SIRT1对ox-LDL诱导的血管内细胞凋亡的影响

Experimental study of SIRT1 reducing intravascular apoptosis induced by ox-LDL

【目的】探讨沉默信息调节因子1(silence information regulator 1,SIRT1)对氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导的血管内皮细胞损伤的影响。【方法】人脐静脉血管内皮细胞CRL-1730转染SIRT1过表达载体(pc DNA3.1-SIRT1)和对照载体(pc DNA3.1),q RT-PCR和Western blotting检测细胞中SIRT1水平。用ox-LDL处理转染后细胞,流式细胞术检测细胞凋亡,黄嘌呤氧化法检测培养液上清中超氧化物歧化酶(superoxide dismutase,SOD)水平,硝酸还原酶法检测培养液上清中一氧化氮(NO)含量,Western blotting检测细胞中活化的含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved cysteinyl aspartate specific proteinase 3,Cleaved Caspase-3)、信号转导与转录因子3(signal transducers and activators of transcription 3,STAT3)、磷酸化的STAT3(p-STAT3)水平。【结果】pc DNA3.1-SIRT1转染后细胞中SIRT1 m RNA和蛋白水平均明显高于转染pc DNA3.1细胞(P<0.05)。ox-LDL处理后CRL-1730细胞凋亡率升高,细胞培养液中SOD水平和NO含量降低,与正常培养的CRL-1730细胞相比,差异具有统计学意义(P<0.05)。转染pc DNA3.1-SIRT1后的CRL-1730细胞经过ox-LDL处理后细胞凋亡率有所降低,细胞培养液中SOD水平和NO含量升高,与转染pc DNA3.1的CRL-1730细胞相比,差异具有统计学意义(P<0.05)。ox-LDL处理后的CRL-1730细胞中Cleaved Caspase-3水平升高,细胞中p-STAT3水平下降,而过表达SIRT1降低Cleaved Caspase-3水平,提高p-STAT3水平。【结论】ox-LDL诱导血管内皮细胞凋亡,降低细胞分泌SOD、NO水平,而SIRT1能够减弱ox-LDL的上述作用,改善血管内皮细胞损伤。

【Objective】To explore the effect of silence information regulator 1（SIRT1） on the vascular endothelial cells induced by Cu-oxidized LDL（ox-LDL）. 【Methods】Human umbilical vein endothelial cells（CRL-1730） were transfected with SIRT1 overexpression vector（pcDNA3.1-SIRT1） and control vector（pcDNA3.1）. The SIRT1 levels in cells were detected by qRT-PCR and Western blotting. The transfected cells were treated with ox-LDL, and apoptosis was detected by flow cytometry. The level of superoxide dismutase（SOD） in the supernatant of culture medium was detected by xanthine oxidation. The content of NO in the supernatant of culture medium was detected by nitrate reductase method. Western blotting was used to detect the levels of cleaved cysteinyl aspartate specific proteinase 3（Cleaved Caspase-3）, signal transducers and activators of transcription 3（STAT3） and p-STAT3 in cells.【Results】The mRNA and protein levels of SIRT1 in pcDNA3.1-SIRT1 transfected cells were significantly higher than those in pcDNA3.1 transfected cells（P〈0.05）. The apoptosis rate increased and the level of SOD and the content of NO decreased in the cell culture medium of CRL-1730 cells treated with ox-LDL, compared with normal CRL-1730 cells,and the difference was statistically significant（P0.05）. The apoptosis rate of CRL-1730 cells transfected with pcDNA3.1-SIRT1 decreased after the ox-LDL treatment, while the level of SOD and the content of NO increased in the cell culture medium, compared with CRL-1730 cells transfected with pcDNA3.1, and the difference was statistically significant（P0.05）. The level of Cleaved Caspase-3 in CRL-1730 cells after the ox-LDL treatment increased, while the level of p-STAT3 in the cells decreased. The level of Cleaved Caspase-3 after the overexpression of SIRT1 decreased, while the p-STAT3 level was improved.【Conclusion】ox-LDL can induce the apoptosis and decrease the level of SOD and NO in vascular endothelial cells, while SIRT1 can weaken the role of oxLDL and improve the damage of vascular endothelial cell.