摘要
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25μmol/L,DAPT终浓度为50μmol/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(
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 25μmol/L and the final concentration of DAPT was 50μmol/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,
作者
于婧文
郭敏芳
张冰心
穆秉桃
孟涛
张慧宇
马存根
殷金珠
宋丽娟
尉杰忠
Yu Jingwen;Guo Minfang;Zhang Bingxin;Mu Bingtao;Meng Tao;Zhang Huiyu;Ma Cungen;Yin Jinzhu;Song Lijuan;Yu Jiezhong(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;Department of Neurosurgery,Tongmei General Hospital/Key Laboratory of Prevention and Treatment of Neurological Diseases,Shanxi Provincial Health Commission,Datong 037003,Shanxi Province,China;The Fifth People’s Hospital of Datong,Datong 037009,Shanxi Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2024年第31期5022-5028,共7页
Chinese Journal of Tissue Engineering Research
基金
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。
