基于UPP/NF-κB信号通路调控P2Y12受体研究大黄蛰虫丸抗血小板作用及其机制

Clopidogrel resistance (CR) is a hot and difficult issue in the prevention and treatment of stroke. Traditional Chinese medicine for promoting blood circulation and removing blood stasis has some effect on CR, but its mechanism is still unclear. In our preliminary study, we found a significant decrease in platelet aggregation in CR patients treated with Dahuang Zhechong Wan (P=0.028), which mean Dahuang Zhechong Wan had a combined antiplatelet effect with clopidogrel. Dahuang Zhechong Wan was found to have the function of not only down-regulating the expression of ADP receptor P2Y12, but also blocking the UPP/NF-κB pathway by inhibiting ubiquitin protein Ub in previous research. Considering the activation of nuclear factor-κB (NF-κB) can up-regulate the expression of P2Y12 receptor, we hypothesize that Dahuang Zhechong Wan may inhibit the synthesis of ubiquitin protein Ub, the upstream signal of the UPP/NF-κB pathway, which can cause the expression of downstream P2Y12 receptor blocked. Thus, Dahuang Zhechong Wan has synergistic antiplatelet effect with the P2Y12 receptor antagonist clopidogrel. This project intends to use the advanced platelet function analyzer VerifyNow to detect the antiplatelet effect of Dahuang Zhechong Wan, and verify the previous hypothesis by detecting the upstream and downstream key signal molecules of the UPP/NF-κB pathway. The above study is expected to reveal the antiplatelet mechanism of Dahuang Zhechong Wan, and to establish the efficacy evaluation system of antiplatelet drugs, which may provide a new target and scientific basis for the antiplatelet therapy of integrated traditional Chinese and western medicine.

氯吡格雷抵抗(CR)是卒中防治的热点和难点。传统活血化瘀中药对CR有一定干预作用，但其机制尚不清楚。申请人前期研究发现CR患者联用大黄蛰虫丸后血小板聚集有明显下降(P=0.028)，提示大黄蛰虫丸有协同抗血小板作用。进一步研究发现大黄蛰虫丸可下调血小板P2Y12受体的表达，亦可抑制泛素蛋白Ub合成进而阻断UPP/NF-κB信号通路。考虑到激活NF-κB能上调P2Y12受体表达，我们推测大黄蛰虫丸可能通过抑制UPP/NF-κB信号通路上游Ub合成，阻断下游P2Y12受体表达，从而与P2Y12受体拮抗剂氯吡格雷发挥协同抗血小板作用。为此，本课题拟采用VerifyNow血小板功能分析仪明确大黄蛰虫丸的抗血小板作用，并同步检测UPP/NF-κB信号通路上下游关键信号分子进而验证上述假说。上述研究有望揭示大黄蛰虫丸抗血小板机制，有望建立抗栓药物疗效评价体系，为中西医结合抗栓治疗提供新靶点和科学依据。

缺血性卒中患者对抗血小板药物P2Y12受体拮抗剂氯吡格雷存在显著个体差异性，部分患者存在氯吡格雷抵抗，这是目前国内外缺血性卒中西药抗栓治疗的难点和热点。中医经典名方大黄蛰虫丸具有活血通络、祛瘀生新之功，具有一定的抗血小板作用，与氯吡格雷联用具有协同抗血小板作用，但其作用机制有待研究。在临床研究中，课题组成功入组126例新发缺血性卒中血瘀证患者，采用VerifyNow血小板功能分析仪检测患者血小板聚集功能，利用RT-PCR和western blot检测患者P2Y12 mRNA、P2Y12受体蛋白表达量以及P2Y12受体下游信号分子血管扩张刺激磷蛋白磷酸化(p-VASP)和丝氨酸-苏氨酸蛋白激酶B磷酸化(p-Akt)水平，其中121例患者完成了3个月随访。临床研究发现传统中药大黄蛰虫丸通过抑制ADP途径，与氯吡格雷发挥协同抗血小板作用，大黄蛰虫丸主要通过下调ADP受体P2Y12蛋白的表达从而发挥抗血小板作用，大黄蛰虫丸能够降低血小板聚集功能，改善神经功能预后，降低中医证候积分，降低缺血事件再发风险。在基础研究中，课题组采用改良线栓法构建脑缺血再灌注损伤(MCAO/R)大鼠模型，在大鼠CD61+巨核细胞上采用western blot检测UPP/NF-κB信号通路上游泛素蛋白Ub及下游P2Y12受体的表达，利用RT-PCR检测NF-κB mRNA、IκB mRNA和P2Y12 mRNA的表达；同时在血小板上采用western blot检测下游P2Y12受体的表达，使用全血浆检测p-VASP和p-Akt水平。研究发现，大黄蛰虫丸通过抑制UPP/NF-κB信号通路上游泛素蛋白Ub合成，上调IκB的表达从而下调NF-κB mRNA水平，阻断UPP/NF-κB信号通路下游P2Y12受体的表达，从而发挥抗血小板作用。本研究为抗栓药物提供了新的治疗靶点，同时建立临床可行的抗血小板药物疗效评价体系，为精准医疗下中西医结合个体化抗血小板治疗奠定一定循证医学基础。