中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (31): 5022-5028.doi: 10.12307/2024.726

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

黄芪甲苷可抑制炎性诱导星形胶质细胞激活及炎症反应

于婧文1，郭敏芳1，张冰心1，穆秉桃1，孟  涛1，张慧宇1，马存根1，2，殷金珠3，宋丽娟2，3，尉杰忠1，2，4   

  1. 1山西大同大学脑科学研究所/分子细胞免疫学大同市重点实验室/附属第一医院神经科，山西省大同市   037009；2山西中医药大学国家中医药管理局多发性硬化益气活血重点研究室/神经生物学研究中心，山西省晋中市   030619；3国药同煤总医院神经外科/山西省卫健委神经疾病防治研究重点实验室，山西省大同市   037003；4山西大同市第五人民医院，山西省大同市   037009
  • 收稿日期:2023-09-06 接受日期:2023-11-01 出版日期:2024-11-08 发布日期:2024-01-22
  • 通讯作者: 尉杰忠，博士，教授，山西大同大学脑科学研究所/分子细胞免疫学大同市重点实验室/附属第一医院神经科，山西省大同市 037009；山西中医药大学国家中医药管理局多发性硬化益气活血重点研究室/神经生物学研究中心，山西省晋中市 030619；山西大同市第五人民医院，山西省大同市 037009 宋丽娟，博士，副教授，山西中医药大学国家中医药管理局多发性硬化益气活血重点研究室/神经生物学研究中心，山西省晋中市 030619；国药同煤总医院神经外科/山西省卫健委神经疾病防治研究重点实验室，山西省大同市 037003
  • 作者简介:于婧文，女，1985年生，山西省大同市人，汉族，2010年山西大学毕业，硕士，高级实验师，主要从事中医药防治神经炎性变性疾病方面的研究。
  • 基金资助:
    山西省基础研究计划(20210302123337)，项目负责人：于婧文；山西省基础研究计划(20210302123478)，项目负责人：张慧宇；山西大同大学校级科研项目(2022K17)，项目负责人：于婧文；山西省大学生创新创业训练计划项目(XDC2022119)，项目负责人：张冰心；山西省卫健委医学科技领军团队(2020TD05)，项目负责人：马存根；国家中医药管理局张仲景传承与创新专项(GZY-KJS-2022-048-1)，项目负责人：宋丽娟；2022年山西省科技创新青年人才团队(202204051001028)，项目负责人：宋丽娟；山西省卫健委2022年度中医药科研课题立项计划(2022ZYYC090)，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；山西中医药大学2022年度科技创新团队(2022TD2010)，项目负责人：马存根，宋丽娟

Astragaloside inhibits astrocyte activation and inflammatory response induced by inflammation

Yu Jingwen1, Guo Minfang1, Zhang Bingxin1, Mu Bingtao1, Meng Tao1, Zhang Huiyu1, Ma Cungen1, 2, Yin Jinzhu3, Song Lijuan2, 3, Yu Jiezhong1, 2, 4   

  1. 1Institute of Brain Science/Key Laboratory of Molecular Cellular Immunology in Datong City/Department of Neurology of First Affiliated Hospital, Shanxi Datong University, Datong 037009, Shanxi Province, China; 2Key Research Laboratory of Benefiting Qi for Acting Blood Circulation Method to Treat Multiple Sclerosis of State Administration of Traditional Chinese Medicine/Research Center of Neurobiology, Shanxi University of Chinese Medicine, Jinzhong 030619, Shanxi Province, China; 
  • Received:2023-09-06 Accepted:2023-11-01 Online:2024-11-08 Published:2024-01-22
  • Contact: Yu Jiezhong, MD, Professor, Institute of Brain Science/Key Laboratory of Molecular Cellular Immunology in Datong City/Department of Neurology of First Affiliated Hospital, Shanxi Datong University, Datong 037009, Shanxi Province, China; Key Research Laboratory of Benefiting Qi for Acting Blood Circulation Method to Treat Multiple Sclerosis of State Administration of Traditional Chinese Medicine/Research Center of Neurobiology, Shanxi University of Chinese Medicine, Jinzhong 030619, Shanxi Province, China; The Fifth People’s Hospital of Datong, Datong 037009, Shanxi Province, China Song Lijuan, MD, Associate professor, Key Research Laboratory of Benefiting Qi for Acting Blood Circulation Method to Treat Multiple Sclerosis of State Administration of Traditional Chinese Medicine/Research Center of Neurobiology, Shanxi University of Chinese Medicine, Jinzhong 030619, Shanxi Province, China; Department of Neurosurgery, Tongmei General Hospital/Key Laboratory of Prevention and Treatment of Neurological Diseases, Shanxi Provincial Health Commission, Datong 037003, Shanxi Province, China
  • About author:Yu Jingwen, Master, Senior experimentalist, Institute of Brain Science/Key Laboratory of Molecular Cellular Immunology in Datong City/Department of Neurology of First Affiliated Hospital, Shanxi Datong University, Datong 037009, Shanxi Province, China
  • Supported by:
    Shanxi Basic Research Program, No. 20210302123337 (to YJW); Shanxi Basic Research Program, No. 20210302123478 (to ZHY); University Level Research Project of Shanxi Datong University, No. 2022K17 (to YJW); Innovation and Entrepreneurship Training Program of Shanxi College Students, No. XDC2022119 (to ZBX); Medical Science and Technology Leading Team of Shanxi Provincial Health Commission, No. 2020TD05 (to MCG); Zhang Zhongjing Inheritance and Innovation Project of National Administration of Traditional Chinese Medicine, No. GDY-KJS-2022-048-1 (to SLJ); 2022 Shanxi Science and Technology Innovation Young Talent Team, No. 202204051001028 (to SLJ); 2022 Traditional Chinese Medicine Research Project Plan of Shanxi Provincial Health Commission, No. 2022ZYYC090 (to MCG); Young Scientist Training Project of Shanxi University of Chinese Medicine, No. 2021-PY-QN-09 (to SLJ); 2022 Annual Science and Technology Innovation Team of Shanxi University of Chinese Medicine, No. 2022TD2010 (to MCG, SLJ)

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摘要：


文题释义：

黄芪甲苷：是从传统益气中药黄芪中提取出来的具有生物活性成分的单体，是评价黄芪药材质量优劣的标准。课题组前期研究发现黄芪甲苷对中枢神经系统具有免疫调节、抗炎、抗氧化、抗凋亡以及保护线粒体等功能，可以促进神经的修复和再生。
星形胶质细胞：是中枢神经系统中最丰富的一类胶质细胞，生理状态下具有营养、支持和代谢功能。近年研究发现，星形胶质细胞也参与免疫调节。在炎性环境下，星形胶质细胞被激活，会分化为A1和A2两个亚型，A1型会释放炎性因子，导致神经元退行性病变，A2型可以释放神经营养因子，对神经元起保护作用，但炎症激活下A1型要多于A2型，两者失衡会导致许多疾病：如缺血性脑卒中、帕金森症、阿尔茨海默症等。研究炎性环境下星形胶质细胞的表型改变对于了解这些疾病具有重要的意义。


背景：星形胶质细胞在中枢神经系统疾病的病理中起着重要作用。星形胶质细胞的表型变化及功能改变，提示其可能是中枢神经系统疾病的有效治疗靶点。前期研究证实，黄芪甲苷可以抑制脂多糖(lipopolysaccharide，LPS)诱导的星形胶质细胞炎性反应，其是否可以通过Notch-1及其下游信号通路调控星形胶质细胞表型和功能，尚不清楚。

目的：探讨黄芪甲苷对炎性诱导的星形胶质细胞激活及炎症反应的影响及可能机制。
方法：体外培养新生C57BL/6小鼠大脑皮质星形胶质细胞，CCK-8法检测星形胶质细胞活力确定黄芪甲苷及Notch活性抑制剂DAPT的最适浓度；然后将星形胶质细胞分为5组：PBS组、LPS组、LPS+黄芪甲苷组、LPS+DAPT组和LPS+DAPT+黄芪甲苷组，ELISA法检测炎性因子分泌水平，Griess法检测一氧化氮水平，用Transwell小室将星形胶质细胞与脾脏单个核细胞共培养，观察CD4 T细胞的迁移情况，免疫荧光染色检测星形胶质细胞活化标志物GFAP、星形胶质细胞的A1标记物C3、A2标记物S100A10以及信号通路相关Notch-1、Jag-1的表达，Western blot法检测CFB、C3、S100A10、PTX3、Notch-1、Jag-1和Hes的表达。

结果与结论：①根据CCK-8结果，筛选黄芪甲苷终浓度为25 μmol/L，DAPT终浓度为50 μmol/L进行后续实验；②与PBS组相比，LPS组白细胞介素6、白细胞介素12和一氧化氮分泌水平显著升高(P < 0.05，P < 0.05，P < 0.01)；与LPS组相比，LPS+黄芪甲苷组、LPS+DAPT组、LPS+DAPT+黄芪甲苷组白细胞介素6(均为P < 0.05)、白细胞介素12(P > 0.05，P < 0.05，P < 0.05)和一氧化氮(P < 0.05，P < 0.01，P < 0.01)分泌显著减少；③与PBS组相比，LPS组星形胶质细胞激活明显，GFAP表达明显增多，CD4 T细胞的迁移数量明显增多(P < 0.01)；与LPS组相比，LPS+黄芪甲苷组、LPS+DAPT组、LPS+DAPT+黄芪甲苷组星形胶质细胞激活受到显著抑制，CD4 T细胞迁移减少(P < 0.05，P < 0.05，P < 0.01)；④与PBS组相比，LPS组A1型标记物C3和CFB的表达增加(P < 0.01，P < 0.05)，而A2型标记物S100A10和PTX3的表达减少(P < 0.01，P < 0.05)；与LPS组相比，LPS+黄芪甲苷组、LPS+DAPT组、LPS+DAPT+黄芪甲苷组C3(均为P < 0.01)和CFB(均为P < 0.05)的表达显著减少，S100A10(均为P < 0.01)和PTX3(P < 0.05，P < 0.05和P > 0.05)的表达增加；⑤与PBS组相比，LPS组Jag-1、Notch-1和Hes表达明显增加(均为P < 0.01)；与LPS组相比，LPS+黄芪甲苷组、LPS+DAPT组、LPS+DAPT+黄芪甲苷组Jag-1(均为P < 0.01)、Notch-1(均为P < 0.01)和Hes(P < 0.05，P < 0.01，P < 0.01)表达显著减少；⑥结果表明：黄芪甲苷可通过调控Notch-1信号通路促进星形胶质细胞由A1型向A2型转化，减少炎性因子的分泌和CD4 T细胞的迁移，从而抑制星形胶质细胞的激活和炎性反应。

https://orcid.org/0000-0002-3983-7954 (于婧文) 

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: 黄芪甲苷, 脂多糖, 星形胶质细胞, Notch-1/Hes信号通路, 神经炎性反应, DAPT

Abstract: BACKGROUND: Astrocytes play an important role in the pathology of central nervous system diseases. The phenotypic and functional changes in astrocytes suggest that it may be an effective therapeutic target for central nervous system diseases. Our previous studies have confirmed that astragaloside can inhibit the lipopolysaccharide-induced astrocyte inflammatory response. Whether astragaloside can regulate the phenotype and function of astrocytes through Notch-1 and its downstream signaling pathway remains unclear.
OBJECTIVE: To explore the effect of astragaloside on astrocyte activation and inflammatory response induced by inflammation and its possible mechanism.
METHODS: Cerebral cortex astrocytes derived from neonatal C57BL/6 mouse were cultured in vitro. CCK-8 assay was used to determine the optimum concentration of astragaloside and Notch active inhibitor DAPT. The astrocytes were divided into five groups: PBS group, lipopolysaccharide group, lipopolysaccharide + astragaloside group, lipopolysaccharide + DAPT group and lipopolysaccharide + DAPT + astragaloside group. The secretion level of inflammatory factors was detected by ELISA, and the level of nitric oxide was detected by Griess method. The astrocytes and splenic mononuclear cells were co-cultured in Transwell chamber to observe the migration of CD4T cells. The expression of astrocyte activation marker GFAP, A1 marker C3 and A2 marker S100A10 as well as Notch 1 and Jag-1 was detected by immunofluorescence staining. The expressions of CFB, C3, S100A10, PTX3, Notch-1, Jag-1, and Hes were detected by western blot assay.
RESULTS AND CONCLUSION: (1) According to the results of CCK8 assay, the final concentration of astragaloside was selected as 25 μmol/L and the final concentration of DAPT was 50 μmol/L for follow-up experiments. (2) Compared with PBS group, interleukin-6, interleukin-12 and nitric oxide secretion levels in the lipopolysaccharide group were significantly increased (P < 0.05, P < 0.05, P < 0.01). Compared with the lipopolysaccharide group, interleukin-6 (all P < 0.05), interleukin-12 (P > 0.05, P < 0.05, P < 0.05) and nitric oxide (P < 0.05, P < 0.01, P < 0.01) secretion significantly reduced in the lipopolysaccharide + astragaloside group, lipopolysaccharide +DAPT group, lipopolysaccharide + DAPT + astragaloside group. (3) Compared with the PBS group, the expression of GFAP that is the marker of activated astrocytes and the migration of CD4 T cells were significantly increased in the lipopolysaccharide group (P < 0.01). Compared with the lipopolysaccharide group, astrocyte activation was significantly inhibited and CD4 T cell migration was significantly reduced in the lipopolysaccharide + astragaloside, lipopolysaccharide +DAPT, lipopolysaccharide + DAPT + astragaloside group (P < 0.05, P < 0.05, P < 0.01). (4) Compared with the PBS group, the expressions of A1 markers C3 and CFB in the lipopolysaccharide group were increased (P < 0.01, P < 0.05), and the expressions of A2 markers S100A10 and PTX3 were decreased (P < 0.01, P < 0.05). Compared with the lipopolysaccharide group, C3 (all P < 0.01) and CFB (both P < 0.05) were significantly reduced and S100A10 (all P < 0.01) and PTX3 (P < 0.05, P < 0.05 and P > 0.05) were increased in the lipopolysaccharide + astragaloside, lipopolysaccharide +DAPT, lipopolysaccharide + DAPT + astragaloside group. (5) Compared with the PBS group, the expressions of Jag-1, Notch-1 and Hes in the lipopolysaccharide group were significantly increased (all P < 0.01). Compared with the lipopolysaccharide group, the expressions of Jag-1 (all P < 0.01), Notch-1 (all P < 0.01) and Hes (P < 0.05, P < 0.01 and P < 0.01) were significantly reduced in the lipopolysaccharide + astragaloside, lipopolysaccharide +DAPT, lipopolysaccharide + DAPT + astragaloside group. (6) The results indicate that astragaloside can promote the transformation of astrocytes from A1 to A2 by regulating Notch-1 signaling pathway, reduce the secretion of inflammatory factors and the migration of CD4 T cells, and thus inhibit astrocyte activation and inflammatory response. 

Key words: astragaloside, lipopolysaccharide, astrocyte, Notch-1/Hes signaling pathway, neuroinflammatory response, DAPT

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