细胞内游离钙在辛伐他汀诱导大鼠血管平滑肌细胞凋亡中的作用

Effect of intracellular free calcium on simvastatin induced vascular smooth muscle cells apoptosis in rats

目的 研究 3 羟 3 甲戊二酰辅酶A (HMG CoA)还原酶抑制剂辛伐他汀诱导血管平滑肌细胞(VSMC)凋亡的机制。方法 以荧光染料Fura 2 /AM负载后荧光分光光度计法检测细胞内游离钙浓度 ,以DNA琼脂糖凝胶电泳、流式细胞仪PI/膜联蛋白 (an nexin)V染色及半胱天冬酶 3激活来检测细胞凋亡。结果 辛伐他汀 30 μmol·L- 1孵育VSMC后 ,细胞内游离钙浓度显著升高 ,6h时达对照的 3倍以上 (P <0 .0 1) ,维拉帕米 80 μmol·L- 1与辛伐他汀 30 μmol·L- 1共同孵育VSMC 6h后细胞内游离钙浓度为 (14 4± 34)nmol·L- 1(P <0 .0 1)。辛伐他汀可诱导细胞凋亡率增高、“DNA梯状”样改变及半胱天冬酶 3的激活 ,这些变化均可被维拉帕米所逆转。结论辛伐他汀通过使细胞外钙大量内流而诱导VSMC凋亡。

AIM To investigate the mechanisms involved in simvastatin induced apoptosis in vascular smooth muscle cells(VSMC). METHODS Cultured VSMC was treated with simvastatin. Intracellular free calcium concentration ([Ca 2+ ] i) was measured by fluorescent Ca 2+ sensitive probe fura 2 acetoxylmethyl ester(Fura 2/AM), apop totic changes were distinguished by annexin Ⅴ binding , DNA fragment and caspase 3 activation. RESULTS When incubated with 30 μmol·L -1 simvastatin, [Ca 2+ ] i in VSMC increased with time and reached to (336±52) nmol·L -1 at 6 h, more than 3 fold of control (P<0.01 , n=5). Verapamil (80 μmol·L -1 ), a membrane voltage dependent Ca 2+ channel blocker, inhibited the increase of free calcium concentration induced by simvastatin from (336±52) nmol·L -1 to (144±34)nmol·L -1 (P<0.01) . Caspase 3 also activated by simvastatin after 12 h. Verapamil could efficiently inhibit simvastatin induced caspase 3 activation. Furthermore, 80 μmol·L -1 verapamil could decreased simvastatin induced apoptosis rate from (24.2±1.7)% to (7.9±0.6)% (P< 0.01) and also prevented simvastatin induced DNA laddering. CONCLUSION Simvastatin could increase [Ca 2+ ] i mainly through calcium influx from extracellular solution and then induces apoptosis.