I型干扰素通路介导的清肺口服液类黄酮组分抗RSV的机制研究

Phlegm-heat obstructing lung is a common syndrome in children with viral pneumonia. Qingfei oral liquid, which comes from Maxing Shigan decoction, has good curative effects in clinic, while its mechanism is not clear. Recent studies have shown that flavonoid bacterial metabolites can play a role in clearing pathogens through the type I interferon signaling pathway. On the other hand, it has known that flavonoids account for 32% of the active ingredients of Qingfei oral liquid. So in our early work, we identified 13 flavonoid components in Qingfei oral liquid by LC-MS technology, and the lung tissue proteomics showed that Qingfei oral liquid could up-regulate type I interferon pathway protease OAS1, Based on these data, we put forward the hypothesis that "Qingfei Oral Liquid mainly activates the type I interferon pathway by the flavonoid component to exert antiviral effects". Here, qualitative and quantitative analysis of flavonoids in Qingfei oral liquid will be conducted by LC-MS technology. Then the flavonoids and their bacterial metabolites will be screened by luciferase cell line, to determine their anti-virus effects. Based on the effect evaluation by molecular biology techniques, the regulatory role of the flavonoid components from Qingfei oral liquid in Type I interferon signaling pathway will be further studied, through phagocytic scavengers and IFNAR blockers in RSV-induced WT mouse pneumonia model. This will reveal the main antiviral components and the mechanism of Qingfei oral liquid.

痰热闭肺证是小儿病毒性肺炎的常见证型。由麻杏石甘汤化裁而来的清肺口服液对其有良好疗效，但作用机制尚不清楚。近期研究表明，类黄酮的细菌代谢产物可通过I型干扰素信号通路发挥清除病原体的作用。而在清肺中，类黄酮占了活性物质的32%。基于此，课题组前期通过LC-MS技术鉴定出清肺中13种类黄酮成分，且肺组织蛋白质组学研究显示，清肺可以上调I型干扰素通路上的蛋白酶OAS1。因此提出了“清肺口服液主要通过类黄酮组分激活I型干扰素通路，发挥抗病毒效应”的假说。本项目拟通过LC-MS技术，定性定量分析清肺总黄酮提取物中的类黄酮组分，并利用荧光素酶细胞株筛选类黄酮及其细菌代谢产物，确定复方抗病毒效应的类黄酮组分；再通过RSV诱导的WT小鼠肺炎模型结合抗生素复合物、IFNAR阻滞剂，在效应评价基础上，运用多种分子生物学技术验证类黄酮组分对I型干扰素通路的调控作用，从而揭示清肺的主要抗病毒活性成分及其效应机制。

背景：呼吸道合胞病毒（RSV）肺炎是我国儿科常见病和多发病之一，西医仍无成熟有效的疫苗和理想的抗RSV药物。前期的随机单盲平行对照研究，证实清肺口服液是治疗小儿RSV肺炎痰热闭肺证的安全有效药物，但其作用的物质基础及机制是不明确的。.主要研究内容：通过醇提复方的方式得到清肺口服液冻干粉，并进一步进行工艺设计，明确了从清肺口服液冻干粉中提取清肺口服液黄酮类化合物的最优工艺条件，并采用此药物对RSV小鼠模型进行干预。为了进一步明确其中的效应物质基础，我们采用ISRE-CBG99Luc报告基因系统，对其中的化合物进行激活ISRE的高通量筛选，以此进一步组合完善了清肺口服液黄酮类化合物组分，并在RSV小鼠模型中进行了效应机制探究。.重要结果：明确了清晰准确的从清肺口服液冻干粉中提取清肺口服液黄酮类化合物的最优工艺条件。同时证实了清肺口服液黄酮类化合物通过调控I型干扰素通路发挥抗RSV效应的深层次机制。通过药物筛选，完善了清肺口服液黄酮类化合物组分，并完成了其抗RSV的药效机制研究。同时，理清了清肺口服液黄酮类化合物组分-肠道菌-I型干扰素通路在抗RSV中三者之间的关联效应。.科学意义：研究结果为进一步阐明复方清肺口服液抗RSV的效应机制提供了基础研究数据支持，同时，也将对同领域内的中医药抗病毒的成分及机制研究提供借鉴参考。