Khoi-Nguyen Ha1, Yan Chen, Jiyang Cai, Paul Sternberg. 1. Department of Ophthalmology and Visual Sciences, Vanderbilt Eye Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-8808, USA.
Abstract
PURPOSE: To determine the molecular mechanisms underlying the protective effects of zinc against oxidative stress in cultured retinal pigment epithelial (RPE) cells. METHODS: Cultured ARPE-19 cells were treated with different concentrations of zinc for various times. Cellular glutathione (GSH) and glutathione disulfide (GSSG) levels were measured by high-performance liquid chromatography (HPLC). Glutamate-cysteine ligase (GCL) expression was measured by quantitative reverse transcription-PCR (RT-PCR). Nuclear factor erythroid2-related factor (Nrf2) activity was measured in a dual luciferase assay after transfection of reporter plasmids containing the antioxidant response element (ARE). The small interference (si)RNA approach was used to knock down the expression of Nrf2. RESULTS: Zinc significantly increased GSH levels in ARPE-19 cells through induction of the de novo synthesis pathway. At 150 microM, zinc increased the GSH level by 70%. At similar concentrations, zinc upregulated the mRNA level of GCL and activated the ARE-Nrf2 pathway. The effects of zinc on ARE activation and GSH synthesis were inhibited by knockdown of Nrf2 expression using the siRNA approach. CONCLUSIONS: Induction of the ARE-Nrf2 pathway by zinc provides powerful and prolonged antioxidation and detoxification that may explain the beneficial effects of zinc observed in the treatment of age-related macular degeneration (AMD).
PURPOSE: To determine the molecular mechanisms underlying the protective effects of zinc against oxidative stress in cultured retinal pigment epithelial (RPE) cells. METHODS: Cultured ARPE-19 cells were treated with different concentrations of zinc for various times. Cellular glutathione (GSH) and glutathione disulfide (GSSG) levels were measured by high-performance liquid chromatography (HPLC). Glutamate-cysteine ligase (GCL) expression was measured by quantitative reverse transcription-PCR (RT-PCR). Nuclear factor erythroid2-related factor (Nrf2) activity was measured in a dual luciferase assay after transfection of reporter plasmids containing the antioxidant response element (ARE). The small interference (si)RNA approach was used to knock down the expression of Nrf2. RESULTS: Zinc significantly increased GSH levels in ARPE-19 cells through induction of the de novo synthesis pathway. At 150 microM, zinc increased the GSH level by 70%. At similar concentrations, zinc upregulated the mRNA level of GCL and activated the ARE-Nrf2 pathway. The effects of zinc on ARE activation and GSH synthesis were inhibited by knockdown of Nrf2 expression using the siRNA approach. CONCLUSIONS: Induction of the ARE-Nrf2 pathway by zinc provides powerful and prolonged antioxidation and detoxification that may explain the beneficial effects of zinc observed in the treatment of age-related macular degeneration (AMD).
Authors: Vera L Bonilha; Brent A Bell; Mary E Rayborn; Ivy S Samuels; Anna King; Joe G Hollyfield; Chengsong Xie; Huaibin Cai Journal: Free Radic Biol Med Date: 2017-01-11 Impact factor: 7.376