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Andrea R. Carvalho, Anna Salas Torras, Miguel Zapata, Laura Distefano, Emilio Segovia, Jose Garcia-Arumi; The Effects of EPA and EPA/DHA Combination in Cultured Human RPE Cells Under Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5349.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies demonstrated that omega-3 long-chain polyunsaturated fatty acids within the eye have an anti-angiogenic, neuroprotective and anti-inflammatory effects. As the first and most important cell population damaged in AMD is the retinal pigment epithelium, the aim of this study was evaluate the effect of eicosapentaenoic acid (EPA) and a combination of EPA and docosahexaenoic acid (EPA/DHA) in ARPE-19 cultured cells under oxidative stress.
To better mimic aged-RPE, cultured ARPE-19 cells were exposed to 10µM of H2O2 for a week to induce adaptive response. Adapted cells were than exposed to 300µM of H2O2 with or without EPA (10, 50 and 100µM) and EPA/DHA (150, 75 and 37µM final concentration) in 0.03% dimethyl sulphoxide (DMSO) serum free media. It was evaluated cellular metabolism by alamar blue assay, apoptosis using apoptag staining, nitric oxide formation (Griess assay), and VEGF release in media by ELISA. Data represent the average of two or three experiments ± SD. Statistical significance was determined by Student’s two-tailed t-test. A p value less than 0.05 were considered significant.
Viability results were expressed as an optical density percentage in relation to the controls. Cellular viability curves were drawn up on 96-well plates by showing about 20.000 cells per well. EPA and EPA/DHA treated cells showed 15% ± 3% and 27,4% ± 1% respectively more metabolic activity in a chemical reduction of alamar blue than H2O2 treated cells; there were no statistical difference between the different concentrations tested.Immunofluorescence to detect apoptotic cells showed no difference in treated and control groups regarding number of stained cells. NO2- in media was bellow limit of detection (125pmol) in all samples tested.VEGF levels in culture media after 3h of treatment were 41.7pg/mL ± 7.7; 30.2pg/mL ± 3.8; 16.9pg/mL ± 5.7; 62.9pg/mL ± 1.9; and 131.5pg/mL ± 3.9 for EPA 10µM, EPA 50µM, EPA 100µM, control and H2O2 300µM treated cells respectively. Statistical values between EPA treated cells with controls and H2O2 treated cells were significant in all concentrations. In EPA/DHA treated cells VEGF levels were 53.27pg/mL ± 5.4; 45.57pg/mL ± 0.9; 39.8pg/mL ± 2.7; 64.2pg/mL ± 9.2; and 131.5pg/mL ± 3.9 for 37, 75, 150µM final concentration of EPA/DHA, control and H2O2 300µM treated cells respectively. Only concentrations of 75µM (p= 0.029) and 150µM (p= 0.04) showed statistical significance in relation of H2O2 treated cells.
EPA and EPA/DHA improved metabolism of cells under oxidative stress. EPA and EPA/DHA (higher concentrations) decreased VEGF release from oxidative stressed cells. EPA and EPA/DHA can have a valuable anti-angiogenic effect in AMD treatment.
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