基于TLR7/MyD88/NF-κB信号通路探讨薄荷、荆芥药对协同增效配伍规律及其作用机制

Every year, virus infective disease affected by influenza virus is going around all over the world, China has a high incidence of influenza. The anti-flu virus effect of Traditional Chinese Medicine is outstanding with the advantage of multicomponent, multiple targets. Mentha haplocalyx and Schizonepeta, commonly used Chinese medicine, are all active against virus when used alone. As the classic medicine, they are used not only directly in independent prescription “Jinghesan", but also as a core Chinese medicine pair in the numerous prescriptions against influenza. Exploring the antiviral mechanism of TCM based on related inflammatory pathways of toll-like receptors is a new strategy. TLR7 is a important receptors in TLR in influenza virus infection. In this project, clinical commonly used medicines of Mentha haplocalyx and Schizonepeta pairs are regarded as the research object with TLR7/MyD88/NF-κB signaling pathway as the research core, and the method of spectrum ralationship and different compatibility of Mentha haplocalyx and Schizonepeta antiviral effect of system research, to illustrate the spectrum efficiency relations. So, in this project, based on the theory of Chinese medicine compatibility,we will clarify the material basis and mechanism to reveal the metabolic process by vivo animal and cell culture experiments in vitro with modern technologies of Molecular Biology、Chemometrics 、Flow cytometry and the methods on spectrum-effect relationship. Based on TLR7/MyD88/NF-κB signaling pathway , we will explore the synergies compatibility rule and mechanism of action on Mentha haplocalyx and Schizonepeta pairs, which will not only develop the theory of Chinese medicine compatibility but also contribute to laying a foundation for the innovation of the influenza virus drug development.

流感病毒引起的感染性疾病，每年都在全世界流行，我国是流感高发国。中医药抗流感病毒疗效确切，有多组分、多靶点的优势。薄荷、荆芥均有明确抗流感病毒作用，作为经典药对，不仅直接独立成方《荆荷散》，而且在众多抗流感方剂中，作为核心药对发挥作用。基于Toll样受体相关炎症通路探讨中医药抗病毒机制是新策略，TLR7是与流感病毒感染相关的TLR中重要受体。本项目以中医临床常用药对“薄荷-荆芥”为研究对象，以TLR7/MyD88/NF-κB信号通路为研究核心，采用谱效关系研究方法，对薄荷、荆芥不同组分配伍抗病毒作用进行系统研究，阐明谱效关系；采用分子生物学、化学计量学、流式细胞术等技术手段，进行整体动物实验和细胞水平药效筛选与机理研究，基于TLR7/MyD88/NF-kB信号通路探讨荆芥、薄荷药对协同增效配伍规律及其作用机制。为指导薄荷、荆芥临床合理配伍应用提供依据，为抗流感病毒创新药物开发奠定基础。

本研究以中医临床常用药对“薄荷-荆芥”为研究对象，采用谱效关系研究方法，对薄荷、荆芥不同组分配伍组合抗病毒作用进行系统研究，采用分子生物学、化学计量学、代谢组学等技术手段，在薄荷、荆芥单味药及其药对全成分半定量分析基础上，进行整体动物实验和细胞水平药效筛选与机理研究，基于TLR7/MyD88/NF-kB信号通路探讨荆芥、薄荷药对协同增效配伍规律及其作用机制。采用GC-MS和UPLC-Q/TOF-MS/MS色质联用技术对薄荷、荆芥单味药及其药对物质基础进行研究，鉴定薄荷挥发性成分50种，非挥发性成分70种；荆芥挥发性成分41种，非挥发性成分67种；薄荷‒荆芥药对挥发性成分60种化合物，非挥发性成分74种。并对薄荷、荆芥共有51种非挥发性化学成分进行定性与半定量分析，结果显示，薄荷、荆芥配伍对其化学成分及含量均有一定影响。. 本研究分别采用聚酰胺树脂、D101大孔树脂吸附制备薄荷、荆芥总黄酮，鉴别其化学成分。并通过对薄荷挥发油、薄荷黄酮、荆芥挥发油、荆芥黄酮不同配伍组合对甲型流感病毒A/PR8/34（H1N1）感染小鼠进行整体动物实验和细胞水平药效筛选与机理研究，发现一些薄荷、荆芥不同组分配伍的组合生物效应较好，具有良好的抗病毒作用。本研究首次联合评价了薄荷-荆芥单味药及其药对挥发油GC-MS指纹图谱中所标示的化学成分与生物效应的关系，将药理数据与指纹图谱测定结果相关联，研究了69种化学成分相对质量分数的改变与抑菌效果、抗病毒小鼠肺指数、甲型流感病毒感染小鼠后对IL-6、IL-1β、TNF-α表达的影响。通过谱效关系研究，找到了发挥生物效应的主要药效成分。. 薄荷-荆芥作为经典药对在中医药理论指导下，一直沿用至今，并取得了较好的临床疗效。本研究证实了薄荷-荆芥作为核心药对具有确切的抗流感病毒作用，TLR7相关通路的调控作用可能是其主要作用机制。本研究采用谱效关系的研究思路，基于TLR7/MyD88/NF-kB信号通路探讨荆芥、薄荷药对协同增效配伍规律及其作用机制，为指导薄荷、荆芥临床合理配伍应用提供依据，为抗流感病毒创新药物开发奠定基础。