熊胆药效成分抗流感病毒侵入的转录机制研究

Chinese herbal medicine compound preparation containing bear bile has long been clinically used for the treatment of influenza virus infection. Due to the complexity of bear bile composition, the underlying anti-virus constituent and the mechanism of action remain to be defined. We have previously found that among four known bioactive components of bear bile, only Tauroursodeoxycholic acid (TUDCA) exhibited an anti-influenza virus activity, which is associated with an inhibition of endosomal acidification involved in enveloped virus entry. Moreover, the protection of TUDCA against influenza virus was largely dependent on the expression of bile acid receptor farnesoid X Receptor (FXR). Here, we hypothesize that activation of FXR by TUDCA transcriptionally suppresses endosomal acidification, leading to the blockage of influenza virus entry. Through fluorescence-labeled virus and FITC-labeled bear bile components, together with on-line cell imaging, quantification of endosomal acidification and SPR techniques, we plan to figure out the essential inhibitory steps of TUDCA involved in influenza virus entry and its association with endosomal acidification. Based on above and further molecular exploration, we intend to clarify that the disturbance of endosomal acidification by TUDCA is mediated by FXR activation, which leads to an alteration of endosomal ion channels. These results may be an explanation of why TUDCA is the only effective anti-influenza virus component among the four bear bile bioactive substances. The current study will provide experimental evidence for the application of bile acid in the prevention and control of infectious diseases, and for the development and application of bear bile alternatives due to the lack of medical animal sources.

熊胆制剂临床抗流感病毒有效，但熊胆成分复杂，抗流感病毒药效物质及分子机制尚不明确。我们前期工作发现熊胆四种主要活性成分中只有“牛磺熊去氧胆酸”TUDCA具有抗流感病毒功效，表现为抑制病毒侵入宿主细胞。此抑制作用与内吞泡失酸化及核受体转录因子“法尼酯衍生物X受体”FXR密切相关。据此我们提出科学假说：熊胆成分TUDCA通过FXR对内吞泡酸化过程的转录抑制，从而阻止流感病毒侵入宿主细胞。我们将通过荧光标记病毒及熊胆药效成分、活细胞成像、内吞泡酸化定量及SPR等技术方法，揭示TUDCA抑制病毒进入的关键环节及其与内吞泡失酸化的关系，阐明TUDCA是通过激活FXR介导的转录调控，改变维持内吞泡酸化稳态的离子通道蛋白，发挥区别于其他三种熊胆药效成分抑制流感病毒侵入宿主细胞的分子机制。此项研究将为熊胆在重大传染病早期防控的广泛应用提供循证依据；为开发熊胆替代品，解决动物药药源不足的难题奠定科学基础。

熊胆制剂临床抗流感病毒有效，但熊胆成分复杂，抗流感病毒药效物质及分子机制尚不明确。我们前期工作发现熊胆四种主要活性成分中只有“牛磺熊去氧胆酸”TUDCA具有抗流感病毒功效，表现为抑制病毒侵入宿主细胞。本课题重点围绕TUDCA抑制流感病毒侵入的作用机制，先后从体内动物实验、体外细胞实验及构建iVLP模型分子层面等开展了融扎根pH依赖的内吞理论、突破iVLP构建关键技术与探索M2蛋白新功能于一体的创新性探索研究。我们首先通过acid-bypass实验设计，证实了TUDCA在抑制流感病毒侵入环节的关键作用；继而通过构建四种包含至少两个流感病毒外壳蛋白的iVLP模型，揭示了TUDCA对流感病毒M2蛋白的破坏是介导其抑制病毒侵入的重要因素；其后在确证M2蛋白具有细胞穿透肽的功能后，进一步明确了TUDCA通过对M2蛋白的解聚，使其失去质子泵及细胞穿透肽功能，从而发挥抗病毒侵入的作用机制。基于上述课题计划实施内容，我们也对M2细胞穿透肽的功能进行了氨基酸序列层面的拓展解析，证明了流感病毒M2蛋白的细胞穿透功能与其非跨膜区的45-62氨基酸形成的两亲性螺旋结构相关。同时，课题组集成物理化学领域的常用技术，尝试了对流感病毒及其与TUDCA分子相互作用的拉曼光谱表征。此项研究在分子层面深入解析了熊胆主要药效成分TUDCA抗流感病毒的药效机制，为熊胆在重大传染病早期防治的广泛应用提供了基础实验依据；为开发熊胆替代品，解决动物药药源不足的难题奠定了科学基础；为研究广谱抗病毒新药提供理论依据。同时，明确TUDCA抗流感病毒的作用机制将为进一步研发基于TUDCA的抗流感产品提供借鉴；iVLP的模型构建也将为体外高通量筛选抗流感病毒药物提供具有高生物安全级别的简单、易行、仿真、可靠的生物学分析工具。