基于不同饲养环境下流感小鼠肠道微生态及肠道黏膜免疫的变化探讨湿性粘滞的本质

The evil of dampness is the most common cause of external infection in Chinese medicine. The nature of wet-stickness means that: wet would cause stickness, which means long duration or defecation viscous of diseases and the like. So far scholars mainly study wet through water metabolism or digestive function, however, they did not discuss the molecular mechanism of wet pathogenic characteristics. The results of our preliminary study showed that: dampness could suppress the immune recognition and immune response of RLH signal pathway, and cause intestinal microflora and Th/Tregs imbalance. That the above results could explain part of the mechanism of wet pathogenic characteristics. However, what is the molecular mechanism that wet evil could cause immune response disorders and intestinal flora imbalance? What is the exact qualitative and quantitative change that wet evil influence on intestinal microflora? What is the relationship between the intestinal microflora imbalance, immune recognition disorders and Th/Tregs imbalance caused by wet? Therefore, the proposal intends to solve these problems from the following two aspects: By comparing the changes of the intestinal mucosal immunity and Th/Tregs in normal mice and mice with flu FM1 (C57BL、Foxp3-/-、Stat6-/-) under the different reared environments.Through metagenomic sequencing technology systematically study the specific qualitative and quantitative change of intestinal flora spectrum. Furthermore, revealing the scientific nature of "wet-stickness".

湿邪属六淫，是常见外感病因之一。湿性粘滞是指：湿邪致病往往缠绵难愈，排泄粘滞不爽等。迄今学者们对其研究主要集中在湿邪对机体免疫功能、脾胃运化、及水液代谢等方面的影响，未对其致病特点的分子机制做深入探讨。本课题组在以往国家基金资助下，发现：湿邪对机体免疫识别的主要信号通路之一RLRs有明显抑制作用，湿邪可引起T细胞分化受抑，及肠道菌群失调；认为上述结果可能是湿性粘滞的部分机制。那么湿邪是通过什么途径导致机体免疫识别障碍及肠道菌群失调的呢？湿邪对肠道菌谱的具体定性定量影响如何？湿邪所致的肠道菌群失调与机体免疫识别障碍及T细胞分化受抑是否有关联呢？为了回答上面问题，我们拟通过不同饲养环境下流感小鼠(分别使用C57BL、Foxp3-/-、Stat6-/-基因敲除鼠)肠道黏膜免疫及Th/Tregs的成熟分化变化，并且使用宏基因芯片技术对上述各组小鼠的肠道菌谱具体定性定量，依此进一步揭示湿性粘滞本质。

湿邪属中医的“六淫”之一，中医理论认为湿性粘滞，其致病特点主要表现在：一方面使病情反复发作，缠绵不愈，病程延长；另一方面表现为症状粘滞不爽，如大便不爽，小便不畅，舌苔粘腻等。迄今学者们对其研究主要集中在湿邪对机体免疫功能、脾胃运化、及水液代谢等方面的影响，未对其致病特点的分子机制做深入探讨。.本项目对湿环境饲养下的正常小鼠及FM1流感小鼠的肠道菌群进行了16s rDNA分析，同时也检测了肠道黏膜通透性相关指标，并与其它外邪及正常饲养环境相比较，讨论了不同饲养环境对小鼠肠道微生物菌谱的影响；通过检测不同环境下小鼠sIgM等表达的差异，分析了湿邪对小鼠肠黏膜免疫系统免疫球蛋白的影响；本项目还进行了湿邪环境和正常环境对小鼠肠黏膜免疫系统B细胞相关pIgR和NLRs信号通路的影响研究；并完成了不同环境饲养下正常小鼠及FM1流感小鼠的免疫平衡最重要的两对T细胞亚群Th1/Th2和Th17/Treg的变化以及它们所介导的免疫通路相关基因与蛋白的检测。.通过本项目的研究，我们发现：1.湿邪环境能影响机体肠道微生态，导致肠道菌群的组成结构、菌群丰度和多样性失衡，并可导致肠道微生物代谢产物含量发生变化，从而改变肠道菌群短链脂肪酸受体（FFAR2、FFAR3、HCAR3）和吲哚化合物受体（AhR）等表达升高，造成肠黏膜通透性增加。这可能是湿邪致病所表现出的大便黏滞、臭秽等症状的病理机制。2. 湿邪环境能影响肠粘膜免疫系统，导致pIgR、sIgA和IgA的表达降低，使机体无法清除肠道黏膜局部易位的菌群和相关的病原体。并且湿邪环境导致肠道菌群失衡后，还能导致肠道中NLRs免疫识别信号通路NOD1、NOD2、NF-kB和pIgR等相关基因表达升高，导致肠道附近相关炎症因子增多，加重局部炎症，造成肠黏膜通透性增加，从而致使感受湿邪后疾病缠绵难愈，病程变长。.基于此，我们认为湿性粘滞所导致的排便粘滞不爽等症状可能与肠道微生态失衡有关；湿性粘滞所导致的疾病缠绵不愈，可能与肠黏膜免疫系统调控失常等密切相关。上述机制可能是湿性粘滞的本质。