中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (7): 1057-1062.doi: 10.12307/2023.766

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

黄芪甲苷对实验性自身免疫性脑脊髓炎小鼠T细胞免疫调节的影响

穆秉桃1，于婧文1，刘春云1，郭敏芳1，孟  涛1，杨鹏伟2，魏文悦1，宋丽娟3，4，尉杰忠1，马存根1，3   

  1. 1山西大同大学脑科学研究所，山西省大同市   037009；2晋城市第二人民医院，山西省晋城市   048000；3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；4山西医科大学生理学系，山西省太原市   030032
  • 收稿日期:2022-10-24 接受日期:2022-12-27 出版日期:2024-03-08 发布日期:2023-07-17
  • 通讯作者: 马存根，博士，教授，博士生导师，山西大同大学脑科学研究所，山西省大同市 037009；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
  • 作者简介:穆秉桃，女，1976年生，山西省大同市人，硕士，讲师，主要从事神经系统疾病方面的研究。
  • 基金资助:
    国家自然科学青年基金项目(82004028)，项目负责人：宋丽娟；中国科学院遗传与发育生物学研究所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：山西省卫健委医学科技领军团队(2020TD05)，项目负责人：马存根；山西中医药大学青年科学家培育项目(2021-PY-QN-09)，项目负责人：宋丽娟；山西省黄芪资源产业化及产业国际化协同创新中心项目(HQXTCXZX2016-022)，项目负责人：马存根；大同市应用基础研究计划项目(2020145)，项目负责人：于婧文

Immunomodulatory effect of astragaloside IV on T cells of experimental autoimmune encephalomyelitis mice

Mu Bingtao1, Yu Jingwen1, Liu Chunyun1, Guo Minfang1, Meng Tao1, Yang Pengwei2, Wei Wenyue1, Song Lijuan3, 4, Yu Jiezhong1, Ma Cungen1, 3   

  1. 1Institute of Brain Science, Shanxi Datong University, Datong 037009, China; 2The Second People’s Hospital of Jincheng, Jincheng 048000, China; 3Research Center of Neurobiology, Shanxi University of Chinese Medicine/The Key Research Laboratory of Benefiting Qi for Acting Blood Circulation Method to Treat Multiple Sclerosis, National Administration of Traditional Chinese Medicine, Jinzhong 030619, China; 4Department of Physiology, Shanxi Medical University, Taiyuan 030032, China
  • Received:2022-10-24 Accepted:2022-12-27 Online:2024-03-08 Published:2023-07-17
  • Contact: Ma Cungen, PhD, Professor, Doctoral supervisor, Institute of Brain Science, Shanxi Datong University, Datong 037009, Shanxi Province, China; Research Center of Neurobiology, Shanxi University of Chinese Medicine/The Key Research Laboratory of Benefiting Qi for Acting Blood Circulation Method to Treat Multiple Sclerosis, National Administration of Traditional Chinese Medicine, Jinzhong 030619, Shanxi Province, China
  • About author:Mu Bingtao, Master, Lecturer, Institute of Brain Science, Shanxi Datong University, Datong 037009, Shanxi Province, China
  • Supported by:
    the National Natural Science Foundation of China (Youth Program), No. 82004028 (to SLJ); Open Project of the State Key Laboratory of Molecular Developmental Biology, Institute of Genetic and Developmental Biology, Chinese Academy of Science, No. 2020-MDB-KF-09 (to SLJ); the Leading Team of Medical Science and Technology of Shanxi Provincial Health Commission, No. 2020TD05 (to MCG); Young Scientist Program of Shanxi University of Chinese Medicine, No. 2021-PY-QN-09 (to SLJ); Shanxi Province Astragalus Resources Industrialization and Industrial Internationalization Collaborative Innovation Center Project, No. HQXTCXZX2016-022 (to MCG); Datong Applied Basic Research Project, No. 2020145 (to YJW)

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摘要：


文题释义：

黄芪甲苷：又称黄芪苷，是治疗气虚证的代表性药物黄芪中生物活性最好的有效成分，可以显著增强细胞代谢功能，减少体内炎性反应，同时可以增强机体特异性免疫与非特异性免疫。课题组前期研究结果表明黄芪内有效成分黄芪糖蛋白能够调节实验性自身免疫性脑脊髓炎小鼠体内免疫反应，并有效保护其血脑屏障，抑制中枢神经系统炎症浸润。
实验性自身免疫性脑脊髓炎：多发性硬化是一种与自身免疫异常密切相关的慢性炎症性疾病，其病理特点为中枢神经系统白质多发性髓鞘脱失，疾病发展过程中复发与缓解相交替，最终导致少突胶质细胞死亡、轴突崩解，甚至出现神经元丢失，神经系统功能出现不可逆损伤。


背景：多发性硬化初始阶段，中枢免疫细胞激活并释放大量炎症因子，引起白质脱髓鞘甚至累及灰质神经元。CD4+T细胞不同亚群之间的分化平衡在实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis，EAE)的病程进展中发挥着重要作用。课题组前期研究结果表明黄芪内有效成分黄芪甲苷能够调节EAE小鼠体内免疫反应，其是否对T细胞亚群分化具有调节作用尚未明确。

目的：探究黄芪甲苷对EAE小鼠治疗效果及其对T细胞的免疫调控机制。
方法：将C57BL/6雌性小鼠分为正常对照组、EAE疾病模型组和黄芪甲苷治疗组，每组8只，后2组使用髓鞘少突胶质细胞糖蛋白35-55(MOG35-55)制备EAE模型，免疫后第10-28天，黄芪甲苷治疗组以40 mg/(kg•d)灌胃给药。免疫当天至第28天，记录各组小鼠的体质量及临床评分；免疫后第28天取小鼠脊髓制成冰冻切片行苏木精-伊红染色、固蓝染色观察脊髓病理改变，流式细胞术检测脾脏T细胞亚群百分比，Western blot法检测脊髓组织中γ干扰素、白细胞介素17、白细胞介素6的蛋白表达，ELISA检测脾细胞上清液中γ干扰素、白细胞介素17、白细胞介素6、白细胞介素4水平。

结果与结论：①与EAE疾病模型组相比，黄芪甲苷治疗能够减少EAE小鼠体质量丢失(P < 0.05)，缓解临床症状(P < 0.05)，减轻脊髓炎症细胞浸润及髓鞘脱失病理改变(分别为P < 0.01和P < 0.05)；②与EAE疾病模型组相比，黄芪甲苷治疗可抑制表达γ干扰素和白细胞介素17的CD4+T细胞亚群比例(分别为P < 0.001和P < 0.001)，上调表达白细胞介素10和转化生长因子β的CD4+T细胞亚群百分比(分别为P < 0.001和P < 0.01)；③黄芪甲苷可下调脊髓和脾脏中γ干扰素(分别为P < 0.05和P < 0.01)、白细胞介素17(分别为P < 0.05和P < 0.05)、白细胞介素6(分别为P < 0.05和P < 0.05)的表达，上调脾脏中抑炎因子白细胞介素4的表达(P < 0.01)；④结果说明，黄芪甲苷可以减轻EAE小鼠的临床症状，其机制与调节脾脏免疫细胞亚群进而抑制炎症细胞向中枢浸润、减少髓鞘脱失有关。

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：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 中枢神经系统, 多发性硬化, 实验性自身免疫性脑脊髓炎, 髓鞘少突胶质细胞糖蛋白35-55, 黄芪甲苷, T细胞, 炎症

Abstract: BACKGROUND: In the initial stage of multiple sclerosis, central immune cells activate and release a large number of inflammatory factors, causing white matter demyelination and even involving gray matter neurons. The equilibrium of differentiation between different subsets of CD4+ T cells plays an important role in the progression of experimental autoimmune encephalomyelitis. The previous results of the research group showed that the active ingredient astragalus glycoprotein in astragalus can regulate the immune response in experimental autoimmune encephalomyelitis mice, and whether it has a regulatory effect on the differentiation of T cell subsets has not been determined.  
OBJECTIVE: To explore the therapeutic effects and immune regulatory mechanisms of astragaloside IV on experimental autoimmune encephalomyelitis mice.
METHODS: Female C57BL/6 mice were divided into the normal control group, experimental autoimmune encephalomyelitis disease model group, and astragaloside IV treatment group (n=8 per group). Myelin oligodendrocyte glycoprotein peptides 35-55 were used for experimental autoimmune encephalomyelitis model induction in the last two groups. On day 10 to 28 after immunization, the astragaloside IV treatment group was treated with 40 mg/kg per day astragaloside IV intragastrically. Body weight and clinical scores of mice in each group were recorded from the immunization day to the 28th day. On the 28th day after immunization, the mouse spinal cord was taken and made into frozen sections for hematoxylin-eosin staining and Lux fast blue staining to observe pathological changes in the spinal cord. Percentage of splenic T cell subsets was detected using flow cytometry. Western blot assay was used to determine the protein expression of interferon-γ, interleukin-17 and interleukin-6 in the spinal cord. Levels of interferon-γ, interleukin-17, interleukin-6 and interleukin-4 in supernatants of cultured splenocytes were determined by ELISA.  
RESULTS AND CONCLUSION: (1) Compared with the experimental autoimmune encephalomyelitis disease model group, astragaloside IV could reduce the degree of weight loss in experimental autoimmune encephalomyelitis mice (P < 0.05), ameliorate clinical symptoms (P < 0.05), inhibit the infiltration of inflammatory cells and alleviate myelin loss (P < 0.01, P < 0.05). (2) Compared with the experimental autoimmune encephalomyelitis disease model group, astragaloside IV could inhibit the proportion of CD4+T cell subsets expressing interferon-γ (P < 0.001) and interleukin-17 (P < 0.001), but increase percentages of CD4+ interleukin-10+ (P < 0.001) and CD4+ transforming growth factor-β+ (P < 0.01) T cell subsets. (3) Astragaloside IV could inhibit the expression of interferon-γ (P < 0.05, P < 0.01), interleukin-17 (P < 0.05, P < 0.05), and interleukin-6 (P < 0.05, P < 0.05) in the spinal cord and spleen, and up-regulate the expression of interleukin-4 (P < 0.01) in spleen. (4) These findings confirm that astragaloside IV alleviates clinical symptoms in experimental autoimmune encephalomyelitis mice, which may be related to regulating the splenic T cell subsets, therefore, inhibiting the infiltration of inflammatory cells into the center and reducing the demyelination.

Key words: central nervous system, multiple sclerosis, experimental autoimmune encephalomyelitis, myelin oligodendrocyte glycoprotein peptides 35-55, astragaloside IV, T cell, inflammation

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