基于Nrf2通路探讨环黄芪醇调控H_(2)O_(2)致HT22细胞氧化应激损伤的作用

Anti-Oxidative Stress Injury Effect of Cycloastragenol on HT22 Cells Induced by H_(2)O_(2) Based on Nrf2 Pathway

目的:探讨环黄芪醇对H_(2)O_(2)处理的HT22细胞氧化应激损伤的保护作用,并对其潜在机制进行研究。方法:首先采用MTT法检测经不同浓度环黄芪醇处理的HT22细胞活性差异,对环黄芪醇的有效浓度进行筛选。将细胞分为5组,其中空白组和H_(2)O_(2)组不加入环黄芪醇,其余3组根据筛选结果分别加入不同浓度的环黄芪醇(50、75、100μmol/L)培养24 h。然后除空白组外,其余4组均加入H_(2)O_(2)作用4 h。采用DCFH-DA活性氧探针检测各组细胞中ROS的生成水平;免疫荧光染色检测各组细胞中Nrf2的入核情况;蛋白免疫印迹法检测各组细胞凋亡相关蛋白的表达情况。结果:本实验中,使用环黄芪醇作用于细胞的有效浓度分别为50、75、100μmol/L。随着环黄芪醇浓度的增加,环黄芪醇的保护作用也随之增强。环黄芪醇可抑制细胞中ROS的产生,促进Nrf2的入核,降低凋亡相关蛋白Caspase-3、Bax的表达,增加CleavedCaspase-3、Bcl-2的表达,减少细胞色素C的释放,并表现出浓度依赖性。结论:环黄芪醇对H_(2)O_(2)导致的HT22细胞的氧化应激损伤呈现出保护作用,这种保护作用可能是通过激活Nrf2通路来实现的。

Objective:To investigate the protective effect of cycloastragenol on oxidative stress injury of HT22 cells induced by H_(2)O_(2),and to explore its potential mechanism.Methods:MTT was used to screen the effective concentration of cycloastragenol by detecting the cell viability of HT22 treated by different concentrations of cycloastragenol.The cells were divided into five groups,different concentrations of cycloastragenol(50μmol/L,75μmol/L,100μmol/L)were added into the medication groups but for the blank group and the H_(2)O_(2) group.Apart from the blank group,the other fours groups were intervened with H_(2)O_(2).The ROS production level in cells was detected by DCFH-DA probe to evaluate the antioxidant effect of cycloastragenol.Immunofluorescence staining was used to detect the entry of Nrf2 into the nucleus,and the regulatory effect of that was evaluated.Western Blot was used to detect the expressions of apoptosis-related proteins and to evaluate the protective effect of cycloastragenol on oxidative stress injury of HT22 cells intervened by H_(2)O_(2).Results:In the experiment,the effective concentrations of cycloastragenol on cells were 50μmol/L,75μmol/L and 100μmol/L.With the increased concentration of cycloastragenol,the protective effect was enhanced.Cycloastragenol could inhibit the production of ROS in cells,promote Nrf2 entering into the nucleus,decrease the expression of apoptosis-related proteins of Caspase-3 and Bax,increase the expressions of Cleaved Caspase-3 and Bcl-2 and reduce the release of cytochrome C in a concentration-dependent manner.Conclusion:Cycloastragenol has a protective effect on oxidative stress injury of HT22 cells induced by H_(2)O_(2),and the protective effect may be realized by regulating the activation of Nrf2 pathway.