CES1A1基因的DNA甲基化在氯吡格雷抵抗中的作用及其分子机制研究

Clopidogrel is one of the gold standard drugs of antiplatelet therapy of cardiovascular and cerebrovascular disease. About 5 to 44% patients will have thrombosis clinical events because of clopidogrel resistance, and the mechanism of clopidogrel resistance has become a hot. However, nearly 80% of clopidogrel resistance mechanisms are unclear. The previous research findings of the applicant find that: CES1A1 hypomethylation promotes the hydrolytic inactivation of clopidogrel and its active metabolite by up-regulating CES1A1 expression, which causse clopidogrel resistance. Based on the fact that the activation of PXR is the major regulator of CES1A1 expression and DNMTs inhibition is the main mechanism to induce gene demethylation, we put forward the hypothesis: DNMTs mediates the demethylation of CES1A1, raises the CES1A1 expression through activation of PXR and promotes the hydrolytic inactivation of clopidogrel and its active metabolite, which leads to clopidogrel resistance. With DNA methylation as the breakthrough point, the project aims to clarify the various subtypes of DNMTs which mediates CES1A1 methylation differences and the interaction of PXR/RXR and CES1A1 promoter, leading to the modulated regulation of CES1A1 and eventually clopidogrel resistance. The project reveals the molecular mechanisms of clopidogrel resistance from clinical, animal and cellular levels. It may reveal the potential drug targets to overcome clopidogrel resistance with important clinical significance.

氯吡格雷是心脑血管病抗血小板治疗的金标准药物之一，但仍有5～44%患者因氯吡格雷抵抗导致血栓等临床事件，其抵抗机制研究已成为热点，但目前仍有约80%抵抗机制不明确。申请人前期研究发现：CES1A1基因低甲基化通过上调CES1表达，促进氯吡格雷及其活性代谢物水解，从而导致氯吡格雷抵抗。鉴于PXR活化是调控CES1A1的主要途径，DNMTs抑制是基因去甲基化调控的主要机制，我们提出假说：DNMTs介导CES1A1去甲基化，通过激活PXR而上调CES1A1表达，促进氯吡格雷及其活性代谢物水解失活，从而导致氯吡格雷抵抗。本项目以DNA甲基化为切入点，拟研究DNMTs介导CES1A1甲基化状态差异，阐明影响PXR/RXR与CES1A1启动子相互作用而调控CES1A1表达并诱导氯吡格雷抵抗的关键因素，从临床、动物和细胞水平揭示抵抗的分子机制，预期结果可能揭示逆转抵抗的潜在药物靶标，具有重要的临床意义。

1.项目背景：.目前仍有5～44%患者因氯吡格雷抵抗导致血栓等临床事件，但仍有约80%抵抗机制不明确。.2.研究内容：.（1）研究缺血性脑卒中患者CES1A1、ABCB1、CYP2C19甲基化在氯吡格雷吸收、分布、代谢和抵抗中的作用。.（2）研究CES1A1甲基化在氯吡格雷吸收、分布、代谢和临床抵抗中的作用,并通过影响PXR/RXR与CES1A1启动子相互作用而调控 CES1A1 表达。.（3）结合患者临床多特征数据，预测氯吡格雷抗血小板治疗后的临床不良结局的发生风险。..3.重要结果：.（1）在缺血性脑卒患者中，氯吡格雷抵抗患者血浆CES1A1甲基化程度显著偏低， mRNA表达水平显著增加，血浆中氯吡格雷及其活性代谢物（R-130964，CAM ）的血药浓度显著降低，非活性羧酸代谢物浓度（SR26334） 显著增加，说明CES1A1低甲基化和mRNA高表达在氯吡格雷抵抗中发挥了重要的作用；.（2）氯吡格雷抵抗患者CES1A甲基化水平显著降低，CES1A表达水平会增高，SR26334血药浓度显著增加，而CAM血药浓度显著下降；表明：CES1A低甲基化会上调 CES1A1表达，促进氯吡格雷及CAM水解，导致CAM浓度下降，是氯吡格雷抵抗的重要因素。.（3）肝细胞CES1A低甲基化会增加表达水平；去甲基化试剂呈会降低肝细胞CES1A甲基化水平，并上调CES1A基因的转录水平和蛋白表达水平。 .（4） PXR调控CES1A1表达， PXR表达与CES1A1表达完全一致，利福平可促进CES1A1表达。 CES1A1表达受到PXR调控。.（5）结合CES1A1、CYP2C19和临床多特征数据，预测患者氯吡格雷抗血小板治疗后3个月、6个月和12个月的临床预后以及卒中性相关肺炎的发生风险。..4.科学意义.本研究将从多个角度来揭示氯吡格雷抵抗的分子机制，预期结果可能揭示逆转抵抗的潜在药物靶标，具有重要的临床意义。