呼吸道M细胞及黏膜RIG-I/MAVS/IRF3/7信号通路触发的天然免疫应答对肾阳虚外感病证的调控作用及经方干预机制研究

Influenza is an acute respiratory infectious disease caused by influenza virus. The prevention and treatment of influenza in high risk population which susceptible to influenza is always urgent problem to be solved in modern medicine, and this kind of syndrome belongs to Kidney-yang deficiency with exterior symptom according to traditional Chinese medicine. Based on lots of research evidences, we hypothesis that Kidney-yang deficiency with exterior symptom is a kind of influenza virus infection at throat part respiratory M cells in the immunodeficiency condition firstly, then to have response disorder with corresponding mucosal immunity RIG-I receptor, causing cascaded amplification effect triggered by excess inflammatory factors and serious pathologic injury. Mouse model of Kidney-yang deficiency with exterior symptom has been established by our research group through intraperitoneal injection with benzoic acid estradiol combined with intranasal instillation of the H1N1/rPR8-GFP virus. We will start with important effect cell and molecular from respiratory M cells and RIG-I receptor, studying the expression of mRNA and protein with transcription factor IRF3/7, type I interferon, TNF-alpha, IL-6, IL-10 etc. Mahuang-xixin-fuzi decoction would be used for intervention with RIG-I receptor which triggers natural immune response pathway, and mucosal immunity regulation pathway and mechanism will be investigated. Key link to the onset of Kidney-yang deficiency with exterior symptom and the target of MXF in this immunity response will be explored further in the project.

流感是流感病毒引起的急性呼吸道传染病，对其高危人群防治一直是现代医学亟待解决难题，此类人群多属于中医“肾阳虚外感证”。我们依据大量研究证据推测，肾阳虚外感证是因机体免疫力低下后流感病毒首先集中侵染到咽喉部位呼吸道M细胞，导致相应黏膜免疫RIG-I受体应答失常，产生过量炎性因子触发级联放大效应而造成机体严重病理损伤。本课题病证结合，在前期已建立成熟肾阳虚外感小鼠模型（苯甲酸雌二醇腹腔注射，鼻腔感染H1N1/rPR8-GFP流感病毒）基础上，从呼吸道M细胞、RIG-I受体重要的效应细胞和分子入手，研究转录因子IRF3/7，下游I型干扰素及TNF-alpha、IL-6、IL-10等促炎和抗炎细胞因子mRNA和蛋白水平表达；并以麻黄细辛附子汤（MXF）干预RIG-I受体所触发天然免疫应答通路，探讨MXF对黏膜免疫调控途径与机制。深入研究该免疫应答在肾阳虚外感证发病的始动和关键环节及MXF干预靶点。

流感是流感病毒引起的急性呼吸道传染病，每年都多次爆发，由于流感的高危人群容易引起重症和危症，因而对其高危人群防治一直是现代医学有待解决难题，此类人群多属于中医“肾阳虚外感证”。. 针对肾阳虚外感证的防治，本研究主要开展以下三个方面研究：（1）课题组创新性地采用气管插管方式染毒，对比了正常小鼠感染流感病毒和肾阳虚证小鼠感染流感病毒后咽喉黏膜免疫的差异。首次发现了肾阳虚状态下滴鼻染毒方式对机体的损伤程度要严重于气管插管方式感染病毒。提示了机体在肾阳虚状态下咽喉黏膜免疫应答在流感病毒感染中的重要性。（2）为了更好地开展针对肾阳虚外感证模型的基因调控研究，本项目采用目前主流的geNorm, BestKeeper, NormFinder, Bio‑Rad Maestro等软件系统地筛选了肾阳虚外感证肺、心、肝和肾组织的10个文献上常见的管家基因表达，创新性地提出了采用组间t检验分析和绝对值比较策略来筛选稳定的双基因组合的管家基因，该研究已申请国家发明专利。（3）从咽喉黏膜RIG-I/MAVS/IRF3/7信号通路角度开展流感病毒感染研究时，我们发现，在病毒感染初期肾阳虚证小鼠咽喉M细胞对流感病毒的摄取及其引发类似抗原提呈作用要高于正常小鼠咽喉M细胞的反应，而在感染后期肾阳虚证小鼠和正常小鼠咽喉M细胞对流感病毒的类似抗原提呈作用差异不明显。炎症因子、蛋白表达数据证实麻黄细辛附子汤可通过干预TLR7-MyD88-IRF7信号通路，从而抑制肾阳虚外感证小鼠肺内炎症反应过度，减轻肺组织损伤，发挥其抗流感作用。. 科学意义：本项目首次创新性地开展了气管插管染毒方式，这将为呼吸道类病原体对咽喉黏膜免疫应答影响提供了重要的思路和解决方法；在管家基因筛选方法中创新性地提出了应用组间t检验分析和绝对值比较策略来筛选稳定的管家基因（双基因组合），无论从准确性还是操作方便性方面都要优于目前主流的管家基因筛选软件；从病毒感染的初始重要环节（咽喉黏膜免疫应答角度）开展经典名方的抗病毒研究，为这类病毒性疾病防控提供了早期干预、尽早截断病势的重要参考策略。