褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Protective effects of melatonin on cultural human retinal pigment epithelial cells against oxidative damage in vitro

目的探讨褪黑素（Mel）对过氧化氢（H2O2）氧化损伤的人视网膜色素上皮（hRPE）细胞的保护作用及其作用机制。方法实验研究。采用600μmol／LH2O2建立体外培养的hRPE细胞氧化损伤模型。实验分为6组：溶剂对照组、600μmol／L H2O2＋溶剂组（H2O2损伤模型组）、600μmol／L H2O2＋10^-7mol／L Mel组、600μmol／L H2O2＋10^-6mol／L Mel组、600μmol／L H2O2＋10^-5mol／L Mel组、600μmol／L H2O2＋10^-4mol／L Mel组。通过四甲基偶氮唑盐（MTT）法检测细胞活性；测定细胞内超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量以反映细胞氧化损伤程度；分别用DNALadders电泳法和流式细胞仪检测细胞的凋亡情况。溶剂对照组与600μmol／L H2O2组间均数比较采用随机区组设计的t检验；600μmol／L H2O2组以及600μmol／L H2O2＋不同浓度Mel组间均数比较采用单因素5水平设计的方差分析，组间两两比较采用LSD—t检验。结果H2O2模型组较对照组细胞活性明显降低、SOD活性降低、MDA含量增加、凋亡率升高，差异均有统计学意义（t=2．25，39．50，68．42；P〈0．05）；Mel干预组较模型组细胞活性升高、SOD活性升高、MDA含量减少、凋亡率降低，差异有统计学意义（P〈0．05），并与药物浓度的变化呈正相关趋势。结论Mel对H2O2诱导的RPE的氧化损伤具有保护作用，其机制可能与影响细胞活性、增强抗氧化酶活性。

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium （RPE） against oxidative damage induced by hydrogen dioxide （H2O2 ） and its mechanism. Methods The RPE ceils were seeded and divided into normol control group, oxidative damage group and the treatment group （treated with melatonin at the concentration of 1 × 10^-7 mol/L, 1 × 10^-6 moL/L, 1×10^-5 mol/L and 1 ×10^-4 mol/L）. The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium （MTT） method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase （SOD） and maleic dialdehyde （MDA）. Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothod, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate（t = 2. 25,39. 50,68.42;P 〈0.05）. Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low MDA contents, and low apoptosis rate （P 〈 0. 05）. Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.