The protective effect of quercetin against oxidative stress in the human RPE in vitro

Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.