迷迭香酸抗过氧化氢诱导血管平滑肌细胞凋亡作用的研究

目的探讨迷迭香酸对过氧化氢(H2O2)诱导血管平滑肌细胞凋亡的保护作用及其机制。方法流式细胞术检测细胞凋亡率;吖啶橙(acridine orange,AO)染色观察细胞形态学变化;MTT法检测细胞活性;Western blot检测细胞中Bcl-2、Bax、Fas和FasL蛋白的表达。结果经500μmol·L-1H2O2处理24h后,细胞凋亡率为34.9%±2.55%,出现核固缩、核碎裂等典型的凋亡形态学改变,所以随后实验选择500μmol·L-1H2O2为诱导血管平滑肌细胞凋亡的最佳浓度。①500μmol·L-1的H2O2处理血管平滑肌细胞24h能引起细胞活性明显降低;细胞凋亡率明显增加,达35.7%±1.33%;细胞中Bcl-2蛋白表达降低,Bax蛋白表达升高,Bcl-2/Bax蛋白比值减少,Fas和FasL蛋白表达升高。②用迷迭香酸(10、20、40μmol·L-1)预处理细胞30min,可以增加细胞活性;降低细胞凋亡率,呈浓度依赖性;细胞中Bcl-2/Bax蛋白比值升高,Fas和FasL蛋白表达降低。结论迷迭香酸能拮抗H2O2诱导血管平滑肌细胞凋亡;其作用机制可能与升高细胞中Bcl-2/Bax蛋白比值,减少Fas、FasL蛋白表达有关。

Inhibition of rosmarinic acid on the apoptosis of vascular smooth muscle cells induced by hydrogen peroxide

Aim To investigate the effects of rosmarinic acid on H2O2-induced apoptosis in rat VSMCs and its related mechanisms. Methods Flow cytometry was used to determine apoptotic rate of VSMCs. nuclear staining by acridine orange for morphologic change,MTT assay for cell viability and Western blot for expression of Bcl-2,Bax, fas and FasL. Results After being treated by H2O2(0,250,500,750 μmol·L-1),apoptotic rate of VSMCs was 3.71%±0.56%,17.6%±6.92%,34.9%±2.55 %, and 85.6%±5.22% by FACS analysis,and VSMCs appeared nuclear condense and nuclear fragmentation after being treated by 500 μmol·L-1 H2O2 for 24 hours,which were typical morphological changes of apoptosis. ① VSMCs treated with 500 μmol·L-1 H2O2 for 24 hours had a significantly decrease on cell viability compared with control,and the apoptosis rate was increased to 35.7%±1.33%; Bcl-2 protein expression decreased,Bax protein expression increased, Bcl-2/Bax protein ratio decreased and Fas receptor and Fas ligand expression also increased.② Pre-incubation with rosmarinic acid(10, 20, 40 μmol·L-1)for 30 min enhanced the cell viability, and decreased the apoptotic rate to 31.1%±1.38%,21.2%±1.18%,13.6%±0.51% in a dose-dependent manner. Moreover, Bcl-2/Bax protein ratio increased and Fas and FasL expression decreased. Conclusions ① Rosmarinic acid antagonizes H2O2-induced apoptosis of VSMCs. ② The antagonism of rosmarinic acid on apoptosis may be correlated with an increase Bcl-2/Bax protein ratio and a decrease expression of Fas and FasL protein.