基于肠道菌群移位触发炎症因子及水通道蛋白机制的岭南湿热证实质研究

The relationship of inflammatory mediator reactions induced by endotoxin/Toll-like receptor4(TLR4) pathway with the heat of dampness-heat syndrome(DHS), and the relationship of aquaporin (AQP) with the dampness of DHS are both close. Meanwhile, the relationship between AQP and endotoxin was also proved..So pathogenesis of DHS may be that over-expressions of endotoxin-TLR4 pathway and AQP, which are started doubly-line and synchronously by endotoxin released by bacterial translocation(BT) from gut, accordingly inducing cascade reactions, leading to body inflammatory mediators reactions and metabolic disorders in water-humor, and then developing into DHS. .BT correlative index differences between two kinds of mouse models with DHS, and difference of “hot”and“cold” objective indexes were all detected to explore the relationship and its essence among BT, inflammatory factor and AQP, using mouse models with DHS with predominant dampness or heat respectively as the research cores. As well as, the intervene experiment of Artemisia capillaries, Fructus forsythia and probiotics, the relative experiment of AQP and inflammatory factor, and the translocation experiments of germ-free mousse were executed to verifiably explore the relationship among BT, inflammatory factor and AQP further, and by strive to prove that BT from gut inducing inflammatory factor and AQP is the core mechanism of DHS, to provide the new approaches and new target for explaining the mechanism.

内毒素-Toll样受体4（TLR4）通路诱导的炎症介质反应与湿热证之“热”、水通道蛋白（AQP）与湿热证之“湿”关系密切，AQP与内毒素亦存在被证实的联系，故湿热证病机可能在于肠道菌群移位释放的内毒素双线同步启动内毒素-TLR4通路和AQP的过表达，从而促发瀑布式级联反应，导致机体炎症介质反应及水液代谢紊乱，发展为湿热证证型。以湿重于热及热重于湿小鼠湿热证模型为核心，探索两种湿热证小鼠肠道菌群移位相关指标的差异、及“湿”“热”客观指标的差异，研究菌群移位、炎症因子、AQP三者的关系及其实质；并通过茵陈蒿、连翘及益生菌干预实验、AQP与炎症因子相关实验、无菌小鼠菌群移植实验，进一步验证性的研究菌群移位、炎症因子、AQP三者关系，力图证实肠道菌群移位触发炎症因子及AQP是湿热证的核心机制，为阐明其机理提供新途径、新靶点。

岭南地理气候环境潮湿炎热，日照时间长、气温高、雨水充沛，易致地湿上蒸，湿热证极为多见；加之近年来随着生活水平的提高，饮食结构发生剧变，岭南地区海产品丰富，又有“无鸡不成宴”的传统，导致饮食偏重高蛋白，又嗜食甘醴，可致脾气受损，阳气阻遏，湿邪内生，内外相引，而成湿热。现代医学研究发现内毒素-Toll样受体4（TLR4）通路诱导的炎症介质反应与湿热证之“热”、水通道蛋白（ AQP）与湿热证之“湿”关系密切，AQP与内毒素亦存在被证实的联系。本课题是基于肠道菌群移位触发炎症因子及水通道蛋白机制的岭南湿热证实质研究，在该课题的资助下，在原有湿热证模型的基础上，我们发现湿热模型小鼠干扰素 γ( INF － γ) 、肿瘤坏死 因子 α( TNF － α)、NF － κBp65、TLＲ4、 AQP3 的表达升高，肠杆菌属、肠球菌属、梭菌属等条件致病菌过度增长; 而双歧杆菌属、乳杆菌属等益生菌的含量受湿热发病机制的影响出现含量表达的差异。我们还发现了湿热环境可以导致小鼠肠道菌群失调和肠道炎症，通过 16S rDNA检测发现肠道菌群的显著改变，表现为Bifidobacterium、Allobaculum、 Butyricimonas、Mucispirillum、Sutterella等11种细菌丰度发生了改变。因此，我们推测湿热环境导致肠道菌群的改变，诱导了细菌移位，从而诱发了肠道炎症反应。我们通过抗生素的干预验证了此假设，抗生素干预可以改善湿热环境所诱导的菌群改变和肠道炎症。我们进一步发现湿热环境可以使肠组织线粒体DNA减少，P62/SQSTM1升高、LC3Ⅱ总量降低抑制肠组织自噬。除此之外，我们发现潮湿环境使肠道细菌Helicobacter、AF12的丰度降低，这两类细菌都是产丁酸的细菌，而丁酸对于调控血糖是有益的糖，因此潮湿环境可能影响糖代谢。