阿司匹林丁香酚酯抗氧化应激致血管内皮细胞凋亡的分子机制

Vascular endothelial cells (VECs) play an important role in prevention of thrombosis, atherosclerosis and other cardiovascular diseases. The previous study results showed that the novel medicinal compound aspirin eugenol ester (AEE) had the preventive effect on VECs apoptosis induced by oxidative stress. However, the molecular mechanism of AEE protecting VECs from abnormal apoptosis is unclear. Therefore, human umbilical vein endothelial primary cells are selected as investigative material in this study, and the methods of cell biology and molecular biology are used to reveal the effects of AEE on apoptosis pathways and regulation factors at the molecular level. Under oxidative stress, the influences of AEE on mRNA, proteins and metabolites profiles of VECs will be investigated at integral level through transcriptomics, proteomics and metabonomics based on the technologies of next generation sequencing, iTRAQ and LC-MS, respectively. Then the effects of AEE on pathways will be analyzed through bioinformatics methods to probe the mechanism of AEE against apoptosis. The results from different levels will be analyzed integrately to establish the regulatory network of AEE anti-apoptosis. The relative regulatory pathways will be verified to illustrate the mechanism of AEE against VECs apoptosis induced by oxidative stress. This study will provide a theoretical basis for AEE to prevent thrombosis and atherosclerosis.

血管内皮细胞（VECs）对于预防血栓和动脉粥样硬化等心血管疾病具重要作用。前期研究表明，新型药用化合物阿司匹林丁香酚酯（AEE）对氧化应激致VECs凋亡具有保护作用，但其抗凋亡的分子机制尚不明确。本项目拟以原代人脐静脉内皮细胞为对象，通过细胞生物学和分子生物学方法，研究AEE对VECs凋亡通路和调控因子的调节作用；采用基于二代测序、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，研究氧化应激后AEE对VECs的mRNA、蛋白质和代谢物的影响，借助生物信息学方法，探讨AEE的抗凋亡机制；综合分析不同层面的结果，构建AEE的调控网络，进行相关调控通路的验证，以期较为系统地阐明AEE抗氧化应激致VECs凋亡的分子机制，为AEE预防血栓和干预动脉粥样硬化提供理论基础。

血管内皮细胞（VECs）对于预防血栓和动脉粥样硬化等心血管疾病具重要作用。前期研究表明，新型药用化合物阿司匹林丁香酚酯（AEE）对氧化应激致VECs凋亡具有保护作用，但其抗凋亡的分子机制尚不明确。项目以人脐内静脉内皮细胞（HUVECs）为对象，建立了H2O2诱导的血管内皮氧化损伤模型。采用western blotting、RT-qPCR、细胞免疫组织化学、流式细胞术和基因干预等技术和手段，从细胞层面揭示了AEE 抗细胞氧化损伤的调控机制，并在整体层面系统考察了AEE的相关调控机制，同时通过不同模型在体外和体内对结果进行了验证。结果显示，AEE能够通过对线粒体-溶酶体轴的调节，显著降低H2O2诱导的HUVECs氧化应激，Bcl2是AEE保护细胞免受氧化应激的重要调节靶点；AEE可显著改善H2O2诱导的HUVECs内NO调节系统紊乱，Nrf2在此过程中发挥了重要作用；AEE能够通过ASK1信号通路，减轻由H2O2诱导的细胞凋亡和百草枯（PQ）诱导的大鼠主动脉氧化损伤。转录组学、蛋白组学、乙酰化蛋白组学和代谢组学结果联合分析及验证试验结果显示，AEE抗氧化损伤的机制主要与MAPK信号通路、AMPK/mTOR信号通路、PI3K/Akt信号通路以及铁死亡-过氧化酶等途径有关，综合分析发现，整体层面与细胞水平的结果在一定程度上得到了相互验证。本研究为将AEE开发为预防血栓形成的药物奠定了理论基础。