ABCB1基因在P2Y12受体拮抗剂类抗血小板药物引发消化道出血中的作用及其分子机制研究

P2Y12 inhibitors, including clopidogrel, prasugrel and ticagrelor, not only show definite antithrombotic effects for patients with acute coronary syndromes (ACS) undergoing percutaneous coronary intervention (PCI), but also have associations with increased risk of bleedings. Gastrointestinal bleedings occur with higher frequency than other kind of bleedings and do great harm to patients. However, the mechanism of gastrointestinal bleedings remains unclear because both cardiologists and gastroenterologists may not attach importance to it. Our research group has demonstrated that the key gene affecting the absorption of P2Y12 inhibitors, ABCB1, harbors single nucleotide polymorphisms (SNP) rs1045642 significantly associated with gastrointestinal bleedings in patients with PCI in clinical level. Our study includes experiments of cultured cells and mice. With the hypothesis that the mutant allele carriers of rs1045642 may have increased absorption of P2Y12 inhibitors, we will do research on the following aspects to elucidate the molecular mechanism of gastrointestinal bleedings caused by P2Y12 inhibitors: (1) to investigate whether the increased absorption of P2Y12 inhibitors could disrupt intestinal epithelial barrier by damaging the structure of tight junctions, inducing autophagy and apoptosis, finally result in gastrointestinal bleedings in vivo and in vitro; (2) to demonstrate whether the increased exposure to P2Y12 inhibitors could suppress the platelet aggregation excessively to cause higher risk of bleeding. The aim of this study is to provide foundation for prevention and treatment of gastrointestinal bleedings caused by P2Y12 inhibitors in patients with ACS and PCI, guide the clinical medication, and decrease the adverse drug reactions on the individual difference.

P2Y12拮抗剂在抗缺血方面使冠心病患者明确获益，但其引发的出血并发症尤其是消化道出血，常因属于心血管和消化的交叉学科内容，导致重视不足，更未见到相关机制研究。我们的前期临床研究发现肠道药物吸收相关基因ABCB1的多态性位点rs1045642与P2Y12拮抗剂引发的冠脉支架术后消化道出血显著相关。本项目以"该基因多态性位点可能会增加P2Y12拮抗剂肠道吸收"的科学假设为切入点，拟从细胞水平、整体动物水平深入研究P2Y12拮抗剂引发消化道出血的分子机制：（1）通过损伤肠道上皮细胞间紧密连接结构与自噬两个层面，诱导细胞凋亡，进而直接损伤肠道上皮屏障，引发溃疡后出血；（2）通过升高血药浓度，过度抑制血小板而增加出血风险；并最终以临床验证为研究结点。本项目将为P2Y12拮抗剂引发消化道出血的防治提供一种新思路、新靶点，为最大程度减少由于个体差异而导致的药物不良反应提供基础理论和研究依据。

P2Y12 拮抗剂在抗缺血方面使冠心病患者明确获益，但其引发的出血并发症尤其是消化道出血，常因属于心血管和消化的交叉学科内容，导致重视不足，更未见到相关机制研究。本项目以ABCB1基因多态性位点可能会增加P2Y12 拮抗剂肠道吸收的科学假设为切入点，从细胞水平、整体动物水平深入研究P2Y12 拮抗剂引发消化道出血的分子机制。.细胞水平研究发现，高浓度氯吡格雷通过激活p38-MAPK通路下调紧密连接蛋白occludin表达，导致小肠上皮细胞凋亡增加。p38-MAPK通路抑制剂能减弱氯吡格雷处理48h后细胞凋亡比例。此外，我们建立了含有ABCB1基因人源型野生位点和阳性位点rs1045642的小肠上皮细胞模型，可用于探讨该位点是否通过增加氯吡格雷吸收浓度损伤消化道，从而引发出血。.动物试验方面，我们掌握了建立可行的P2Y12拮抗剂损伤胃肠道的模型的方法，完成了ABCB1a和ABCB1b双基因敲除动物首建大鼠品系，并可以顺利繁殖后代。此外，我们正在构建ABCB1ab双基因敲除合并ABCB1人源野生型大鼠，ABCB1ab双基因敲除合并ABCB1人源ABCB1基因rs1045642基因突变大鼠品系。Western blot检测及病理免疫荧光法检测发现随P2Y12拮抗剂浓度升高，凋亡通路磷酸化p38-MAPK表达量有升高趋势。.临床验证方面，我们对510名STEMI患者进行了1年的随访，根据患者的ABCB1及PON1等基因的多态性，构建了STEMI患者术后出血风险的基因风险评分系统。研究结果表明携带ABCB1基因rs1045642，rs7779562；P2RY12基因rs6809699；PON1基因rs854560是STEMI患者PCI术后出血的独立预测因子。基因风险评分可以在一定程度上预测STEMI患者PCI术后BARC≥3型出血事件。基因风险评分联合传统临床高危出血因素可以提高对出血风险的预测能力。.我们的发现为未来冠状动脉经皮介入术后患者消化道出血的防治提供了新的思路，为最大程度减少由于个体差异而导致的药物不良反应提供基础理论和研究依据。