辅酶Q10对人视网膜色素上皮细胞氧化应激损伤的保护作用

摘要

Background Multiple factors are associated with the pathogenesis of age-related macular degeneration (AMD),and the oxidative stress injury of retinal pigment epithelial (RPE) cells is thought to play a key role.Coenzyme Q10 (CoQ10) is verified to be a powerful antioxidant.Exploring if CoQ10 has a protective effect on RPE cells from oxidative stress injury is of important significance for the prevention and treatment of AMD.Objective This study was conducted to investigate the protective efficacy of CoQ10 on RPE cells against oxidative stress and its potential mechanisms.Methods Human RPE cell line,D407,was used for the study.The RPE cells were subjected to oxidative stress with 400 μmol/L tert-butyl hydroperoxide (TBHP) in the absence or presence of CoQ10 at different concentrations (0.01,1,100 μmol/L).The cell viability was determined using a cell counting kit8 (CCK-8) assay.Apoptosis was detected by flow cytometry(FCM) using annexin V-FITC and PI staining.The levels of oxidative stress in the cells that were exposed to different oxidizing conditions were determined using a Thiobarbituric Acid-reactive Substances (TBARS) Assay Kit.Reactive oxidative species (ROS) of the cells were detected with FCM measurements of peroxide-dependent oxidation of 2'-7'-dichlorofluorescein-diacetate (DCFHDA).Expressions of the pro-apoptotic gene Fas mRNA and Fas protein were assayed by real-time PCR and Western blot analysis,respectively.Results The survival RPE cells were obviously less in the only TBHP treatment group than those in the normal control group.However,the number of the survival cells was gradually increased in the 0.01,1,100 μmol/L CoQ10 groups in comparison with the only TBHP treatment group.Compared with only TBHP treatment group,the proliferative value (A450) was significantly enhanced in the 1 μmol/L CoQ10 group and 100 μmol/L CoQ10 group (0.52±0.10 vs.0.25 ±0.03,0.59±0.06 vs.0.25 ±0.03) (P =0.041,0.002),and the cell apoptosis rate was significantly lower than that of RPE cell([72.1 ±6.6] ％ vs.[91.7±2.3] ％,[69.0±4.4] ％ vs.[91.7±2.3]％) (P=0.032,0.004).The lipid peroxidation levels were gradually declined in the 0.01,1,100 μmol/L CoQ10 groups compared with only TBHP treatment group (P=0.047,0.030,0.015),and the ROS in the cells were lowed in the 0.01,1,100 μmol/L CoQ10 groups,showing significant differences between the 1 μmol CoQ10 group or 100 μmol CoQ10 group and only TBHP treatment group (5.25±0.90 vs.11.39±2.30,7.91±0.80 vs.11.39±2.30)(P=0.028,0.007).Compared with only TBHP treatment group,the expressions of Fas mRNA in the cells were considerably decreased in the 1 μmol/L,100 μ mol/L CoQ10 groups (P=0.049,0.008),and the expressions of Fas protein in the cells were considerably decreased in the 0.01,1,100 μmol/L CoQ10 groups (P =0.001,0.000,0.000).Conclusions CoQ10 appears to has protective effects on RPE cells from oxidative stress damage in vitro in a dose-dependent manner probably by down-regulating the pro-apoptotic protein.%背景 有多种因素参与年龄相关性黄斑变性(AMD)的发病,其中视网膜色素上皮(RPE)细胞的氧化应激损伤与AMD的发病密切相关.实验和临床研究表明,辅酶Q10是一种强抗氧化剂,探讨其对人RPE细胞的氧化应激损伤是否有保护作用对于AMD的防治有重要的临床意义. 目的 探讨辅酶Q10对体外培养的人RPE细胞氧化应激损伤的保护作用及其作用机制. 方法 体外培养人RPE细胞,分为正常对照组(单纯培养基培养)、不同浓度(0.01、1、100 μmol/L)辅酶Q10预处理组和单纯叔丁基过氧化氢(TBHP)处理组,其中各浓度辅酶Q10预处理组以辅酶Q10预处理后暴露于TBHP,光学显微镜下观察各组RPE细胞的形态；用细胞计数试剂盒-8(CCK-8)法检测各组RPE细胞活性的变化；Annexin V-FITC/PI染色后用流式细胞仪检测细胞凋亡情况；TBARS法检测细胞的脂质过氧化水平；用DCFH-DA荧光法检测细胞内活性氧簇(ROS)水平；实时定量PCR法检测RPE细胞中促凋亡基因Fas mRNA的表达；Western blot法检测细胞中Fas蛋白的表达.结果 单纯TBHP处理组RPE细胞贴壁数量较正常对照组明显减少,而0.01、1、100 μmol/L辅酶Q10预处理组贴壁细胞数较单纯TBHP处理组增加,高浓度辅酶Q10预处理组细胞形态的改善和细胞存活的数目更接近正常对照组.与单纯TBHP处理组比较,1μmol/L、100 μmol/L辅酶Q10预处理组RPE细胞吸光度(A450)值明显增高(0.52±0.10 vs.0.25 ±0.03、0.59±0.06 vs.0.25±0.03),差异均有统计学意义(P=0.041、0.002);RPE细胞的凋亡率明显下降[(72.1±6.6)％vs.(91.7±2.3)％、(69.0±4.4)％ vs.(91.7±2.3)％],差异均有统计学意义(P=0.032、0.004)；0.01、1、100 μmol/L辅酶Q10预处理组的细胞脂质过氧化水平依次降低,与单纯TBHP处理组比较差异均有统计学意义(P=0.047、0.030、0.015),与单纯TBHP组相比,0.01、1、100 μmol/L辅酶Q10预处理组细胞内ROS水平逐渐下降,1μmol/L、100tμmol/L辅酶Q10预处理组细胞内ROS水平明显低于单纯TBHP处理组(5.25±0.90 vs.11.39±2.30、7.91 ±0.80 vs.11.39±2.30),差异均有统计学意义(P=0.028、0.007)；与单纯TBHP处理组相比,辅酶Q10预处理组细胞中Fas mRNA表达量均有不同程度的下降,1 μmol/L、100 μmol/L辅酶Q10预处理组细胞中Fas mRNA表达量的差异均有统计学意义(P=0.049、0.008)；0.01、1、100μmol/L辅酶Q10预处理组细胞中Fas蛋白表达量均明显下降,差异均有统计学意义(P=0.001、0.000、0.000). 结论 辅酶Q10对体外培养的人RPE细胞具有保护作用,其作用在一定范围内呈浓度依赖性,这种保护作用可能与减少细胞的氧化应激损伤和抑制细胞凋亡有关.