奥美拉唑上调MRP3表达在氯吡格雷抵抗中的作用及机制研究

Clopidogrel is the gold standard drug for antiplatelet therapy of coronary heart disease. However, there are still 15 – 40 % of patients who have clopidogrel resistance leading to severe cardiovascular events. Accumulating evidence has demonstrated that a higher mRNA expression level of multidrug resistance-associated protein 3 (also known as MRP3) may be associated with clopidogrel resistance. Results of our preliminary experiment indicated that modification of MRP3 expression could regulate clopidogrel transport and its antiplatelet effect. Clinical research has shown that combination use of clopidogrel and omeprazole may lead to occurrence of clopidogrel resistance. However, the precise mechanism remains unkonwn. Our recent work showed that omeprazole induced the expression of hepatic transporter MRP3 and also the level of nuclear factor erythroid 2-related factor 2 (also known as Nrf2), which can upregulate the expression of MRP3. Thus, we hypothesized that omeprazole activates Nrf2/MRP3 pathway, which results in increase of clopidogrel efflux, less formation of clopidogrel active metabolite, and consequently clopidogrel resistance. This work would be used to clarify the potential role of omeprazole-induced upregulation of MRP3 in clopidogrel resistance in vitro and vivo experiments, with a special focus on “omeprazole→Nrf2→MRP3→clopidogrel transport” pathway. These efforts would provide theoretical basis of personalized medicine of clopidogrel in patient care.

氯吡格雷是冠心病患者抗血小板治疗的“金标准”，15%–40%的患者因氯吡格雷抵抗引发严重心血管事件。研究表明，MRP3 mRNA表达增加与氯吡格雷抵抗有关。我们预实验发现MRP3表达及活性的改变能够影响氯吡格雷的转运代谢及其抗血小板作用。临床研究显示，联用奥美拉唑可引起氯吡格雷抵抗的发生，但具体机制尚不明确。我们的前期研究发现：奥美拉唑可诱导MRP3的表达，亦能上调Nrf2水平，鉴于Nrf2能增加MRP3的表达，我们提出假说：奥美拉唑通过激活Nrf2，诱导MRP3上调，使氯吡格雷外排增加，细胞内氯吡格雷减少，其活性产物随之下降，从而引起氯吡格雷抵抗。本项目拟以细胞模型和敲除动物模型，运用分子生物学、药代动力学等技术来探明“奥美拉唑→Nrf2→MRP3→氯吡格雷转运”的调节通路在氯吡格雷抵抗中的作用及机制，为阐明氯吡格雷抵抗机制提供新的理论依据，并对临床冠心病患者个体化用药具有指导意义。

氯吡格雷是冠心病抗血小板治疗的“金标准”，临床研究显示，联用奥美拉唑可引起氯吡格雷抵抗。近期研究显示，奥美拉唑可增加HepG2中MRP3的水平，且CLP-G为MRP3底物。为了单独观察奥美拉唑对氯吡格雷的影响，我们连续7天给予小鼠奥美拉唑75 mg/kg/day，后给予氯吡格雷24 h，研究发现，奥美拉唑可增加小鼠肝脏中MRP3的蛋白表达，增加胞核Nrf2的蛋白水平，但对胞浆Nrf2和Keap1无影响。奥美拉唑亦可增加HepG2细胞胞核中Nrf2的水平，且给予Nrf2 siRNA干扰后，奥美拉唑诱导的MRP3的上调受到了抑制。提示奥美拉唑通过增加核Nrf2水平诱导MRP3的蛋白表达。LC-MS/MS法检测显示，奥美拉唑可显著增加血液中CLP-G的血药浓度、Cmax和AUC，降低CLP-G的肝/血浆比值，还可提高CAM H4的AUC，但Cmax在两组间并无差异，而且，连续给予奥美拉唑并未影响氯吡格雷的抗血小板作用。我们还对影响氯吡格雷活化相关的一相代谢酶CYP450及活化中间代谢产物2-oxo-clopidogrel的水平进行了测定。结果显示，奥美拉唑增加了参与氯吡格雷第二步氧化反应的代谢酶Cyp3a11的mRNA和蛋白水平，且对2-oxo-clopidogrel无影响。以上研究结果表明，连续给予奥美拉唑一方面可以通过增加胞核Nrf2水平诱导MRP3蛋白表达，促进CLP-G从肝细胞内转运至血液中；另一方面，奥美拉唑亦可增加Cyp3a11的mRNA和蛋白水平，从而增加CAM H4的系统暴露，但对Cmax无影响，因此未改变氯吡格雷的抗血小板作用。本研究在小鼠模型上阐述了奥美拉唑对氯吡格雷的影响，为奥美拉唑与氯吡格雷抵抗的关系提供了新的观点，对临床冠心病患者个体化用药具有指导意义。