雌马酚对血管内皮细胞凋亡和增殖影响

Impact of equol on apoptosis and proliferation of vascular endothelial cells

目的探讨大豆异黄酮(SI)代谢物雌马酚对血管内皮细胞凋亡和增殖的影响。方法体外培养血管内皮细胞(ECV-304),采用不同浓度雌马酚(0、6.25、12.50、25.00、50.00和100.00μmol/L)处理24 h,收集细胞,采用流式细胞术分析细胞内活性氧(ROS)、细胞凋亡和细胞周期;采用免疫组织化学技术分析凋亡相关蛋白Bax、Bcl-2和caspase-3表达;采用噻唑蓝(MTT)法分析雌马酚对ECV-304细胞增殖影响。结果与对照组比较,6.25μmol/L雌马酚组ECV-304细胞内ROS含量下降,100.00μmol/L雌马酚组ECV-304细胞内ROS含量升高;与对照组比较,25.00、50.00、100.00μmol/L雌马酚组ECV-304细胞早、晚期凋亡率明显升高,并可导致细胞G1期阻滞(P<0.05);MTT实验结果显示,0、6.25、12.50、25.00、50.00、100.00μmol/L雌马酚组ECV-304细胞增殖抑制率分别为-(8.23±2.21)%、-(3.29±1.07)%、(6.75±1.73)%、(25.46±4.82)%和(36.93±4.66)%,低剂量雌马酚对细胞生长具有促进作用,高剂量雌马酚对细胞生长具有抑制作用;与对照组比较,25.00μmol/L雌马酚组作用24 h后,细胞内Bax和caspase-3表达增加,而Bcl-2表达减少。结论低浓度雌马酚可降低细胞内ROS含量,促进ECV-304细胞增殖;高浓度雌马酚可导致细胞凋亡、细胞周期阻滞,抑制细胞增殖。

Objective To study the effect of equol（ Eq）,a metabolite of soybean isoflavones,on apoptosis and proliferation of vascular endothelial cells. Methods Vascular endothelial cells（ ECV-304） were cultured in vitro and treated with different concentrations of Eq（ 0,6. 25,12. 5,25,50,and 100 μmol / L） for 24 hours and then the cells were collected. Reactive oxygen species（ ROS）,apoptosis and cell cycle were determined with flowcytometry. Apoptosis-related proteins（ Bax,Bcl-2,and caspase-3） were detected with immunohistochemical method. Eq-related cell proliferation inhibition rate（ IR） was measured with 3-（ 4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（ MTT） reduction assay.Results Compared with that of the control group,lower concentration of Eq（ 6. 25 and 12. 5 μmol / L） reduced intracellular ROS,whereas the higher concentration（ 100 μmol / L） of Eq increased intracellular ROS. The apoptosis rates of the cells treated with Eq of higher concentration（ 25,50 and 100 μmol / L） were increased（ 9. 67%,12. 17%,and 12. 87%）.Lower concentration of Ep did not affect the cell cycle also,while higher concentration of Ep caused G1 arrest. The IR of the ECV-304 cells treated with different concentrations of Eq（ 6. 25,12. 5,25,50,and 100 μmol / L） were- 8. 23 ±2. 21%,- 3. 29 ± 1. 07%,6. 75 ± 1. 73%,25. 46. 23 ± 4. 82%,and 36. 93 ± 4. 66%,respectively. Lower concentration of Eq promoted but the higher concentration inhibited the proliferation of the cells. Compared with the control group,the expression of Bax and caspase-3 in the cells treated with Eq（ 25 μmol / L） for 24 hours increased,while the expression of Bcl-2 decreased. Conclusion Lower concentration of Eq could reduce intracellular ROS,promote cell proliferation,while higher concentration of Eq could induce cell apoptosis and cell cycle arrest.