法舒地尔通过调节Nrf2/HO-1信号通路抑制脂多糖诱导的星形胶质细胞损伤  

Fasudil inhibits lipopolysaccharide-induced astrocytic injury by regulating Nrf2/HO-1 signaling pathway

作　　者：郭敏芳[1] 张慧宇 章培军 白振军 于婧文[1] 王玉银 魏文悦 宋丽娟 柴智 尉杰忠[1,4] 马存根 Guo Minfang;Zhang Huiyu;Zhang Peijun;Bai Zhenjun;Yu Jingwen;Wang Yuyin;Wei Wenyue;Song Lijuan;Chai Zhi;Yu Jiezhong;Ma Cungen(Institute of Brain Science,Shanxi Datong University/Key Laboratory of Shanxi Province for Basic and Applied Research on Neuroinflammation and Degenerative Diseases,Datong 037009,Shanxi Province,China;Key Laboratory of Multiple Sclerosis,Nourishing Qi and Activating Blood,National Administration of Traditional Chinese Medicine/Neurobiology Research Center,Shanxi University of Chinese Medicine,Jinzhong 030619,Shanxi Province,China;Department of Neurology,First Clinical Medical College of Shanxi Medical University,Taiyuan 030000,Shanxi Province,China;Department of Neurology,The Fifth People’s Hospital of Datong,Datong 037009,Shanxi Province,China)

机构地区：[1]山西大同大学脑科学研究所/神经炎症及变性疾病基础与应用研究山西省重点实验室,山西省大同市037009 [2]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,山西中医药大学,山西省晋中市030619 [3]山西医科大学第一临床医学院神经科,山西省太原市030000 [4]大同市第五人民医院神经科,山西省大同市037009

出　　处：《中国组织工程研究》2021年第31期5012-5017,共6页Chinese Journal of Tissue Engineering Research

基　　金：山西省平台基地专项(201805D111009),项目负责人:马存根;山西省平台基地专项(201805D131005),项目负责人:尉杰忠;山西省高等学校科技创新项目(2020L0484),项目负责人:郭敏芳;神经炎症及变性疾病基础与应用研究山西省重点实验室开放课题(KF2019007),项目负责人:郭敏芳。

摘　　要：背景:越来越多的证据表明,ROCK信号通路参与了炎症过程和氧化应激。法舒地尔是一种有效的Rho激酶抑制剂,课题组前期的系列研究证明法舒地尔具有神经保护作用。然而,法舒地尔是否对星形胶质细胞的氧化损伤具有保护作用及其可能的机制尚不明确。目的:探讨法舒地尔对脂多糖诱导的星形胶质细胞氧化应激损伤的保护作用及其机制。方法:将培养的C57BL/6小鼠星形胶质细胞分为PBS对照组、脂多糖模型组(1 mg/L)、脂多糖(1 mg/L)+法舒地尔(15 mg/L)干预组。药物作用24 h后,MTT法检测星形胶质细胞的活性,试剂盒测定星形胶质细胞中丙二醛水平和超氧化物歧化酶活性以评估细胞的氧化应激水平,免疫荧光染色法检测凋亡相关蛋白cleaved-caspase-3的表达,免疫荧光染色法检测星形胶质细胞中核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)的核内转移及血红素加氧酶1(heme oxygenase-1,HO-1)的表达,Western blot法检测总Nrf2、核内Nrf2和HO-1的蛋白水平。结果与结论:①与PBS对照组比较,脂多糖组星形胶质细胞活性降低,法舒地尔改善脂多糖诱导的细胞损伤;②与PBS对照组比较,脂多糖导致星形胶质细胞中超氧化物歧化酶活性明显降低,丙二醛水平明显升高(P<0.01);法舒地尔能够显著提高超氧化物歧化酶活性(P<0.01),减少丙二醛水平(P<0.01);③与PBS对照组比较,脂多糖组细胞cleaved-caspase-3表达明显增加;与脂多糖组比较,法舒地尔干预组cleaved-caspase-3表达降低;④法舒地尔可以促进Nrf2A的核内转位,明显增强脂多糖损伤后总Nrf2、核内Nrf2和HO-1的表达;⑤结果表明,法舒地尔能够通过调节Nrf2/HO-1信号通路抑制脂多糖诱导的星形胶质细胞损伤。BACKGROUND:More and more evidences show that ROCK signaling pathway is involved in inflammation and oxidative stress.Fasudil is an effective Rho kinase inhibitor.Our previous series of studies have proved that fasudil has neuroprotective effects.However,it is still unclear that whether fasudil has protective effect on oxidative damage to astrocytes and its possible mechanisms.OBJECTIVE:To explore the protective effect and mechanism of fasudil on lipopolysaccharide-induced oxidative injury in astrocytes.METHODS:Primary C57BL/6 mouse astrocytes were cultured and divided into PBS control group,lipopolysaccharide stimulation group(1 mg/L),lipopolysaccharide(1 mg/L)combined with fasudil(15 mg/L)treatment group.After 24 hours of treatment,MTT assay was used to detect the viability of astrocytes.The content of malondialdehyde and the activity of superoxide dismutase in astrocytes were measured using the kit to evaluate the level of oxidative stress.Immunofluorescence staining was used to detect the expression of cleaved-caspase-3,the nuclear translocation of nuclear factor erythroid 2-related factor 2(Nrf2)and the expression of heme oxygenase-1(HO-1)in astrocytes.Western blot assay was used to detect the total Nrf2,nuclear Nrf2 and HO-1 protein levels.RESULTS AND CONCLUSION:(1)Compared with the PBS control group,the astrocyte viability in the lipopolysaccharide stimulation group was reduced,and fasudil treatment improved the cell damage induced by lipopolysaccharide.(2)Compared with the PBS control group,lipopolysaccharide resulted in a significant decrease in the activity of superoxide dismutase and a significant increase in the level of malondialdehyde in astrocytes(P<0.01).Fasudil significantly increased the activity of superoxide dismutase(P<0.01)and reduced the level of malondialdehyde(P<0.01).(3)Compared with the PBS control group,the expression of cleaved-caspase-3 was significantly increased in the lipopolysaccharide stimulation group.Compared with the lipopolysaccharide stimulation group,the expression of cleav

关 键 词：星形胶质细胞 脂多糖 法舒地尔 核因子E2相关因子2 氧化应激 凋亡 实验 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]