钙依赖中性蛋白酶和半胱天冬酶-3在辛伐他汀诱导大鼠血管平滑肌细胞凋亡中的作用

Apoptosis induced by simvastatin in rat vascular smooth muscle cells through calpain and caspase-3-dependent pathways

目的 :观察辛伐他汀 (simvastatin)诱导大鼠血管平滑肌细胞 (VSMC)凋亡及可能参与其中的凋亡信号通路的研究 ,探讨他汀类药物可能的非调脂抗动脉粥样硬化作用机制。方法 :体外培养VSMC ,荧光分光光度仪检测钙依赖中性蛋白酶 (calpain)活性 ;Western印迹杂交法检测半胱天冬酶 - 3(caspase - 3)的激活 ;基因组DNA凝胶电泳、流式细胞仪PI/AnnexinV双重染色等鉴定细胞凋亡。结果 :30 μmol/Lsimvastatin刺激 8h后 ,calpain活性显著高于对照组 (P <0 0 5 ,n =4 ) ,12h后达对照的 3倍以上 (P <0 0 1) ;caspase - 3的 2 0kD活性亚基在 30 μmol/Lsimvas tatin刺激 12h后直至 4 8h均可检测到 ,而对照组及simvastatin刺激 2h均未见caspase - 3的 2 0kD条带 ;calpain特异性抑制剂PD15 0 6 0 6可显著抑制simvastatin诱导的VSMC凋亡 ,10 0 μmol/LPD15 0 6 0 6与 30 μmol/Lsimvastatin共同刺激细胞 2 4h后 ,流式细胞仪检测凋亡率为 9 5 %± 1 9% ,显著低于simvastatin单独刺激的 2 4 2 %± 1 7% (P <0 0 1,n =4 )。DNA梯度现象亦被抑制。同时PD15 0 6 0 6也能有效抑制simvastatin诱导的caspase - 3激活。结论 :Simvastatin诱导大鼠VSMC凋亡可能是通过calpain并进而激活caspase -

AIM: Hydroxymethylglutaryl CoA (HMG-CoA) reductase inhibitors, such as simvastatin, have been shown to reduce atherosclerotic cardiovascular morbidity and mortality by mechanisms unrelated to its lipid-lowering effect. Several studies have shown that simvastatin induces apoptosis in a varieties of cell lines including vascular smooth muscle cells (VSMC). The aim of this study was to investigate the signal pathways involved in apoptosis induced by simvastatin. METHODS: Cultured VSMC were treated with simvastatin. Calpain activity was determined by measuring Ca 2+ ionophore-specific calpain substrate (suc-LLVY-AMC), caspase-3 activation was detected by Western blot, and apoptotic changes were distinguished by annexin Ⅴ binding and DNA laddering. RESULTS: After incubated with 30 μmol/L simvastatin for 8 h, calpain activity had a marked increase ( P <0 05, n= 4) and reached to more than 3-fold of control at 12 h ( P< 0 01) Caspase-3 also activated by simvastatin after 12 h PD150606, a cell-permeable selective calpain inhibitor, decreased simvastatin-induced apoptosis rate from 24 2%±1 7% to 9 5%±1 9% ( P< 0 01) and also prevented simvastatin-induced DNA laddering. Furthermore, 100 μmol/L PD150606 efficiently inhibited simvastatin-induced caspase-3 activation. CONCLUSION:Simvastatin induces apoptosis by activating caspase-3 via calcium-dependent protease calpain.