Tanshinone IIA reduces apoptosis induced by hydrogen peroxide in the human endothelium-derived EA.hy926 cells
ABSTRACT 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.
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.
The EA.hy926 cells were incubated for 24h with different concentrations of TSN IIA (5, 10 and 20 μg/μL ) or DMEM. Subsequently, cells were treated with 300 μmol/L H(2)O(2) for another 4h. Then, the percentage of cell viability was evaluated by 3-(4, 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.
Loss of cell viability and excessive cell apoptosis were observed in EA.hy926 cells after 4h of challenge with H(2)O(2) (300 μmol/L). However, cell apoptosis was attenuated in different concentrations of TSN IIA (5, 10 and 20 μg/μL) pretreated cells. Furthermore, TSN IIA markedly inhibited the elevation of ROS evoked by H(2)O(2). 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 H(2)O(2).
These observations provide preliminary evidence that TSN IIA protects EA.hy926 cells against H(2)O(2) 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.
- SourceAvailable from: Xue-Jie Yu
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- "Labeled cells were observed under a LSM 780 Laser Scanning Confocal Microscope (Carl Zeiss SAS, Jena, Germany). In order to further discriminate the early and late cell apoptosis and perform a quantitative analysis, flow cytometry with Annexin V-FITC/PI double staining was employed as previously described . Briefly, PC12 cells were detached with 0.125% trypsin, centrifuged at 1,000 rpm for 5 min, and then washed twice with PBS. "
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