叶酸通过DNA甲基化减轻同型半胱氨酸诱导的大鼠心肌细胞损伤

Folic acid relieves rat cardiomyocyte injury induced by homocystein through DNA methylation

目的研究同型半胱氨酸(Hcy)对体外培养的大鼠心肌细胞的影响,并分析叶酸对DNA甲基化的作用以探讨叶酸对H9C2心肌细胞Hcy暴露的保护作用及其机制。方法用(0、 0.5、 1、 2)mmol/L Hcy处理H9C2细胞24 h,观察细胞形态,采用CCK-8法检测细胞活力,流式细胞术检测细胞凋亡情况;设置2 mmol/L Hcy处理组、 0.1 mmol/L叶酸联合2 mmol/L Hcy处理组、 0.1 mmol/L叶酸处理组及DMSO对照组。处理24 h,采用以上方法检测细胞活力及细胞凋亡,MethylFlash总DNA甲基化ELISA试剂盒检测DNA甲基化水平,反转录PCR检测DNA甲基化转移酶1(DNMT1)、 DNMT3a、 DNMT3b的mRNA水平,Western blot法检测DNMT1、 DNMT3a、 DNMT3b的蛋白水平。结果不同浓度Hcy处理24 h的H9C2细胞数随Hcy浓度增大而减少。与对照组相比,2 mmol/L Hcy处理组细胞活力降低、细胞凋亡增加,叶酸联合Hcy处理组细胞数、存活情况较Hcy处理组明显增加;与其余各组相比,Hcy处理组的细胞总凋亡率增加,甲基化水平明显降低,叶酸联合Hcy处理组DNA甲基化水平增加;DNMT1 mRNA水平仅在叶酸处理组明显升高,而DNMT1、 DNMT3a、 DNMT3b蛋白水平均未出现明显改变。结论叶酸能通过DNA甲基化减轻Hcy对大鼠心肌细胞的损伤。

Objective To investigate the effect of homocysteine(Hcy) on the cardiomyocytes cultured in vitro, and to analyze the role of folic acid in DNA methylation to explore the protective effect and mechanism of folic acid during Hcy exposure of H9C2 cardiomyocytes. Methods H9C2 cells were treated with Hcy at different concentrations(0, 0.5, 1, 2) mmol/L for 24 hours. Cell viability was tested by CCK-8 assay. The apoptosis was detected by flow cytometry. H9C2 cells were divided into 2 mmol/L Hcy group, 0.1 mmol/L folic acid combined with 2 mmol/L Hcy group, 0.1 mmol/L folic acid group and DMSO control group. The above corresponding treatment lasted 24 hours. Then we detected the cell viability and apoptosis. The total DNA methylation level was determined by MethylFlash ELISA kit. DNMT1, DNMT3a, DNMT3b mRNA and protein expression were detected by real-time quantitative PCR and Western blot analysis. Results The number of H9C2 cells treated with different concentrations of Hcy for 24 hours decreased with the increase of Hcy concentration. Compared with the control group, the activity and apoptosis of the cells in the 2 mmol/L Hcy treatment group were reduced, and the number of cells in the folic acid combined with Hcy treatment group was significantly higher than that in the Hcy treatment group. Compared with the other groups, the total apoptosis rate of Hcy treatment group increased, methylation level decreased significantly, and the level of DNA methylation increased in the folic acid combined with Hcy treatment group. The level of DNMT1 mRNA significantly increased only in the folic acid treatment group, and the levels of DNMT1, DNMT3a and DNMT3b were not significantly changed. Conclusion Folic acid can relieve the damage of Hcy to myocardial cells by DNA methylation.