基于甲基化DNA含量的单核细胞分型对胆固醇致冠心病风险的预警机制研究

Overloaded cholesterol induces inappropriate inflammation to promote coronary artery disease (CAD). However, the mechanism of the inflammation is still unclear, that is not conducive for CAD prevention. We identified the decline of 5-mC (methylcytosine) contents in white blood cells (WBC) as an independent risk factor for CAD. The decline of 5-mC contents in WBC was mainly due to the decrease of that in mononuclear cells, and associated with high total cholesterol levels. Preliminary experiment results suggested monocyte subsets might be associated with genomic 5-mC contents. Recently, it is also reported that there is epigenetic programming in monocyte-to-macrophage differentiation and among immune cells subsets, and monocyte subsets are related to the risk of CAD. We speculated that the overloaded cholesterol might affect the DNA methylation to alter monocyte subsets, and then to promote CAD. Therefore, we want i) in blood samples, to compare the monocyte subsets, and analyze its associations with CAD processes, CAD risk factors, DNA methylation, expressions of DNMT1 and ten-eleven translocation methylcytosine dioxygenase (TET), intermediate products of one-carbon unit metabolism, and inflammation cytokines; ii) in cell lines, to screen and identify the key molecule in the DNA methylation pathway motivated by overloaded cholesterol, which altering the monocyte subsets; iii）in animal models, to validate the function of the identified target molecule on atherosclerosis and investigate the mechanism underlying. Eventually, we wish to find a new target for early warning and treatment of CAD and establish a novel method for detecting monocyte subsets based on decreased DNA methylation.

高胆固醇引起不恰当的炎症反应致CAD的机制尚不清楚,不利于预防。我们发现白细胞基因组5-mC含量下降是CAD的独立风险因素，这种下降源于单个核细胞并和胆固醇水平关联。预实验结果提示单核细胞分型可能与基因组5-mC含量相关。近期文献也报道单核细胞巨噬化和免疫细胞分型中均存在表观遗传学重编程，而且单核细胞分型与CAD风险相关。我们提出高胆固醇可能影响DNA甲基化,介导单核细胞分型致CAD，5-mC下降反映了一段时间内高胆固醇状态下单核细胞的炎症压力。为此,拟在血液样本中检测单核细胞分型,分析其与CAD进程、CAD风险因素、基因组甲基化、DNMT1和TET表达、炎症趋化因子和代谢中间产物之间的关系;在单核巨噬泡沫化细胞模型中,筛选胆固醇影响DNA甲基化介导单核细胞分型的关键分子;在动物模型中验证和探索该分子在AS中的功能和机制;寻找CAD新的预警和治疗靶点，建立单核细胞分型的DNA甲基化新方法。

我们曾报道血浆胆固醇与外周血白细胞（PBLs）基因组DNA甲基化水平（5-mC含量）负相关，然而血浆代谢物是否影响PBLs的功能从而在冠心病（CAD）中发挥作用还不明确。本研究发现：(1)与对照比较，CAD患者中间型单核细胞比例增加且CCR2表达水平显著增加，PBLs中5-mC含量和DNMT1表达显著减少，中间型单核细胞的5-mC含量最低，中间型单核细胞数量与PBLs的5-mC含量负相关（r = -0.1782，P = 0.0167）；患者组中ARID5B的cg25953130位点甲基化增加，表达水平降低且与单核细胞分型及CCR2的表达显著相关，受年龄、TG 、HDL-C以及Hcy等CAD风险因素的影响；(2)过表达ARID5B降低THP-1细胞CCR2、MCP-1及TNF-α的表达，促进早期凋亡，抑制迁移和粘附，促进巨噬细胞从M0型向M2型极化和早期凋亡并能抑制MCP-1的表达，而敲除ARID5B则相反；ox-LDL、Hcy能够显著降低单核细胞5-mC含量及DNMT1、ARID5B的表达水平，升高g25953130位点甲基化水平、中间型单核细胞比例和CCR2表达，而叶酸则相反；(3)在动物模型中，进一步验证了叶酸拮抗高脂或高Hcy诱导的ApoE-/-小鼠的高血脂和高Hcy，抑制中间型单核细胞比例以及斑块的形成；高脂和高Hcy还能够降低ApoE-/-小鼠PBLs基因组5-mC含量、DNMT1和ARID5B的表达，却升高MCP-1和TNF-α的表达，补充叶酸能够抑制这一作用。本研究揭示了胆固醇、叶酸和同型半胱氨酸对动脉粥样硬化炎症途径的表观遗传学调控机制，为CAD的预防和诊断提供了新靶点。