Effects of oxysterols on cell viability, inflammatory cytokines, VEGF, and reactive oxygen species production on human retinal cells: cytoprotective effects and prevention of VEGF secretion by resveratrol
Effects of oxysterols on cell viability, inflammatory cytokines, VEGF, and reactive oxygen species production on human retinal cells: cytoprotective effects and prevention of VEGF secretion by resveratrol
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DOI：
10.1007/s00394-010-0102-2
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发表时间：
2010-10-01
影响因子：
5
通讯作者：
Lizard, Gerard
中科院分区：
文献类型：
--
作者：
Dugas, B.;Charbonnier, S.;Lizard, Gerard
氧化甾醇被认为在老年性黄斑变性中起着重要作用，老年性黄斑变性是导致失明的主要原因。因此，我们研究了氧化甾醇(7-β-羟基胆固醇(7-β-OH)、7-酮胆固醇(7KC)、25-羟基胆固醇(25-OH))对人视网膜ARPE-19细胞的毒性、氧化、炎症和血管生成活性，并评价了白藜芦醇(RSV：1MU M)对ARPE-19细胞的保护作用。用台盼蓝和四甲基偶氮唑蓝比色法检测细胞存活率。用电子显微镜和荧光标记的半胱氨酸天冬氨酸酶抑制剂原位检测激活的半胱氨酸天冬氨酸酶来表征细胞死亡。用氢乙胺法测定活性氧(ROS)生成量。用ELISA法和流式细胞仪检测与炎症相关的细胞因子(IL-8、IL-1β、IL-6、IL-10、IL-12p70、肿瘤坏死因子-α、单核细胞趋化蛋白-1)和VEGF.7β-OH和7KC触发的caspase非依赖性细胞死亡过程，与引起磷脂沉积的多层细胞质结构、ROS产生增加和IL-8分泌有关。7β-OH可促进血管内皮生长因子的分泌。25-OH对ROS的产生、单核细胞趋化蛋白-1(MCP-1)和血管内皮生长因子的分泌无明显的细胞毒作用。氧固醇组未检测到IL-10、TNF-α和IL-12p70的分泌。25-OH通过MEK/ERKA1/2信号通路诱导IL-8的分泌，RSV具有细胞保护作用并抑制血管内皮生长因子的分泌。7β-OH、7KC和25-OH对ARPE-19细胞具有细胞毒、氧化、炎症和/或血管生成活性。由于RSV对氧化甾醇诱导的细胞死亡和血管内皮生长因子的分泌有一定的保护作用，因此在ARMD的治疗中具有一定的应用价值。
Oxysterols are assumed to play important roles in age-related macular degeneration, a major cause of blindness. So we characterized the cytotoxic, oxidative, inflammatory, and angiogenic activities of oxysterols (7 beta-hydroxycholesterol (7 beta-OH), 7-ketocholesterol (7KC), 25-hydroxycholesterol (25-OH)) in human retinal ARPE-19 cells, and evaluated the protective effects of resveratrol (Rsv: 1 mu M), a polyphenol from red wine.ARPE-19 cells were treated with 7 beta-OH, 7KC, or 25-OH (5-40 mu g/mL; 24-48 h) without or with Rsv. Cell viability was determined using trypan blue and the MTT assay. Cell death was characterized by electron microscopy and in situ detection of activated caspases with fluorochrome-labeled inhibitors of caspases. Reactive oxygen species (ROS) production was measured with hydroethidine. ELISA methods and a cytometric bead assay were used to quantify cytokines involved in inflammation (IL-8, IL-1 beta, IL-6, IL-10, IL-12p70, TNF-alpha, MCP-1) and VEGF.7 beta-OH and 7KC triggered a caspase-independent cell death process associated with the presence of multilamellar cytoplasmic structures evocating phospholipidosis, increased ROS production, and IL-8 secretion. 7 beta-OH enhanced VEGF secretion. No cytotoxic effects were identified with 25-OH, which highly stimulated ROS production, MCP-1, and VEGF secretion. With oxysterols, no IL-10, TNF-alpha, and IL-12p70 secretion were detected. 25-OH induced IL-8 secretion through the MEK/ERKA1/2 signaling pathway, and Rsv showed cytoprotective activities and inhibited VEGF secretion.7 beta-OH, 7KC, and 25-OH have cytotoxic, oxidative, inflammatory, and/or angiogenic activities on ARPE-19 cells. As Rsv has some protective effects against oxysterol-induced cell death and VEGF secretion it could be valuable in ARMD treatment.