Sreekumar Parameswaran G, Ishikawa Keijiro, Spee Chris, Mehta Hemal H, Wan Junxiang, Yen Kelvin, Cohen Pinchas, Kannan Ram, Hinton David R
Arnold and Mabel Beckman Macular Research Center, Doheny Eye Institute, Los Angeles, California, United States.
Department of Ophthalmology, University of Southern California, Los Angeles, California, United States.
Invest Ophthalmol Vis Sci. 2016 Mar;57(3):1238-53. doi: 10.1167/iovs.15-17053.
To investigate the expression of humanin (HN) in human retinal pigment epithelial (hRPE) cells and its effect on oxidative stress-induced cell death, mitochondrial bioenergetics, and senescence.
Humanin localization in RPE cells and polarized RPE monolayers was assessed by confocal microscopy. Human RPE cells were treated with 150 μM tert-Butyl hydroperoxide (tBH) in the absence/presence of HN (0.5-10 μg/mL) for 24 hours. Mitochondrial respiration was measured by XF96 analyzer. Retinal pigment epithelial cell death and caspase-3 activation, mitochondrial biogenesis and senescence were analyzed by TUNEL, immunoblot analysis, mitochondrial DNA copy number, SA-β-Gal staining, and p16INK4a expression and HN levels by ELISA. Oxidative stress-induced changes in transepithelial resistance were studied in RPE monolayers with and without HN cotreatment.
A prominent localization of HN was found in the cytoplasmic and mitochondrial compartments of hRPE. Humanin cotreatment inhibited tBH-induced reactive oxygen species formation and significantly restored mitochondrial bioenergetics in hRPE cells. Exogenous HN was taken up by RPE and colocalized with mitochondria. The oxidative stress-induced decrease in mitochondrial bioenergetics was prevented by HN cotreatment. Humanin treatment increased mitochondrial DNA copy number and upregulated mitochondrial transcription factor A, a key biogenesis regulator protein. Humanin protected RPE cells from oxidative stress-induced cell death by STAT3 phosphorylation and inhibiting caspase-3 activation. Humanin treatment inhibited oxidant-induced senescence. Polarized RPE demonstrated elevated cellular HN and increased resistance to cell death.
Humanin protected RPE cells against oxidative stress-induced cell death and restored mitochondrial function. Our data suggest a potential role for HN therapy in the prevention of retinal degeneration, including AMD.
研究人胰岛素（HN）在人视网膜色素上皮（hRPE）细胞中的表达及其对氧化应激诱导的细胞死亡、线粒体生物能量学和衰老的影响。
通过共聚焦显微镜评估人胰岛素在RPE细胞和极化RPE单层中的定位。在不存在/存在HN（0.5 - 10μg/mL）的情况下，用人视网膜色素上皮细胞用150μM叔丁基过氧化氢（tBH）处理24小时。用XF96分析仪测量线粒体呼吸。通过TUNEL、免疫印迹分析、线粒体DNA拷贝数、SA-β-Gal染色以及ELISA分析视网膜色素上皮细胞死亡和半胱天冬酶-3激活、线粒体生物发生和衰老。在有和没有HN共处理的情况下，研究氧化应激诱导的跨上皮电阻变化。
在hRPE的细胞质和线粒体区室中发现了人胰岛素的显著定位。人胰岛素共处理抑制了tBH诱导的活性氧形成，并显著恢复了hRPE细胞中的线粒体生物能量学。外源性HN被RPE摄取并与线粒体共定位。人胰岛素共处理可防止氧化应激诱导的线粒体生物能量学下降。人胰岛素处理增加了线粒体DNA拷贝数，并上调了线粒体转录因子A，一种关键的生物发生调节蛋白。人胰岛素通过STAT3磷酸化和抑制半胱天冬酶-3激活保护RPE细胞免受氧化应激诱导的细胞死亡。人胰岛素处理抑制了氧化剂诱导的衰老。极化的RPE显示细胞内人胰岛素水平升高且对细胞死亡的抵抗力增加。
人胰岛素保护RPE细胞免受氧化应激诱导的细胞死亡并恢复线粒体功能。我们的数据表明HN疗法在预防包括年龄相关性黄斑变性（AMD）在内的视网膜变性方面具有潜在作用。
Zotero 插件
