阿司匹林丁香酚酯预防血栓的调控机制研究

To overcome the disadvantages of aspirin and eugenol, aspirin eugenol ester (AEE) as a novel medicinal compound was designed and synthesized according to the prodrug principles of structure recombination. The previous research results had showed that AEE not only had the effects of anti-inflammation, antipyretic, analgesia, antioxidant and lowering blood-lipid, but also had the prevention effect of thrombosis. AEE had more remarkable inhibition activities than compatibility of aspirin and eugenol for those enzymes related with thrombosis. Thrombosis prevention effect of AEE and the control drugs, which contained aspirin, eugenol, and mixture of aspirin and eugenol (molar ratio, 1:1), will be investigated in this study. To illustrate mechanism of prevention thrombosis, effects of AEE on inhibition platelet aggregation and protection vascular endothelial cells from oxidative injury will be investigated in this study. In particular, influences of mRNA, proteins and metabolic profiles also will be researched to explore thrombosis prevention mechanism of AEE through transcriptomics, proteomics and metabonomics based on the technologies of whole rat genome oligo microarray, isobaric tags for relative and absolute quantitation (iTRAQ) and LC-MS, respectively. Then the effects of AEE on pathways will be analyzed through bioinformatics method. Thus, the mechanism and the difference of AEE on thrombosis prevention from those control drugs may be revealed on the levels of platelet, vascular endothelial cell and systems biology. The study will promote the development of AEE as a new thrombosis prevention drug.

依据前药原理，设计合成了新型药用化合物阿司匹林丁香酚酯（AEE），以克服两原药的缺点。研究表明，其不但具有抗炎、解热、镇痛、抗氧化和降血脂等作用，还有预防血栓的作用；且AEE对血栓相关的酶具有显著的抑制活性，效果优于阿司匹林和丁香酚配伍。本项目拟以阿司匹林、丁香酚、阿司匹林+丁香酚（摩尔比为1:1）为对照，进一步明确AEE的预防血栓作用；并对其抗血小板聚集作用，对氧化损伤的血管内皮细胞的保护作用进行研究；特别是采用基于全基因组表达谱芯片、iTRAQ和LC-MS技术的转录组学、蛋白质组学和代谢组学等系统生物方法，研究AEE干预后，对血栓模型动物mRNA、蛋白质和内源性代谢物的影响，借助生物信息学的方法，阐明AEE对通路的影响。以期从血小板、血管内皮细胞和系统生物学3个层面，探讨AEE预防血栓的调控机理，并阐明AEE与阿司匹林和/或丁香酚的差异，为将其开发成预防血栓的新药奠定基础。

阿司匹林丁香酚酯（AEE）是依据前药原理设计合成的新型药用化合物，其不但具有抗炎、解热、镇痛、抗氧化和降血脂等作用，还有预防血栓的作用。在前期研究基础上，项目进一步确认了AEE 预防血栓形成作用，从抗血小板聚集、对血管内皮细胞氧化损伤保护及系统生物学等方面阐明AEE预防血栓的调控机理。结果表明：AEE能够显著改善血栓和血瘀模型大鼠的全血和血浆粘度等血液流变学参数及APTT、PT、TT、纤维蛋白原等凝血系统的参数。在体内外，AEE均可抑制不同激动剂诱导的血小板聚集，其机理与对PI3K/Akt、MAPK和Sirt 1/CD40L等通路的调节有关，主要包括抑制TXA2的产生和促进6-keto-PGF1α生成，降低胞内钙离子浓度、抑制对ERK2的活性，升高Sirt 1蛋白表达、降低CD40L表达，抑制ATP释放和JNK1磷酸化。AEE可通过升高GSH/GSSG比值及SOD、GSH-Px活性增强血管内皮细胞（HUVEC）的抗氧化能力，通过抑制iNOS表达降低NO的生成，通过调节线粒体膜电位和ROS生成改善线粒体功能障碍，促进抗凋亡蛋白Bcl2、Bcl-xL的表达并抑制促凋亡蛋白Bax、Bid、Cas3的表达等途径，实现对H2O2诱导的HUVEC氧化损伤的保护作用，还可通过抑制粘附分子E-selectin的表达而降低HUVEC对THP-1单核细胞的粘附。AEE能够调节cAMP、细胞粘附分子、NF-κB等通路相关基因的转录，调节能量代谢、脂蛋白结合、氧化还原、补体和凝血级联反应及血小板激活等通路相关蛋白的表达，调节亚油酸、花生四烯酸、脂肪酸和氨基酸等通路的内源性代谢物生成，产生预防血栓的作用。多组学整合分析显示，AEE产生药效作用主要与能量代谢和血小板激活等通路有关。多组学分析结果与体内外的试验结果基本一致，有很好的对应关系。研究结果初步阐明了AEE预防血栓的调控机制，为进一步探讨AEE抗氧化应激致血管内皮细胞凋亡的分子机制及其他研究提供了依据，为将其开发成预防血栓的新药奠定基础。