从诱生IFN-α/β的TLR7/8-MyD88信号通路研究最佳煎煮方式之麻杏石甘汤抗A型流感病毒的效应机制

MXSGT is commonly used in the treatment of pyretic pulmonary syndrome caused by exogenous pathogens. Also MXSGT is a classical Chinese herb prescription that effective for toxic-heat-invading lung and syndrome of heat-toxicity-obstructing lung caused by influenza. However, the current decocting method of MXSGT is totally different from the original method described in "Treatise on Febrile Diseases". The approaches for this project: First, obtaining a series of combinations through orthogonal test by analyzing the herbs of MXSGT from the original text. Associated with the influenza A virus infected cells models, exploring the influence of herb ingredients serums of extraction groups on virus proliferation from several aspects of viral load, cell activity, cell lesions and so on, and then selecting the best decocting method. stimulating monocytes / macrophages with influenza A virus, and then detecting the expression level of target genes and proteins, the activation and function of cells in the experiments by using fluorescence quantitative RT-PCR, Western Blotting and laser confocal microscopy to study the best decocting method of MXSGT, as well as regulation effect of MXSGT on main "elements" of TLR7/8-MyD88 pathway induced IFN-α/β. using the animal models of lung infection, explaining the MXSGT's mechanism by studying the correlation between MXSGT's regulation effect of TLR7/8-MyD88 signal pathway and its anti-influenza A virus effect from the aspects of pathway signal, virus proliferation and antiviral immunity. The main goals for this project are to enrich the scientific connotation and to broaden the clinical application of MXSGT.

麻杏石甘汤是治疗外感发热肺热证的常用方，也是治疗流感热毒袭肺证和热毒壅肺证的基本方，但其应用过程中的煎煮方式与《伤寒论》原文描述截然不同。本项目先以正交试验组合"原文"中该方煎煮方式之变量，形成系列组合，用A型流感病毒细胞感染模型，从病毒载量、细胞活性、细胞病变研究各组合提取物之含药血清对病毒增殖的影响，筛选最佳煎煮方式；再用A型流感病毒刺激单核/巨噬细胞，以荧光定量RT-PCR、Western blot、激光共聚焦显微镜等技术，从基因与蛋白表达水平、活化与功能状况研究最佳煎煮方式之麻杏石甘汤对诱生IFN-α/β的TLR7/8-MyD88信号通路各主要"元件"的调控作用；最后用A型流感病毒肺部感染动物模型，从通路信号、病毒增殖、抗病毒免疫研究最佳煎煮方式之麻杏石甘汤对TLR7/8-MyD88信号通路的调控作用与其抗流感病毒效应的相关性，注释效应机制。目的是丰富该方科学内涵，拓展其临床应用。

麻杏石甘汤是治疗外感发热肺热证的常用方，也是治疗流感热毒袭肺证和热毒壅肺证的基本方。基于该方应用中煎煮方式与《伤寒论》原文描述截然不同的现状，本项目开展以下研究：①麻杏石甘汤体外抗A型流感病毒最佳煎煮方式的筛选及其综合效应分析；② A型流感病毒感染对TLR7/8介导的IFN-α/β分泌水平的影响与最佳煎煮方式之麻杏石甘汤调控作用的研究；③A型流感病毒感染对诱生IFN-α/β的TLR7/8-MyD88信号通路的影响与最佳煎煮方式之麻杏石甘汤调控靶点的研究；④最佳煎煮方式之麻杏石甘汤对诱生IFN-α/β的TLR7/8-MyD88信号通路的调控作用与其抗A型流感病毒效应相关性的研究。结果是：①麻杏石甘汤含药血清对A型流感病毒对A型流感病毒感染的细胞有保护作用，麻黄先煎部分时间点之含药血清的作用优于四药同煎组；②麻杏石甘汤含药血清对流感病毒感染巨噬细胞IFN-a、IFN-β的分泌水平有明显的调节作用，麻黄先煎20分钟或25分钟组的效果优于四药同煎组；③麻杏石甘汤含药血清对A型流感病毒感染巨噬细胞TLR7/8-MyD88信号通路中TLR7、TLR8、MyD88、IFN-α、IFN-β基因表达、蛋白表达水平有明显调节作用，在干预24h时间点，麻黄先煎组对TLR7、TLR8、MyD88、IFN-α、IFN-βmRNA表达水平的调节作用优于四药同煎组，在干预48h时间点，麻黄先煎25分钟组对TLR7、TLR8、MyD88、IFN-β的mRNA表达水平的调节作用优于四药同煎组。④麻杏石甘汤对A型流感病毒感染小鼠有明显的保护作用，麻黄先煎25分钟组效果优于四药同煎组，其抗流感病毒效应与MyD88、TLR7、TLR8蛋白表达水平存在一定的相关性。研究结果注释了麻杏石甘汤抗流感病毒的部分效应机制，丰富了《伤寒论》中该方煎煮方式原文描述的科学内涵，为其正确煎煮方式推广应用提供实验支撑。