迷迭香酸通过激活Nrf2/HO-1通路对Aβ_(1-42)所致星形胶质细胞损伤的保护作用  

Protective effect of rosmarinic acid on Aβ_(1-42)-induced astrocyte injury by activating Nrf2/HO-1 pathway

作　　者：张亚苹 隋海娟 闫恩志 ZHANG Yaping;SUI Haijuan;YAN Enzhi(Department of Pharmacology,School of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121001,China)

机构地区：[1]锦州医科大学基础医学院药理学教研室,辽宁锦州121001

出　　处：《吉林大学学报（医学版）》2023年第1期22-30,共9页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金项目(81400875);辽宁省科技厅计划项目(2019-ZD-0823);辽宁省锦州市科技局科学技术计划项目(JZ2022B040)。

摘　　要：目的:探讨迷迭香酸(RA)对β淀粉样蛋白1-42(Aβ_(1-42))引起的星形胶质细胞损伤的保护作用及炎症因子释放的抑制作用,并阐明其相关机制。方法:原代培养星形胶质细胞,将细胞分为对照组,Aβ_(1-42)组(5μmol∙L^(-1)),不同浓度(20,50和100μmol∙L^(-1))RA+Aβ_(1-42)组,核因子E2相关因子2(Nrf2)抑制剂(ML385)组,ML385+RA+Aβ_(1-42)组。星形胶质细胞以不同浓度(20、50和100μmol∙L^(-1))RA预处理24 h,再给予终浓度5μmol∙L^(-1)Aβ_(1-42)处理24 h,ML385组在加入RA前先加入终浓度10μmol∙L^(-1)ML385预处理6 h。MTT法检测各组细胞活性,乳酸脱氢酶(LDH)试剂盒检测各组细胞LDH释放率,酶联免疫吸附试验(ELISA)试剂盒检测各组细胞中白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)水平,Griess法检测各组细胞培养液中一氧化氮(NO)水平,免疫荧光法检测各组细胞中胶质原纤维酸蛋白(GFAP)和Nrf2表达情况,Western blotting法检测各组细胞中GFAP、Nrf2、血红素加氧酶1(HO-1)蛋白表达水平。结果:与对照组比较,Aβ_(1-42)组细胞活性明显降低(P<0.01),LDH释放率及细胞中IL-1β、TNF-α和NO水平明显升高(P<0.01),GFAP荧光强度明显增强,Nrf2荧光强度明显减弱,细胞中GFAP蛋白表达水平明显升高(P<0.01),Nrf2和HO-1蛋白表达水平明显降低(P<0.01);与Aβ_(1-42)组比较,不同浓度RA+Aβ_(1-42)组细胞活性明显升高(P<0.05或P<0.01),LDH释放率及细胞中IL-1β、TNF-α和NO水平明显降低(P<0.01),GFAP荧光强度明显减弱,Nrf2荧光强度明显增强,细胞中GFAP蛋白表达水平明显降低(P<0.01),Nrf2和HO-1蛋白表达水平明显升高(P<0.01);与RA(50μmol∙L^(-1))+Aβ_(1-42)组比较,ML385+RA+Aβ_(1-42)组细胞活性明显降低(P<0.01),LDH释放率及细胞中IL-1β、TNF-α和NO水平明显升高(P<0.01),GFAP荧光强度明显增强,Nrf2荧光强度明显减弱,细胞中GFAP蛋白表达水平明显升高(P<0.01),Nrf2和HO-1蛋白表达水平明显降低(P<0.01)。结论:RA对AβObjective:To investigate the protective effect of rosaminic acid(RA)on astrocyte injury induced byβ-amyloid protein 1-42(Aβ_(1-42))and the inhibitory effect of the inflammatory factor release,and to clarify the related mechanism.Methods:After primary culture,the astrocytes were divided into control group,Aβ_(1-42)group(5μmol∙L^(-1)),different concentrations(20,50,100μmol∙L^(-1))of RA+Aβ_(1-42)groups,nuclear factor E2-related factor 2(Nrf2)inhibitor ML385 group,and ML385+RA+Aβ_(1-42)group.The astrocytes were pretreated with different concentrations(20,50,100μmol·L^(-1))of RA for 24 h and then treated with Aβ_(1-42)at a final concentration of 5μmo·l L^(-1)for 24 h,and the final concentration of 10μmol·L^(-1)ML385 was pretreated for 6 h before added RA in ML385 group.The cell activities in various groups were detected by MTT method,the lactate dehydrogenase(LDH)release rates of the cells in various groups were detected by LDH kits,the levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in the cells in various groups were detected by enzyme linked immunosorbent assay(ELISA)kits,the levels of nitric oxide(NO)in culture medium in various groups were detected by Griess method,the expressions of glial fibrillary acidic protein(GFAP)and Nrf2 in the cells in various groups were detected by immunofluorescence method,and the expression levels of GFAP,Nrf2 and heme oxygenase-1(HO-1)proteins in the cells in various groups were detected by Western blotting method.Results:Compared with control group,the cell activity in Aβ_(1-42)group was significantly decreased(P<0.01),the LDH release rate and the levels of IL-1β,TNF-α,and NO were significantly increased(P<0.01),the fluorescence intensity of GFAP was significantly enhanced,the fluorescence intensity of Nrf2 was significantly reduced,the expression level of GFAP protein in the cells was significantly increased(P<0.01),and the expression levels of Nrf2 and HO-1 proteins were significantly decreased(P<0.01).Compared with Aβ_(1-42)group,the ce

关 键 词：迷迭香酸 星形胶质细胞 Β淀粉样蛋白 核因子E2相关因子2 血红素加氧酶1 

分 类 号：R742[医药卫生—神经病学与精神病学] R285.5[医药卫生—临床医学]