基于Angptl4-PI3K/AKT/mTOR自噬调控PMVECs通透性探讨麻杏石甘汤治疗流感病毒性肺炎的作用机制研究

Intervened influenza virus pneumonia by regulating autophagy has became the research hotspot in international at present. Based on the maxingshigan(MXSG) decoction clinical application of anti-inflammation and the treatment of Xie Re Yong Fei Syndrome of patients with influenza, referenced to anti-inflammation of regulating autophagy and the therapy of clearing away lung heat way in Shanghan Lun, and based on the study of aggravation inflammation injury in lung through Angptl4（Ang4）, combined with the study basis that MXSG effect of anti- influenza virus pneumonia, we put forward that “The hypothesis of Ang4 on regulation of autophagy activity and PMVECs membrane passage via PI3K/AKT/mTOR, and The hypothesis of MXSG on regulation of autophagy to intervene in influenza virus pneumonia to improve lung injury”. The study will be utilized the technique of electron microscope、RT-PCR、western-blot、immunohistochemistry，on the animal models of pneumonia lung injury infected with influenza virus FM1 in mice and the PMVECs transfected with Ang4 and shRNA-Ang4 gene, then the cell model that stimulated with influenza virus; drug intervention will be applied with MXSG. There will be observed with the lung tissue of mice, the expressed inflammatory factor in cell, the protein related to autophagy signal pathway and the cellular morphology change of PMVECs. The purpose is to explore the protective mechanism and new target for clinical treatment of autophagy in influenza virus pneumonia, and to provide new methods and experimental basis on the effective treatment of lung injury of influenza viral pneumonia with classic recipe of chinese medicine.

调节自噬干预流感病毒性肺炎是目前国际上研究的热点。基于麻杏石甘汤(MXSG)抗炎、治疗邪热壅肺型肺炎的临床应用，借鉴自噬抗炎及Angptl4(Ang4)加剧肺部炎症损伤的研究，结合MXSG抗流感病毒性肺炎研究基础。我们提出“Ang4通过PI3K/Akt/mTOR自噬调节PMVECs细胞膜通透性，MXSG抑制Ang4调节自噬干预流感病毒性肺炎实现改善肺损伤作用”。本研究拟采用电镜、RT-PCR、western-blot、免疫组化等技术，在流感病毒FM1感染性肺炎的小鼠动物模型加Ang4、PI3K、mTOR抑制剂，和在PMVECs中加shRNA-Ang4并用流感病毒刺激的细胞模型上；应用MXSG药物干预，观察小鼠肺组织及细胞上的表达的炎症因子、自噬相关因子及信号通路上的蛋白和PMVECs细胞膜结构改变；探讨自噬在流感病毒性肺炎的保护机制和新靶点，为经方防治流感病毒性肺炎提供新的新的方法和依据。

病毒性肺炎是最常见的呼吸系统疾病之一，由于一直缺乏积极有效的抑制流感感染后期引发的过度炎症反应的治疗手段和措施，病毒性肺炎的治疗一直是国际上的医学难题。基于血管生成素样蛋白4 (angiopoietin-like protein 4，Angptl4，以下简称Ang4)介导的肺微血管内皮细胞(PMVECs)通透性改变是急性肺损伤的主要发病环节之一，炎症反应是病毒性肺炎的核心病理学特征，通过干预自噬来治疗流感病毒性肺炎已成为目前国际上研究的热点。本研究首先制备流感病毒感染PMVECs细胞细胞模型和动物模型，分别用PI3K inhibitors干预、shRNA-Angptl4转染和麻杏石甘汤含药血清处理，流式细胞技术、RT-PCR、Western-blot、ELISA、HE染色、免疫组化检测等进行相关指标的检测。结果显示，PI3K抑制剂LY294002可抑制H1N1诱导的细胞通透性增加、促进自噬相关蛋白的表达；沉默Ang4基因可以降低H1N1诱导的细胞通透性、促进自噬蛋白表达、降低炎症细胞因子水平；麻杏石甘汤含药血清处理可降低动物模型肺部的病理变化、促进细胞自噬、降低炎症细胞因子水平。可得到如下结论：流感病毒感染机体后，刺激炎症因子TNF-α释放促进肺部炎症损伤形成，Ang4既可降低F-actin蛋白诱发细胞骨架改变或促进Ang4又可通过PI3K/AKT/mTOR抑制自噬，进而降低血管渗透性，增加炎症因子渗出到间质。麻杏石甘汤抑制Ang4的分泌，通过调节PI3K/AKT/mTOR自噬，改善肺PMVECs细胞通透性发挥减轻病毒性肺炎所致肺损伤的作用。本研究探寻了麻杏石甘汤调控自噬的机制或途径，将有利于揭示流感病毒感染后引发肺损伤条件下自噬功能的调节规律，对发现积极有效的病毒性肺炎治疗靶点具有重要的理论意义和应用价值，对于中医药的广泛应用具有重要的社会意义。