中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (2): 232-238.doi: 10.12307/2022.038

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

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

魏文悦1，2，3，王玉银2，3，郭敏芳3，张  婧2，3，谷青芳3，宋丽娟1，2，柴  智1，2，3，尉杰忠1，3，4，马存根1，2，3   

  1. 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
  • 收稿日期:2020-11-10 修回日期:2020-11-13 接受日期:2020-12-07 出版日期:2022-01-18 发布日期:2021-10-27
  • 通讯作者: 马存根，男，博士，教授，博士生导师，山西医科大学，山西省太原市 030001 尉杰忠，硕士，教授，博士生导师，大同市第五人民医院神经科，山西省大同市 037009
  • 作者简介:魏文悦，女，山西省临汾市人，山西医科大学在读硕士，主要从事神经退行性变疾病的基础和应用研究。 王玉银，女，安徽省马鞍山市人，硕士研究生，主要从事神经退行性变疾病的基础和应用研究。
  • 基金资助:
    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

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

Wei Wenyue1, 2, 3, Wang Yuyin2, 3, Guo Minfang3, Zhang Jing2, 3, Gu Qingfang3, Song Lijuan1, 2, Chai Zhi1, 2, 3, Yu Jiezhong1, 3, 4, Ma Cungen1, 2, 3   

  1. 1Department of Neurology, First Affiliated Hospital, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China; 2Research 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; 3Institute of Brain Science, Shanxi Key Laboratory of Inflammatory Neurodegenerative Diseases, Shanxi Datong University, Datong 037009, Shanxi Province, China; 4Datong Fifth People’s Hospital, Datong 037009, Shanxi Province, China

  • Received:2020-11-10 Revised:2020-11-13 Accepted:2020-12-07 Online:2022-01-18 Published:2021-10-27
  • Contact: Ma Cungen, MD, Professor, Doctoral supervisor, 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 Yu Jiezhong, Master, Professor, Doctoral supervisor, Department of Neurology, First Affiliated Hospital, Shanxi Medical University, Taiyuan 030001, 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
  • About author:Wei Wenyue, Master candidate, 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 Wang Yuyin, Master candidate, 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
  • Supported by:
    the National Natural Science Foundation of China (General Program), No. 81473577 (to MCG); Open project of the State Key Laboratory of Molecular Developmental Biology, Chinese Academy of Sciences, No. 2020-MDB-KF-09 (to SLJ); Shanxi Provincial Applied Basic Research Project, No. 201901D211538 (to SLJ); Shanxi Provincial Colleges and Universities Science and Technology Innovation Project, No. 2019L0734 (to SLJ); Basic and Applied Research of Neuroinflammation and Degenerative Diseases, Shanxi Provincial Key Laboratory Open Project, No. KF2019002 (to SLJ); Science and Technology Innovation Project of Shanxi Provincial Colleges and Universities, No. 2020L0484 (to GMF) 

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摘要：

文题释义：
法舒地尔(Fasudil)：法舒地尔[六氢-1-(5-异喹啉磺酰基)-1H-1，4-二氮杂卓]是唯一应用于临床的选择性Rho激酶抑制剂(Rho-associated kinase，ROCK)，可以通过抑制肌球蛋白轻链磷酸化缓解蛛网膜下腔出血后的脑血管痉挛。随着研究的深入，法舒地尔在防治中枢神经系统疾病方面展现了巨大的潜力，近期研究发现其具有减轻神经炎症反应、促进神经营养因子的释放及促进神经再生等作用。
线粒体动力学：线粒体在线粒体分裂蛋白和线粒体融合蛋白的调控下，不断进行分裂融合以维持细胞稳态的过程被称为线粒体动力学。目前研究表明线粒体动力学异常在神经系统退行性疾病、心血管疾病、肿瘤等疾病中普遍存在，通过调节线粒体动力学的方法可能成为防治多种疾病的新策略。
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。

https://orcid.org/0000-0002-2637-2924 (魏文悦) ；https://orcid.org/0000-0003-0049-1658(马存根)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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

Abstract: 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 function in APP/PS1 mice, which may be related to restoring the mitochondrial fission and fusion imbalance so as to inhibit neuronal apoptosis.


Key words: Alzheimer’s disease, Fasudil, APP/PS1 mice, cognitive disorder, apoptosis, mitochondrial dynamics, mitochondria fission and fusion, neuroprotection

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