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Abstract
Purpose
Grape seed-derived polyphenols (GSPs) provide a concentrated source of polyphenols having antioxidant capacity. In this study we investigated the cytoprotective effect of GSP against oxidative stress-induced cell damage in cultured human reti-nal pigment epithelial (RPE) cells.
Methods
Cultured adult retinal pigment epithelium (ARPE)-19 cells were incubated with GSP from Vitis vinifera (0.1, 0.5, 1, 5 or 10 μ g/mL) for 24 hours and treated with hydrogen peroxide (H2 O2, 0.4 mM) for 24 hours to induce oxidative stress. Cell viability was measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Intracellular reactive oxygen species (ROS) was quantified using 2′,7′-dichlorofluorescein diacetate (DCF-DA) fluorescence.
Results
The percentage of viable RPE cells was significantly lower in cultures treated with H2 O2 0.4 mM than in control cultures. GSP significantly reduced H2 O2-induced cell death in a dose dependent manner. GSP at 0.1, 0.5, 1, 5 and 10 μ g/mL significantly reduced cell mortality due to the treatment with H2 O2. Intracellular ROS production increased significantly in cultures treated with H2 O2 0.4 mM compared with control. There was a significant dose-dependent decrease in intracellular ROS levels after treat-ment of RPE with GSP.
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Figure 1.
Cytotoxic effect of grape seed-derived polyphenols (GSP). The ARPE-19 cells were incubated with different concen-tration of GSP for 24 hours. (A) GSP showed cytotoxicity at 100 μ g/mL. (B) The asterisk (*) indicates p < 0.05 versus untreated control. ARPE = adult retinal pigment epithelium.
Figure 2.
Grape seed-derived polyphenols (GSP) protected against H2 O2 cell death in ARPE-19 cells. (A) The ARPE-19 cells were treated with H2 O2 at dose of 200, 400, and 600 μ M for 24 hours. (B) The cells were pretreated with GSP for 24 hours then exposed to H2 O2 (400 μ M) for 24 hours. Data was expressed as a percentage of the untreated control. The aster-isk (*) indicates p < 0.05 versus untreated control (A), or H2 O2-induced cells without GSP pretreatment (B). ARPE = adult retinal pigment epithelium.
Figure 3.
Grape seed-derived polyphenols (GSP) reduced H2 O2-induced intracellular reactive oxygen species (ROS) generation in ARPE-19 cells. (A) The ARPE-19 cells were treated with H2 O2 at dose of 200, 400, and 600 μ M for 24 hours. (B) The cell were pretreated with GSP for 24 hours then treated with 400 μ M for 4 hours. The intracellular ROS was examined by a fluorescence spectrophotometer using DCF- DA. Data was expressed as a percentage of the untreated control. The asterisk (*) indicates p < 0.05 versus untreated control (A), or H2 O2-induced cells without GSP pretreatment (B). ARPE = adult retinal pigment epithelium; DCF-DA = 2′,7′-dichlorofluorescein diacetate.
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