雷帕霉素保护实验性自身免疫性脑脊髓炎小鼠脊髓神经元的作用途径  

Mechanism by which rapamycin protects spinal cord neurons in experimental autoimmune encephalomyelitis mice

作　　者：谢阳 张淑江 刘梦兰 罗映 杨洋 李作孝 Xie Yang;Zhang Shujiang;Liu Menglan;Luo Ying;Yang Yang;Li Zuoxiao(Department of Neurology,the First Affiliated Hospital of Southwest Medical University,Luzhou 646000,Sichuan Province,China)

机构地区：[1]西南医科大学附属第一医院神经内科,四川省泸州市646000

出　　处：《中国组织工程研究》2021年第5期695-700,共6页Chinese Journal of Tissue Engineering Research

基　　金：泸州市人民政府基金课题(2018LZXNYD-ZK17),项目负责人:李作孝。

摘　　要：背景:当前就自噬的有关报道较多,但神经元中自噬水平改变与神经元保护机制的关系未有明确定论。目的:探究mTOR自噬通路抑制剂雷帕霉素是否通过调控P70S6K及mTOR蛋白水平激活自噬发挥对实验性自身免疫性脑脊髓炎小鼠脊髓神经元的保护作用。方法:将54只雌性C57BL/6小鼠随机分为正常组、模型组及雷帕霉素组,每组18只,模型组及雷帕霉素组注射含MOG35-55的完全弗氏佐剂和百日咳菌稀释液进行实验性自身免疫性脑脊髓炎造模,雷帕霉素组小鼠在免疫当日开始腹腔注射雷帕霉素[1 mg/(kg·d)],正常组与模型组则每天同一时间注射等量生理盐水,将小鼠在发病高峰期时处死,正常组和其余未发病小鼠在饲养4周后处死并取出脊髓组织,分离出腰膨大段组织,分别进行尼氏染色行脊髓组织病理观察,免疫荧光双标染色观察脊髓组织中自噬标志物LC3与NeuN的表达及共定位情况,Western blot检测脊髓组织中mTOR、P70S6K蛋白及其磷酸化水平。结果与结论:①正常组小鼠均未发病,模型组和雷帕霉素组小鼠均不同程度发病,雷帕霉素组与模型组比较,潜伏期有延长(P<0.01),进展期缩短明显(P<0.01),高峰期时的神经功能障碍评分有所下降(P<0.05);②模型组尼氏小体数量在发病高峰期时相较于正常组明显降低(P<0.05),而雷帕霉素组尼氏小体数量相较于模型组明显增加,但仍然低于正常组(P<0.05);③模型组小鼠脊髓神经元中自噬标志物LC3散乱分布无明显点状聚集现象,而雷帕霉素组小鼠脊髓神经元中LC3呈明显的点状聚集,LC3与NeuN的分布基本一致;④模型组和雷帕霉素组mTOR及P70S6K蛋白的磷酸化水平高于正常组,但雷帕霉素组mTOR及P70S6K蛋白的磷酸化水平却低于模型组;⑤结果表明,雷帕霉素可能通过抑制P70S6K及mTOR蛋白磷酸化水平从而激活自噬,对实验性自身免疫性脑脊髓炎小鼠脊髓神经元起保护作用。BACKGROUND:There are increasing reports about autophagy,but the relationship between the level of autophagy in neurons and the neuroprotection mechanism is not clear.OBJECTIVE:To investigate whether rapamycin,an mammalian target of rapacmycin(mTOR)autophagy pathway inhibitor,could activate autophagy by mediating the P70s6k and mTOR protein levels to protect spinal cord neurons in experimental autoimmune encephalomyelitis mice.METHODS:Fifty-four healthy female C57BL/6 mice were divided into three groups:control group,model group and treatment group,with 18 mice in each group.Mice in the model group and treatment group were injected with complete Freund’s adjuvant containing MOG35-55 and pertussis diluent for establishing models of experimental autoimmune encephalomyelitis.At the same time,the mice in the treatment group were given rapamycin(1 mg/kg per day),and those in the model and control groups were given the same amount of normal saline.The mice in the model and treatment were sacrificed at the peak of the onset,and the non-morbid mice,including those in the control group,were sacrificed after 4 weeks of feeding.The spinal cord tissue from each animal was taken to isolate the intumescentia lumbalis of the spinal cord.Nissl staining was used for pathological observation of the spinal cord tissue.Immunofluorescence double staining was used to observe the expression and co-localization of autophagy markers LC3 and NeuN in spinal cord tissue.Western blot was used to detect mTOR,P70S6K proteins and their phosphorylation levels in spinal cord tissue.RESULTS AND CONCLUSION:No mice in the control group had an attack,but those in the other groups developed experimental autoimmune encephalomyelitis to different extents.Compared with the model group,the treatment group had prolonged incubation time(P<0.01),shortened progressive stage(P<0.01),and decreased neurologic dysfunction score(P<0.05).Compared with the control group,the model group had the significantly less number of Nissl bodies(P<0.05),while the number of Ni

关 键 词：雷帕霉素 变态反应性脑脊髓炎 脊髓 神经元 尼氏小体 自噬 蛋白 鼠 实验 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]