Department of Ophthalmology, Hallym University College of Medicine, Seoul, Republic of Korea.
Curr Eye Res. 2011 Dec;36(12):1116-22. doi: 10.3109/02713683.2011.614372. Epub 2011 Oct 14.
To investigate the protective effect of rapamycin on oxidative stress-induced cell death of human corneal endothelial cells (HCECs).
HCECs were cultured according to previously published methods. With treatment of 0 mM or 5 mM of tert-butyl hydroperoxide (tBHP) with various concentrations (0, 25 and 50 nM) of rapamycin, reactive oxygen species (ROS) production was measured using an oxidation-sensitive fluorescent probe, 2'7'-dichlorofluorescin diacetate (DCFH-DA, USA) methods. Cell viability was assayed by the method of Cell Counting Kit-8 (CCK-8, Wako). The levels of cellular glutathione were also assessed enzymatically with glutathione reductase by using a commercial glutathione (GSH) assay kit (Cayman Chemical, USA).
Rapamycin reduced 2'7'-dihydrodichlorofluorescein oxidation and increased GSH in HCECs. Rapamycin significantly inhibited tBHP-induced ROS production. Cells treated with rapamycin showed higher viability compared to control at 5 mM tBHP. Rapamycin effectively protected HCECs from ROS-induced cell death through increasing intracellular GSH.
Our data suggest that rapamycin protects HCECs from oxidative injury-mediated cell death via inhibition of ROS production and enhancement of GSH.
研究雷帕霉素对人角膜内皮细胞（HCEC）氧化应激诱导细胞死亡的保护作用。
根据先前发表的方法培养 HCEC。用不同浓度（0、25 和 50 nM）的雷帕霉素处理 0 mM 或 5 mM 叔丁基过氧化物（tBHP），使用氧化敏感荧光探针 2'7'-二氯荧光素二乙酸酯（DCFH-DA，美国）方法测量活性氧（ROS）的产生。通过细胞计数试剂盒-8（CCK-8，和光纯药，日本）方法测定细胞活力。还使用商业谷胱甘肽（GSH）测定试剂盒（Cayman Chemical，美国）通过谷胱甘肽还原酶酶法评估细胞内谷胱甘肽（GSH）的水平。
雷帕霉素降低了 2'7'-二氢二氯荧光素的氧化作用，并增加了 HCEC 中的 GSH。雷帕霉素显著抑制了 tBHP 诱导的 ROS 产生。与 5 mM tBHP 处理的对照组相比，用雷帕霉素处理的细胞显示出更高的活力。雷帕霉素通过增加细胞内 GSH 有效保护 HCEC 免受 ROS 诱导的细胞死亡。
我们的数据表明，雷帕霉素通过抑制 ROS 产生和增强 GSH 来保护 HCEC 免受氧化损伤介导的细胞死亡。
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