二十二碳六烯酸抑制氧化应激状态下人视网膜色素上皮细胞凋亡

Docosahexaenoic acid protects human retinal pigment epithelial cells against oxidative stress-induced apoptosis

目的：观察二十二碳六烯酸（docosahexaenoic acid,DHA）对外源性H2O2诱导人视网膜色素上皮细胞凋亡的影响及分子机制。方法：体外培养人视网膜色素上皮细胞系ARPE-19,加入终浓度为12.5 mol/L的H2O2诱导氧化应激,随后用30-100μmol/L DHA作用细胞4-24 h;real-time PCR和Western blot分别检测血红素氧合酶-1（heme oxygenase-1,HO-1）mRNA和蛋白的表达;比色法分析HO-1酶活性;荧光探针检测活性氧簇（reactive oxygen species,ROS）的产生;免疫荧光检测转录因子NF-E2相关因子2（NF-E2-related factor 2,Nrf2）的核转位。最后通过HO-1 siRNA干扰后,流式细胞术观察其对ARPE-19细胞凋亡的影响。结果：DHA能以浓度依赖性方式诱导ARPE-19细胞表达HO-1 mRNA和蛋白,同时,HO-1的酶活性也随着DHA浓度的递增而增强;DHA处理也能诱导Nrf2核转位。此外,H2O2处理可促进ARPE-19细胞凋亡,并诱导其产生ROS。同时给予100μmol/L DHA处理后,细胞凋亡率和ROS生成显著降低。转染HO-1 siRNA或用HO-1抑制剂Zn PP处理后,可明显降低DHA对细胞凋亡率和ROS的抑制作用。结论：DHA可能通过Nrf2途径诱导视网膜色素上皮细胞表达HO-1,从而发挥对细胞的保护作用。

AIM：To observe the effect of docosahexaenoic acid（ DHA） on H2O2-induced apoptosis in human retinal pigment epithelium cells and its molecular mechanism.METHODS：Human retinal pigment epithelium cell line ARPE-19 was cultured in vitro,and 12.5 mmol/L H2O2 was used to mimic the oxidative stress condition.The cells were treated with 30 - 100 μmol/L DHA for 4 - 24 h.The expression of heme oxygenase-1（ HO-1） at mRNA and protein levels was detected by real-time PCR and Western blot,respectively.The enzymic activity of HO-1 was measured by colorimetry.Production of reactive oxygen species（ ROS） was determined by fluorescent probe.Activation of NF-E2-related factor 2（ Nrf2） was examined by immunofluorescence method.Apoptosis of ARPE-19 cells was analyzed by flow cytometry.RESULTS：The mRNA and protein expression and the enzymic activity of HO-1 were significantly increased in the ARPE-19 cells after DHA treatment.Meanwhile,nuclear translocation of Nrf2 was also observed.Apoptosis appeared and ROS was produced upon H2O2 incubation.In contrast,DHA at 100 μmol/L significantly abrogated H2O2-induced apoptosis and ROS production.Furthermore,silencing of HO-1 by specific siRNA,or treatment with Zn PP,an inhibitor of HO-1,partly counteracted the protective effect against H2O2-induced apoptosis and ROS production.CONCLUSION：DHA protects retinal pigment epithelial cells against oxidative stress via induction of heme oxygenase-1 expression after Nrf2 activation.