【摘要】 研究葡萄皮多酚类化合物是否具有保护RPE细胞免受对脂褐素损伤的作用,并且探讨这种保护作用是否与维持自噬活性相关。脂褐素沉积是细胞衰老标志之一,当脂褐素在视网膜色素上皮细胞(RPE)中累积到一定水平,将增加年龄相关性黄斑变性(AMD)的风险。但是,脂褐素在RPE细胞内的作用机制尚不清晰。目前尚无有效预防、治疗AMD的手段,通过抗氧化剂清除自由基,维持细胞自噬活性可能会成为预防AMD的新策略。采用化学合成脂褐素主要荧光基团Nretinyledin-N-retinylethanolamin(A2E)建立RPE细胞累积脂褐素模型,用共聚焦显微镜和液相色谱-质谱联用检测A2E在RPE细胞中累积。细胞活力及凋亡检测结果表明A2E对细胞活性具有抑制作用。Western blotting结果表明A2E提高自噬相关蛋白LC3-Ⅱ的累积,并且自噬阻断剂bafilomycin A1和A2E共同处理RPE细胞条件下LC3-Ⅱ表达量较单独用A2E处理条件下没有显著性差异,说明A2E能够阻断自噬流。此外,葡萄皮提取物能够减少A2E诱导的LC3-Ⅱ累积,提示葡萄皮多酚化合物对自噬流的保护作用。并且,葡萄皮提取物能够抑制A2E诱导RPE细胞凋亡,这种抑制作用在一定程度上归因于对自噬流的保护作用。更多还原

【Abstract】 We aimed to investigate whether grape skin polyphenols can protect RPE cells from lipofuscin-induced damage,and whether any protective effects are related to preserving autophagic processes,the accumulation of autofluorescent lipofuscin in the retinal pigment epithelium(RPE)is one of the hallmarks of aging,and is related to the increased risk of age-related macular degeneration(AMD).So far,there isn’t efficient AMD therapy or prevention method yet.Dietary antioxidants may be useful.In present study,we established an experimental model that mimics lipofuscin accumulation in RPE,using synthetic N-retinyledin-N-retinylethanolamin(A2E),the major fluorophore of lipofuscin.Internalized A2 Ein ARPE-19 was observed using confocal microscopy and quantified by liquid chromatography-mass spectrometry(LC-MS).Results of cell proliferation and apoptosis indicated that the viability of RPE cells was inhibited by A2 E.Furthermore,Western blotting showed A2 Etreatment alone increased LC3-Ⅱ levels which wasn’t significantly different from that in the presence of bafilomycin A1.These results collectively indicated that A2 Econfers detrimental effects on the RPE by partially blocking autophagic flux.Additionally,grape skin polyphenols decreased LC3-Ⅱ accumulation induced by A2 E,suggesting that grape skin polyphenols preserved autophagic flux.These grape skin polyphenols protected the RPE from A2E-induced apoptosis.The ability of grape skin polyphenols precting RPE cells may be partly attributed to preserving autophagic flux.更多还原