法舒地尔抑制APP/PS1小鼠神经元凋亡的线粒体动力学作用机制  

Fasudil inhibits neuronal apoptosis via regulating mitochondrial dynamics in APP/PS1 mice

作　　者：魏文悦 王玉银 郭敏芳[3] 张婧 谷青芳[3] 宋丽娟 柴智 尉杰忠 马存根[1,2,3] Wei Wenyue;Wang Yuyin;Guo Minfang;Zhang Jing;Gu Qingfang;Song Lijuan;Chai Zhi;Yu Jiezhong;Ma Cungen(Department of Neurology,First Affiliated Hospital,Shanxi Medical University,Taiyuan 030001,Shanxi Province,China;Research Center of Neurobiology,The Key Research Laboratory of Benefiting Qi for Acting Blood Circulation Method to Treat Multiple Sclerosis of State Administration of Traditional Chinese Medicine,Shanxi University of Chinese Medicine,Jinzhong 030619,Shanxi Province,China;Institute of Brain Science,Shanxi Key Laboratory of Inflammatory Neurodegenerative Diseases,Shanxi Datong University,Datong 037009,Shanxi Province,China;Datong Fifth People’s Hospital,Datong 037009,Shanxi Province,China)

机构地区：[1]山西医科大学第一临床医学院,山西省太原市030001 [2]山西中医药大学神经生物学研究中心/国家中医药管理局多发性硬化益气活血重点研究室,山西省晋中市030619 [3]山西大同大学脑科学研究所/中枢神经炎症变性疾病新药创制省市共建山西省重点实验室培育基地,山西省大同市037009 [4]大同市第五人民医院,山西省大同市037009

出　　处：《中国组织工程研究》2022年第2期232-238,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(81473577),项目负责人:马存根;中国科学院分子发育生物学国家重点实验室开放课题(2020-MDB-KF-09),项目负责人:宋丽娟;山西省应用基础研究计划项目(201901D211538),项目负责人:宋丽娟;山西省高等学校科技创新项目(2019L0734),项目负责人:宋丽娟;神经炎症及变性疾病基础与应用研究山西省重点实验室开放课题(KF2019002),项目负责人:宋丽娟;山西省高等学校科技创新项目(2020L0484),项目负责人:郭敏芳。

摘　　要：背景:线粒体动力学异常已被证实与阿尔茨海默病的发生密切相关,课题组前期研究发现,法舒地尔具有神经保护作用,但其是否对线粒体动力学具有调控作用尚未明确。目的:探究ROCK抑制剂法舒地尔对阿尔茨海默病小鼠认知功能、神经元凋亡的影响以及可能的调控机制。方法:将淀粉样前体蛋白/早老素1(APP/PS1)小鼠随机分为法舒地尔组[25 mg/(kg·d)]和生理盐水组,腹腔注射治疗2个月,并以C57BL/6野生型小鼠作为正常对照。应用Morris水迷宫和Y迷宫测试评价小鼠空间认知功能,尼氏染色检测神经元数量与形态,TUNEL染色观察神经元凋亡情况,Western blot法检测海马组织NeuN、Bax、Bcl-2、Cleaved Caspase-3、动力相关蛋白1(DRP1)、线粒体分裂蛋白1(FIS1)、视神经萎缩因子1(OPA1)、线粒体融合蛋白1(Mfn1)、线粒体融合蛋白2(Mfn2)的表达,免疫荧光染色检测NeuN、动力相关蛋白1表达。结果与结论:(1)法舒地尔干预明显改善APP/PS1小鼠损伤的认知障碍,提高其学习、记忆和探索功能;(2)与正常对照组相比,APP/PS1小鼠神经元数量减少,凋亡率增加,海马组织成熟神经元标志物(NeuN)及抗凋亡蛋白(Bcl-2)表达减少,促凋亡蛋白(Bax、Cleaved Caspase-3)表达增加,法舒地尔治疗组得到明显改善;(3)法舒地尔减少线粒体分裂蛋白(动力相关蛋白1、线粒体分裂蛋白1)表达,增加线粒体融合蛋白(视神经萎缩因子1、线粒体融合蛋白1、线粒体融合蛋白2)表达;(4)结果说明,法舒地尔具有改善APP/PS1小鼠认知的功能,其机制可能与修复线粒体分裂-融合失衡进而抑制神经元凋亡有关。BACKGROUND:The pathogenesis of Alzheimer’s disease is closely related to abnormal mitochondrial dynamics.Our previous research demonstrated that Fasudil has neuroprotective effect.However,it is needed to explore whether Fasudil has beneficial effect on regulating mitochondiral dynamics.OBJECTIVE:To investigate the effect and mechanism of ROCK inhibitor on cognitive function and neuronal apoptosis in Alzheimer’s disease mice.METHODS:Amyloid precursor protein/presenilin-1(APP/PS1)transgenic mice were randomly divided into the Fasudil group(25 mg/kg/d)and normal saline group(equivalent volume normal saline),and wild-type C57BL/6 mice at the same age and gender served as normal controls(same volume normal saline).Administration in each group was given via intraperitoneal injection once daily for 2 months.Spatial cognition of mice was detected by Morris water maze test and Y maze test.Nissl staining was used to observe and analyze the number and morphology of neurons.TUNEL staining was applied to observe neuronal apoptosis.The protein levels of NeuN,Bax,Bcl-2,Cleaved Caspase-3,dynamin-related protein 1(DRP1),mitochondrial fission protein 1(FIS1),optic atrophic protein 1(OPA1),mitofusin 1(Mfn1),and mitofusin 2(Mfn2)in hippocampus tissue were determined by western blot test.The expression of NeuN and DRP1 was detected by immunofluorescence staining.RESULTS AND CONCLUSION:Fasudil ameliorated cognitive impairment and improved loss of learning,memory and exploration function in APP/PS1 mice.Compared with the normal control group,the number of survived neurons was decreased,the apoptotic rate of neurons was increased,the expression of Bax and Cleaved Caspase-3 was increased,but the expression of NeuN and Bcl-2 was decreased in APP/PS1 mice.These changes were all strongly reversed by a 2-month treatment of Fasudil.Fasudil markedly down-regulated the expression of DRP1 and FIS1,but remarkably up-regulated the expression of OPA1,Mfn1,and Mfn2.These findings indicate that Fasudil significantly improves the spatial cognitive

关 键 词：阿尔茨海默病 法舒地尔 APP/PS1小鼠 认知障碍 凋亡 线粒体动力学 线粒体分裂融合 神经保护 

分 类 号：R453[医药卫生—治疗学] R338.2[医药卫生—临床医学]