阿托伐他汀对同型半胱氨酸作用下人脐静脉内皮细胞Bcl-2启动子区甲基化水平的影响及其抗动脉粥样硬化机制

Influence of atorvastatin in Bcl-2 methylation in cultured human umbilical endothelial cells treated with homocysteine and its mechanism of anti-arteriosclerosis

目的:探讨阿托伐他汀对同型半胱氨酸(Hcy)作用下人脐静脉内皮细胞(HUVECs)Bcl-2基因启动子区甲基化水平及其表达的影响,阐明阿托伐他汀抗动脉粥样硬化(AS)的可能机制。方法:HUVECs分别采用含有0、2、4、8、16和32mmol·L-1 Hcy的RPMI 1640培养液作用48h,采用MTT实验检测细胞增殖抑制率及半数抑制浓度(IC50),根据IC50结果将本实验分为对照组(0 mmol·L-1 Hcy)、Hcy组(9.00mmol·L-1 Hcy)、阿托伐他汀组(9.00mmol·L-1 Hcy+1×10-3 mmol·L-1阿托伐他汀)。各组细胞培养48h后,流式细胞术检测细胞凋亡率,荧光定量PCR法检测Bcl-2基因的mRNA表达,Western blotting法检测Bcl-2蛋白表达水平,甲基化特异性PCR(MSP)联合巢式降落式PCR法检测Bcl-2启动子区甲基化水平。结果:与对照组比较,Hcy组细胞凋亡率上升(P<0.01),Bcl-2基因mRNA及蛋白表达水平下降(P<0.01),Bcl-2基因启动子区甲基化水平降低(P<0.01);与Hcy组比较,阿托伐他汀组内皮细胞凋亡率下降(P<0.01),Bcl-2基因mRNA及蛋白表达水平增加(P<0.05),Bcl-2基因启动子区甲基化水平升高(P<0.05)。结论:阿托伐他汀可通过调节Hcy作用下的HUVECs Bcl-2基因甲基化水平抑制细胞凋亡。

Objective To investigate the influence of atorvastatin in methylation and expression level of Bcl-2 in human umbilical endothelial cells（HUVECs）treated with homocysteine（Hcy）and to expound potential mechanism of atorvastatin resisting arteriosclerosis.Methods After HUVECs were treated with 0, 2, 4, 8, 16, and 32 mmol·L-1 Hcy for 48 h,MTT was used to measure the inhibitory rates of HUVECs and the half inhibitory concentration （IC50 ）. According to the experimental results, the HUVECs cultured in vitro were divided into control group （0.00 mmol · L-1 Hcy ）, Hcy group （9.00 mmol·L-1 Hcy ）, and atorvastatin group （9.00 mmol·L-1 Hcy＋1×10-3 mmol·L-1 atorvastatin）.After treated for 48 h,flow cytometry was used to detect the apoptotic rate of cells, the mRNA expression of Bcl-2 was analyzed by fluorescence quantitative PCR,the protein expression of Bcl-2 was detected by Western blotting method, and the methylation level of Bcl-2 promoter region was determined by nest touch-down PCR combined with methylation specific PCR （MSP ）. Results Compared with control group,the apoptotic rate of HUVECs in Hcy group was increased（P〈0.01）,the mRNA and protein expression levels of Bcl-2 were significantly decreased（P〈0.01）,and the Bcl-2 promoter region methylation level was also decreased（P〈0.01）.Compared with Hcy group,the apoptotic rate of HUVECs in atorvastatin group was decreased（P〈0.01）,the mRNA and protein expression levels of Bcl-2 gene were increased （P〈0.05）, and the Bcl-2 promoter region methylation level was also increased （P〈0.05）. Conclusion Atorvastatin can prevent the apoptosis of HUVECs induced by Hcy through regulating Bcl-2 methylation.