Rosen Richard B, Hu Dan-Ning, Chen Min, McCormick Steven A, Walsh Joseph, Roberts Joan E
Department of Ophthalmology, New York Eye and Ear Infirmary, New York Medical College, New York, NY, USA.
Mol Vis. 2012;18:1640-8. Epub 2012 Jun 20.
Recently, we reported finding that circulating melatonin levels in age-related macular degeneration patients were significantly lower than those in age-matched controls. The purpose of this study was to investigate the hypothesis that melatonin deficiency may play a role in the oxidative damage of the retinal pigment epithelium (RPE) by testing the protective effect of melatonin and its receptor antagonist on RPE cells exposed to H(2)O(2) damage.
Cultured human RPE cells were subjected to oxidative stress induced by 0.5 mM H(2)O(2). Cell viability was measured using the microculture tetrazoline test (MTT) assay. Cells were pretreated with or without melatonin for 24 h. Luzindole (50 μM), a melatonin membrane-receptor antagonist, was added to the culture 1 h before melatonin to distinguish direct antioxidant effects from indirect receptor-dependent effects. All tests were performed in triplicate.
H(2)O(2) at 0.5 mM decreased cell viability to 20% of control levels. Melatonin showed dose-dependent protective effects on RPE cells against H(2)O(2). Cell viability of RPE cells pretreated with 10(-10), 10(-8), 10(-6), and 10(-4) M melatonin for 24 h was 130%, 160%, 187%, and 230% of cells treated with H(2)O(2) alone (all p<0.05). Using cells cultured without H(2)O(2) as the control, cell viability of cells treated with H(2)O(2) after pretreatment with 10(-10)-10(-4) M melatonin was still significantly lower than that of the controls, suggesting that melatonin significantly decreased but did not completely abolish the in vitro cytotoxic effects of H(2)O(2). Luzindole completely blocked melatonin's protective effects at low concentrations of melatonin (10(-10)-10(-8) M) but not at high concentrations (10(-6)-10(-4) M).
Melatonin has a partial protective effect on RPE cells against H(2)O(2) damage across a wide range of concentrations (10(-10)-10(-4) M). This protective effect occurs through the activation of melatonin membrane receptors at low concentrations (10(-10)-10(-8) M) and through both the direct antioxidant and indirect receptor activation effects at high concentrations (10(-6)-10(-4) M).
最近，我们报告发现年龄相关性黄斑变性患者的循环褪黑素水平显著低于年龄匹配的对照组。本研究的目的是通过测试褪黑素及其受体拮抗剂对暴露于过氧化氢损伤的视网膜色素上皮（RPE）细胞的保护作用，来研究褪黑素缺乏可能在RPE细胞氧化损伤中起作用的假说。
将培养的人RPE细胞暴露于0.5 mM过氧化氢诱导的氧化应激中。使用微量培养四氮唑蓝试验（MTT）测定细胞活力。细胞在有或没有褪黑素的情况下预处理24小时。在加入褪黑素前1小时，向培养物中加入50 μM的褪黑素膜受体拮抗剂鲁辛朵，以区分直接抗氧化作用和间接受体依赖性作用。所有测试均重复三次。
0.5 mM的过氧化氢使细胞活力降至对照水平的20%。褪黑素对RPE细胞抵抗过氧化氢具有剂量依赖性保护作用。用10⁻¹⁰、10⁻⁸、10⁻⁶和10⁻⁴ M褪黑素预处理24小时的RPE细胞活力分别是单独用过氧化氢处理细胞的130%、160%、187%和230%（所有p<0.05）。以未用过氧化氢培养的细胞作为对照，用10⁻¹⁰ - 10⁻⁴ M褪黑素预处理后用过氧化氢处理的细胞活力仍显著低于对照，这表明褪黑素显著降低但并未完全消除过氧化氢在体外的细胞毒性作用。鲁辛朵在低浓度褪黑素（10⁻¹⁰ - 10⁻⁸ M）时完全阻断了褪黑素的保护作用，但在高浓度（10⁻⁶ - 10⁻⁴ M）时则没有。
褪黑素在广泛的浓度范围（10⁻¹⁰ - 10⁻⁴ M）内对RPE细胞抵抗过氧化氢损伤具有部分保护作用。这种保护作用在低浓度（10⁻¹⁰ - 10⁻⁸ M）时通过激活褪黑素膜受体发生，在高浓度（10⁻⁶ - 10⁻⁴ M）时通过直接抗氧化作用和间接受体激活作用共同发生。
