Department of Biochemistry and Molecular Biology, Liaoning University of Traditional Chinese Medicine, Shenyang 110847, China.
J Ethnopharmacol. 2012 Aug 30;143(1):100-8. doi: 10.1016/j.jep.2012.06.007. Epub 2012 Jun 28.
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.
ETHNOPHARMACOLOGICAL 相关性：丹参（也称为中药丹参）是一种广泛使用的中药。丹参酮 IIA（TSN IIA）被认为是丹参中最重要的生物活性成分，具有抗动脉粥样硬化活性。
评估 TSN IIA 对过氧化氢体外损伤的人内皮 EA.hy926 细胞的保护作用及其可能的机制。
将 EA.hy926 细胞用不同浓度的 TSN IIA（5、10 和 20 μg/μL）或 DMEM 孵育 24 小时。然后，用 300 μmol/L H（2）O（2）处理细胞 4 小时。然后，通过 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化盐（MTT）测定评估细胞活力百分比。通过 AnnexinV-FITC/PI 双重染色和激光扫描共聚焦技术检测 EA.hy926 细胞的凋亡。通过流式细胞术分析细胞内活性氧（ROS）的产生。通过实时逆转录聚合酶链反应（实时 RT-PCR）检测 caspase-3、Bcl-2 和 Bax 的 mRNA 表达。通过 Western blot 测定 Bcl-2 和 Bax 的蛋白表达。还使用标准方法测量 MDA 水平、NO 产生、LDH 漏出、SOD 和 caspase-3 活性。
用 300 μmol/L H（2）O（2）孵育 4 小时后，EA.hy926 细胞活力下降，细胞凋亡过度。然而，用不同浓度的 TSN IIA（5、10 和 20 μg/μL）预处理细胞可减轻细胞凋亡。此外，TSN IIA 显著抑制 H（2）O（2）引起的 ROS 升高。实时 RT-PCR 和 Western blot 分析表明，TSN IIA 显著降低促凋亡蛋白（Bax 和 caspase-3）的表达，同时显著增加抗凋亡蛋白 Bcl-2 的表达，导致 H（2）O（2）诱导的 EA.hy926 细胞中 Bax/Bcl-2 比值明显降低。
这些观察结果为 TSN IIA 保护 EA.hy926 细胞免受 H（2）O（2）损伤提供了初步证据，这主要与 ROS 生成有关，随后是 Bax/Bcl-2 比值失衡和 caspase-3 激活导致凋亡。
