基于高通量测序技术的绞股蓝调控肠道微生态防治NAFLD的机制研究

Modern research shows that, the major signal transduction pathway which leads intestinal flora disturbance to chronic systemic inflammation, IR, and finally NALFD may be mediated by TLR4.Intestinal flora regulator has gradually become a new target for NAFLD. Gynostemma pentaphyllum has a good effect on NAFLD. And it has a broad spectrum of antimicrobial activity.According to our previous research, Gynostemma pentaphyllum can improve NALFD patients’ common symptoms of unfinished sensation during bowl movement and sticky stools obviously，which are closely related to intestinal flora disturbance. Besides,it can regulate the structure of intestinal flora of NALFD mice. As a result,we put forward the hypothesis that by regulating intestinal flora,Gynostemma pentaphyllum decreases gut derived infection and then prevents the process of NAFLD.To test this hypothesis,Gynostemma pentaphyllum intervened NALFD mice are put as research objects. By using new biological techniques, such as high-throughput sequencing,key intestinal flora, the permeability of intestinal walls and inflammatory reaction which related to Gynostemma pentaphyllum intervention are explored. Through this study, theoretical foundation of Gynostemma pentaphyllum in preventing and treating non alcoholic fatty liver disease based on intestinal microbiome theory is hope to be provided.

现代研究表明，肠道菌群可能主要通过TLR4 介导的信号转导途径导致慢性系统性炎症，进而引起胰岛素抵抗（IR）和非酒精性脂肪性肝病（NAFLD），肠道菌群调节剂逐渐成为防治NAFLD的新靶点。绞股蓝对NAFLD疗效确切，且有广谱抗菌活性，但其“解毒”功效与防治NAFLD的关系目前尚不明确。前期研究发现，绞股蓝可明显改善NAFLD患者常伴有的排便不尽感、黏腻便，后者与肠道微生态结构变化关系密切。既往研究发现，绞股蓝可调节NAFLD小鼠肠道部分菌群的数量。由此，我们提出“绞股蓝可能通过调节肠道微生态，减轻肠源性全身慢性低度炎症，进而改善IR和NAFLD”这一假说。鉴于此，本研究以绞股蓝干预的NAFLD模型小鼠为研究对象，运用高通量测序等技术，探索与绞股蓝干预相关的关键肠道菌属，探讨绞股蓝对关键菌属所致肠壁通透性增加及炎症反应的作用，为从肠道微生态角度阐述绞股蓝防治NAFLD的机制提供理论依据。

一、背景：.肠道菌群调节剂逐渐成为防治NAFLD的新靶点，绞股蓝（GP）对NAFLD疗效确切，且有广谱抗菌活性，但其“解毒”功效与防治NAFLD的关系目前尚不明确。.二、主要研究内容：.1. 探讨GP干预对NAFLD及IR的作用：分别测定GP高、中、低干预组，模型组与对照组大鼠肝脏酶学指标、胰岛素水平，同时通过HE染色与油红染色法观察各组大鼠肝脏组织病理学的表现，明确GP对NAFLD及IR 的改善作用。.2. 探讨与GP干预相关的NAFLD发病关键肠道菌属：运用高通量测序平台对各组大鼠盲肠内容物中细菌16S rRNA V3 -V5区进行定性分析，全面深入探索各组大鼠肠道菌群结构多样性的特征。.3. 探讨GP干预对肠壁通透性的影响：通过HE染色与透射电镜观察各组大鼠肠黏膜病理组织学与超微结构的表现，明确GP对肠壁通透性的影响。.4. 探讨GP干预对TLR4 信号通路介导的炎症反应的作用：通过RT-qPCR法检测各组大鼠TLR-4、TNF-α、IL-1β、IL-6等炎性因子mRNA 水平，用WB法检测TLR-4S水平，明确GP对TLR4 信号通路介导的炎症反应的作用。.三、重要结果：.1. GP对高脂血症的作用：GP降低了NAFLD大鼠TC、TG和LDL-C水平，升高HDL-C水平，呈剂量依赖性，且优于DLPC组。.2. GP对肝功能、肝脏组织学的作用：GP降低了NAFLD大鼠ALT、AST水平，呈剂量依赖性，且优于DLPC组；HE染色与油红染色显示，GP可改善NAFLD大鼠肝细胞脂肪变性情况，呈剂量依赖性，且优于DLPC组。.3. GP对炎症、内毒素血症及IR的作用：GP降低了TNF-α、IL-1β和IL-6的水平，降低了NAFLD大鼠的内毒素血症和胰岛素抵抗，而DLPC改善情况不显著。.4. GP对肠壁通透性与肠道菌群结构的作用：在高脂饮食（HFD）诱导的NAFLD大鼠中，GP维持了肠道完整性并调整了肠道菌群结构，其降低了厚壁菌门的相对丰度，增加了拟杆菌门的相对丰度，使两者比值下降。GP治疗也丰富了有益细菌（如乳球菌属）的相对丰度，并减少了肠内致病细菌（如瘤胃球菌属）的相对丰度。.四、科学意义：.本项研究结果显示，绞股蓝改善NAFLD的机制可能与调节肠道菌群结构有关，GP可能是一种治疗NAFLD的潜在肠道菌群调节剂。