TLRs/NF-κB/P65信号通路在肾阳虚外感病证中的作用及麻黄细辛附子汤干预机制研究

The prevention and treatment of influenza in high risk population which susceptible to influenza virus is always some problems crying out for solutions of modern medicine，and this kind of syndrome belongs to exogenous fever with kidney-yang deficiency according to traditional Chinese medicine. It is estimated on the basis of lots of research evidence that exogenous fever combined with kidney-yang deficiency is a kind of viral infection in the condition of immunodeficiency resulting in TLRs／NF-κB/P65 signal pathway losing their normal control in the innate immune system, lots of inflammatory factors being produced and the balance between pathological damage and repair being broken. Mice model of exogenous fever combined with deficiency of kidney-yang has been established by the our research group through benzoic acid estradiol intraperitoneal injection combined with nasal vaccination influenza viruses FM1. Using the above model, effect of Mahuang-Fuzi-Xixin decoction extraction (MFX) on TLR3, TLR7, NF-κB, MyD88, TRIF, downstream cytokines TNF-α and INF-β and P65/P50 protein translation will be studied through up-stream of cytokine expression and protein kinase expression, TLRs／NF-κB/P65 signal pathway. Key link to the onset of exogenous fever combined with kidney-yang deficiency and intervention target of MXF with this signal pathway will be investigated further in this project. It will be of profound interest to elucidate the molecular mechanism of this syndrome and provide evidence for the effective pharmacological intervention on the disease.

流感病毒易感高危人群的防治一直是现代医学亟待解决难题，此人群所患流感多属于中医肾阳虚外感病证。我们根据大量研究证据推测，肾阳虚外感是因机体自身免疫力低下并被流感病毒传染，导致TLRs／NF-κB/P65途径发挥先天抗病毒免疫反应通路失控及产生大量炎性因子而造成病理损伤与修复之间的平衡被破坏。本课题依据前期已建立的成熟方法（苯甲酸雌二醇腹腔注射，鼻腔接种甲型流感病毒FM1）病证结合，复制肾阳虚外感小鼠模型。以此为工具，从细胞因子、蛋白激酶表达的源头TLRs／NF-κB/P65通路入手，观察经方"麻黄细辛附子汤"（MXF）对其一些重要环节TLR3、TLR7、NF-κB及其中间环节MyD88、TRIF和下游细胞因子TNF-α、INF-β及蛋白翻译控制P65/P50的作用；深入研究该通路在肾阳虚外感病证发病关键环节及MXF干预作用靶点；为深入了解该病证发病的分子机制并进行有效药物干预提供科学依据。

流感病毒易感高危人群的防治一直是现代医学亟待解决难题，体弱多病、免疫力低下的慢性基础病患者绝对是历次流感的高危人群，此人群所患流感多属于中医肾阳虚外感病证。.本课题主要研究内容包括：.1. 不同因素所致肾阳虚外感模型的筛选：腹腔注射苯甲酸雌二醇/氢化可的松+滴鼻感染甲型流感病毒H1N1/H3N2建立四种肾阳虚外感模型，采用偏最小二乘法综合分析各项指标评价并筛选最佳模型。.2. 选用了文献上不同配比的麻黄细辛附子汤进行干预肾阳虚外感证药效比较，优化出最优比例，并进行多种细胞因子（IL-6、IL-10、TNF-α、MCP-1）检测和抗氧化（SOD、MDA）作用研究。.3. 运用Western-Blot、RT-PCR等技术检测小鼠肺组织中甲型流感病毒H1N1、TLR3、TLR7、TRIF、MyD88及NF-κB (P50)的mRNA及蛋白表达，分析麻黄细辛附子汤干预肾阳虚外感可能作用的靶点。.4. 基于代谢组学方法，采用HPLC-TOF/MS和QE对病证结合模型动物的血清和肺组织进行定性分析，使用R语言，MPP，SIMCA-P等软件对数据进行处理，得到模型动物血清中内源性物质的变化和肺组织中脂质的差异；在MXF的整体药效作用基础上，应用HPLC-TOF/MS仪器分析MXF复方中主要化学成分及药效物质，探索MXF干预所建立病症模型的作用机理。.课题组研究发现：.1. 偏最小二乘法（PLS）可较好地建立中医病证结合动物的数学模型，可用于综合药效评价。.2. 经方治疗外感性疾病的基础研究应该是中医证候研究的一个重要的切入点。.3. 麻黄细辛附子汤降低TLRs/NF-κB/P65信号通路炎性因子的表达而起到降低因体内免疫炎性应答反应对机体的损伤，从而降低流感高危人群可能的恶化风险性，达到治疗效果。.4. 《伤寒论》上的经方的剂量及配比并不是最优化的，经研究可发现有剂量更小，配比更合适的处方，相关结果已获得发明专利，并将具有较好的应用前景。.5. 代谢组学研究发现，麻黄细辛附子汤主要影响到模型动物的氨基酸代谢、原发性胆汁酸代谢、糖皮激素代谢和甘油磷脂代谢。脂质类代谢物可能是肾阳虚外感模型病证小鼠的一类潜在标志物。