforDextomaximallysuppressIgE-inducedmediatorreleaseinMCs[20,21].Hence,asa?rststeptowardcharacterizingtheeffectsofDexonIL-33-mediatedMCactivation,weinvestigatedtheeffectsofpretreatmenttimingbeforeIL-33stimulation.BMMCsisolatedfromC57BL/6JmiceweregivenDexforupto24hbeforeIL-33.AlthoughallpretreatmenttimesreducedIL-33-mediatedTNFandIL-6secretion,maximumsuppressionwasnoted,with0C8hpre-treatment(Fig.1A).TodeterminewhetherDexexhibitsitssuppressiveeffectsafteractivationbegins,wetreatedMCs2or4hafterIL-33addition.Althoughdrug-treatedcellstendedtoproducelessIL-6andTNFwhenDexwasadded2hafterIL-33,suppressiondidnotreachsigni?cance.Thesedataindicatedrapidandrelativelyshort-actingeffectsofDex,promptingouruseofsimultaneousadditioninfurtherexperiments.We?rstcalculatedtheIC50forDex-mediatedeffects.Usingabroadrangeofthedrug,wefoundthatIC50forsuppressionofIL-6,TNF,andIL-13was#5nM,whereasforMCP-1,50nM(Fig.1B).PreviousstudiesinourlabindicatedthatTGFb1or?uvastatinsuppressMC-mediatorreleaseonC57BL/6backgroundbutnotonthe129/SvJbackground[25,28]indicatingdifferentialsensitivityamongdifferentstrains.However,129/SvJBMMCsshowednosigni?cantdifferenceinIC50forDexrespon-sivenesswhencomparedtoC57BL/6BMMCs(datanotshown).Reducedcytokinereleasecanberelatedtoadecreaseinproductionorsecretion.Todifferentiatethis,weperformedin-cellstainingand?owcytometryforTNFandIL-6onIL-33-activatedBMMCstreatedwithvehicleorDex(Fig.1C;datanotshown).WeobservedthatthefractionofTNF-producingcellsbecamenegligibleinthepresenceofDex.ThesedatasuggestthatDexactsatthelevelofcytokineinductionratherthanitssecretion.Tofurtherinvestigatethis,wemeasuredcytokinemRNAsynthesisbyRT-qPCR.IL-33inducedIL-6andTNFmRNA4C8-foldvs.controlcells,aneffectthatwassigni?cantlyreducedbyDex.WealsonotedreducedbasalTNFmRNAlevelsamongDex-treatedBMMCs(Fig.1D).Tocon?rmthatDexactsviatheGR,BMMCsweretreatedwiththeGRantagonistRU-486beforeaddingDex.RU-486com-pletelyreversedDex-mediatedsuppressionofcytokines(Fig.2A),indicatingthatthesuppressiveeffectsrequiredGRfunction.AlthoughBMMCsareprimarycells,theirinvitrodifferentiationmayalterresponsivenesstoDex.Therefore,weemployedmurineperitonealMCsexpandedexvivo.AswithBMMCs,IL-33-mediatedIL-13and-6productionwassigni?cantlyreducedbyDex(Fig.2B).ThesecollectivedatashowthatDexisapotentandrapidinhibitorofIL-33-mediatedMCcytokineproduction,actingatleastpartlytosuppressIL-33-mediatedcytokinetranscription.Figure2.EffectsofDexarespeci?candareconsistentwithperitonealMCs.(A)BMMCsweretreatedwithRU-486(0.5mM)for1hbeforeIL-33(50ng/ml)andDex(0.1mM)wereadded.Supernatantscollected6hafteractivationwereanalyzedbyELISA.(B)SupernatantsobtainedfrommurineperitonealMCsactivatedwithIL-33(50ng/ml)andsimultaneouslytreatedwithvehicleorDex(1mM)for6hwereanalyzedbyELISA.Datashownarerepresentativeof3independentexperimentsperformedintriplicate.Tukey’smultiple-comparisontestwasusedtocalculatetheP-values.****P,0.0001.4JournalofLeukocyteBiologyVolume100,October2016www.jleukbio.orgParanjapeetal.DexamethasoneinhibitsIL-33effectsonmastcellsDex-mediatedsuppressionofcytokineproductionisindependentofchangesinST2receptorsurfaceexpressionApossibleexplanationfortheeffectsofDexisthatitsuppressesexpressionofthereceptorST2,whichisrequiredforMCresponsestoIL-33[11].Toassessthispossibility,BMMCsweretreatedwithDexforupto24hbefore?owcytometricanalysis.TheresultsrevealedmodestsurfaceST2receptordown-regulationstarting6hafterDextreatment(Fig.3A).BecausedexamethasoneeffectsonST2receptorexpressionoccurredmoreslowlythanitseffectsoncytokineproduction,ST2suppressioncannotbethemainexplanationforinhibitioncausedbysimultaneousadditionofthedrugwithIL-33.However,regulationofST2receptorispoorlyunderstood,anditsdown-regulationmaybepartlyresponsiblefortheeffectsofDexthatoccuratlatertimepoints.Usingthe24htimepoint,we?rstfoundthattheeffectsofDexweredosedependent,withST2receptorsuppressionmoreappar-entatconcentrationsgreaterthan10nM(Fig.3B).DexhadlittleimpactonthepercentageofcellsexpressingST2receptor,whichstayedatorabove80%.Rather,drug-treatedBMMCsshowedanearly60%reductioninST2receptorstainingintensity,indicatingfewersurfacereceptors/cellwereavailableafterDexexposure.WenextdeterminedtherelationshipbetweenST2receptorandcytokineproteinexpression.ThestainingintensityofFigure3.DexdecreasesST2receptorexpression.(A,B)FlowcytometryanalysisofsurfaceexpressionofST2receptoronBMMCstreatedwith1mM(A)orindicatedconcentrationsofDex(B)fortheindicatedtimes(A)orfor24h(B).(C)Stainingintensitiesforcytokinesvs.ST2receptorexpressiononBMMCspretreatedwithDex(1mM)for24handactivatedwithIL-33(50ng/ml).Dataarerepresentativeof3independentexperimentsperformedintriplicates.Dunnett’smultiple-comparisontestwasperformedtocomparedrugandvehicle-treatedgroupsin(B).*P,0.05;**P,0.01;***P,0.001;****P,0.0001.www.jleukbio.orgVolume100,October2016JournalofLeukocyteBiology5包含总结汇报、计划方案、高中教育、农林牧渔、党团工作、求职职场、自然科学、出国留学、IT计算机、教学研究、表格模板以及Dexamethasone rapidly suppresses IL-33-stimulated mast cell function等内容。本文共2页
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