Ha Khoi-Nguyen, Chen Yan, Cai Jiyang, Sternberg Paul
Department of Ophthalmology and Visual Sciences, Vanderbilt Eye Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-8808, USA.
Invest Ophthalmol Vis Sci. 2006 Jun;47(6):2709-15. doi: 10.1167/iovs.05-1322.
To determine the molecular mechanisms underlying the protective effects of zinc against oxidative stress in cultured retinal pigment epithelial (RPE) cells.
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.
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.
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).
确定锌对培养的视网膜色素上皮（RPE）细胞氧化应激产生保护作用的分子机制。
用不同浓度的锌处理培养的ARPE-19细胞不同时间。通过高效液相色谱法（HPLC）测量细胞内谷胱甘肽（GSH）和谷胱甘肽二硫化物（GSSG）水平。通过定量逆转录-聚合酶链反应（RT-PCR）测量谷氨酸-半胱氨酸连接酶（GCL）的表达。在转染含有抗氧化反应元件（ARE）的报告质粒后，通过双荧光素酶测定法测量核因子红细胞2相关因子（Nrf2）的活性。使用小干扰（si）RNA方法敲低Nrf2的表达。
锌通过诱导从头合成途径显著提高ARPE-19细胞中的GSH水平。在150微摩尔时，锌使GSH水平提高了70%。在相似浓度下，锌上调了GCL的mRNA水平并激活了ARE-Nrf2途径。使用siRNA方法敲低Nrf2表达可抑制锌对ARE激活和GSH合成的作用。
锌诱导的ARE-Nrf2途径提供了强大且持久的抗氧化和解毒作用，这可能解释了在年龄相关性黄斑变性（AMD）治疗中观察到的锌的有益作用。
