基于肠道微生物对肠粘膜屏障影响的健脾泄浊法干预NAFLD作用机制研究

The imbalance of intestinal microbial flora distribution damage the function and structure of intestinal mucosal barrier, it plays an important role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). TCM theory holds that the main pathogenesis is spleen had been blocked, phlegm had been formed, deficiency in origin and excess in superficiality, thus Jianpi Xiezhuo methods has been established, which can guide clinical practice. We hereby put forward the scientific hypothesis: the regulation of intestinal microflora distribution, .intestinal mucosal barrier integrity maintenance is an important and effective mechanism of Jianpi Xiezhuo prescription. This project intends to Jianpi Xiezhuo prescription, concise, precise matching of Jinkui Zexie decoction prepared rhubarb as the research object, treating NAFLD rats model fed by high fat diet, using high-throughput sequencing method, analyses metagenomics characteristics of rats intestinal microbial; fecal suspension of NAFLD rats model will be inoculated in germ free mice, intestinal mucosa barrier structure, occludin, ZO-1 protein expression will be observed, microbial particles deposition will be estimated by electron microscopic, and detection of liver and peripheral inflammatory factor level will be carried out. Investigating the mechanism of Jianpi Xiezhuo method prescription treatment NAFLD from the angle of intestinal microbial, intestinal mucosal barrier, provide new ideas and methods for the modern study of TCM in NAFLD.

肠道微生物分布比例失衡破坏肠粘膜屏障结构和功能，在非酒精性脂肪性肝病（NAFLD）发病中起到重要作用。中医理论认为本病主要病机为水谷精微不归正化，酿成痰浊，阻滞肝脾，本虚标实，确立的健脾泄浊治疗大法对临床有较好指导作用。据此我们提出科学假设：调节肠道微生物分布比例，维护肠粘膜屏障是健脾泄浊法方药治疗NAFLD一个重要取效机制。本项目拟以健脾泄浊法方药干预NAFLD高脂饮食模型大鼠，以药物简练、配伍精当的金匮泽泻汤合制大黄为研究对象，运用高通量测序方法，分析大鼠肠道微生物宏基因组学特点；模型大鼠粪便混悬滤液接种无菌小鼠，观测小鼠肠粘膜屏障结构蛋白occludin、ZO-1表达、电镜观察肠粘膜微生物颗粒沉着，检测肝脏及外周血炎症因子水平。从肠道微生物-肠粘膜屏障角度探讨健脾泄浊法方药治疗NAFLD的作用机制，为NAFLD中医方药现代研究提供新的思路和方法。

肠道微生物分布比例失衡破坏肠粘膜屏障结构和功能，在非酒精性脂肪性肝病（NAFLD）发病中起到重要作用。中医理论认为本病主要病机为水谷精微不归正化，酿成痰浊，阻滞肝脾，本虚标实，确立的健脾泄浊治疗大法对临床有较好指导作用。本项目研究发现加味泽泻汤在改善NAFLD小鼠血清肝功能、肝脏TG含量及肝脏组织学积分上效应明显，作用机制可能是通过改善脂质相关炎症反应。进一步研究发现其可降低IL-1β、TNFα及F4/80mRNA的表达。在对肠道微生物丰度、多样性的研究中，Ace指数、Chao指数、Shannon指数均显示模型组、给药组较空白组下降；Simpson指数模型组较空白组升高，给药组较模型组指数有所下降，提示模型组及药物组较空白组肠道微生物丰度及多样性下降。在门的分类学水平上，加味泽泻汤组较空白组Bacteroidetes升高；厚壁菌门（Firmicute）在模型组及给药组均出现了下降；变形菌门（Proteobacteria）在模型组出现升高，经加味泽泻汤干预后占比下降。在属的分类学水平上，脱硫弧菌属（Desulfovibrio）OTU数量在模型组显著增长，经加味泽泻汤治疗后下降到与空白组一致的水平；普雷沃氏菌属（Prevotella）在模型组数量增加，加味泽泻汤治疗后可降低；在罗氏菌属（Roseburia）及Lachnospiraceae_Incertae_Sedis属的数量表现也与Desulfovibrio、Prevotella一致。肠粘膜屏障完整性实验发现模型组回肠与结肠occludin表达量显著下降，加味泽泻汤治疗后可提升其表达水平。通过伪无菌小鼠粪菌接种实验发现健脾泄浊方可通过影响肠道微生物进而改善肝脏功能，空回肠电镜观察提示健脾泄浊方菌粪移植组小鼠肠道粘膜表面上皮细胞间各种连接结构较为清晰，紧密连接结构正常。毒理研究实验显示加味泽泻汤给ICR小鼠灌胃最大给药量约为临床患者拟用剂量的111倍(>100倍)，雌性及雄性小鼠在肝脏及肾脏病理上均未出现器质性改变。物质基础研究发现加味泽泻汤中存在及大黄素、大黄酸、芦荟大黄素等，质谱图谱分析加味泽泻汤中还可能存在没食子酸、大黄酚、大黄素甲醚等。本研究从肠道微生物-肠粘膜屏障角度探讨健脾泄浊法方药治疗NAFLD的作用机制，为NAFLD中医方药现代研究提供新的思路和方法。