TLR4信号通路介导DFMG抗AS作用机制研究

It was proved that 7-Difluoromethyl-5,4'-dimethoxygenistein(DFMG) with Chinese Patent No.ZL200710104389.4 had the anti-atherosclerosis effect in vivo and in vitro by the applicant. We have found that DFMG could reduce the level of phosphorylated TNR receptor-2 and expression of TLR-4 protein in injured HAECs induced by oxidative stress, but the molecular mechnism by which modulate the anti-atherosclerosis effect still is unknown. In this research project, we will plan to explore the effect of DFMG on TLR4 asscociated proteins,TLR-4 MyD88-dependent signaling pathway, TLR4 TRIF-dependent signaling pathway and expression of inflammatory cytokines and adhesion molecules in the cultured human endothelial cells, macrophages and vascular smooth muscle cells by gene regulation of TLR-4;then, further validate the effect of DFMG on TLR-4 MyD88-dependent and TRIF-dependent signaling pathway in TLR-4 knockout mice.The findings of this research will reveal the detailed molecular mechanisms of DFMG by which regulate TLR-4 signal transduction against the atherosclerosis, which will provide a scientific basis to exploit and utilize DFMG in prevention and treatment of atherosclerosis in hunman.

申请人证实7-二氟甲氧基-5,4'-二甲氧基金雀异黄素（DFMG）具有体内外抗动脉粥样硬化(AS)效应（专利号：ZL200710104389.4）,且新近发现DFMG降低氧化应激人主动脉内皮细胞TNF-R2磷酸化水平和下调TLR-4蛋白表达，但DFMG抗AS作用的分子调控机制仍未知。本项目拟在AS相关细胞(内皮细胞、血管平滑肌细胞和巨噬细胞)，针对TLR-4进行基因调控(沉默/过表达)，研究DFMG对TLR-4相关蛋白及其触发MyD88和TRIF依赖信号传导途径和这些信号传导调控细胞粘附和炎症因子分泌功能的影响；并在TLR-4基因敲除小鼠模型上，进一步验证DFMG对TLR-4信号传递相关的MyD88依赖性、TRIF依赖性途径的影响，探讨DFMG体内外抗AS作用的分子机制。研究结果有助于阐明DFMG调控TLR-4信号传导发挥抗AS作用的分子机制，为DFMG防治AS研究提供实验和理论依据。

7-二氟甲氧基-5,4'-二甲氧基金雀异黄素(7-difluoromethyl-5,4'-dimethoxygenistein, DFMG)是自主设计和合成的具有血管内皮细胞氧化应激损伤保护作用的抗动脉粥样硬化(atherosclerosis, AS)候选药物。本项目在溶血磷脂酰胆碱（lysophosphatidylcholine, LPC）诱导HUVEC-12细胞损伤模型证明：DFMG干预TLR4-MyD88和TLR4-TRIF信号转导发挥保护HUVEC-12免受炎症损伤的作用。证据是：DFMG与SiRNA-TLR4一样具有降低LPC诱导HUVEC-12细胞损伤模型TNF-α和IL-6等炎症因子分泌，下调MyD88及TRIF蛋白及相应mRNA的表达，降低平滑肌细胞的迁移率和巨噬细胞活化等。本项目在载脂蛋白基因敲除（ApoE-/-）小鼠动脉粥样硬化模型及TLR4-/-鼠证实：DFMG在TLR4缺失的条件下具有更好的防止动脉粥样硬化发生、发展的作用。其表现为在双敲除（ApoE-/-TLR4-/-）小鼠使用DFMG，血清中总胆固醇（TC）水平、低密度脂蛋白胆固醇（LDL-C）水平和血清中载脂蛋白B（APO-B）水平明显降低，血清中高密度脂蛋白胆固醇（HDL-C）水平明显上升；胸主动脉、腹主动脉粥样硬化斑块面积、血管壁平滑肌细胞表型转化和血管新生等指标明显降低。总而言之，DFMG具有抗AS的作用，其作用机制与干预TLR4信号通路有关。