Yang X L, Tian J, Liang Y, Ma C J, Yang A N, Wang J, Ma S C, Cheng Y, Hua X, Jiang Y D
Department of Pathophysiology, Basic Medical School, Ningxia Medical University, Yinchuan, Ningxia, China.
Key Laboratory of Cardio-Cerebro-Vascular Diseases, Ningxia Medical University, Yinchuan, Ningxia, China.
Genet Mol Res. 2014 May 16;13(2):3787-99. doi: 10.4238/2014.May.16.2.
Homocysteine (Hcy) is an independent risk factor of atherosclerosis through its involvement with the methionine cycle. In this study, we aimed to determine the blood vessel global methylation rate in Hcy-induced atherosclerosis in apolipoprotein-E-deficient (ApoE-/-) mice, and to explore the possible mechanism of this change in endothelial cells. ApoE-/- mice were divided into a hyperlipidemia (HLP) group, a hyperhomocysteinemia (HHcy) group, and an HHcy + folate + vitamin B12 (HHcy+FA+VB) group. Wild-type C57BL/6J mice were prepared as controls. Total Hcy, lipids, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) contents in serum were measured with an automatic biochemistry analyzer and high-performance liquid chromatography. Methylation of B1 repetitive elements in blood vessels was tested using nested methylation-specific-polymerase chain reaction (nMS-PCR). Endothelial cells (ECs) were pretreated with Hcy or by adding FA and VB. Lectin-like oxidized LDL receptor-1 (LOX-1) expressions were determined by quantitative PCR, Western blot, and nMS-PCR. The HHcy group displayed severe HLP and HHcy. SAM and SAH contents were also elevated in the HHcy group compared with other groups. Methylation of B1 repetitive elements was significantly increased in the HHcy group (0.5050 ± 0.0182) compared to the HLP (0.5158 ± 0.0163) and control (0.5589 ± 0.0236) groups. mRNA and protein expressions of LOX-1 increased (0.2877 ± 0.0341, 0.6090 ± 0.0547), whereas methylation expression decreased (0.5527 ± 0.0148) after 100 μM Hcy stimulation in ECs. In conclusion, Hcy-induced atherosclerosis was closely associated with induced hypomethylation status in the blood vessel, and this process was partially mediated by LOX-1 DNA methylation.
同型半胱氨酸（Hcy）通过参与蛋氨酸循环，是动脉粥样硬化的一个独立危险因素。在本研究中，我们旨在确定载脂蛋白E缺陷（ApoE-/-）小鼠中Hcy诱导的动脉粥样硬化的血管整体甲基化率，并探讨内皮细胞中这种变化的可能机制。将ApoE-/-小鼠分为高脂血症（HLP）组、高同型半胱氨酸血症（HHcy）组和HHcy + 叶酸 + 维生素B12（HHcy+FA+VB）组。制备野生型C57BL/6J小鼠作为对照。用自动生化分析仪和高效液相色谱法测定血清中总Hcy、脂质、S-腺苷甲硫氨酸（SAM）和S-腺苷同型半胱氨酸（SAH）的含量。使用巢式甲基化特异性聚合酶链反应（nMS-PCR）检测血管中B1重复元件的甲基化。用Hcy或添加FA和VB预处理内皮细胞（ECs）。通过定量PCR、蛋白质印迹和nMS-PCR测定凝集素样氧化低密度脂蛋白受体-1（LOX-1）的表达。HHcy组表现出严重的HLP和HHcy。与其他组相比，HHcy组的SAM和SAH含量也升高。与HLP组（0.5158±0.0163）和对照组（0.5589±0.0236）相比，HHcy组中B1重复元件的甲基化显著增加（0.5050±0.0182）。在ECs中，100μM Hcy刺激后，LOX-1的mRNA和蛋白质表达增加（0.2877±0.0341，0.6090±0.0547），而甲基化表达降低（0.5527±0.0148）。总之，Hcy诱导的动脉粥样硬化与血管中诱导的低甲基化状态密切相关，并且这个过程部分由LOX-1 DNA甲基化介导。
