基于VIP/PKA/AQP5通路的石荠苧总黄酮抗流感病毒作用机制研究

Traditional Chinese medicine has unique advantages for the treatment of influenza. Mosla Scabra is widely used as a traditional medicinal herb in Jiangsu-Zhejiang region and is regulated in local standards. It is often used as an antipyretic and antiviral drug for lung diseases and has been found to be effective in colds, fever and influenza. In our past researches, we found that its active constituents- - total flavonoids (MF) exhibit anti-influenza virus activities in vivo and in vitro. In particular, MF could ameliorate viral pathological damage through up-regulation of VIP and AQP5 expressions instead of the inhibition of canonical TLRs signal pathway. To clarify this new mechanism, this research, taking VIP/PKA/AQP5 pathways as the breakthrough point, is to investigate its accommodation on abnormal developing water metabolism, and to observe its signal transduction mechanism and spatial variation regularity by using gene silencing and serum pharmacology methods in lethal murine experimental influenza virus infection model and in vitro cell model. This research also reveals regulatory mechanism of MF in the neuro-immune network and major targets to expound its "ventilating lung and eliminating phlegm" scientific connotation from water metabolism, viral load and signal transduction. It not only provides evidence for the quality-control of Mosla Scabra and the discovery of new antiviral lead compounds, but also provides a new method or idea for the functional research of the Chinese anti-influenza virus medicine. Moreover, our research may promote the production of this medicinal plant and increase farmers' income.

中医药治疗流感具有独特优势。石荠苧为江浙民间常用中药，地方标准收载药材，主治时疫感冒、风热外感等症。项目组前期证实其药效物质黄酮具有体内外抗流感病毒作用，可通过调控VIP和AQP5表达而改善病毒性病理损伤，而非对TLRs经典通路的阻断作用。为阐明这一新的药效机制，本项目以VIP/PKA/AQP5通路信号转导为切入点，通过建立流感感染小鼠模型和离体细胞模型，借用血清药理学和基因沉默的研究方法，从水液代谢、病毒抑制和信号转导3个方面，动态观察石荠苧总黄酮对感染组织水液代谢异常的调控作用，阐明其抗流感病毒效应的信号转导机制和时空变化规律，揭示其神经-免疫网络调控机制和主要作用靶点，阐明其"清肺化痰"的科学内涵，为石荠苧药材的质量评价，药效新靶点的发现，及抗流感中药新药的研发提供依据，也为中药抗流感病毒作用机制的研究提供新的思路和方法。同时对促进石荠苧药材产业化种植、提高三农经济具有积极作用。

石荠苧为临床常用中草药，具有抗菌、抗病毒、抗氧化、抗炎和免疫调控作用，主治感冒、风寒、咳嗽、肺水肿、肺气肿和慢性支气管炎等症。项目组前期证明石荠苧提取物具有较好的抗流感病毒作用。为进一步阐明其药效机制，本项目(1) 通过大孔树脂吸附分离技术，纯化石荠苧药效活性成分总黄酮（MF）并确定其最佳分离纯化条件为：在室温下，将浓度为5.55 mg/mL (pH=3.0)的石荠苧总黄酮提取液，以2.0 mL /min速上经预处理后的树脂柱，吸附后先以80 mL蒸馏水洗脱树脂柱，再用70 mL 60%乙醇以2.0 mL/min流速洗脱。此工艺条件下石荠苧总黄酮的回收率达 89.12%，纯度为90.77%。.(2) 采用甲型流感病毒（IAV）感染MDCK和A549细胞建立体外细胞模型，证明50～200 μg/mL MF可明显提高IAV感染MDCK和A549细胞的存活率，并呈明显的量效关系和时效关系；流式细胞分析结果表明，MF可明显抑制IAV诱导的MDCK和A549细胞凋亡，逆转IAV诱导的S期细胞周期阻断，呈剂量依赖性。此外，细胞免疫荧光检测发现，MF可上调IAV感染细胞AQP5蛋白的表达，拮抗甲型流感病毒诱导的细胞病变。.(3) 以AQP5和PRRs信号通路为切入点，通过鼻腔攻毒建立流感病毒感染小鼠模型，给予小鼠MF灌胃治疗，以及VIP基因活体转染技术，证明可明显降低IAV感染小鼠的肺指数，缓解IAV感染引起的肺损伤和细胞凋亡；可降低机体外周血IL-1β、IL-6和TNF-α水平，升高IFN-α水平来改善机体免疫功能；并MF可增加流感病毒感染小鼠肺组织TLR7、TRAF6、VIP、PKCa、AQP5 mRNA和AQP5蛋白的表达，降低Caspase-3和NF-κB蛋白表达，证明MF可能是通过TLR7，RIG-1和AQP5通路来起作用。分子对接结果显示，VIPR1的结合空腔相对较大，MF中的6个配体均可与VIPR1结合，其中，5-羟基-6,7-二甲氧基黄酮是VIPR1较为理想的配体，其结合能为-83.88 kcal/mol，可能为MF抗IAV主要药效物质基础。上述研究为抗流感病毒药效新靶点的发现，以及抗流感中药新药的研发提供了依据，也为抗流感病毒中药的作用机制研究提供新的方法和思路。.现已发表论文12篇，其中 SCI论文3篇，授权专利1项。