The Protective Effect of Quercetin against Oxidative Stress in the Human RPE In Vitro

Abstract

To investigate the possible protective effect of the dietary antioxidant quercetin on retinal pigment epithelial (RPE) cell dysfunction and cellular senescence occurring in age-related macular degeneration (AMD). The major flavonoid quercetin was studied on RPE cells in vitro. Cultured human RPE cells were incubated with different concentrations of quercetin for 24 hours. Cells were then treated with 150 to 300 microM hydrogen peroxide for 2 hours. Mitochondrial function was measured by using MTT assay and cell vitality by live-dead staining assay. Intracellular levels of glutathione were determined by using a glutathione assay kit. Apoptosis was quantified by a caspase-3 assay, and cellular senescence was quantified by beta-galactosidase staining. Expression of the senescence-associated transmembrane protein caveolin-1 was investigated by Northern and Western blot analyses. Hydrogen peroxide treatment caused significant decreases in mitochondrial function (52%) and in cell vitality (71%), whereas preincubation with 50 microM quercetin diminished this decrease in a dose-dependent manner. Quercetin treatment did not show any notable effect on intracellular levels of glutathione in either used concentration of quercetin. Hydrogen peroxide-induced activation of caspase-3 was reduced by 50 microM quercetin, from 1.9- to 1.4-fold, compared with untreated control (P < 0.001). Hydrogen peroxide caused a large (>90%) dose-dependent increase in beta-galactosidase-positive cells, whereas in the untreated control only single cells expressed this enzyme (<5%). This increase in cellular senescence was significantly attenuated by quercetin in a dose-dependent manner. The highest attenuation was reached at 50 microM quercetin. Quercetin caused a significant dose-dependent reduction of caveolin-1 mRNA 48 hours after treatment with hydrogen peroxide. After 96 hours of incubation, caveolin-1 protein levels were also reduced. The data demonstrate that quercetin is able to protect RPE cells from oxidative damage and cellular senescence in vitro in a dose-dependent manner. The authors suggest that this increase in antioxidative capacity is--among other mechanisms, such as the intracellular redox state--also mediated by inhibiting the upregulation of caveolin-1. Downregulation of caveolin-1 may be important for the retinal pigment epithelium to prevent apoptotic cell death in response to cellular stress, a condition implicated in the early pathogenesis of AMD. Therefore, the authors believe that the use of antioxidative dietary flavonoids such as quercetin is a promising approach in the prevention of early AMD.