基于不同饲养环境下流感小鼠肠道微生态的变化及脑肠互动探讨“湿性重浊”的病生机制

Dampness, with characteristic of “Sticky-stagnant” and “Heavy-turbid”, is one of the six pathogenic factors of TCM. “Sticky-stagnant” means long duration or repetition of diseases. “Heavy-turbid” can lead to symptoms such as heaviness and lassitude of the head and body, or turbid excreta and secreta and so on. With the support of previous national funds, our group found that dampness can cause down regulation of immune recognition and immune response, and also cause imbalance of intestinal flora, which is probably the pathological mechanism of “Sticky-stagnant”. However, our group has not done further research on "Heavy-turbid ". Based on the previous research, this project puts forward the following scientific problems: What is the correlation between the “Turbid” (turbid excreta and secreta) caused by dampness and the qualitative and quantitative analysis of intestinal microenvironment? When dampness causes "Heavy" (heaviness and lassitude of the head and body), what are the changes of the body’s neuroendocrine and main neural signal? Whether the pathological correlation between "Heavy" and "Turbid" is due to "brain-gut interaction"? And what is the intrinsic mechanism between "Heavy" and "Turbid"? To clarify the scientific problems above, the approach taken in this project is to use metagenomic sequencing to analyze the intestinal microflora of mice model raised in different environments and to detect the changes of related indices such as major neurotransmitters, behavioral science and so on. By applying the methods above, this project aims to explore how the dampness to affect sense, emotion and endocrine system through changing Intestinal microenvironment, and finally reveals the pathophysiological mechanism of “dampness being heavy and turbid”.

湿邪属中医六淫之一，其性粘滞、重浊。湿性粘滞主要指病情反复缠绵难愈；湿性重浊是指感受湿邪可出现头身困重，排泄物、分泌物秽浊不清等症状。本课题组在以往多个项目资助下，发现湿邪可以下调机体免疫识别与免疫应答等,并可导致肠道菌群失调，其可能是湿性粘滞的病理机制；但对“湿性重浊”还未深入研究。基于前期研究基础，本项目的科学问题是：湿邪所导致的“湿性重浊”所出现的“浊”（排泄物、分泌物秽浊不清）与肠道微环境的具体定性定量关系如何？湿邪致 “重”（头身困重等），其机体的神经内分泌、主要神经信号变化如何？“重 ”与“浊”之间的病理关联是否由于“脑-肠互动”所致？两者之间的内在关联机制如何？为了阐明上述问题，我们拟采用宏基因测序检测不同饲养环境下小鼠的肠道微环境，并检测神经递质、行为学等相关指标的变化，以期探求湿邪是如何通过改变肠道微环境来影响感官、情绪和神经信号系统的，并最终揭示“湿性重浊”的病生机制。

湿邪属中医六淫之一，具有“湿性粘滞”和“湿性重浊”两个特点。湿邪之“浊”主要表现为：排泄物、分泌物秽浊不清，湿邪之“重”主要表现为：头身肢体困重、精神倦怠、情绪变化等。本课题使用人工气候箱和高脂饮食分别模拟外湿、内湿之邪，并以20%LD50 FM1作为致病因素，分别建立湿邪及其他证候流感小鼠模型，探讨湿邪“重”与“浊”的物质基础及二者之间的内在关联。①观察模型的生存状况，并通过RT-PCR和HE染色等，明确“环境+饮食+生物因子”造模可成功建立湿证、湿邪病证结合模型。②借助神经行为学技术，检测湿邪模型小鼠的精神情志等相关行为学，及肌肉力量的改变，发现：湿邪所导致的机体精神情志及肌肉力量改变，可能是湿邪致病“湿性重浊”--“重”所表现的头身困重、四肢沉重以及行为情变化等症状的生物学依据。③采用16s rDNA测序，具体分析湿邪模型小鼠肠道微生物的变化，并检测肠黏膜通透性相关蛋白及免疫因子受体mRNA的表达，结合肠道结构的实质性改变，发现：湿邪可导致肠道菌群的组成结构、菌群丰度和多样性失衡，并改变肠道紧密连接蛋白及相关炎症因子受体的表达和分布，造成肠黏膜通透性增加，进一步引起机体肠道微生态失衡。这可能是湿邪致病“湿性重浊”--“浊”所表现出的大便黏滞、臭秽等症状的生物学基础。④结合代谢组学技术，检测湿邪模型小鼠肠道菌群代谢产物的变化，对肠道菌群及其差异代谢产物、神经行为学、以及主要神经递质等变化进行关联分析，发现：湿邪可导致肠道微生物及其代谢产物的改变，造成与神经递质、神经内分泌激素、以及氨基酸等相关代谢物的显著变化，伴随肠黏膜屏障和血脑屏障通透性的增加，进而影响血清及脑组织中相关神经递质、炎症因子含量的变化。这有可能是湿邪模型小鼠机体进行肠-脑互动的重要物质基础。⑤通过ELISA检测脑组织及血清中相关神经递质（如，多巴胺、5-羟色胺、γ-氨基丁酸、谷氨酸）的变化趋势，并采用RT-PCR、Western Blot技术，进一步检测关键通路PKA/PI3K/AKT/CREB的蛋白表达，并以免疫荧光观察脑源性神经营养因子（BDNF）在皮质层的表达，从而验证湿邪小鼠机体“肠脑互动”的可能途径，最后阐明湿性重浊的两个证素群的内在关联，揭示“湿性重浊”的病机本质。