Tanshinol protects human umbilical vein endothelial cells against hydrogen peroxide-induced apoptosis

The present study was designed to investigate the effect of tanshinol on hydrogen peroxide (H2O2)-induced apoptosis in human umbilical vein endothelial cells (HUVECs), and to determine the underlying mechanisms. HUVECs were pre-incubated with tanshinol (25-200 mu M) for 24 h, followed by an incubation with 600 mu M H2O2 for 6 h. The cell viability was assessed using MTT reagent and the level of cell death was determined by measuring lactate dehydrogenase (LDH) activity. Superoxide dismutase (SOD) activity, levels of malondialdehyde (MDA), reactive oxygen species (ROS) production and NADPH oxidase activity were measured spectrophotometrically using commercially available kits. The apoptotic rate of the HUVECs was detected using Annexin-V/propidium iodide (PI) staining, followed by flow cytometry analysis using a fluorescence microscope. The protein expression of SOD-1, SOD-2, B-cell lymphoma-2 (Bcl-2), cytochrome c and caspase-3 was determined by western blot analysis. Pretreatment with tanshinol resulted in a significant increase in the cellular viability of HUVECs and SOD activity, and a decrease of cell apoptosis, MDA levels and ROS production, induced by H2O2. These findings were accompanied by the upregulation of Bcl-2 protein expression, reduction in the release of cytochrome c from the mitochondria to the cytosol and a downregulation of caspase-3 protein expression. This study showed that tanshinol protects against atherosclerosis by preventing H2O2-induced apoptosis of HUVECs. These effects appear to be mediated by enhancing the antioxidant defenses and preserving the mitochondrial function of the cells.