基于GM-CSF的信号传导调节下呼吸道菌群的黄芩“行上焦，清湿热”的机制研究

Lower respiratory tract infections (LRIs) are a serious threat to human health. It is believed that LRIs are closely related to the microbiota in lower respiratory tract. And, the microbiota can help the hosts enhance the defense against respiratory infections based on GM-CSF signaling. The pathogenesis and clinical manifestations of syndrome of dampness-heat in upper jiao in Traditional Chinese Medicine and LRIs are exactly similar. Traditional Chinese medicine Scutellaria baicalensis is good at going upper-jiao to clear dampness-heat and it has many applications in the treatment of respiratory infections. However, the mechanism for its action of “going upper-jiao and playing the role of clearing heat and dampness” is poorly defined. Therefore, under the guidance of the traditional Chinese medicine theory, this project organically correlates the effect of Scutellaria baicalensis on anti-respiratory infection together with the lower respiratory tract microbiota, and put forward a hypothesis “Scutellaria baicalensis play a role in going upper-jiao and clearing dampness-heat is based on GM-CSF signaling to regulate the lower respiratory microbiota. We take GM-CSF signal transduction as the breakthrough point. And then 16S rRNA gene-sequencing technology and UPLC-Q-TOF-MS will be used to identify the microbiota species that Scutellaria baicalensis regulated and the related active components in Scutellaria baicalensis. Next, we use GM-CSF gene knockout mice, pseudo-sterile mice and invitro culture of bacteria and cells and employ laser scanning confocal microscope, flow cytometry, Western Blot, qRT -PCR to study the anti-infection mechanism of Scutellaria baicalensis in vitro and in vivo. We aim at interpreting the mechanism of the effect of Scutellaria baicalensis on “going upper-jiao and playing the role of clearing heat and dampness” from the perspective of “whole-microbiota-cell-molecule”. It is possible to provide new target and research idea for exploring the mechanism of traditional Chinese medicine in the treatment of syndrome in upper-jiao.

下呼吸道感染已严重威胁人类健康，而下呼吸道菌群可基于GM-CSF的信号传导增强对下呼吸道感染的防御。中医中的上焦湿热证与下呼吸道感染病因及临床表现相似。黄芩长于行上焦而清湿热，对呼吸道感染的治疗应用颇多。但其“行上焦，奏清热燥湿之效”的作用机制不清。因此，本项目在中医药理论的指导下，将黄芩抗呼吸道感染的作用与下呼吸道菌群进行有机关联，提出“黄芩‘行上焦，清湿热’的作用是基于GM-CSF的信号传导调节下呼吸道菌群来实现的”的假说。以GM-CSF的信号传导为切入点，利用16S rRNA测序技术和UPLC-Q-TOF-MS明确黄芩调控的菌群物种及其物质基础；借助GM-CSF基因敲除小鼠同时采用激光共聚焦、FCM、Western Blot和qRT-PCR等手段，从“整体-菌群-细胞-分子”水平，阐明黄芩“行上焦，奏清热燥湿之效”的科学内涵，为探索中药防治上焦病证的机制研究提供新的靶点和思路。

下呼吸道菌群与下呼吸道感染密切相关，可调控GM-CSF信号增强对呼吸道感染的防御。中医中上焦湿热证与下呼吸道感染病因及症状相似。黄芩长于行上焦而清湿热，常用于治疗呼吸道感染，但其机制不明。因此，本项目在中医药理论的指导下，建立下呼吸道感染模型，结合抗生素处理和呼吸道菌群再移植的方式，利用宏基因组、UPLC-Q/TOF-MS、生物信息学及分子生物学等技术，探讨黄芩调控下呼吸道菌群的物种特征、药效物质及其基于 GM-CSF 信号调控呼吸道菌群从而“行上焦，清湿热”的作用机制。研究发现：1）肺炎克雷伯菌和肺炎链球菌感染均可改变小鼠下呼吸道菌群的物种组成和结构，而黄芩给药可明显提高流产衣原体、衣原体属、不动杆菌属、γ‐变形菌丰度并降低罗尔斯顿菌属、肺炎克雷伯菌、链球菌属、鼠杆菌及肺炎链球菌等菌群丰度，能将模型小鼠失衡的微生物群落调控到与空白对照组相当的水平，可较好地恢复感染小鼠呼吸道微生物菌群多样性及维持菌群物种组成平衡，表现出调节下呼吸道菌群微生态平衡及生物学功能的能力。此外，黄芩对肺炎链球菌感染模型菌群的调控强于肺炎克雷伯菌感染模型。2）明确了黄芩调控菌群的体内效应物质为汉黄芩素、蓟黄素、白杨素和千层纸素A等成分。3）黄芩可减轻肺炎小鼠肺部炎症和病理损伤；4）GM-CSF可调控ERK的信号来调控呼吸道菌群的平衡，从而发挥其在呼吸道感染后机体先天免疫反应中的关键信号作用。而黄芩则可以菌群依赖的方式发挥其基于GM-CSF和ERK之间的信号传导防治下呼吸道感染的双向调控作用；即在呼吸道菌群正常的情况下对机体的先天免疫反应起一定的控制作用，以免其过度增强，而在呼吸道菌群耗尽后，起到调解（适当增强）机体先天免疫反应的作用。我们的研究揭示了黄芩基于GM-CSF信号传导调控下呼吸道菌群的分子机制，阐明了黄芩“行上焦，奏清热燥湿之效”的中医药科学内涵，为黄芩治疗呼吸道感染提供了新靶点，为其确切的临床疗效提供理论基础，也为其现代化的开发利用提供了科学依据。