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DOO YOUNG CHO, Young Joo Shin, Jong Mo Seo, Tae Young Chung, Joon Young Hyon, Won Ryang Wee; Rapamycin Reduces Reactive Oxygen Species in Cultured Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6454.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the protective effect of rapamycin on oxidative stress-induced cell death of human corneal endothelial cells
Human corneal endothelial cells (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 CCK-8 (Cell Counting Kit-8, Wako). The levels of cellular glutathione were also assessed enzymatically with glutathione reductase by using a commercial glutathione assay kit (Cayman Chemical, USA)
In vitro studies showed that rapamycin reduced 2',7'-dihydrodichlorofluorescein oxidation and increased glutathione. Rapamycin significantly inhibited tBHP-induced ROS production. Cells treated with rapamycin had higher viability compared to control at 5 mM tBHP and rapamycin effectively protected HCEC from ROS-induced cell death through increasing intracellular glutathione.
Our data suggest that rapamycin protect HCEC at high concentrations from oxidative injury-mediated cell death via inhibition of ROS production.
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