不同治法方药对不同饲养环境下FM1流感小鼠TLR7/NF-κB、RLH信号转导调控机制的研究

Traditional Chinese medicine (TCM) shows its originality in prevention and treatment of influenza. Currently, the study of TCM is focusing on screening of antiviral drugs, however, the molecular mechanisms under the Chinese medicine theory guidance is still not clear. To well understand the mechanism of influenza and its therapy which is associated with the interaction between environment, host and influenza virus, and briefly explain the principle of "treating same disease with different methods" by common resolving superficies method, we will conduct TLR?/?, MyD88?/? and RIG-I?/? mice and normal mice infected by FM1 virus. Furthermore, different feeding environment of cold, hot and moist will be provided artificially, and the mice will be treated by three basic treatments according to TCM guidance for superficies syndrome of exogenous disease formula for relieving superficies syndrome with pungent and warm natured drugs, pungent and cool natured drugs and desiccating formula respectively. We will use the knowledge of molecular biotechnology, flow cytometry (FCM) and confocal microscopy to analyze the injury and restoration of TLR7/NF-κB signal pathway, signal proteins involved in TLR7 pathway, RLH signal pathway and its signal proteins effect. From such multi-way and multi-target profiling, we analyze the regulation of cell signaling in immune signal pathways promoting recovery from FM1 virus infected model treated by different resolving superficies method.

中药对流感防治有其独到之处，目前中药防治流感研究集中在对抗流感病毒药物的筛选，缺乏在中医理论指导下的分子机制研究。本研究采用TLR7-/-、MyD88-/-、RIG-I-/-小鼠和正常小鼠感染流感病毒株FM1，制造小鼠流感模型，并且人为制造寒、热、湿饲养环境，使用中医治疗外感表证的三种基本治法：辛温解表、辛凉解表、祛湿解表给予治疗。使用分子生物学、流式细胞仪及激光共聚焦显微镜等检测在不同饲养环境下，上述三种解表法对流感小鼠的病毒损伤恢复重要免疫通路TLR7/NF-κB及TLR7信号通路中信号蛋白的影响；细胞质的类维甲酸诱导基因-I样解旋酶（RLHs）及RLH信号通路中信号蛋白的影响。多途径、多靶点探讨不同解表法对流感小鼠的病毒损伤恢复重要免疫通路的细胞信号调控作用。其目的是探讨环境-机体-病原体三者相互作用对流感病情本身及治疗的影响机制，初步阐明几种常用解表法治疗该病的"同病异治"科学原理。

中药对流感防治有其独到之处，目前中药防治流感研究集中在对抗流感病毒药物的筛选，缺乏在中医理论指导下的分子机制研究。本课题研究不同中药治疗原则对甲型流感病毒FM1株感染三种不同环境小鼠肺部TLR7和RLH信号通路的影响，探讨其抗病毒的作用机制。本研究采用敲基因鼠（TLR7−/−小鼠）、敲基因鼠（MAVS−/−小鼠）和正常野生型C57BL/6小鼠随机为6组，正常对照组、病毒对照组、奥司他韦组、银翘散组、桂枝麻黄各半汤组、新加香薷饮组，饲养于正常环境、寒环境和湿热环境。三种不同环境小鼠造模11天，第11天通过滴鼻建立小鼠流感模型，24小时后分别用奥司他韦、银翘散、桂枝麻黄各半汤、新加香薷饮治疗流感小鼠，每天一次，共5天，第17天处死小鼠取材。每天观察并记录小鼠的状态和体重等。通过流式细胞术检测小鼠脾免疫细胞Th1/Th2、Th17/Treg的比例。肺组织病理观察小鼠肺部炎症状况。RT-qPCR和Western-blot检测小鼠肺部免疫细胞TLR7信号通路中TLR7, MyD88, NF-κBp65和RLH信号通路中RIG-I、MAVS、NF-κBp65的表达水平。银翘散和桂枝麻黄各半汤可减轻正常环境C57BL/6小鼠肺部炎症，桂枝麻黄各半汤可减轻寒环境C57BL/6小鼠肺部炎症，新加香薷饮可减轻湿热环境C57BL/6小鼠肺部炎症。流式细胞术结果显示奥司他韦和三组中药可不同程度下调Th1/Th2和Th17/Treg比值。RT-qPCR和Western-blot检测结果显示在正常环境中，桂枝麻黄各半汤和银翘散可下调流感C57BL/6小鼠肺中TLR7通路中TLR7，MyD88和NF-κBp65和RLH信号通路中RIG-I、MAVS、NF-κBp65的表达；在寒环境中，桂枝麻黄各半汤可下调流感C57BL/6小鼠肺中TLR7通路中TLR7，MyD88和NF-κBp65和RLH信号通路中RIG-I、MAVS、NF-κBp65的表达；在湿热环境中，新加香薷饮可下调流感C57BL/6小鼠肺中TLR7通路中TLR7，MyD88和NF-κBp65和RLH信号通路中RIG-I、MAVS、NF-κBp65的表达。本实验依据环境-机体-病原体三者相互作用对病情本身的影响，初步阐明中医使用不同解表法治疗流感的机理，为“同病异治”治疗流感的原理提供依据。