Dihydrotestosterone protects human vascular endothelial cells from H 2O 2-induced apoptosis through inhibition of caspase-3, caspase-9 and p38 MAPK

Abstract

Oxidative stress is proved to be harmful to the vascular endothelial cells which are important in preventing the formation and progression of atheromatous plaque. This study was designed to investigate the protective effect and potential mechanisms of dihydrotestosterone (DHT) against H 2O 2-induced apoptosis of human umbilical vein endothelial cells (ECV-304). ECV-304 cells were pretreated with different concentrations of DHT (0.01, 0.1 and 1 μM) for 2 h, followed by exposure to 100 μM H 2O 2 for 18 h. 3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate cell viability. To detect apoptosis, the cells were assessed by morphologic examination and Annexin V-propidium iodide double staining with flow cytometry. Finally, the expression of caspase-3, caspase-9 and phospho p38 MAPK was assayed by Western blot to investigate the possible molecular mechanisms. We found that H 2O 2 treatment for 18 h significantly decrease the viability of ECV-304 cells characterized by a high percentage of apoptotic cells. DHT could antagonize the apoptosis inducing effect of H 2O 2 in a dose-dependent manner. Consistently, DHT also significantly inhibit the expression of caspase-3, caspase-9 and phospho p38 MAPK induced by H 2O 2. In summary, pretreatment with DHT can inhibit apoptosis of ECV-304 cells induced by H 2O 2. The protective effect of DHT was associated with the inhibition of caspase-3, caspase-9 and phospho p38 MAPK expression.