基于RTK信号通路的新双环酮类化合物purpurquinone B抗甲型流感病毒作用机制研究

Receptor tyrosine kinase (RTK) and its downstream signaling pathways play important roles in the process of infection and replication of influenza A virus (IAV), thus RTK signaling molecules are expected to become the novel anti-IAV targets. Our preliminary studies indicated that the novel bicyclic ketone purpurquinone B (PPQ-B), derived from the acid-resistant fungus, had significant anti-influenza A virus effects in vitro and in vivo, and it might inhibit the replication of influenza A virus by interfering the RTK signaling pathways. On that basis, the project intends to identify the IAV infection stages which PPQ-B affects by using flow cytometry and electron microscopy technology; To explore the inhibitory effects of PPQ-B on the Raf/MEK/ERK, NF-κB and p38MAPK signaling pathways in IAV infected cells by using quantitative RT-PCR, western blot and immunofluorescence methods, in order to reveal the anti-IAV targets of PPQ-B against influenza A virus; To investigate the inhibition mechanisms of PPQ-B against the immune injury in mice by using immunohistochemistry and ELISA technology, and eventually fully clarify the antiviral mechanisms of PPQ-B against influenza A virus. In summary, the project will not only provide the theoretical reference for the development of novel natural anti-IAV drugs, but also provide a reference for the anti-IAV therapy targeting RTK signaling pathway.

受体酪氨酸激酶（RTK）及其下游信号通路在甲型流感病毒（IAV）的感染和复制过程中发挥着重要作用，而RTK相关信号分子有望成为新的抗IAV靶点。项目组前期研究发现来源于耐酸真菌的新双环酮类化合物purpurquinone B（PPQ-B）具有显著的体内外抗IAV作用，且其可能是通过干扰RTK信号通路来抑制IAV的复制。本项目拟利用流式细胞术和电镜等技术鉴定PPQ-B作用的IAV感染阶段；利用定量RT-PCR、western blot和免疫荧光等方法探讨PPQ-B对于IAV感染细胞内Raf/MEK/ERK、NF-κB和p38MAPK等RTK信号通路的抑制作用，以揭示其抗IAV作用靶点；利用免疫组化和ELISA方法探究PPQ-B对小鼠免疫损伤的抑制作用机制，以期全面阐明PPQ-B抗IAV作用机制。本项目不仅为新型天然抗IAV药物研发提供理论参考，还将为靶向RTK信号通路的抗IAV治疗提供借鉴。

受体酪氨酸激酶（RTK）及其下游信号通路在甲型流感病毒（IAV）的感染和复制过程中发挥着重要作用，而RTK相关信号分子则有望成为新的抗IAV靶点。项目组前期研究发现来源于耐酸真菌的新双环酮类化合物purpurquinone B（PPQ-B）具有显著的体内外抗IAV作用，且其可能是通过干扰RTK通路来抑制IAV的复制。基于此，本项目从RTK信号通路入手，针对新双环酮类化合物PPQ-B抗IAV作用靶点和分子机制进行了深入研究。研究发现PPQ-B具有很好的体外抗甲型流感病毒增殖的作用（治疗指数SI > 20.0），且其抗IAV作用具有广谱性，对H1N1亚型的抑制效果略优于H3N2亚型。PPQ-B对IAV有直接灭活作用，但不抑制病毒吸附而是作用于病毒吸附后的感染过程。PPQ-B能够延缓流感病毒RNP的出核过程，从而在相同时间内降低病毒的增殖量。PPQ-B在10 mg/kg/day的剂量下口服给药能够显著减少肺病毒滴度，提高小鼠生存率，减轻体重下降趋势，与奥司他韦效果相当。PPQ-B还能显著地抑制IAV感染小鼠中炎症因子TNF-α, IL-6, RANTES和KC的分泌，减轻肺部病变，且可能与其对NF-κB 通路的抑制作用有关。PPQ-B还能通过下调细胞内RTK下游的Raf/MEK/ERK，p38MAPK和NF-κB等信号通路来抑制病毒的复制。因此，PPQ-B主要通过干扰RTK通路的激活来抑制IAV的体内外增殖和IAV感染引起的炎症反应。此外，本项目还建立了靶向宿主炎性反应和病毒侵入过程的细胞模型用于其它天然小分子化合物的活性筛选。总之，这些研究成果不仅为微生物来源的天然抗IAV药物的研发提供物质基础和理论参考，还可为靶向宿主RTK信号通路的新型抗IAV治疗提供借鉴。