摘要
背景 氧化应激是年龄相关性黄斑变性发生的重要机制.研究表明,二十二碳六烯酸（DHA）在视网膜光感受细胞的发育中发挥重要作用,并可上调血红素氧合酶1（HO-1）的表达,从而发挥抗氧化效应,但DHA是否能影响人视网膜色素上皮（RPE）细胞表达HO-1尚未阐明. 目的 观察DHA对体外培养的RPE中HO-1表达的影响及其分子机制. 方法 对人RPE细胞系ARPE-19进行培养,分别用30、50、100和120 μmol/L DHA作用4～24 h,以不含DHA培养的细胞作为对照组.采用乳酸脱氢酶（LDH）法检测DHA对细胞的毒性;分别采用实时荧光定量PCR和Western blot法检测各组细胞中HO-1 mRNA及其蛋白的相对表达;采用比色法分析各组细胞中HO-1酶活性变化情况;采用荧光探针H2DCFDA检测细胞中活性氧簇（ROS）的相对比例,并采用免疫荧光技术检测各组培养细胞中核转录因子-E2相关因子2（Nrf2）的核转位情况;分别采用ROS抑制剂N-乙酰半胱氨酸（NAC）干预法和Nrf2小干扰RNA（siRNA）转染法作用于培养的细胞,采用Western blot法检测细胞中Nrf2蛋白的表达,并观察其对细胞中HO-1蛋白表达量的影响. 结果 0、30、50、100和120 μmol/L DHA作用于ARPE-19细胞后24 h,培养上清液中LDH漏出率的总体比较差异有统计学意义（F=8.14,P＜0.05）,其中120 μmol/L DHA作用后细胞中LDH漏出率明显高于0、30、50、100 μmol/L DHA组,差异均有统计学意义（均P＜0.05）.不同浓度DHA作用ARPE-19细胞后8h,HO-1mRNA及其蛋白的相对表达量以及HO-1的酶活性总体比较差异均有统计学意义（F=16.24,P＜0.05;F=11.34,P＜0.05;F=11.81,P＜0.05）,其中30、50、100 μmol/L DHA组细胞中HO-1 mRNA及其蛋白的相对表达量以及HO-1的酶活性均明显高于0μmol/L DHA组,差异均有统计学意义（均P＜0.05）.不同浓度DHA作用ARPE-19细胞后4h细胞内ROS相对荧光强度、细胞核Nrf2阳性细胞比例的总体比较差异均有统计学意义（F=11.08,P＜0.05;F=16.42,P＜0.05）,其中30、50和100μmol/L DHA组细胞中ROS相对荧光强度、细胞核Nrf2阳性细胞比例均明显高于0μmol/L DHA组,差异均有统计学意义（均P＜0.05）.100 μ.mol/L DHA处理条件下,NAC预处理后细胞中HO-1蛋白的相对表达量和细胞核Nrf2阳性细胞比例均明显低于单纯100μmol/L DHA组,Nrf2 siRNA转染组细胞中HO-1的相对表达量和细胞核Nrf2阳性细胞比例均明显低于空白siRNA转染组,差异均有统计学意义（均P＜0.05）. 结论 低于100 μmol/L的DHA可能通过ROS/Nrf2途径诱导RPE细胞表达HO-1,从而发挥对细胞的保护作用.
Background Oxydative stress is an important pathogenesis of age-related macular degeneration.Resent evidences indicate that docosahexaenoic acid （DHA） plays an important role during the development of retinal photoreceptor cells and protect the cells against oxydative stress by inducing the expression of heme oxygenase-1 （HO-1）.However,whether DHA can induce the expression of HO-1 in human retinal pigment epithelium （RPE） cells is unelucidated.Objective This study was to investigate the effect of DHA on the expression of HO-1 in RPE cells and its molecular mechanism.Methods Human RPE cell line ARPE-19 was cultured in vitro and treated with 30,50,100 and 120 μmol/L DHA for 4 to 24 hours,respectively,and the cells were cultured without DHA as the control group.The cytotoxicity of DHA was detected by lactate dehydrogenase（LDH）,and the expression of HO-1 mRNA and protein were detected by real-time PCR and Western blot assay,respectively.The enzymatic activity of HO-1 was detected by colorimetry.The reactive oxygen species （ROS） proportion in the cells was detected using fluorescence probe H2 DCFDA,and immunofluorescence technology was adopted to detect the nuclear translocation of nuclear facotor-E2-related factor 2 （Nrf2）.The expression of Nrt2 protein in the cells was detected by Western blot after intervention of ROS inhibitor N-acetylcysteine （NAC） and transfection of Nrf2 small interfering RNA （siRNA）.Results The LDH leakage rate was significantly different after 0,3,50,100 and 120 μmol/L DHA treated the cells for 24 hours （F=8.14,P〈0.05）,and the LDH leakage rate in the 120 μmol/L DHA group was significantly higher than that of 0,30,50 and 100 μmol/L DHA group （all at P〈0.05）.The relative expression levels of HO-1 mRNA and HO-1 protein or HO-1 enzymatic activity in the cells were significantly different among different concentrations of DHA group in 8 hours after treatment （F=16.24,P〈0.05;F=11.34,P〈0.05;F=11.81,P〈0.05）,and the expressions of these factors were considerably higher in the 30,50 and 100 μ mol/L DHA group than those in the 0 μmol/L DHA group （all at P〈0.05）.The ROS relative fluorescence intensity and nuclear Nrf2 positive cells proportion were statistically significant among different concentrations of DHA groups （F =11.08,P 〈 0.05;F=16.42,P〈0.05）,and the ROS relative fluorescence intensity and nuclear Nrf2 positive cells proportion were evidently higher in the 30,50 and 100 μmol/L DHA group than those in the 0 μmol/L DHA group （all at P〈0.05）.The relative expression levels of HO-1 protein and the proportion of nuclear Nrf2 positive cells were significantly lower in the NAC pretreated 100 μmol/L DHA group than those in the 100 μmol/L DHA group.In addition,the HO-1 relative expression level and the positive cells proportion of nuclear Nrf2 were significantly lower in the of Nrf2 siRNA transfection group than those in the blank siRNA transfection group （both at P〈0.05）.Conclusions DHA with concentration below 100 μ mol/L can protect RPE cells from oxidative stress by inducting the expression of HO-1 in the cells via ROS/Nrf2 pathway.
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2016年第8期677-683,共7页
Chinese Journal Of Experimental Ophthalmology
基金
十堰市科学技术研究与开发计划项目（14Y40）
