基于TLR4/MyD88通路对Beclin1-Bcl2的影响研究麻杏石甘汤调控流感病毒诱导的自噬机制

Beclin1-Bcl2 complex is an important inhibitor for autophagy,the dissociation of Beclin1 from Bcl2 is essential for autophagy and is regulated by many proteins and signal pathways,the TLR4-MyD88 signal pathway as an important pathway of the host cell inflammation caused by IAV infected,through promoting MyD88 interacts with Beclin1, reducing Polymerization of Beclin1 and Bcl2, promote autophagy, eventually aggravate the pathogenesis of IAV. The project combines the nowdays hotspot（host cell autophagy）with Our preliminary studies（Maxingshigan decoction can reduce the influenza virus infection in mice lung inflammation, reduce the expression of TLR4 protein）,The interaction of dissociation by BiFC and Co-IP technology for detection of influenza virus infection inducedpulmonary macrophage autophagy in Beclin1-Bcl2 dimer and the two proteins and intervention in Maxingshigan Decoction. By using molecular biology technique for detection of influenza virus infection after lung macrophages and inflammatory factor key protein TLR4-MyD88 pathway on the expression of the relation between autophagy and intervention in Maxingshigan Decoction. Based on The form-function-Molecules, the TLR4-MyD88 signal pathway, with the pulmonary macrophage of mice by IAV infected, explore the intervention effect of Maxingshigan Decoction on the dissociation of Beclin1-Bcl2 Complex in autophagy of the pulmonary macrophage by IAV infected, for more likely target to provide the basis for Maxingshigan Decoction anti-IAV, futher provide the experimental evidences for its clinical application.

Beclin1与Bcl2聚合是抑制细胞自噬的关键。病毒感染时被活化的TLR4-MyD88信号通路可以通过促进Beclin1-Bcl2聚体解离而调控自噬。本项目结合时下热点（病毒感染可诱导细胞自噬）与系列前期研究基础（麻杏石甘汤能下调TLR4蛋白表达和抑制流感病毒核酸表达），首先以显微技术观察麻杏石甘汤含药血清对流感病毒感染诱导的肺巨噬细胞自噬的影响；继而采用双分子荧光互补（BiFC）分析技术和免疫共沉淀（Co-IP）技术技术检测该方对流感病毒感染诱导的肺巨噬细胞自噬中Beclin1-Bcl2聚体解离的干预作用；最后采用分子生物学技术检测该方是否通过影响TLR4-MyD88信号通路抑制Beclin1-Bcl2聚体的解离进而调控流感病毒诱导的肺巨噬细胞自噬，最终实现干预流感病毒的致病作用的效应机制？目的是从形态-功能-分子三个方面揭示麻杏石甘汤抗流感病毒的新作用靶点，为其临床应用提供实验依据。

流感性感冒（简称流感）是目前尚未得到有效控制的全球性急性呼吸道传染病，A型流感病毒是人和动物流感的主要病原体。体外研究表明：A型流感病毒感染时被活化的TLR4-MyD88信号通路可以通过上调Beclin1，下调Bcl2的表达，解离Beclin1-Bcl2聚体，促进自噬的发生，上调炎性细胞因子的分泌，关键通路元件的表达，最终加剧流感病毒的细胞损伤作用和肺部炎症，而麻杏石甘汤能有效抑制自噬的发生，下调IL-1β、IL-6、GCSF和TNF-α的分泌和自噬关键蛋白Beclin1和LC3的表达，上调Bcl2的表达，最终缓解流感病毒的细胞损伤。体内研究表明：TLR4基因缺失可以有效地延缓A型流感病毒感染小鼠所致的急性肺损伤的进程，且A型流感病毒诱导的小鼠肺部炎症可能的作用机制是通过激活TLR4-MyD88-TRAF6信号通路，启动炎性细胞因子的转录、翻译和大量释放。而麻杏石甘汤上调A型流感病毒感染小鼠的体质量、脾指数、胸腺指数，改善肺组织病理损伤，降低肺泡灌洗液中TNF-α、IL-1β和IL-6水平，下调肺组织中MyD88、TRAF6 mRNA和蛋白表达量，且存在一定的量效关系。同时麻杏石甘汤能有效修复免疫抑制型流感病毒感染小鼠模型的免疫功能，调节机体的免疫力，使机体对抗病原微生物的能力提高，缓解流感病毒引起的肠道炎症反应，形成“正气内存，邪不可干”的良好状态，而实现抗流感病毒作用。麻杏石甘汤这一多靶点效应，具有良好的社会效益和临床应用前景。同时对不同比例配伍的麻杏石甘汤抗流感病毒感染小鼠肺部炎症与病理变化的改善作用进行研究发现优化麻杏石甘汤各药物组成比例有助于将该方最大化发挥药效，可望为中药方剂现代化研究提供一条新思路。