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

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。

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

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 (［Ca²⁺］_i) was measured by fluorescent Ca²⁺-sensitive probe fura-2 acetoxylmethyl ester(Fura-2/AM), apoptotic changes were distinguished by annexin Ⅴ binding, DNA fragment and caspase-3 activation. RESULTS When incubated with 30 μmol·L^-1 simvastatin, ［Ca²⁺］_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 Ca²⁺ 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 ［Ca²⁺］_i mainly through calcium influx from extracellular solution and then induces apoptosis.