Tanshinone IIA reduces apoptosis induced by hydrogen peroxide in the human endothelium-derived EA.hy926 cells

Ethnopharmacological relevance: Salvia Miltiorrhiza Bunge (also known as herb Danshen in Chinese) is a widely used Chinese herbal medicine. Tanshinone IIA (TSN IIA) is considered to be the most important bioactive ingredient in Danshen and exhibits an anti-atherosclerotic activity. Aim of study: To evaluate the protective effect of TSN IIA on the human endothelial EA.hy926 cells injured by hydrogen peroxide in vitro and its possible mechanism. Materials and methods: The EA.hy926 cells were incubated for 24 h with different concentrations of TSN IIA (5, 10 and 20 mu g/uL) or DMEM. Subsequently, cells were treated with 300 mu mol/L H2O2 for another 4 h. Then, the percentage of cell viability was evaluated by 344, 5-di-methylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The apoptosis of EA.hy926 cells was detected by flow cytometry with AnnexinV-FITC/PI double staining and laser scanning spectral confocal technique. The generation of intracellular reactive oxygen species (ROS) generation was analyzed by flow cytometry. The mRNA expressions of caspase-3, Bcl-2 and Bax were tested by real time-reverse transcription polymerase chain reaction (real time RT-PCR). The protein expression of Bcl-2 and Bax was determined by Western blotting. MDA levels, NO production, LDH leakage, and SOD as well as caspase-3 activities were also measured using standard methods. Results: Loss of cell viability and excessive cell apoptosis were observed in EA.hy926 cells after 4 h of challenge with H2O2 (300 mu mol/L). However, cell apoptosis was attenuated in different concentrations of TSN IIA (5, 10 and 20 mu g/mu L) pretreated cells. Furthermore, TSN IIA markedly inhibited the elevation of ROS evoked by H2O2. Real time RT-PCR and Western blotting analysis showed that TSN IIA significantly decreased the expressions of pro-apoptotic proteins (Bax and caspase-3) while significantly increased the expression of anti-apoptotic protein Bcl-2, and resulted in obvious reduction of Bax/Bcl-2 ratio in EA.hy926 cells induced by H2O2. Conclusion: These observations provide preliminary evidence that TSN IIA protects EA.hy926 cells against H2O2 damage, which is mainly associated with the ROS generation, followed by the imbalance of the Bax/Bcl-2 ratio, and caspase-3 activation leading to apoptosis. (C) 2012 Elsevier Ireland Ltd. All rights reserved.