基于UPLC-Q-Exactive-Orbitrap-MS整合网络药理学探讨金银花抗RSV肺炎的作用机制

目的： 基于超高效液相色谱-四极杆-静电场轨道阱高分辨质谱（UPLC-Q-Exactive-Orbitrap-MS）技术、网络药理学和整体动物实验药理学的方法，初步探讨金银花抗RSV肺炎的作用机制。方法： 采用滴鼻法建立呼吸道合胞病毒（respiratory syncytial virus，RSV）肺炎小鼠模型，设立空白组、模型组、阳性对照组、金银花低、中、高剂量给药组，以小鼠体质量、肛温、肺指数以及IFN-γ、TNF-α、MCP-1、IL-10、IL-6、IFN-β的变化为指标，探讨金银花抗RSV肺炎的作用机制。采用UPLC-Q-Exactive-Orbitrap-MS对金银花中成分进行表征。采用网络药理学，将已定性成分-靶点网络与RSV肺炎-靶点网络进行映射，构建成分-RSV肺炎靶点网络。同时利用STRING数据库筛选网络中核心靶点，导入Cytoscape 3.6.1软件，构建核心靶点PPI网络，计算拓扑参数，推测核心靶点。结果： 与模型组比较，各金银花给药组小鼠体质量均增加，肛温及肺指数均下降，炎性因子（IFN-γ、TNF-α、MCP-1、IL-10、IL-6）含量增加或减少，且有显著性差异；对金银花中39个成分进行表征，结合网络药理学获得成分对应靶点277个，疾病靶点390个，去重后生成PPI网络，PPI网络中根据度值确定核心靶点为TNF、AKTI、TLR4、IL-10、IL-2。结论： 通过网络药理技术和整体动物实验推测，金银花可以通过下调TLR4/NK-κB通路，减少TNF-α、IL-6、IL-10等炎性因子的表达；作用于T细胞、自然杀伤细胞进而上调免疫因子IFN-γ的表达；降低MCP-1含量，减少炎性损伤、巨噬细胞的产生来协同发挥抗RSV感染肺炎的作用。

Integrate network pharmacology to explore the anti-RSV pneumonia mechanism of Lonicera Japonica Thunb.based upon UPLC-Q-Exactive-Orbitrap-MS

OBJECTIVE To employ ultra-performance liquid chromatography-Q-Exactive-Orbitrap-mass spectrometry(UPLC-Q-Exactive-Orbitrap-MS) technology,network pharmacology and overall animal experimental pharmacology methods for preliminarily exploring the anti-respiratory syncytial virus(anti-RSV) mechanism action of Lonicera Japonica Thunb. METHODS A murine model of RSV pneumonia was established by nasal dripping.Blank,model,positive control and low/medium/high-dose groups of Lonicera Japonica Thunb.were established.Body weight,rectal temperature,lung index and the changes of IFN-γ,TNF-α,MCP-1,IL-10,IL-6 and IFN-β were utilized as indicators for exploring the mechanism of honeysuckle against RSV pneumonia.UPLC-Q-Exactive-Orbitrap-MS was employed for characterizing the components in Lonicera Japonica Thunb.The qualitative component-target network was mapped with the RSV pneumonia-target network by using network pharmacology for constructing a component-RSV pneumonia target network.At the same time,the STRING database was accessed for screening the core targets in the network and the Cytoscape 3.6.1 software was imported.After calculating the topological parameters,the core targets were inferred. RESULTS Compared with model group,body weight of mice in Lonicera Japonica Thunb.dosing groups increased,rectal temperature and lung index decreased and the content of inflammatory factors(IFN-γ,TNF-α,MCP-1,IL-10 & IL-6) fluctuated and there were significant differences.A total of 39 components in Lonicera Japonica Thunb.were characterized and 277 corresponding targets and 390 disease targets obtained.After deduplication,a PPI network was generated and core targets were determined according to the degree value.The core targets were TNF,AKTI,TLR4,IL-10 and L-2. CONCLUSION Through network pharmacological technology and whole animal experiments,it is speculated that Lonicera Japonica Thunb.may down-regulate the TLR4/NK-κB pathway to lower the expressions of TNF-α,IL-6,IL-10 and other inflammatory factors.And it up-regulates the level of IFN-γ by acting on T cells and natural killer cells.Also it down-regulates the content of MCP-1 and minimizes inflammatory damage and the production of macrophages to play a synergistic role against RSV pneumonia.