Zeitz Oliver, Schlichting Lars, Richard Gisbert, Strauss Olaf
Universitätsklinikum Hamburg-Eppendorf, Klinik und Poliklinik ffir Augenheilkunde, Martinistr. 52, 20246 Hamburg, Germany.
Graefes Arch Clin Exp Ophthalmol. 2007 Feb;245(2):276-81. doi: 10.1007/s00417-006-0384-5.
Oxidative damage to the retinal pigment epithelium might be involved in the pathogenesis of age related macular degeneration. Thus antioxidative protection represents a rationale for a causative therapy or prophylaxis. The aim of the present study is to evaluate antioxidative properties of vitamin C and pyruvate at retinal pigment epithelial (RPE) cells exposed to oxidative stress.
The ability of vitamin C and pyruvate to quench hydroxyl radicals was tested using the di-hydro-rhodamine (DHR) assay. Cells of the human RPE cell line ARPE-19 were exposed for 8 min to hydroxyl radicals generated by the Fenton reaction from 2.25 mM H2O2 and 30 microM Fe3+ -nitrilo-tri-acetate. This was done in the absence and presence of 0.3-3.0 mM pyruvate and vitamin C, respectively. Cell survival was analysed by vitality staining (life-dead-assay) and expressed as cell survival ratio. A survival ratio <1.0 indicates cell loss.
At concentrations from 0.1 to 1.0 mM vitamin C and pyruvate quench hydroxyl radicals in the DHR assay in absence of living matter. In the presence of 0.1- 0.3 mM vitamin C and pyruvate, ARPE-19 showed a reduced survival ratio (0.87 +/- 0.01 to 0.89 +/- 0.02 after 6 h) which was not the case at the higher concentrations between 1 and 3 mM. The exposure of ARPE-19 cells to hydroxyl radicals reduced the survival ratio to 0.92 +/- 0.02. At concentrations at which vitamin C and pyruvate exert toxic effects, a potentiation of radical induced cell death can be observed (survival ratio 0.79 +/- 0.02 and 0.82 +/- 0.03, respectively). Higher concentrations of vitamin C or pyruvate had no explicit protective effect to the hydroxyl radical induced damage.
Although vitamin C and pyruvate are potent hydroxyl radical quenchers in vitro they failed to protect cultured ARPE-19 cells from oxidative stress induced cell death. In contrast, when applying the scavengers at low concentrations a potentiation of cell damage was observed.
视网膜色素上皮的氧化损伤可能参与年龄相关性黄斑变性的发病机制。因此，抗氧化保护是一种病因治疗或预防的基本原理。本研究的目的是评估维生素C和丙酮酸在暴露于氧化应激的视网膜色素上皮（RPE）细胞中的抗氧化特性。
使用二氢罗丹明（DHR）测定法测试维生素C和丙酮酸淬灭羟自由基的能力。将人RPE细胞系ARPE-19的细胞暴露于由2.25 mM H2O2和30 μM Fe3 + -次氮基三乙酸的芬顿反应产生的羟自由基中8分钟。分别在不存在和存在0.3 - 3.0 mM丙酮酸和维生素C的情况下进行此操作。通过活力染色（死活测定）分析细胞存活情况，并表示为细胞存活比率。存活比率<1.0表示细胞损失。
在DHR测定中，在无生物物质存在的情况下，浓度为0.1至1.0 mM的维生素C和丙酮酸可淬灭羟自由基。在存在0.1 - 0.3 mM维生素C和丙酮酸的情况下，ARPE-19显示存活比率降低（6小时后为0.87±0.01至0.89±0.02），而在1至3 mM的较高浓度下则并非如此。ARPE-19细胞暴露于羟自由基会使存活比率降至0.92±。在维生素C和丙酮酸发挥毒性作用的浓度下，可以观察到自由基诱导的细胞死亡增强（存活比率分别为0.79±0.02和0.82±0.03）。较高浓度的维生素C或丙酮酸对羟自由基诱导的损伤没有明显的保护作用。
尽管维生素C和丙酮酸在体外是有效的羟自由基淬灭剂，但它们未能保护培养的ARPE-19细胞免受氧化应激诱导的细胞死亡。相反，当以低浓度应用清除剂时，观察到细胞损伤增强。
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