基于核受体FXR活化探讨茵陈蒿汤调控NAFLD胆汁淤积胆汁酸和脂肪代谢效应机制研究

NAFLD cholestasis has double barriers to bile acid metabolism and fat metabolism. Activation of FXR is an important step in improving both bile acids and fat metabolism. The research group confirmed that Yinchenhaotang (YCHT) could effectively regulate the expression of bile acid profiles, bile acid nuclear receptors and transporters, improve the bile acid metabolic disorder, and found that it had the effect of regulating fat metabolism. This project is based on the important role of FXR in bile acids and fat metabolism. It is suggested that "YCHT activates FXR, which can improve the bile acid and fat metabolism disorder, which may be the main mechanism of its treatment of NAFLD cholestasis" scientific hypothesis. Based on the patent work of YCHT component decoction, the pathogens of NAFLD cholestasis induced by FXR-/- mice and ANIT were used to induce HFD mice. The YCHT and its patent were 1) Using UPLC-MS/MS technology to detect bile acids and free fatty acid profiles, elucidate the histological characteristics of YCHT and its components to regulate bile acids metabolism and fat metabolism; 2) use molecular biology techniques to detect the key factors to regulating fat metabolism in mRNA and protein expression, analysis of YCHT and its components on the different aspects of fat metabolism intervention mechanism for the development and application of traditional Chinese medicine components to lay a scientific basis.

NAFLD胆汁淤积存在胆汁酸代谢和脂肪代谢双重障碍。激活FXR是同时改善胆汁酸和脂肪代谢的重要环节。课题组研究证实茵陈蒿汤能够有效调控胆汁淤积胆汁酸谱、胆汁酸核受体及转运体表达，改善胆汁酸代谢紊乱，并初步发现其具有调控脂肪代谢作用。本项目基于FXR在胆汁酸和脂肪代谢中的重要作用，提出“茵陈蒿汤激活FXR，改善胆汁酸和脂肪代谢紊乱，可能是其治疗NAFLD胆汁淤积的主要机制”科学假说。拟以前期茵陈蒿汤组分复方的专利工作为基础，采用FXR-/-小鼠和ANIT诱导HFD小鼠形成NAFLD胆汁淤积病理模型，以茵陈蒿汤及其专利组分复方为研究对象，1）使用UPLC-MS/MS技术，检测胆汁酸和游离脂肪酸谱，阐明茵陈蒿汤调控胆汁酸和脂肪代谢的组学特点；2）使用分子生物学技术，检测调控脂肪代谢关键因子的mRNA和蛋白表达，解析茵陈蒿汤及其组分对脂肪代谢不同环节的干预效应机制，为中药组分复方发展奠定科学基础。

本研究采用HFD诱导NAFLD小鼠模型，剖析茵陈蒿汤及其组分调控NAFLD的胆汁酸谱、脂肪酸谱特征及干预效应机制。发现寻常拟杆菌（Bacteroides_vulgatus）是NAFLD疾病进展的重要菌种。茵陈蒿汤及其组分绿原酸、大黄多糖、没食子酸、栀子苷均可显著降低寻常拟杆菌丰度。其中大黄多糖作用最为显著。脂肪酸、有机酸、胆汁酸、氨基酸代谢紊乱是NAFLD代谢表型的主要特征。棕榈油酸是NAFLD代谢紊乱的生物标记物。茵陈蒿汤及其组分均降低寻常拟杆菌丰度，降低肝组织棕榈油酸浓度，改善NAFLD代谢紊乱状态。茵陈蒿汤及其组分复方能够升高肝组织FXR/SHP，降低肝组织SREBP-1c、CD36蛋白表达，改善NAFLD脂肪酸、胆汁酸代谢。其中，绿原酸、大黄多糖通过调控肝组织FXR/SHP/LXR-a信号通路，抑制胆汁酸合成，促进胆汁酸排泄，减少脂肪生成，加速脂肪酸氧化代谢，是其治疗NAFLD的主要效应机制。栀子苷促进肝FXR表达，抑制胆汁酸合成，改变胆酸池组成，减少胆汁酸从肝血窦面摄入，降低肝细胞内脂肪酸蓄积，促进饱和脂肪酸转换为单不饱和脂肪酸，减轻肝损伤，是其治疗NAFLD的主要效应机制。.在此基础上，采用ANIT灌胃诱导HFD喂养小鼠形成NAFLD合并胆汁淤积病理模型，解析茵陈蒿汤调控NAFLD合并胆汁淤积小鼠胆汁酸和脂肪代谢的效应机制；使用标准饲料和HFD分别喂养Fxr-/-小鼠，考察调控FXR表达是否为茵陈蒿汤发挥治疗作用的关键环节，进而揭示茵陈蒿汤发挥治疗作用的效应机制。研究发现，使用HFD+ANIT能够诱导小鼠发生NAFLD合并胆汁淤积病变。其病理学特征为肝细胞小泡性脂肪变性、汇管区小胆管显著增生、炎性细胞浸润；血清ALT、AST、ALP及TBIL、DBIL、TC、TG水平升高。NAFLD合并胆汁淤积小鼠同时存在胆汁酸代谢和脂肪代谢异常。胆汁酸代谢异常的病理机制主要表现为肝组织胆汁酸转运体表达异常，导致肝细胞性胆汁淤积；脂肪代谢异常的病理机制主要表现为肝细胞脂肪酸合成增加大于氧化代谢，导致肝细胞脂肪蓄积。肝组织FXR表达降低，是NAFLD合并胆汁淤积存在胆汁酸代谢和脂肪代谢异常的关键环节。上调FXR表达是茵陈蒿汤组分大黄多糖、栀子苷、没食子酸及绿原酸调控NAFLD合并胆汁淤积的共同作用环节。FXR是大黄多糖调控NAFLD合并胆汁淤积的关键靶点。