基于“肠-肝轴”和代谢组学探讨黄芪散多靶点干预非酒精性脂肪肝的分子机制

Recent researches have shown that gut microflora is closely related to non-alcoholic fatty liver (NAFLD). The Chinese medicine HuangQi San is a representative formula of strengthening spleen and replenishing Qi. Our previous studies demonstrated that HuangQi San could improve glucose and lipid metabolism, and having functions of lowering lipid and protecting the liver in NAFLD rats. But its mechanism is not fully understood. According to the Traditional Chinese medicine theory of liver-spleen correlation, the new appreciation of modern medicine understandings of "Gut-liver axis", and metabonomics methodology, combined the previous studies, we propose a new hypothesis that Huangqi San may play a role in fighting NAFLD by regulating the gut microflora-Gut-liver axis and associated metabolites. Based on this new hypothesis, this study will establish NAFLD mice model induced by high-fat diet. Using microbial molecular ecology, metabonomics methodology, and multivariate statistical analysis, we will explore the connections between the effects of Huangqi San on gut microflora diversity and the effects of that on hepatic lipopathy and gut mucosal barrier dysfunction in mice. And the function of regulating Gut-liver axis is clarified. We will further analyze the "metabolites spectrum" in serum, urine, liver and faeces, and identifying the biomarkers closely related to the pathogenesis of NAFLD and the mechanism of HuangQi San. This study finally is to analyze the internal relation of "gut microflora - metabolites". The Multi-target molecular mechanism of intervening NAFLD of HuangQi San is explored, and this results provide scientific basis for its clinical application and promotion.

近年研究表明肠道菌群与非酒精性脂肪肝（NAFLD）关系密切。黄芪散是健脾益气代表方，我们前期研究证实其能改善糖脂代谢，对NAFLD具有降脂、护肝作用，但其作用机制尚未明确。本项目基于中医“肝脾相关”理论、现代医学对NAFLD“肠-肝轴”病理机制新认识及代谢组学研究方法，结合前期研究基础提出新假说：黄芪散通过调控肠道菌群-肠-肝轴及其关联的代谢物发挥抗NAFLD作用；并建立高脂饮食诱导NAFLD小鼠模型，采用微生物分子生态学技术结合代谢组学技术，探讨黄芪散对肠道菌群多样性的作用与其对肝脏脂肪病变、肠道粘膜屏障功能改善的关联性，揭示其对肠-肝轴功能的调节作用；分析给药前后血液、尿液、肝脏、粪便的“代谢物谱”，确定与NAFLD发病和黄芪散作用机理密切相关的生物标志物；进一步探析其“肠道菌群-代谢物”内在关联，从整体上揭示黄芪散多靶点抗NAFLD的分子机制，为其临床应用和推广提供科学实验依据。

非酒精性脂肪肝（NAFLD）与肠道菌群关系密切，其发病机制尚未阐明。本研究利用16S rRNA高通量测序技术和代谢组学探讨黄芪散对肠道菌群多样性和代谢产物的作用。采用高脂饮食诱导建立NAFLD小鼠模型，发现黄芪散可显著降低模型小鼠的体重、糖脂水平，改善肝和附睾脂肪组织病理形态，并通过调控肠组织中Occludin和ZO-1基因表达改善肠道粘膜屏障功能。肠道菌群结果发现，模型组小鼠拟杆菌门丰度显著降低，厚壁菌门的丰度显著升高，且厚壁菌门/拟杆菌门（F/B）比例升高。黄芪散可明显改善模型小鼠肠道菌群的紊乱，降低厚壁菌门丰度，升高拟杆菌门丰度，从而降低F/B比例。黄芪散可能通过下调Lactobacillaceae、Oscillospiraceae，上调Bacteroidaceae、Tannerellaceae和Ruminococcaceae等菌科丰度；下调Lactobacillus、Oscillibacter、Peptococcus、Rikenella、Streptococcus，上调Bacteroides、Lachnoclostridium、Parabacteroides等菌属丰度而发挥抗NAFLD作用。代谢组学结果显示，通过对三组代谢物取交集共发现49个血清共有差异代谢物和78个肝脏共有差异代谢物为共同潜在生物标志物，甘油磷脂等代谢通路受到显著影响。黄芪散可对血清中Eicosapentaenoic Acid、Longicamphenylone、4,5-dehydro Docosahexaenoic Acid、Cysteinyl-Glutamine、Isoleucyl-Hydroxyproline、L-Asparagine、4-Hydroxybenzaldehyde、2-Phenylacetamide等代谢物丰度显著回调；可对肝脏中L-Serine、Cortisol、Glycerophosphocholine、LysoPC(18:0)、L-Glutamine、Aldosterone、Stearidonic acid等代谢物丰度显著回调。关联分析显示Bacteroides、Lactobacillus等菌属可能在黄芪散防治NAFLD过程中发挥重要作用。研究结果从肠道微生态-肠-肝轴及代谢组学角度阐明了黄芪散多靶点防治NAFLD的作用机制，为中医药防治代谢性疾病提供新思路。