薤白抑制Lox－1延缓AS血栓形成的作用机制研究

The rupture of atherosclerosis plaque and thrombopoiesis is the pathology basis of acute coronary syndrome, tissue factor of vascular endothelial cell expressed is the initiator of coagulation pathway and a variety of adverse stimulus can increase the expression of TF. Bulbus Allii Macrostemonis in treating coronary heart disease angina symptom often as the main prescription, but the system research is lack and the specific mechanism is still not clear. The study includes the following two aspects: (1) In vivo experiment: using plaques with high rate and high thrombosis rate of ApoE knockout mouse model in our laboratory, to detect the plaque rupture rate, the expressing of related indexes about blood coagulation and inflammatory factor with blood biochemical, molecular biology and immunochemistry technology et al, to observe the effects of different doses of Bulbus Allii Macrostemonis on stabling plaque and inhibit thrombosis; (2) In vitro experiment: to build the endothelial cell injury model stimulated by high lipid environment, construct the pEGFP-Lox-1 of recombinant plasmid, and it was transfected into endothelial cells for highly expressing the Lox-1mRNA, using the Lox-1 specific inhibitor at the same time, combining the immunofluorescence and confocal laser technology, to discuss the effect of Bulbus Allii Macrostemonis on reducing inflammatory cytokine and down-regulation of TF expression by inhibiting the signal pathway of CD40/s CD40L -P38MAPK mediated by Lox-1. The results of the study will provide theoretical basis for the prevention and treatment of AS for Bulbus Allii Macrostemonis.

AS斑块破裂继发血栓形成是急性冠脉综合征发生的病理基础，组织因子（TF）是凝血反应始动者。薤白为治疗冠心病胸痹心痛病症常用中药，但缺乏系统研究且具体作用机制尚不明确。本研究拟从以下两方面展开：1）体内实验：采用本实验室建立的ApoE-/-小鼠斑块破裂和血栓形成模型，应用血生化、组织病理及分子生物学等技术，检测斑块破裂率及相关凝血和炎性因子表达水平，观察不同剂量薤白稳定斑块并抑制血栓形成的作用；2）体外实验：胆固醇刺激损伤内皮细胞构建体外研究模型，转染重组质粒pEGFP-Lox-1至内皮细胞以使其高表达Lox－1，应用特异性阻断剂抑表达,双向分析Lox-1、sCD40L-P38MAPK、TF之间相关性，从细胞分子水平探讨薤白抑制Lox-1介导的sCD40L-P38MAPK信号通路，减少内皮细胞炎性反应，下调促凝因子表达从而延缓血栓形成的机制。预期本研究结果将为薤白防治AS发生发展提供理论依据

AS斑块破裂继发血栓形成是急性冠脉综合征(ACS)发生的病理基础，内皮细胞表达的组织因子（TF）是凝血反应始动者。薤白是治疗冠心病胸痹心痛病症常用中药，可能与其增强AS内皮抗凝作用抑制血栓形成有关。本实验从两方面展开研究：(1)体内实验：采用ApoE-/-小鼠高脂饮食+颈动脉套管+精神应激+凝血酶原基因过表达的方法建立斑块高自发破裂率和高血栓形成率的“双高”小鼠模型，应用影像、血液检测、组织病理学及分子生物学等技术，检测内中膜厚度、凝血相关指标和相关炎性因子Lox-1、促凝因子TF及其途径抑制剂等水平，探讨不同剂量薤白稳定斑块并抑制血栓形成的作用；(2)体外实验：高脂培养建立内皮细胞损伤模型，构建重组质粒pEGFP-Lox-1并转染至内皮细胞以使其高表达Lox-1mRNA，应用特异阻断剂抑表达双向对照方法，结合免疫化学、分子生物学等技术从细胞分子水平深入研究薤白抑制Lox-1介导的CD40/sCD40L-P38MAPK通路，减少内皮细胞炎性反应，下调促凝因子TF表达从而延缓血栓形成的机制。结果提示：（1）成功构建的斑块高自发破裂率和高血栓形成率的“双高”小鼠模型，斑块发生破裂，形成附壁血栓。（2）颈动脉内中膜厚度及血脂在各药物组间未见明显差异；大剂量薤白组FIB、D-D、血小板聚集率及ox-LDL、Lox－1、TF、TFPI、sCD40L、p38MAPK的表达水平与其他剂量组相比均有显著差异（P<0.01）。（3）体外实验发现，单纯pEGFP-Lox-1转染组Lox-1、sCD40L、磷酸化p38MAPK、TF分子及蛋白水平均显著升高，而薤白干预后，Lox-1、sCD40L、磷酸化p38MAPK、TF水平均显著降低（P<0.01）。薤白防治AS的实验多为复方，抗凝的作用机制尚不清晰，且多只针对某一方面的病因或症状进行处理，缺少系统性的机制研究。本研究发现，Lox-1、sCD40L－p38MAPK信号通路与TF、TFPI之间具有相关性，并揭示薤白对其作用机制，为防治AS、稳定斑块并延缓血栓形成提供新的研究靶点。