辛伐他汀对雷帕霉素作用下心肌微血管内皮细胞的影响

Effect of Simvastatin on Rapamycin-treated Cardiac Microvascular Endothelial Cells

目的:研究辛伐他汀(SIM)对雷帕霉素(RAPA)引起的体外心肌微血管内皮细胞(CMECs)损害的保护机制。方法:分离、培养大鼠心肌微血管内皮细胞。经形态学及Dil-ac-LDL吞噬试验进行鉴定后,采用RAPA(100 nM)处理24小时建立CMECs损伤模型,然后加入不同浓度的SIM(0,10-2,10-1,100,101μM)培养24小时后,采用MTT,WST-8及Transwell检测受损后CMECs的增殖和迁移;采用Hoechst 33258及Caspase-3检查各组CMECs的凋亡;采用蛋白免疫印迹(Western blotting)检测Akt/p70 S6K磷酸化的程度;格里斯反应及实时逆转录PCR分别检测一氧化氮(NO)含量及一氧化氮合酶(eNOS)mRNA的表达。结果:①经形态学及Dil-ac-LDL吞噬试验均表明,成功培养CMECs;②低浓度SIM 100μM可显著改善100 nM RAPA对CMECs增殖、迁移和凋亡的影响;③SIM可通过上调PI3K/Akt进而上调p70s6K磷酸化(P<0.05或P<0.01);④SIM通过PI3K/Akt促进CMECs分泌NO及eNOS mRNA的表达(P<0.05或P<0.01)。结论:一定浓度下的SIM可改善RAPA作用下CMECs的增殖,迁移和凋亡,这些保护作用可能是通过其激活PI3K/Akt/mTOR/p70S6K信号通路实现的。

Objective： To investigate the molecular mechanisms underlying the protective effects of simvastatin （SIM） on rapamycin （RAPA）-treated cardiac microvascular endothelial cells （CMECs）. Methods： CMECs isolating from rats left ventricle were cultured and identified with microscopy and Dil-ac-LDL intake assay. CMECs were then treated with rapamycin （100 nM） for 24 hours. CMECs treated with 100 nM rapamycin were added with different concentrations of simvastatin （0, 10^-2,10^-1,10^0,10^1 μM） respectively and then cultured for 24 h. Then cell proliferation and migration were detected by MTT, WST-8 and transwell assays. The apoptosis was determined by Hoechst 33258 staining and Caspase-3 activity assay. Western blotting was performed to assess the phosphorylation of Akt/p70S6K. Nitric oxide （NO） secretion was assessed by Griess reaction. The expression of eNOS （endothelial nitric oxide synthase） mRNA was determined by Real-Time Reverse Transcriptase PCR. Results： ① By observing the morphology and Dil-ac-LDL intake assay indicated that CMECs have cultured successfully. ②Low concentrations of simvastatin （100 μM） significantly improved the viability, migration and apoptosis on CMECs after pretreated with rapamycin. ③The addition of simvastatin in CMECs resulted in rapid phosphorylation ofAkt, p70 S6 kinase （p70 S6K） （P〈0.05 or P〈0.01）. ④After pretreatment with simvastatin, the expression of NO and eNOS increased （P〈0.05 or P〈0.01）, which depends the activation of PI3K/Akt/p70 S6K pathway. Conclusion： Certain concentrations of simvastatin could improve the viability, migration and apoptosis on CMECs after pretreated with rapamycin. These protective effects may achieve through the activation PI3K/Akt/mTOR/p70S6K signaling pathway.