Purpose: To investigate the protective effect of rapamycin on oxidative stress-induced cell death of human corneal endothelial cells (HCECs).
Methods: 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).
Results: 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.
Conclusion: Our data suggest that rapamycin protects HCECs from oxidative injury-mediated cell death via inhibition of ROS production and enhancement of GSH.