Department of Ophthalmology, University of Heidelberg, Heidelberg, Germany.
Invest Ophthalmol Vis Sci. 2012 Aug 9;53(9):5354-61. doi: 10.1167/iovs.12-9845.
Aging of the RPE is associated with a decrease of intracellular ATP levels and increased oxidative stress. We investigated the effects of moderate energy deficit on intracellular glutathione levels, oxidative damage of cellular proteins and DNA, and autophagy rates using an RPE cell culture model. Additionally, phagocytosis of photoreceptor outer segments was assayed as an example of an ATP-dependent normal function of the RPE.
ATP synthesis of primary human RPE cells was moderately inhibited by atractyloside. Oxidative stress was induced by tert-butyl hydroperoxide (tBH). ATP, reduced glutathione (rG), malondialdehyde (MDA) adduct formation and 8-hydroxydeoxyguanosine (8OHdG) levels were measured. Autophagy and phagocytosis of photoreceptor outer segments were assayed by radiometric methods.
Atractyloside-treatment reduced cellular ATP levels by 30%, mimicking the energy status of aged RPE. tBH decreased rG in RPE cells with lowered ATP levels whereas cells with normal ATP content were not affected. tBH-induced oxidative stress resulted in substantial accumulation of MDA protein adducts in cells with lowered ATP while cells with regular ATP levels were only modestly affected. tBH induced more oxidative DNA damage (8OHdG formation) in cells with lowered ATP levels than in cells with regular ATP. In atractyloside-treated cells, autophagy rates decreased 3-fold as compared with controls. Phagocytic capacity for uptake and degradation of photoreceptor segments was reduced in RPE with low ATP.
Moderately decreased ATP levels such as seen in aged individuals might contribute to the vulnerability of RPE to oxidative stress damage and to dysfunction.
RPE 的衰老与细胞内 ATP 水平降低和氧化应激增加有关。我们使用 RPE 细胞培养模型研究了适度能量缺乏对细胞内谷胱甘肽水平、细胞蛋白质和 DNA 的氧化损伤以及自噬率的影响。此外，还测定了光感受器外节的吞噬作用，作为 RPE 一种依赖于 ATP 的正常功能的示例。
用苍术苷适度抑制原代人 RPE 细胞的 ATP 合成。用叔丁基过氢（tBH）诱导氧化应激。测定 ATP、还原型谷胱甘肽（rG）、丙二醛（MDA）加合物形成和 8-羟基脱氧鸟苷（8OHdG）水平。通过放射性方法测定自噬和光感受器外节的吞噬作用。
苍术苷处理将细胞内 ATP 水平降低了 30%，模拟了衰老 RPE 的能量状态。tBH 在降低 ATP 水平的 RPE 细胞中降低了 rG，而正常 ATP 含量的细胞不受影响。tBH 诱导的氧化应激导致降低 ATP 水平的细胞中 MDA 蛋白加合物大量积累，而正常 ATP 水平的细胞仅受到适度影响。tBH 在降低 ATP 水平的细胞中诱导的氧化 DNA 损伤（8OHdG 形成）比正常 ATP 水平的细胞更多。与对照组相比，苍术苷处理的细胞中自噬率降低了 3 倍。RPE 中的低 ATP 会降低摄取和降解光感受器节段的吞噬能力。
如在老年人中所见的适度降低的 ATP 水平可能导致 RPE 对氧化应激损伤和功能障碍的易感性增加。
