摘要
目的:探讨积雪草酸(AA)对脂多糖(LPS)诱导大鼠原代海马神经元炎症和氧化应激损伤的作用,并阐明其作用机制。方法:原代培养大鼠海马神经元(免疫荧光染色法鉴定细胞纯度)分为对照组、LPS组(10 mg·L^(-1)LPS)、LPS+AA组(10 mg·L^(-1)LPS+10、20和40μmol·L^(-1)AA)、AA组(20μmol·L^(-1)AA)、ML385组[10μmol·L^(-1)核因子E2相关因子2(Nrf2)抑制剂]和LPS+ML385+AA组(10mg·L^(-1)LPS+10μmol·L^(-1)ML385+20μmol·L^(-1)AA);给药处理后,噻唑蓝(MTT)法检测各组大鼠海马神经元的存活率,乳酸脱氢酶(LDH)试剂盒检测各组大鼠海马神经元LDH漏出率,酶联免疫吸附试验(ELISA)试剂盒检测各组大鼠海马神经元上清液中炎症因子[白细胞介素(IL)-1β和肿瘤坏死因子(TNF)-α]水平以及各组大鼠海马神经元中超氧化物歧化酶(SOD)活性和丙二醛(MDA)水平,Griess法测定各组大鼠海马神经元上清液中一氧化氮(NO)水平,免疫荧光法检测各组大鼠海马神经元中Nrf2和血红素氧合酶1(HO-1)蛋白表达情况;Western blotting法检测各组大鼠海马神经元中Nrf2、HO-1、核因子κB(NF-κB)和B细胞淋巴瘤2(Bcl-2)蛋白表达水平。结果:与对照组比较,LPS组大鼠海马神经元中的海马神经元存活率、SOD活性和Bcl-2表达水平明显降低(P<0.01),LDH漏出率、IL^(-1)β和TNF-α水平、MDA水平、NO水平以及NF-κB蛋白表达水平均明显升高(P<0.01),Nrf2和HO-1的荧光强度明显减弱,Nrf2和HO-1蛋白表达水平明显降低(P<0.01);与LPS组比较,LPS+10μmol?L^(-1)AA组和LPS+20μmol?L^(-1)AA组大鼠海马神经元存活率、SOD活性和Bcl-2表达水平明显升高(P<0.01),LDH漏出率、IL^(-1)β和TNF-α水平、MDA水平、NO水平以及NF-κB蛋白表达水平明显降低(P<0.05或P<0.01),Nrf2和HO-1的荧光强度明显升高(P<0.01),Nrf2和HO-1蛋白表达水平明显升高(P<0.01);与LPS+20μmol?L^(-1)AA组比较,LPS+ML385+AA组海马神经元中Nrf2和HO-1荧光强度明显减弱(P<0.01),细胞核和细胞质中Nrf2、细胞中HO-1和Bcl-2蛋白表达水平明显降低(P<0.01),NF-κB蛋白表达水平明显升高(P<0.01)。结论:AA可改善LPS诱导的原代培养大鼠海马神经元炎症和氧化应激损伤,其机制可能与激活Nrf2/HO-1信号通路有关。
Objective:To discuss the effect of asiatic acid(AA)on the inflammation and oxidative stress damage induced by lipopolysaccharide(LPS)in the primary cultured hippocampus neurons,and to clarify its mechanism.Methods:The primary cultured rat hippocampus neurons(cell purity identified by immunofluorescence staining)were divided into control group,LPS(10 mg·L^(-1))group,and LPS+AA group(10 mg·L^(-1)LPS+10,20,and 40μmol·L^(-1)AA),AA group(20μmol·L^(-1)AA),ML385 group[10μmol·L^(-1)nuclear factor erythroid 2-related factor(Nrf2)inhibitor],and LPS+ML385+AA group(10 mg·L^(-1)LPS+10μmol·L^(-1)ML385+20μmol·L^(-1)AA).After drug treatment,methylthiazolyldiphenyl-tetrazolium bromide(MTT)method was used to detect the survival rates of the hippocampus neurons in various groups;lactate dehydrogenase(LDH)kit was used to detect the LDH leakage rates of the hippocampus neurons in various groups;enzyme linked immunosorbent assay(ELISA)kit was used to detect the expression levels of inflammatory factors[interleukin(IL)-1βand tumor necrosis factor(TNF)-α]and the activities of superoxide dismutase(SOD)and malondialdehyde(MDA)levels in the hippocampus neurons in various groups;Griess method was used to detect the nitric oxide(NO)levels in supernatant of the hippocampus neurons in various groups;immunofluorescence staining was used to detect the expressions of Nrf2 and heme oxygenase-1(HO-1)proteins in the hippocampus neurons in various groups;Western blotting method was used to detect the expression levels of Nrf2,HO-1,nuclear factor-kappa B(NF-κB),and B-cell lymphoma 2(Bcl-2)proteins in the hippocampus neurons in various groups.Results:Compared with control group,the survival rate of the hippocampus neurons,SOD activity,and Bcl-2 expression level in the cells in LPS group were significantly decreased(P<0.01),while the LDH leakage rate,expression levels of IL^(-1)βand TNF-α,MDA level,and NO level,as well as the expression level of NF-κB protein,were significantly increased(P<0.01);the fluorescence intensities and expression levels of Nrf2 and HO-1 proteins in hippocampus neurons were significantly decreased(P<0.01).Compared with LPS group,the survival rates of hippocampus neurons,SOD activities,and expression levels of Bcl-2 in the cells in LPS+10μmol·L^(-1)AA group and 20μmol·L^(-1)AA group were significantly increased(P<0.01),while the LDH leakage rates,expression levels of IL^(-1)βand TNF-α,MDA levels,and NO levels,as well as expression levels of NF-κB protein,were significantly decreased(P<0.05 or P<0.01),and the fluorescence intensities and protein expression levels of Nrf2 and HO-1 in the cells were significantly increased(P<0.01).Compared with LPS+20μmol·L^(-1)AA group,the fluorescence intensities of Nrf2 and HO-1 in the cells in LPS+ML385+AA group were significantly decreased(P<0.01),and the expression levels of Nrf2 protein in the nucleus and cytoplasm,the expression levels of HO-1 and Bcl-2 proteins in the cells were significantly decreased(P<0.01),while the expression level of NF-κB protein was significantly increased(P<0.01).Conclusion:AA can improve LPS-induced inflammation and oxidative stress damage in the primary cultured rat hippocampus neurons,and its mechanism may be related to the activation of the Nrf2/HO-1 signaling pathway.
作者
白燕燕
周禹彤
隋海娟
刘卓
BAI Yanyan;ZHOU Yutong;SUI Haijuan;LIU Zhuo(Department of Pharmacology,School of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121000,China)
出处
《吉林大学学报(医学版)》
北大核心
2025年第1期85-95,共11页
Journal of Jilin University:Medicine Edition
基金
辽宁省科技厅自然科学基金指导项目(2019-ZD-0818)
锦州市科技局指导性科技计划项目(JZ2023B055)
辽宁省教育厅大学生创新创业训练计划项目(20201016029)。
