Increased glutathione synthesis through an ARE-Nrf2-dependent pathway by zinc in the RPE: Implication for protection against oxidative stress
Increased glutathione synthesis through an ARE-Nrf2-dependent pathway by zinc in the RPE: Implication for protection against oxidative stress
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DOI：
10.1167/iovs.05-1322
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发表时间：
2006-06-01
影响因子：
4.4
通讯作者：
Sternberg, Paul, Jr.
中科院分区：
文献类型：
--
作者：
Ha, Khoi-Nguyen;Chen, Yan;Sternberg, Paul, Jr.
目的.目的探讨锌对视网膜色素上皮（RPE）细胞氧化应激的保护作用及其分子机制。培养的ARPE-19细胞用不同浓度的锌处理不同时间。细胞谷胱甘肽（GSH）和谷胱甘肽二硫化物（GSSG）的水平通过高效液相色谱法（HPLC）测定。通过定量逆转录-PCR（RT-PCR）测定谷氨酸-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，在双荧光素酶测定中测量核因子甲状腺2相关因子（Nrf 2）活性。采用小干扰RNA（siRNA）技术敲低Nrf 2基因的表达。锌通过诱导从头合成途径显著增加ITARPE-19细胞中的GSH水平。在150亩M，锌增加了70%的GSH水平。在相似浓度下，锌上调GCL的mRNA水平并激活ARE-Nrf 2通路。锌对ARE激活和GSH合成的影响可通过使用siRNA方法敲低Nrf 2表达来抑制。锌对ARE-Nrf 2通路的诱导提供了强有力的和长期的抗氧化和解毒作用，这可能解释了锌在治疗年龄相关性黄斑变性中观察到的有益作用。
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 er throid2-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 It ARPE-19 cells through induction of the de novo synthesis pathway. At 150 mu M, 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