有氧运动通过肠道菌群-肝脏轴影响非酒精性脂肪肝的效应及机制

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Given the major functional role of the liver in the maintenance of metabolic homeostasis, discovering the root causes and the underlying mechanisms of the progression of NAFLD is essential for finding the effective prevention and treatment strategies in combating NAFLD. Studies have shown that lifestyle such as physical activity and diet is one of the main contributors for NAFLD. Therefore, using exercise may be the most economical way to combat NAFLD. We have previously found that gut microbiota composition in humans is associated with liver fat accumulation and over expression of several genes related to microbiota-derived inflammation in adipose tissue. Thus, it is possible that NAFLD originates from the gut microbiota. To test our hypothesis that microbiota cause adipose tissue dysfunctionality and increased liver fat content, that exercise could modify the gut microbiota and lipid metabolism in adipose tissue thereby affect liver fat accumulation. The underlying mechanisms will be studied in animal model and verified cultured human adipocytes/hepatocytes. Particularly, 1)to study the effect of 8-week aerobic exercise on gut microbiota composition by 16SrRNA in mice and germfree mice; 2) to study whether microbial lipopolysaccharides (LPS) cause inflammation, increased lipid synthesis and insulin resistance in both animal and cultured human adipocytes by assessing gene and protein expression from cell cultures using quantitative PCR and Western blot, respectively, and measuring cytokines from culture media by ELISA; 3)to understand which are the underlying molecular mechanisms by studying the role of endogenous cannabinoid receptors using receptor antagonists in cell culture experiments; 4) to further explore whether the molecules secreted from adipocytes increase fat accumulation and insulin resistance in hepatocytes by applying adipocyte culture media to hepatocytes and assessing gene and protein expression by quantitative PCR and Western blot. This research project will provide new information about the crosstalk between adipocytes and hepatocytes. If our current hypothesis proves to be true, this will open up the possibility of preventing hepatic fat accumulation and NAFLD by modifying the gut microbiota composition, e.g. through specifically designed exercise interventions, towards a metabolically healthier one.

肠道菌群对维持人体正常代谢和免疫功能具有重要作用。前期研究发现肠道菌群与非酒精性脂肪肝(NAFLD)有关，而有氧运动可改善脂肪组织代谢，降低肝脏脂肪堆积，转归早中期NAFLD。本研究假设有氧运动通过肠道菌群-肝脏轴影响NAFLD的机制：通过增加宿主肠道中有益菌（双歧杆菌和FPrau）含量，降低有害菌（肠杆菌）含量，减少肠道菌群-肝脏轴中的关键中介-内毒素（LPS），经CB1受体信号通路引起血清中游离脂肪酸和甘油三酯水平下降，从而降低肝脏脂肪堆积。我们在前期工作的基础上，以非酒精性脂肪肝小鼠为实验对象，通过16SrRNA基因杂交、EchoMRI、蛋白质印迹、免疫荧光等技术，观察8周中、大强度有氧运动对小鼠肠道菌群组成、LPS、脂肪组织炎症和肝脏脂肪含量的影响，并用人体脂肪细胞/肝细胞培养进行验证。本研究结果为运动干预肠道菌群组成效应提供理论依据，从而为非酒精性脂肪肝的早期防治提供新思路。

首先，我们建立了非酒精性脂肪肝小鼠模型， 对模型小鼠进行有氧运动和膳食干预，干预结束后检测小鼠的体成分、血液及组织样品；并根据所提取DNA 进行肠道菌群基因组测序分析。我们发现8周的膳食干预（ND+SED）和膳食联合运动（ND+EX）干预有效缓解小鼠脂肪肝。我们还发现支链氨基酸（BCAA）的增加与早期肝脂肪积累相关，与肥胖、胰岛素抵抗无关。BCAA分解代谢活性降低、线粒体能量代谢、血清BCAA浓度与肝脏脂肪含量的相关，提示脂肪组织功能障碍可能在NAFLD发病机制中起关键作用。.我们对小鼠肠道菌群进行分析，发现有氧干预改变了肠道菌群多样性，响应干预的菌属与体重、肝重、腹腔脂肪、血糖、高&低密度脂蛋白和肝脏切片空泡面积相关。结果表明有氧运动和膳食干预改变肠道菌群丰度，提高肝脏组织糖代谢，改善肝脏氧化状还原态降。.其次，我们通过体外人体脂肪细胞/肝细胞培养验证脂肪组织在肠道菌群-肝脏轴中的介导作用。我们发现单独FLG或LPS处理对HepG2 细胞内脂质含量没有影响，而FLG与脂肪细胞共培养后再处理HepG2后，显著增加了HepG2细胞内的脂质含量。FLG通过激活TLR5，促进脂肪细胞的甘油分泌，增加培养液中甘油的含量。FLG和LPS直接作用和与脂肪共培养作用都促进肝细胞炎症，并降低了胰岛素信号传导和线粒体功能。因此，我们认为脂肪细胞介导了肠道菌群鞭毛蛋白（FLG）促进的肝细胞脂肪堆积。.结合细胞实验结果，我们推测有氧运动改变肠道菌群丰度，从而减弱脂肪组织脂质代谢进而改善肝脏组织糖脂代谢，最终减少肝脏组织脂质堆积，缓解非酒精性脂肪肝。研究有氧运动与肠道菌群-肝脏轴的关系的结果有利于今后拓展相关疾病防治的新思路。.我们根据研究结果，通过学校科学健身大讲堂和社区健康讲座，为非酒精性脂肪肝患者提供饮食和健身指导。并编写《运动与体重管理》一书，为更多的人群提供运动与体重控制的健康知识。本研究已发表7 篇高水平SCI 论文。部分成果在国内与国际学术会议交流。本项目培养1名博士后、3名博士生和2名硕士生。