中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (26): 4166-4172.doi: 10.12307/2022.819

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

补肾益智抗衰方通过调节小胶质细胞和巨噬细胞的极化转变改善APP/PS1小鼠认知功能

王玉银1，2，魏文悦2，3，郭敏芳2，李红霞2，张 婧1，谷青芳2，刘晓琴2，郭晓萍2，宋丽娟1，3，柴 智1， 马存根1，2，尉杰忠1，2，4   

  1. 1 山西中医药大学神经生物学研究中心，国家中医药管理局益气活血法治疗多发性硬化重点研究室，山西省晋中市   030619；2山西大同大学脑科学研究所，炎性神经退行性疾病山西省重点实验室，山西省大同市   037009；3山西医科大学第一临床医学院神经内科，山西省太原市   030001；4大同市第五人民医院神经内科，山西省大同市   037009
  • 收稿日期:2020-11-04 接受日期:2021-01-16 出版日期:2022-09-18 发布日期:2022-03-08
  • 通讯作者: 尉杰忠，博士，教授，博士生导师，山西中医药大学神经生物学研究中心，国家中医药管理局益气活血法治疗多发性硬化重点研究室，山西省晋中市 030619；山西大同大学脑科学研究所，炎性神经退行性疾病山西省重点实验室，山西省大同市 037009；大同市第五人民医院神内科，山西省大同市 037009 马存根，博士，教授，博士生导师，山西中医药大学神经生物学研究中心，国家中医药管理局益气活血法治疗多发性硬化重点研究室，山西省晋中市 030619；山西大同大学脑科学研究所，炎性神经退行性疾病山西省重点实验室，山西省大同市 037009
  • 作者简介:王玉银，女，1993年生，安徽省马鞍山市人，山西中医药大学在读硕士，主要从事中医药防治脑病的研究。 魏文悦，女，1995年生，山西省临汾市人，山西医科大学第一临床医学院神经内科在读硕士，主要从事神经病学的研究。
  • 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；山西省自然科学基金项目(201805D111009)，项目负责人：马存根；山西省自然科学基金项目(201805D131005)，项目负责人：尉杰忠；山西省应用基础研究计划项目(201901D211538)，项目负责人：宋丽娟；山西省中药现代化关键技术研究振东专项(2106ZD0505)，项目负责人：尉杰忠；山西大同大学校级科学研究项目(2019K18)，项目负责人：李红霞

Bushen Yizhi Anti-aging Prescription improves cognitive function of APP/PS1 mice by regulating microglia and macrophage polarization

Wang Yuyin1, 2, Wei Wenyue2, 3, Guo Minfang2, Li Hongxia2, Zhang Jing1, Gu Qingfang2, Liu Xiaoqin2, Guo Xiaoping2, Song Lijuan1, 3, Chai Zhi1, Ma Cungen1, 2, Wei Jiezhong1, 2, 4   

  1. 1Neurobiology Research Center, Shanxi University of Chinese Medicine, Jinzhong 030619, Shanxi Province, China; 2Institute of Brain Science, Shanxi Datong University, Datong 037009, Shanxi Province, China; 3Department of Neurology, First Clinical College of Shanxi Medical University, Taiyuan 030001, Shanxi Province, China; 4Department of Neurology, Datong Fifth People's Hospital, Datong 037009, Shanxi Province, China
  • Received:2020-11-04 Accepted:2021-01-16 Online:2022-09-18 Published:2022-03-08
  • Contact: Wei Jiezhong, MD, Professor, Doctoral supervisor, Neurobiology Research Center, Shanxi University of Chinese Medicine, Key Laboratory of Replenishing Qi and Activating Blood for the Treatment of Multiple Sclerosis, State Administration of Traditional Chinese Medicine, Jinzhong 030619, Shanxi Province, China; Institute of Brain Science, Shanxi Datong University, Shanxi Provincial Key Laboratory of Inflammatory Neurodegenerative Disease, Datong 037009, Shanxi Province, China; Department of Neurology, Datong Fifth People’s Hospital, Datong 037009, Shanxi Province, China Ma Cungen, MD, Professor, Doctoral supervisor, Neurobiology Research Center, Shanxi University of Chinese Medicine, Key Laboratory of Replenishing Qi and Activating Blood for the Treatment of Multiple Sclerosis, State Administration of Traditional Chinese Medicine, Jinzhong 030619, Shanxi Province, China; Institute of Brain Science, Shanxi Datong University, Shanxi Provincial Key Laboratory of Inflammatory Neurodegenerative Disease, Datong 037009, Shanxi Province, China
  • About author:Wang Yuyin, Master candidate, Neurobiology Research Center, Shanxi University of Chinese Medicine, Key Laboratory of Replenishing Qi and Activating Blood for the Treatment of Multiple Sclerosis, State Administration of Traditional Chinese Medicine, Jinzhong 030619, Shanxi Province, China; Institute of Brain Science, Shanxi Datong University, Shanxi Provincial Key Laboratory of Inflammatory Neurodegenerative Disease, Datong 037009, Shanxi Province, China Wei Wenyue, Master candidate, Institute of Brain Science, Shanxi Datong University, Shanxi Provincial Key Laboratory of Inflammatory Neurodegenerative Disease, Datong 037009, Shanxi Province, China; Department of Neurology, First Clinical College of Shanxi Medical University, Taiyuan 030001, Shanxi Province, China Wang Yuyin and Wei Wenyue contributed equally to this work.
  • 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 Natural Science Foundation, No. 201805D111009 (to MCG) and 201805D131005 (WJZ); Shanxi Provincial Applied Basic Research Project, No. 201901D211538 (to SLJ); Zhendong Special Project of Shanxi Provincial Traditional Chinese Medicine Modernization Key Technology Research, No. 2106ZD0505 (to WJZ); Shanxi Datong University School-level Scientific Research Project, No. 2019K18 (to LHX)

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摘要：

文题释义：
补肾益智抗衰方：由11味补益类中药组成的功能性组方，具有补肾益精填髓之功效。研究显示，补肾益智抗衰方中的单药或某些有效成分具有抗淀粉样β蛋白沉积、抗炎、抗氧化应激等作用。但补肾益智抗衰方对阿尔茨海默病是否具有疗效以及其具体作用机制还不清楚，需要进行深入研究。
巨噬细胞的极化：巨噬细胞具高度可塑性、炎症因子诱导下分化异常等特点。在不同环境的刺激下巨噬细胞可极化为经典活化巨噬细胞(M1型巨噬细胞)和替代性活化巨噬细胞(M2型巨噬细胞)，两者的极化功能几乎相互拮抗。巨噬细胞的极化是一个多因子相互作用的复杂过程，受到多种信号分子及其通路的调控。

背景：研究表明补肾益智抗衰方对阿尔茨海默病具有不同程度的治疗作用，前期研究提示同时促进脾脏巨噬细胞和中枢小胶质细胞向M2型极化有可能明显改善APP/PS1 Tg小鼠的认知功能。
目的：探讨补肾益智抗衰方对阿尔茨海默病模型小鼠(APP/PS1 Tg)的治疗效果及其作用机制。
方法：雄性8月龄APP/PS1双转基因小鼠(阿尔茨海默病模型小鼠，APP/PS1 Tg)随机分为阿尔茨海默病模型组和补肾益智抗衰方治疗组，取同性别8月龄C57BL/6小鼠作野生对照组，治疗8周。采用水迷宫实验和Y迷宫实验评估3组小鼠认知功能；免疫荧光染色法检测M1型小胶质细胞标志物诱导型一氧化氮合酶、白细胞介素6和M2型小胶质细胞标志物精氨酸酶1、白细胞介素10在海马区的表达和分布；Western blot法检测Iba-1、诱导型一氧化氮合酶、精氨酸酶1、白细胞介素6、白细胞介素10 以及Toll样受体4、髓样分化因子88和核因子κB的表达；流式细胞术检测脾脏M1型单核巨噬细胞标志物(CD16/32、白细胞介素12)和M2型巨噬细胞标志物(CD206和白细胞介素10)的表达。实验方案已得到山西大同大学动物实验伦理委员会批准。
结果与结论：①与野生组相比，阿尔茨海默病模型组小鼠认知功能下降；脑内小胶质细胞激活且向M1型转化，同时脾脏单核巨噬细胞也向M1型转化；②经补肾益智抗衰方治疗后，APP/PS1 Tg小鼠的认知功能明显改善(P < 0.05)，脑内小胶质细胞激活被抑制(P < 0.01)，M1型小胶质细胞向M2型转变(P < 0.05)；同时Toll样受体4、髓样分化因子88、核因子κB蛋白的表达下调(P < 0.01)；③结果提示，补肾益智抗衰方可能通过抑制TLR4/Myd88/NF-κB信号通路促进M2型小胶质细胞/巨噬细胞极化从而改善APP/PS1小鼠认知功能。
缩略语：人淀粉样前体蛋白695 /突变型人早老素1δE9双转基因：human amyloid precursor protein 695/ mutant human presenilin 1 delta E9，APP/PS1 Tg；诱导型一氧化氮合酶：inducible nitric oxide synthase，iNOS；精氨酸酶1：Arginase-1，Arg-1

https://orcid.org/0000-0002-2208-0959 (王玉银) ；https://orcid.org/0000-0002-2637-2924 (魏文悦) 

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, 补肾益智抗衰方, 炎性细胞因子, 小胶质细胞, 巨噬细胞

Abstract: BACKGROUND: Bushen Yizhi Anti-aging Prescription exerts different therapeutic effects on Alzheimer′s disease, and our previous studies have suggested that Bushen Yizhi anti-aging prescription can significantly improve the cognitive function of APP/PS1 Tg mice by simultaneously promoting the polarization of spleen macrophages and central microglia to M2 type.  
OBJECTIVE: To explore the therapeutic effect and mechanisms of Bushen Yizhi Anti-aging Prescription on Alzheimer’s disease model mice (APP/PS1 Tg).
METHODS: Totally 20 male APP/PS1 transgene mice aged 8 months were randomly assigned to APP/PS1 transgenic model group and Bushen Yizhi Anti-aging Prescription intervention group. Another age- and sex-matched wild-type male mice were used as wild-type control group. All mice were fed for 8 weeks. Morris Water Maze and Y-maze test were used to evaluate cognitive function of mice in the three groups. M1 microglial surface marker nitric oxide synthase, interleukin-6 and M2 microglial surface marker arginase-1 and interleukin-10 in mouse hippocampus were analyzed by immunohistochemistry. Expressions of Iba-1, inducible nitric oxide synthase, arginase-1, interleukin-6, interleukin-10, Toll-like receptor 4, myeloid differentiation factor 88 and nuclear factor-κB were examined by western blot. Flow cytometry was used to analyze the expression of M1 (CD16/32, interleukin-12) and M2-type mononuclear macrophages (CD206, interleukin-10) in mouse spleen. The study protocol was approved by the Animal Ethics Committee of Shanxi Datong Univerisity. 
RESULTS AND CONCLUSION: Compared the wild-type control group, the model group showed decreased cognitive function and the microglia in the brain were activated and transformed into M1, while the mononuclear macrophages in the spleen also transformed into M1. After treatment with Bushen Yizhi Anti-aging Prescription, the cognitive function of APP/PS1 Tg mice was significantly improved (P < 0.05), the activation of microglia was inhibited (P < 0.01), the transformation from M1 microglia to M2 was promoted (P < 0.05), and the expressions of Toll-like receptor 4, myeloid differentiation factor 88 and nuclear factor-κB were down-regulated (P < 0.01). All these findings indicate that Bushen Yizhi Anti-aging Prescription reverses cognitive impairment and facilitates microglia M2 polarization via the Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-κB signaling pathway in APP/PS1 transgenic mice.

Key words: Alzheimer’s disease, Bushen Yizhi Anti-aging Prescription, inflammatory cytokines, microglia, macrophage

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