【doc】庆大霉素耳毒性作用机制及黄芩的神经保护研究

【doc】庆大霉素耳毒性作用机制及黄芩的神经保护研究 庆大霉素耳毒性作用机制及黄芩的神经保 护研究 中国临床康复第9誊第5期2005—02—07出版 ChinesPJournalofClinicalRehabilitation.February7!!:! Mechanismofgentamicininducedototoxicityandthe neuroprotectiveeffectmediatedbybaicalin? DoiDe ? BAsICRESEARCH? DoiDe,DepartmentofOtorhinolaryngology,AffiliatedHospitalofGuandong MedicalCollege,Zhanjiang524001,GuangdongProvince,China DoiDe?,Male,HanNationality,Bornin1965inChangdeCity,Hunan Provinee.China.GraduatedfromUniversityofIowain2000,Master,As— s0ciateprofessor.Researchdirection:neurotology.daide@126.com Telephone:+86.759.2387420 Supportedby:theGeneralProgramFoundationofGuangdongAdministra- ti0n0fTraditionalChineseMedicine.No.102087 Received:2004—06—07Accepted:2004—09—30(26/SY) Abstract BACKGROUND:Gentamicin(GM)一 inducedototoxicityrelatestotheper— manentinjuryonthespinalganglioncells,whichareimportanttothepro’ cessofhearingandcommunication. OBJECTIVE:Toinvestigatethemechanismunderlyingantioxidanteffectof baicalin(BA)onGM—treatedmousespinalganglioncells(SCGs). DESIGN:Randomizedcontrolledtria1. SETTINGandMATERIALS:TheresearchwascompletedintheGuan? dongMedicalCollegeTwentyKunmingmiceofeithergender(clean grade)weighing(15?2)gwereprovidedbytheExperimentalAnimal CenterofGuangdongMedicalCollege(certificatenumber:2003A026).The micewerekeptattheenvironmenttemperatureof28?withrelativehu— miditvof70%. INTERVENTIoNS:Twentymicewererandomlydividedintocontrol group,GMgroup(withGMtreatmentonly),BAgroup(withBAtreatment only)andGM+BAgroup(withGMcombinedwithBAtreatment).GMwas administeredintheGMandGM4-BAgroupsviaintraperitonealiniection at100mg/kgf0r10consecutivedays.andBAwasgiveninBAandGM+BA groupssimilarlyat100mg/kg.Inthecontrolgroup,themicereceived salineiniectioninequivalentvolumeinthesamemanner. MAINoUTCoMEMEASURES:Theactivitiesoftheantioxidativeen. zymessuperoxidedismutase(T—SOD)andglutathioneperoxidase(GSH— Px) wereevaluatedandthecontentsoflipidperoxidationsubstancemalondi— aldehyde(MDA)measuredwithultraviolet(UV)chromatographyinSCGsin differenttreatments. RESULTS:BAcouldreversetheGM—inducedlowactivitiesofT.SOD(fro m 46.5Ot027.86NU/mL.P<0.01)andGSH—PX(from83.77t042.34U. P<0.01).andlimitedtheGM.inducedhighcontentofMDA(froml1.87 t017.03m0l/L.P<0.0l】inSGC. CoNCLUSIoN:BAproducesneuroprotectiveeffectonSGCsthroughe— liminatingsuperoxideradicalsandinhibitinglipidperoxidationintheevent 0fGMexposure. DaiDMechanismofgenlomicininducedololoxicilyandlheneuroprolecliveeffect medialedbyboicolin.ZhongguoLirwhuangKangfu2005;9(5l:184—5fChi nal 【wwwzglckfcaml INTR0DUCT10N Gentamicin(GM)hasbeenwidelyusedasaclinicallyeffectivean— tibiotic,butitsmechanismsofototoxicityhavenotbeenfullyun— derstood.Previousresearcheshaveidentifiedthecrucialrolesof calciumchannelblockagel】I. nitricoxidegenerationI.andfree oxygenradicalsl3_inGM—inducedototoxicityonvestibularhair cells.Spinalganglioncells(SGCs)arevulnerabletoGM—induced ototoxicity00andthenumberofsurvivingSGCscanbecriticaltothe auditoryfunctionl6I_Evidencefromanimalexperimentssuggeststhe relationofsuperoxidetonephropathyinducedbyGMI.and baicalin(BA),oneofthemaineffectivecomponentsinscutellaria baicalensisgeorgi,isknowntopossessantioxidativeactivity.In thisstudy,weevaluatedtheeffectofGMandBAonoxidativefree radicalsinSGCsandinvestigatedthemechanismofBA—mediated neuroprotectiveonSGCsagainstGM—inducedototoxicity. MATERIALSANDnETHODS nateriais BAwaspurchasedfromGuanghanBiotechCo.Ltd,(Sichuan Pr0vince.China),andGMfromFuqingPharmaceuticalFaeto~ fFujianProvince,China).Superoxidedismutase(T—SOD),glutathione peroxidasefGSH—PX)andmalondialdehyde(MDA)reagentkitswere purchasedfromNanjingBiochemCo.,Ltd.,China. TwentyKunmingmiceofeithergender(cleangrade)weighing (15?2)gwereprovidedbytheExperimentalAnimalCenterof GuangdongMedicalCollege(certificatenumber:2003A026).The micewerekeptattheenvironmenttemperatureof28?withrelative humidityof70%. Methods Animalmodelpreparationoflipidperoxidation Themicewererandomlydividedintocontrolgroup,GMgroup (withGMtreatmentonly),BAgroup(withBAtreatmentonly)and GM+BAgroup(withGMcombinedwithBAtreatment).GMwas administeredintheGMandGM+BAgroupsviaintraperitonealin— jectionat100mg/kgfor10consecutivedays,andBAwasgivenin BAandGM+BAgroupssimilarlyat100mg/kg.Inthecontrol group,themicereceivedsalineinjectioninequivalentvolumeinthe sanTiemarl”lUaer. SGCPreparation SGCswerecollectedandpreparedfollowingtheproceduresina previousreport【. TheactivitiesofT—SODandGSH—PXandthe contentofmal0ndialdehvde(MDA)intheSGCsweremeasuredfo1. 1owingtheinstructionofthereagentkit. Statisticalanalysis:ThedatawereexpressedasMcan?SDandsta— tisticalanalysiswasperformedusingstudent’Sttest. RESULTS EffectsofGMandBAonT-S0DactivitiesinSGCs TheactivitiesofT?SODofthecontrolgroup.GMgroup.BAgroup andGM+BAgroupwas(46.50?3.76),(27.86?5.06),(67.29 ?4.83).and(40.92?4.45)NU/mLrespectively.BAcouldan. tagonizetheeffectofGMonSGCs.T.S0Dactivitiesvariedonlyin. significantlybetweenc0ntr0lgroupandGM+BAgmup(P>0.05, Fure1). 《C0ntmlGMBACM+BA GM:gentamicin;BA:baicalin;T—SOD:s”per0xidedismutase;SGCs:pinaI ga”gli0ncells;尸<0.01sc0ntmlgm”p Figure1Efcomkf23385083@sinacom戴德,等庆大霉素耳毒性作用机 制及黄芩的神经保护研究w.瓤德,寺状堡墨星里垡里堡星兰垄堕 (74.97?7.48)Urespectively.BAantagonizedtheeffectoft;Uon theSGCs.GSH—PXactivitywascomparablebetweencontroland GM+BAgroups(P>0.05.Figure2). 一 量 i6.0 l萋010 EffectsofGnandBAonnDAcontentinSGCs TheMDAcontentincontrol.GM.BA,andGM+BAgroups was(11.87?0.88).(17.03?2.47),(3.91?1.56)and (13.13?0.75)mol/L,respectively.BAantagonizedtheeffect ofGMonMDAcontentintheSGCs.astheMDAcontentwassimilar betweencontrolandGM+BAgroups(P>0.05,Figure3). 325 20 g 15 芒 尝10 ;5盎 =0 ControlGMBAGM+BA GM:gentamicin;BA:baicalin;SGCs:spinalganglioncells;.MDA:malonad i— aldehyde;”P<0.O1.controlgroup Figure3EffectsofGMandBAonMDAcontentinSGCs DlSCUSSION Oxidativefreeradicalscausedamagetothesensorycellsandneu— ionsoftheinnerear【.predominantlybyinducingcellmembrane phospholipidandpolyunsaturatedfattyacidperoxidation…1.MDAis oneofthemainproductsofthisoxidativeprocess.Peroxidationof suchendogenouslipidsascellmembranelipidsresultsinpertur- bationoftheenzymeactivities,functionaldisturbanceofthemito— chondria.andreleaseofseveralcellularfactors. Hydroxylradicalsproducedinthereactionofsupeioxideanionswith H202,whicharethoughtasthemostactiveandtoxicspeciesamong thereactiveoxygenspecies【’.arecapableofinducinglipidperox— idationinSGCs.Theincreaseinintracellularsuperoxideanionsdue todifferentfactors(suchasischemia,excitotoxins.inflammation, andsoon)doesnotonlysignificantlyreducetheactivityofSOD, butalsodiminishGSH—pXt’.anintracellularlipidperoxidation productscavenger.Inthisstudy.theactivitiesofT—SODand treatedSGCsweresignificantlylowerthanthoseof GSH—PXinGM— othergroups,indicatingthatGMexertsitsototoxicitybyincreasing superoxideanionsinSGCs.BAinhibitedGM—inducedototoxicityby raisingT—SODandGSH—PXactivities.asseeninGM+BAgroup. tothelevelofthecontrolgroup. MDAmaybindtotheaminocontainedin.forinstance,anenzyme. toprompttheinactivationoftheenzyme.Besidesdamagetothecell membranetocauseintracellularionicconcentrationdisturbance. MDAalsounderminesthefunctionsofmitochondriabyinhibiting oxidativephosphorylation.Inthissense,MDAcontentactuallyrep— resentsthedegreeofcelldamagecausedbyoxidativefreeradi— cals.WeobservedthatGMsignificantlyincreasedMDAcontentin SGCsinthisstudy,orinotherwords,enhancedlipidpeioxidation inthesecells.SinceMDAcontentandGSH—PXactivitywerecom— parablebetweenGM+BAandcontrolgroups.BAmayantagonize 185 .mediatedototoxicitybyinhibitingthegenerationofMDA. GM— Inconclusion,GMproducesitsototoxlcitybyaffectingtheactivitiesof T-SODandGSH—PXaswellasMDAcontentinSGCsthroughox— idativefreeradicals.BAenhancesthecapacityofthecellstoelimi— nateoxygen—derivedfreeradicalsandinhibitlipidpeioxidationto offerprotectionagainstGM—inducedototoxicity. REFERENCES lLopez—EscamezJA.CanizaresFJ.CrespoPV,etalElectronprobemicroanalysis ofgentamicin—inducedchangesonioniccompositionofthevestibulargelatinous membraneHearRes1994;76:60—6 2TakumidaMAnnikoM.Nitricoxideinguineapigvestibularsensorycellsfol— lowinggentamicinexposureinvitroActaOtolaryngol2001:21:346—50 3ConlonBJ.SmithDWSupplementalironexacerbatesaminoglycosideototoxicity invivo.HearRes1998;l15:1—5 4FetoniAR.SergiB,ScaranoE,eta1.Protectiveeffectsofalpha—tocopherol against gentamicin—inducedOto—vestibufotoxicitv:anexperimentalstudyActaOtolaryngol 7 2oo3:123:192— 5SoneM.SchachernPA.PaparellaMMLossofspiralganglioncellsprimary manifestationofaminoglycosideototoxicityHearRes1998;Il5:217—23 6OtteJ.SchunknechtHF.KerrAGGanglioneellpopulationsinnolqlllaland pathologicalhumancochleaeImplicationsforcochlearimplantation.Laryngoscope 1978;88:1231—46 7CuzzocreaS.Maz998;33:705—7 9LebvrePP.VallDeWaterTR.WebetT.Growthfactorinteractionsincultureof dissociatedadultacousticganglianeuronotrophiceriects.BrainRes1991:567: 306一l2 lOWatersCMolecularmechanismsofcelldeathintheearAnnNYAcadSc 1999;884:4l一51 IlHalliwellB.Howtocharacterizeabiologicalantioxidant.FreeRadicalResCorn— m”nl990;9:l一35 12HalliwellB.Thebiologicalsignificanceofoxygen—drivedspecies.In:Va lentineJS, FooteCS,GreenbergA,etal,edActiveOxygeninBiochemistryBlachie:Aca — 5 demic&Professional1995:3l3— l3SakacV,SakacMFreeoxygenradicalsandkidneydiseases—partI.Med 74 20o0:53:463— 庆大霉素耳毒性作用机制及黄芩的神经保护 研究H 戴德(广东医学院附属医院耳鼻咽喉科,广东省湛江市524001) 戴德?,男,1965年生,湖南省常德市人,汉族,2000年美国爱荷华大 学毕业,硕士,副教授,主要从事耳神经病理学的研究. 广东省中医药局科研基金面上项目(102087)} 摘要 背景:应用庆大霉素治疗时其耳毒性常累及耳蜗螺旋神经节细胞,由于 其与听觉信息的传输和言语识别能力密切相关,受损后难以恢复. 目的:探讨黄芩苷对庆大霉素致小鼠耳蜗螺旋神经节细胞毒性作用的拮 抗机制. :随机对照的实验研究. 地点和材料:实验地点为广东医学院.动物:采用清洁级昆明种小鼠2O 只,体质量(15?2)g,雌雄不限,由广东医学院动物实验中心(实验动物 质量合格证:2003A026)提供,饲养环境温度28?,湿度70%. 干预:将20只小鼠按随机数分为4组:对照组,庆大霉素组,黄芩苷组 和混合组(庆大霉素+黄芩苷).庆大霉素组和混合组每日腹腔注射庆大 霉素100mg/kg,共10d;黄芩苷组和混合组均灌胃黄芩苷100mg/kg, 共7d;对照组给予相同容量生理盐水灌胃和(或)腹腔注射. 主要观察指标:应用紫外分光光度法分别测定各实验组耳蜗螺旋神经节细 胞总超氧化物歧化酶活性,谷胱甘肽过氧化物酶活性以及丙二醛含量. 结果:黄芩苷能抑制庆大霉素所引起的耳蜗螺旋神经节细胞中总超氧化 物歧化酶活性(从46.50减少至27.86NU/mL,P<0.01)和谷胱甘肽 过氧化物酶(从83.77减少至42.34U,P<0.01)活性降低,以及丙二 醛含量升高(从11.87上升至l7,03m01/L.P<0.叭). 结论:黄芩昔通过清除耳蜗螺旋神经节细胞内氧自由基和抑制脂质过氧 化反应,从而拮抗庆大霉索耳毒性作用. 主题词:黄芩;庆大霉素类;螺旋神经节;耳蜗;自由基;脂质过氧化作用 中图分类号:R74文献标识码:A文章编号:1671—5926(2o05)05—0184—02 戴德庆大霉素耳毒性作用机制及黄芩的神经保护研究【J】中国临 床康复, 2OO5,9l5J:184—5{,wwzgIcI(fcOm】 (EdjtedbySongJW/S0ngLP/xiaoxL)