Department of Pharmaceutics, The Third Xiangya Hospital, Central South University, Changsha, China.
Hunan Key Laboratory of Medical Epigenomics, Department of Dermatology, The Second Xiangya Hospital, Central South University, Changsha, China.
Planta Med. 2013 Dec;79(18):1715-9. doi: 10.1055/s-0033-1351017. Epub 2013 Nov 15.
Our previous study showed that hypermethylation of dimethylarginine dimethylaminohydrolase 2 contributes to homocysteine-induced apoptosis of human umbilical vein endothelial cells. Epigallocatechin-3-gallate is a green tea-derived phenol which has been proved beneficial on atherosclerosis. It was demonstrated that epigallocatechin-3-gallate inhibits DNA methyltransferase activity and reactivates methylation-silenced genes in cancer cells. The aim of this study was to address whether epigallocatechin-3-gallate could induce DNA demethylation of the dimethylarginine dimethylaminohydrolase 2 gene, contributing to prevent endothelial cells from apoptosis induced by homocysteine. Human umbilical vein endothelial cells (ATCC, CRL-2480) were treated with homocysteine (1 mM) for 48 hours with or without epigallocatechin-3-gallate (20 µM) or 5-Aza (DNA methyltransferase inhibitor, 5 µM). Apoptosis rate of human umbilical vein endothelial cells was assayed by flow cytometry with an annexin V-FITC apoptosis detection kit. The mRNA and protein expression level of dimethylarginine dimethylaminohydrolase 2 and DNA methyltransferase 1 were detected by real-time PCR and Western blot, respectively. DNA methylation level of dimethylarginine dimethylaminohydrolase 2 was assayed by methylation specific PCR. The binding level of DNA methyltransferase 1 in the promoter of dimethylarginine dimethylaminohydrolase 2 was determined by chromatin immunoprecipitation-quantitative real-time PCR. It was shown that the apoptosis rate was decreased significantly in human umbilical vein endothelial cells treated with homocysteine compared with the control. Furthermore, the mRNA and protein level of dimethylarginine dimethylaminohydrolase 2 were downregulated, the dimethylarginine dimethylaminohydrolase 2 gene promoter was hypermethylated, and the DNA methyltransferase 1 mRNA and protein level were increased in human umbilical vein endothelial cells treated with homocysteine. Chromatin immunoprecipitation-quantitative real-time PCR revealed that homocysteine-induced binding of DNA methyltransferase 1 to the dimethylarginine dimethylaminohydrolase 2 promoter was increased. Pretreatment with epigallocatechin-3-gallate or 5-Aza inhibited such effects of homocysteine. In conclusion, epigallocatechin-3-gallate exerted protective effects on homocysteine-induced apoptosis in human umbilical vein endothelial cells by inhibiting promoter hypermethylation of the dimethylarginine dimethylaminohydrolase 2 gene and inducing dimethylarginine dimethylaminohydrolase 2 expression. These effects may be due to the decreased DNA methyltransferase 1 expression and binding of DNA methyltransferase 1 to the dimethylarginine dimethylaminohydrolase 2 promoter induced by epigallocatechin-3-gallate. This research suggests that modulating the epigenetic processes might be a novel plausible way for treatment of atherosclerosis.
我们之前的研究表明，二甲基精氨酸二甲氨基水解酶 2 的高甲基化导致同型半胱氨酸诱导的人脐静脉内皮细胞凋亡。表没食子儿茶素没食子酸酯是一种绿茶衍生的酚类化合物，已被证明对动脉粥样硬化有益。已经证明表没食子儿茶素没食子酸酯抑制 DNA 甲基转移酶活性并使癌细胞中甲基化沉默的基因重新激活。本研究旨在探讨表没食子儿茶素没食子酸酯是否能诱导二甲基精氨酸二甲氨基水解酶 2 基因的 DNA 去甲基化，从而防止内皮细胞受到同型半胱氨酸诱导的凋亡。用同型半胱氨酸（1 mM）处理人脐静脉内皮细胞（ATCC，CRL-2480）48 小时，同时用表没食子儿茶素没食子酸酯（20 µM）或 5-Aza（DNA 甲基转移酶抑制剂，5 µM）处理。用 Annexin V-FITC 凋亡检测试剂盒通过流式细胞术检测人脐静脉内皮细胞的凋亡率。通过实时 PCR 和 Western blot 分别检测二甲基精氨酸二甲氨基水解酶 2 和 DNA 甲基转移酶 1 的 mRNA 和蛋白表达水平。通过甲基化特异性 PCR 检测二甲基精氨酸二甲氨基水解酶 2 的 DNA 甲基化水平。通过染色质免疫沉淀-定量实时 PCR 测定 DNA 甲基转移酶 1 在二甲基精氨酸二甲氨基水解酶 2 启动子中的结合水平。结果表明，与对照组相比，同型半胱氨酸处理的人脐静脉内皮细胞凋亡率显著降低。此外，同型半胱氨酸处理的人脐静脉内皮细胞中二甲基精氨酸二甲氨基水解酶 2 的 mRNA 和蛋白水平下调，二甲基精氨酸二甲氨基水解酶 2 基因启动子超甲基化，DNA 甲基转移酶 1 的 mRNA 和蛋白水平增加。染色质免疫沉淀-定量实时 PCR 显示，同型半胱氨酸诱导的 DNA 甲基转移酶 1 与二甲基精氨酸二甲氨基水解酶 2 启动子的结合增加。表没食子儿茶素没食子酸酯或 5-Aza 的预处理抑制了同型半胱氨酸的这种作用。总之，表没食子儿茶素没食子酸酯通过抑制二甲基精氨酸二甲氨基水解酶 2 基因启动子的过度甲基化和诱导二甲基精氨酸二甲氨基水解酶 2 的表达，对同型半胱氨酸诱导的人脐静脉内皮细胞凋亡发挥保护作用。这些作用可能是由于表没食子儿茶素没食子酸酯诱导的 DNA 甲基转移酶 1 表达减少和 DNA 甲基转移酶 1 与二甲基精氨酸二甲氨基水解酶 2 启动子的结合减少所致。这项研究表明，调节表观遗传过程可能是治疗动脉粥样硬化的一种新的可行方法。
