基于miR-34a/SIRT1轴研究柚皮素对H_(2)O_(2)诱导的人视网膜色素上皮细胞氧化损伤的保护作用  

The protective effect of naringenin on H_(2)O_(2)-induced oxidative damage of human retinal pigment epithelial cells via the miR-34a/SIRT1 axis

作　　者：魏抗抗[1] 董伟华[1] 帖红艳 何章彪[1] 赵琳[1] WEI Kangkang;DONG Weihua;TIE Hongyan;HE Zhangbiao;ZHAO Lin(Department of Otolaryngology,Shangqiu Medical College,Shangqiu 476006,China;Department of Human Anatomy,School of Basic Medicine,Zhengzhou University,Zhengzhou 450001)

机构地区：[1]商丘医学高等专科学校五官科教研室,河南商丘476006 [2]郑州大学基础医学院人体解剖学教研室,郑州450001

出　　处：《中国比较医学杂志》2022年第4期77-85,共9页Chinese Journal of Comparative Medicine

基　　金：2016年度河南省医学科技攻关计划项目(201602105)。

摘　　要：目的基于miR-34a/沉默信息调节因子1(SIRT1)轴探讨柚皮素对H_(2)O_(2)诱导的人视网膜色素上皮细胞(RPE)氧化损伤的保护作用。方法MTT法检测不同浓度的柚皮素对H_(2)O_(2)诱导的人视网膜色素上皮细胞ARPE-19存活率的影响以选取适宜柚皮素浓度为后续实验浓度。取对数期生长的ARPE-19细胞分为对照组、H_(2)O_(2)组(200μmol/L)、20μg/mL柚皮素组、20μg/mL柚皮素+mimics NC组、20μg/mL柚皮素+miR-34a mimics组。采用RT-qPCR检测细胞中miR-34a和SIRT1 mRNA的表达,MTT法及Annexin V-FITC/PI双染分别检测细胞存活与凋亡情况,荧光探针检测细胞内ROS水平,生化法检测SOD活性、MDA和GSH水平,并采用JC-1法检测线粒体膜电位(MMP),Western blot检测SIRT1及Bcl-2、Bax、Caspase-3蛋白表达。结果与对照组相比,H_(2)O_(2)组ARPE-19细胞中miR-34a表达、ARPE-19细胞凋亡率、ROS、MDA含量、Bax和Caspase-3表达显著升高(P<0.05),SIRT1 mRNA表达、ARPE-19细胞存活率、SOD、GSH含量、MMP水平、SIRT1和Bcl-2表达显著降低(P<0.05);与H_(2)O_(2)相比,柚皮素可改善H_(2)O_(2)诱导的ARPE-19细胞损伤,下调miR-34a表达,降低ROS、MDA、Bax和Caspase-3含量(P<0.05),上调SIRT1、MMP、Bcl-2表达,增加GSH、SOD活性(P<0.05);上调ARPE-19细胞miR-34a的表达,可逆转柚皮素对H_(2)O_(2)诱导的RPE细胞氧化损伤的保护作用。结论柚皮素可能通过下调miR-34a,促进SIRT1的表达,抑制RPE细胞凋亡,保护H_(2)O_(2)诱导的RPE细胞氧化损伤。Objective To investigate the protective effect of naringenin on H_(2)O_(2)-induced oxidative damage of human retinal pigment epithelial(RPE)cells via the miR-34a/silent information regulator 1(SIRT1)axis.Methods MTT assay was used to detect the effect of naringenin at different concentrations on the survival rate of ARPE-19 cells treated with H_(2)O_(2),and the appropriate naringenin concentration for subsequent experiments was identified.ARPE-19 cells in logarithmic phase were divided into the control group,H_(2)O_(2) group(200μmol/L),20μg/mL naringenin group,20μg/mL naringenin+mimics NC group and 20μg/mL naringenin+miR-34a mimics group.RT-qPCR was used to detect the expression of miR-34a and SIRT1 mRNA,MTT assay and Annexin V-FITC/PI double staining were used to detect cell survival and apoptosis,respectively.A fluorescence probe was used to detect the level of ROS,and a biochemical method was used to detect the activity of SOD and levels of MDA and GSH.JC-1 method was used to detect the mitochondrial membrane potential(MMP),and Western blot was used to detect the proteins expression of SIRT1 and Bcl-2,Bax and Caspase-3.Results The expression of miR-34a,apoptosis rate,contents of ROS and MDA and expression of Bax and Caspase-3 in ARPE-19 cells were significantly increased in the H_(2)O_(2) group compared with those in the control group(P<0.05).The expression of SIRT1 mRNA,survival rate of APRE-19 cells,contents of SOD and GSH,level of MMP,expression of SIRT1 and Bcl-2 were significantly decreased in the H_(2)O_(2) group compared with those in the control group(P<0.05).Naringenin improved the damage of ARPE-19 cells induced by H_(2)O_(2),down-regulated the expression of miR-34a,decreased the contents of ROS,MDA,Bax and Caspase-3(P<0.05),up-regulated the expression of SIRT1,MMP and Bcl-2 and increased the activities of GSH and SOD compared with effects in the H_(2)O_(2) group(P<0.05).Up-regulation of miR-34a expression in ARPE-19 cells reversed the protective effect of naringenin on H_(2)O_(2)-induced oxidati

关 键 词：柚皮素 视网膜色素上皮细胞 氧化损伤 MIR-34A 沉默信息调节因子1 

分 类 号：R-33[医药卫生]