基于TLR4/MyD88/NF-κB信号通路、miRNA-146a表达探讨补肾方药抗动脉粥样硬化的效应机制

Atherosclerosis (AS) and induced Cardio-cerebral vascular diseases are the main fatal diseases, and the etiological factor and pathogenesis are not clear. It is demonstrated that TLR4/MyD88/NF-κB signal transduction pathway has a close relationship with AS, and miRNA-146a can regulate the pathway negatively. Earlier research of ours have demonstrated that the curative effect of Shen invigorating compounds Shoushen groule definited. On the basis of preliminary study, AS animal model will be maked by ApoE-/- mice in the project, methods of Real time PCR,Western blot and differential proteomics will be used. Expression of TLR4, MyD88, IRAK, TRAF6, NF-κBp65, miRNA-146a, MCP-1, TNF-α and ICAM-1 will be detected and that intervention of Shen invigorating compounds, and building network interactivity of miRNA-146a and proteins of TLR4/MyD88/NF-κB pathway. To be confirmed: Regulation the expression of key factors of TLR4/MyD88/NF-κB signal transduction pathway maybe the main mechanism of Shen inviorating compounds on AS, and Shen inviorating compounds maybe regulate TLR4/MyD88/NF-κB signal transduction pathway through intervention expression of miRNA-146a negative feedback. All that for prividing the multi-level and multi-target scitific evidence for curing AS related diseases.

动脉粥样硬化（AS）及其引起的心脑血管疾病是目前主要致死性疾病，发病机制尚不明确。研究表明miRNA-146可负向调控与AS关系密切的TLR4/MyD88/NF-κB通路。本项目前期研究证实补肾方药疗效肯定，拟采用ApoE-/-小鼠复制AS模型，结合Real time PCR、Western blot、差异蛋白质组学等技术，观察TLR4、MyD88、IRAK、TRAF6、NF-κBp65、miRNA-146a、MCP-1、TNF-α、ICAM-1的变化及补肾方药的干预作用，构建miRNA-146a与TLR4/MyD88/NF-κB通路靶蛋白相互作用网络。拟证实：调控TLR4/MyD88/NF-κB通路中关键因子的表达可能是补肾方药抗AS的重要机制，补肾方药可能通过干预miRNA-146a表达反馈调控TLR4/MyD88/NF-κB通路，为从补肾治疗AS相关疾病提供多层次、多靶点的科学依据。

目的：探讨补肾中药复方首参颗粒通过干预miRNA-146a表达反馈调控TLR4/MyD88/NF-κB通路抑制动脉粥样硬化（Atherosclerosis, AS）的作用机制。方法：采用高脂饮食诱导apoE-/-小鼠制备AS模型，予组织病理、miRNA芯片、2DE-DIGE、生物信息学、ELISA、Real-time PCR、Western blot等技术，研究AS小鼠主动脉的动态变化规律及首参颗粒的干预效果。结果：高脂饮食可诱导apoE-/-小鼠主动脉AS病变形成，随着高脂饮食喂饲时间点的推移，AS病变程度逐渐加重；首参颗粒可明显减轻AS小鼠动脉粥样硬化病变，降低血脂（TC、LDL-C）含量，下调炎症因子TNF-α、MCP-1、ICAM-1表达，调控NF-κB p65、TLR4、MyD88、IRAK、TRAF6表达。主动脉芯片差异miRNA动态表达结果显示：3m/C57差异表达miR29个，上调24个，下调5个；5m/C57差异表达miR31个，上调26个，下调5个；5m/3m差异表达miR4个，上调2个，下调2个。采用荧光定量RT-PCR法分别对差异表达miR146a、miR-146b、miR-192、miR-194、miR-1971、miR-34a进行验证，发现其表达趋势与miRNA芯片结果一致。2DE-DIGE分离差异蛋白，质谱鉴定， NCBI数据库提取蛋白信息，显著性差异分析揭示主动脉差异蛋白动态表达：C57/apoE(3+0):158,C57/apoE(3+5):56,C57/apoE(3+8):136;apoE(3+5)/apoE(3+0):28,apoE(3+0)/apoE(3+8):37,apoE(3+8)/apoE(3+5):26，深入进行GO功能与KEGG通路注释。生物信息学就差异miRNA、差异蛋白的相关通路进行了深入的富集分析，构建了DEM-DEP调控网络，为miRNA-146a与TLR4/MyD88/NF-κB通路靶蛋白相互作用网络提供了佐证。结论：首参颗粒可调控TLR4/MyD88/NF-κB通路中关键因子TLR4、MyD88、NF-κB、IRAK、TRAF6及MCP-1、TNF-α、ICAM-1表达，干预miRNA-146a表达反馈调控TLR4/MyD88/NF-κB通路可能即是补肾中药复方首参颗粒有效干预AS的关键作用机制。