胆碱肠道菌群依赖代谢物-氧化三甲胺与非酒精性脂肪肝病的关系研究及机制探讨

It is well known that choline has a positive role in the prevention and control of nonalcoholic fatty liver disease (NAFLD). In our finished project of the National Natural Science Foundation of China (NO.81072302), we found that the plasma choline of the NAFLD group was higher compared with the controls, but the reason stays unclear. Dietary PC can produce trimethylamine oxide (TMAO) through the action of gut flora in vivo. As elevated levels of circulating TMAO is identified as a risk factor for cardiovascular disease (CVD), we hypothesis that, the increased circulating choline is induced by high dietary choline intake, and then, it causes the elevation of TMAO. The elevated TMAO increases the risk of NAFLD, which has a common cause with CVD. However, this has not been reported yet. In present study, we first try to confirm that TMAO is the gut flora metabolite of diet choline in Chinese by means of antibiotics intervention and choline challenge. Second, we discuss the association between TMAO and NAFLD, and explore the possible mechanism via a case-control study with the obtained liver samples. Last, we verify the risk and predictive value of TMAO for NAFLD with the data of an established cohort. It is innovative of our study to clarify a brand-new pathogenesis of NAFLD via the gut-liver axis metabolism of choline. Meanwhile, it may contribute to a new targeted-therapeutic strategy for NAFLD.

胆碱对非酒精性脂肪肝（NAFLD）的防制作用已成共识，我们已结题的国自然项目（81072302）却发现NAFLD患者血胆碱高于对照，原因未明。膳食胆碱在肠道菌群作用下可在体内生成氧化三甲胺（TMAO），血TMAO水平升高被证实是心脑血管疾病的危险因素。因此我们推测：NAFLD患者血胆碱升高是高膳食胆碱引起，从而使胆碱依赖肠道菌群的代谢物TMAO增高，引起与心脑血管疾病有共同土壤的NAFLD发病风险增高，但未见报道。因此，本研究首先采用抗生素"鸡尾酒"疗法结合胆碱负荷试验确证TMAO是中国人膳食胆碱依赖肠道菌群的代谢物；其次采用已获得的肝脏样品进行病例对照设计，探索性研究TMAO与NAFLD的关联性及机制；最后采用已建立的人群队列验证TMAO对NAFLD发病风险的预测作用及强度。本研究从胆碱肠道菌群依赖的代谢途径阐明NAFLD发病新机制具有创新性，为NAFLD的预测及靶向治疗开辟一个新领域。

研究内容：本研究通过1）建立NAFLD病例对照研究，探究血清TMAO、胆碱、甜菜碱与NAFLD病理进展的关系，2）在已经建立的中老年人健康队列研究中，回顾性测量基线血清TMAO等的水平，随访3.2年后以腹部B超进行NAFLD的诊断和脂肪肝程度分级，3）给予TMAO干预高脂膳食诱导的NAFLD小鼠以及棕榈酸钠诱导的HepG2脂肪变细胞，探究TMAO在NAFLD发生发展中的作用及机制。.结果：1）病例对照研究发现，在纳入的95名研究对象中（正常组：n=35），NAFLD病例组的血清胆碱和TMAO水平显著高于正常组（11.87μM vs 9.07μM、2.02μM vs 1.05μM），血清胆碱、TMAO水平与肝脏脂肪变性、炎症、NAS评分呈正相关。NAFLD组的血清总胆汁酸水平显著高于正常对照组（8.70μM vs 6.34μM， P=0.018），血清TMAO水平与总胆汁酸水平呈正相关关系。2）队列研究中，通过筛选共纳入1628人，随着血清TMAO水平升高，肝脏脂肪变性程度加重，校正混杂因素后，血清TMAO水平与脂肪肝程度呈正相关（P<0.001）。3）TMAO干预高脂膳食饲养小鼠18周后，与高脂膳食组小鼠相比，肝脏脂肪变加重，肝脏脂质合成相关基因的mRNA及蛋白水平显著升高；在体外实验中，TMAO干预的棕榈酸钠诱导的HepG2脂肪变细胞中出现相似的结果，提示TMAO能加重肝脏脂肪变。TMAO干预后，小鼠肝脏胆汁酸水平显著上升，且肝脏胆汁酸构成改变，具有FXR（farnesoid X receptor）激动作用的胆汁酸子类百分含量降低，肝脏FXR及FXR下游的靶基因SHP（small heterodimer partner）蛋白表达降低。TMAO干预棕榈酸钠诱导的HepG2脂肪变细胞中出现相似的结果，采用siRNA抑制HepG2细胞中胆汁酸合成酶的表达后，发现TMAO干预不能上调HepG2细胞脂质合成相关基因的蛋白水平。表明TMAO通过影响胆汁酸代谢，从而加剧肝脏脂肪变。进一步研究发现FXR激活后可以显著降低TMAO引起的肝细胞脂质合成相关蛋白水平的升高，提示TMAO通过胆汁酸-FXR-SHP-肝脏脂质合成基因的途径，加剧肝脏脂肪变的进展。.科学意义：本研究结合人群、动物、细胞实验探讨了TMAO对NAFLD发生发展的影响，为防治NAFLD提供了新的理论依据和干预靶点。