雷公藤甲素对大鼠脊髓损伤后自噬的影响及机制研究.docx

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1、雷公藤甲素对大鼠脊髓损伤后自噬的影响及机制研究金哲I杨建文2罗春山21贵州医科大学研究生院，贵州贵阳550000：2.贵州省骨科医院脊柱外科，贵州贵阳550002基金项目：贵州省卫生健康委科学技术基金项目(gzwjkj2019-1-135)通讯作者：罗春山,主任医师，E-mai1:o摘要目的探讨雷公藤甲素对大鼠脊髓损伤(SCI)后自噬的影响及作用机制。方法将60只健康大鼠随机分为假手术组、模型组、雷公藤甲素组和阳性对照组，每组各15只。假手术组只进行椎板切除术，不损伤脊椎，其他3组按照Anens法制作SC1模型，雷公藤甲素组腹腔注射0.4mg(kg4)雷公藤甲素溶液，阳性对照组腹腔注射30mg

2、(kgBec1in1及凋亡相关蛋白BC1-2、Caspase-3的表达水平C结果假手术组、模型组、雷公藤甲素组和阳性对照组造模成功率分别为93.33%(14/15)、86.67%(13/15)、93.33%(14/15)、93.33%(1415)o模型组、阳性对照组、雷公藤甲素组术后各时间点BBB评分随时间延长升高(PVO.05)；术后模型组、阳性对照组、雷公藤甲素组BBB评分均低于假手术组(PV0.05)；术后阳性对照组、雷公藤甲素组BBB评分均高于模型组(PV0.05)；术后雷公藤甲素组BBB评分均低于阳性对照组(PV0.05)。术后7d假手术组大鼠未见明显组织学改变；模型组大鼠脊髓细胞肿

3、胀明显；阳性对照组、雷公藤甲素组大鼠损伤脊髓组织神经元损伤和水肿有所缓解。假手术组脊髓组织结构基本正常；模型组脊髓组织肿胀、空泡化十分明显，有大量自噬囊泡形成；阳性对照组存活的神经元胞核形态接近正常，自噬小体数量明显减少：雷公藤甲素组受损神经元细胞病理状态有所减轻，自噬小体数量有所减少。雷公藤甲素组、阳性对照组脊髓组织中1C3B蛋白表达水平高于假手术组、模型组(PVo.05)；模型组、雷公藤甲素组和阳性对照组凋亡相关蛋白BCI-2、CaSPaSe-3表达水平均高于假手术组(PV0.05)；雷公藤甲素和阳性对照组Bc1-2表达水平高于模型组，CaSPaSe-3表达水平低于模型组(PVO.05)c

4、结论雷公藤甲素可能通过上调细胞自噬、抑制细胞凋亡机制在一定程度上促进SCI大鼠运动功能恢复。关键词脊髓损伤；雷公藤甲素；自噬；运动功能Basso-Beattie-Bresnahan评分；1C3B蛋白Studyontheeffectoftripto1ideonautophagyafterspina1cordinjuryinratsanditsmechanismJINZhe,YANGJian-wen21UOChun-Shan2(GraduateSchoo1ofGuizhouMedica1University,GuizhouProvincefGuiyang550000.China;SPina1Sur

5、geryDepartment,GuizhouOrthopaedicHospita1GuizhouProvince1Guiyang550002,China)AbstractObjectiveTbinvestigatetheeffectsandmechanismsofactionofrapamycinonautophagyafterspina1cordinjury(SCI)inrats.MethodsSixtyhea1thyratswererandom1ydividedintoasham-operatedgroup,amode1group,arennetgroupandapositivecontr

6、o1group,with15ratsineachgroup.Inthesham-operatedgroup,on1yIaminectomywasperformedwithoutinjurytothespine,andintheotherthreegroups,SCImode1sweremadeaccordingtoA11ensmethod.0.4mg(kg-d)ofFaphenmetrazineso1utionwasadministeredintraperitonea11ytotheraphenmetrazinegroup,30mg(kg-d)ofmethy1predniso1oneso1ut

7、ionwasadministeredintraperitonea11ytothepositivecontro1group,and3m1(kg-d)of5%dimethy1su1foxide-sa1ineso1utionwasadministeredintraperitonea11ytothesham-operatedandmode1groupsfor7d.Themotorfunction(BBB)scoresoffourgroupsofratswereeva1uatedbefore,12h,1d,3d,and7daftersurgery.Histopatho1ogica1changesinth

8、espina1cordofratswereobservedbyhematoxy1in-eosin(HE)stainingaftertakingsectionsoftheinjuredspina1cordat7dpostoperative1y,andtheautophagicstructuresofratspina1cordneuronswereobservedbytransmissione1ectronmicroscopy,andtheexpression1eve1sofautophagy-re1atedproteins1C3B,Bec1in1andapoptosis-re1atedprote

9、insBc1-2andCaspase-3inratspina1cordtissuesweredetectedbyWesternb1ot.Resu1tsThemode1ingsuccessrateswere93.33%(14/15),86.67%(13/15),93.33%(14/15),and93.33%(14/15)forthesham-operatedgroup,themode1group,therapamycingroup,andthepositivecontro1group,respective1y.BBBscoresincreasedwithtimeateachpostoperati

10、vetimepointinthemode1group,positivecontro1group,andtretinoingroup(P0.05).PostoperativeBBBscoreswere1owerinthemode1group,positivecontro1group,andrega1citoningroupthaninthesham-operatedgroup(P0,05),PostoperativeBBBscoreswerehigherinthepositivecontro1groupandtheregimenmethy1enegroupthaninthemode1group(

11、尸0.05).PostoperativeBBBscoreswere1owerinboththeregimenmethy1enegroupthaninthepositivecontro1group(P0.05).Noobvioushisto1ogica1changeswereobsen,edinthesham-operatedratsat7daftersurgery,thespina1cordce11sinthemode1groupwereobvious1yswo11en,theneurona1damageandedemaintheinjuredspina1cordtissuesoftherat

12、sinthepositivecontro1groupandtherhodopsingroupwerere1ieved.Thespina1cordtissuestructureofthesham-operatedgroupwasbasica11ynorma1,theswe11ingandvacuo1izationofthespina1cordtissueofthemode1groupwereveryobvious,anda1argenumberofautophagicvesic1eswereformed,thenuc1eusmorpho1ogyofthesurvivingneuronsofthe

13、positivecontro1groupwasc1osetonorma1,andthenumberofautophagicvesic1eswassignificant1yreduced,thepatho1ogica1stateofthedamagedneurona1ce11softheratsintherhodopsingroupwasreduced,andthenumberofautophagicvesic1eswasreduced.Theexpression1eve1of1C3Bproteininspina1cordtissueswashigherintheraffinosemethy1e

14、negroupandpositivecontro1groupthaninthesham-operatedgroupandmode1group(P0,05),theexpression1eve1sofapoptosis-re1atedproteinsBc1-2andCaspase-3werehigherinthemode1group,rakuganetingroupandpositivecontro1groupthaninthesham-operatedgroup(P0.05),theexpression1eve1sofBc1-2werehigherintherakuganetinandposi

15、tivecontro1groupsthaninthemode1group,andtheexpression1eve1sofCaspase-3were1owerthaninthemode1group.Theexpression1eve1sofCaspase-3were1owerthanthoseofthemode1group(P0,05).Conc1usionsRega1axanthinmaypromotetherecoveryofmotorfunctioninSCIratstosomeextentthroughUpregu1atingce11autophagyandinhibitingapoptosismechanism.KeywordsSpina1cordinjury;Ryanodine;Autophagy;MotorfunctionBasso-Beattie-Bresnahanscore;1C3Bprotein脊髓损伤(spina1cordinjury,SCI)是一种多种因素造成的中枢神经系统损伤疾病，分为原发性损伤和继发性损伤,原发性损伤是创伤直接造成脊髓轴突神经细胞坏死或退行性变,而继发性损伤是在原发性损伤的基础上出现包括水肿、炎症反应、局部缺血等一系列病理生理改变，最终导致神经细胞凋亡，影响损伤节段脊髓的功能恢复，从而造