Effects of melatonin and its receptor antagonist on retinal pigment epithelial cells against hydrogen peroxide damage

摘要

Purpose: 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 H2O2 damage. Methods: Cultured human RPE cells were subjected to oxidative stress induced by 0.5 mM H2O2. 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. Results: H2O2 at 0.5 mM decreased cell viability to 20% of control levels. Melatonin showed dose-dependent protective effects on RPE cells against H2O2. 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 H2O2 alone (all p0.05). Using cells cultured without H2O2 as the control, cell viability of cells treated with H2O2 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 H2O2. 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). Conclusions: Melatonin has a partial protective effect on RPE cells against H2O2 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).