叶黄素对H2O2诱导的人视网膜Muller细胞氧化应激干预的作用机制  

Molecular mechanism of lutein protecting Miiller cells against oxidative stress induced by H2O2

作　　者：杨旭[1] 王明臣[1] 孙艳艳[2] 安慧娟[1] 李庆福[2] 鲍玉洲[3] 

机构地区：[1]郑州大学基础医学院生物化学与分子生物学教研室,450001 [2]河南中医学院基础医学院,郑州450008 [3]河南省立眼科医院河南省眼科研究所,郑州450003

出　　处：《中华实验眼科杂志》2015年第11期1009-1014,共6页Chinese Journal Of Experimental Ophthalmology

摘　　要：背景氧化应激是年龄相关性黄斑变性（AMD）的主要因素之一。研究表明叶黄素对AMD有预防作用，但是其抗氧化机制仍不明确。Mμller细胞是视网膜氧化应激反应的靶细胞之一，研究叶黄素对MUller细胞的氧化应激是否具有保护作用及其作用机制对于视网膜疾病的治疗具有重要意义。目的研究叶黄素对视网膜Mμller细胞核因子E2相关转录因子2／抗氧化反应原件（Nrf2／ARE）信号途径的影响。方法人Muller细胞株进行常规培养，将对数生长期的细胞接种于96孔培养板，在培养液中分别加入不同浓度（40、80、160、320、640ixmol／L）HO2，绘制Mμller细胞的凋亡曲线，取H2O2的半数致死量，即160μmol／L制备Mμller细胞氧化应激模型。将模型细胞分为模型对照组和不同质量浓度（12．5、25．0、50．0mg／L）叶黄素组，根据分组情况分别在培养液中加入相应质量浓度的叶黄素，用常规培养的细胞作为空白对照组。采用MTT比色法测定各组中Maller细胞增生值（吸光度，A值）和凋亡率；采用流式细胞仪检测各组细胞中活性氧簇（ROS）的荧光强度；采用实时荧光定量PCR检测细胞中Nrf2mRNA和血红素加氧酶-1（HO-1）mRNA的相对表达量；采用Westernblot法检测各组细胞中Nrf2和HO-1蛋白的表达水平（灰度值）。结果MTT比色法检测显示，随着H2O2浓度的增加，细胞增生抑制率逐渐增加，各组间总体比较差异有统计学意义（F=43．890，P〈0．01）。空白对照组、模型对照组及12．5、25．0、50．0mg／L叶黄素组细胞凋亡率的总体比较差异有统计学意义（F=346．770，P=0．000），其中随着叶黄素质量浓度的增加，细胞凋亡率逐渐下降，组间比较差异均有统计学意义（均P〈0．05）。空白对照组、模型对照组及12．5、25．0、50．0mg／L叶黄素组细胞中ROS含量分别为1．92±0．18、64．89±2．86、52．70±2．80、3Background Oxidative stress is a main cause Lutein has a preventive role for AMD, but its antioxidant mechanism of age-related macular degeneration （ AMD）. remains unclear. Objective Present study was to investigate the effect of lutein on oxidative stress of Mtiller cells and its signaling pathway. Methods Human MUller cells （ human MUller cell strain） were cultured, and the cells at logarithmimie growth phase were incubated in 96 well plate overnightly. Oxidative stress cell models were established by adding 160 μmol/L H2O2, a median lethal dose for Muiller cells. The models were divided into the model control group and 12.5,25.0,50. 0 mg/L lutein groups,and the different concentrations of lutein were used to culture the cells for 24 hours, respectively. The routine cultured cells served as the blank control group. Growth of the cells was assayed by MTT method （absorbancy） ;the reactive oxygen species （ROS） content in the cells was assayed by flow cytometry;the mRNA and protein levels of nuclear factor-E2-related factor 2 （Nrf2） and heme oxygenase-1 （HO-1） in the ceils were detected by quantitative real-time PCR and Western blot, respectively. Results The inhibitory effects on the cells were gradually enhanced with the increase of H2 02 concentrations,showing a significant difference among the groups （F= 43. 890,P〈0. 01 ）. A significant difference was found in apoptotic rate of the cells among the blank control group, model control group and 12.5,25.0,50.0 mg/L lutein groups （ F = 346. 770, P = 0. 000 ） , and the apoptosis rate was significant elevated with the increase of lutein dose （ all at P〈0. 05 ）. The ROS contents in the cells were 1.92-＋0. 18, 64.89~2.86,52.70~2.80,32.61＋4.20 and 5.68＋1.35 in the blank control group,model control group and 12.5, 25.0,50.0 mg/L group, respeetively, with significant differenee among the groups （ F = 324. 900, P = 0. 000 ） , and the ROS eontent was gradually reduced as the increase of lutein dose （all at P〈0. 05）. Th

关 键 词：氧化应激/生理 叶黄素/药理 MULLER细胞 年龄相关性黄斑变性 核因子E2相关转录 因子2/代谢 血红素加氧酶-1/代谢 

分 类 号：R285[医药卫生—中药学]