从“肠-肝对话”角度探究疏肝要药柴胡基于SLC10A2抑制效应调控胆固醇-胆汁酸代谢干预NAFLD的作用机制

Bupleuri Radix (Bupleurum) as a vitally important liver-soothing herb is commonly used to prevent and treat NAFLD. However, the pharmacokinetic characteristics of Bupleurum such as extremely low concentration of effective components in body circulation and target organ, are contradict to the hepatoprotective effect, which is difficult to comprehensively explain its mechanism. Cholesterol metabolic disorder is a crucial inducement of NAFLD, while bile acids reabsorption occurs in the terminal ileum via active transport by the apical sodium-dependent bile acid transporter SLCA2 (ASBT)，which is closely related to cholesterol-bile acid metabolic pathways. In previous studies, a rapid classification and identification method applied to the analysis of glycosides in Bupleuri by UPLC-Q-TOF-MS/MS coupled with data post-processing technology have been established, and those has been found to exhibit high affinity with ASBT. Therefore, a scientific hypothesis is put forward in this study, " The enterohepatic cycling of bile acid are interrupted by Bupleurum based on the initial ASBT inhibition effect, leading to the feedback regulation of cholesterol-bile acid metabolism in the liver to activate FXR and this is expected to decrease lipids accumulation ". With enterohepatic circulation of bile acid as breakthrough point, the correlation between ileal reabsorption of bile acid and cholesterol metabolism change will be investigated to confirm the intestine target of Bupleurum in different angle, and further the response mechanism of “gut-liver crosstalk ” will be explored by means of targeted bile acid metabolomics with dual luciferase reporter. Additionally, target magnetic beads /UPLC-Q-TOF-MS/MS couple with ITC technology as a powerful approach will be used to accomplish rapid screening of active ingredients of Bupleurum, and those potential active ingredients will be verified by the cell model previously constructed. Through the research above, biological mechanisms and pharmacodynamic material basis of Bupleurum improved NAFLD could be clarified. This may contribute to providing a new perspective for comprehending the mechanism of poorly absorbed Bupleurum with hepatoprotective effect or something like that.

疏肝要药柴胡是防治NAFLD的常用中药，但有效成分体循环及靶器官浓度低，PK-PD不相关难以全面解释其药效机制。胆固醇代谢紊乱是导致NAFLD的重要诱因，而SLC10A2(ASBT)介导的胆汁酸重吸收与胆固醇-胆汁酸代谢密切相关。前期研究利用UPLC-Q-TOF-MS/MS集成数据后处理技术定性柴胡成分并发现柴胡与ASBT具有较高的亲和力，因此提出“柴胡通过抑制ASBT的始动效应反馈调节胆固醇-胆汁酸代谢，改变胆汁酸谱而激活FXR减少脂质蓄积”的假说。拟以胆汁酸肝肠循环为切入点，多角度考察抑制胆汁酸肠道重吸收与胆固醇代谢变化的关联性明确柴胡的作用靶点，靶向胆汁酸代谢组学结合双荧光酶报告基因探明其“肠-肝对话”的作用机制，靶磁珠-液质联用/ITC法筛选活性成分并借助前期已构建的细胞模型进行验证，阐明柴胡干预NAFLD的生物学机制和药效物质基础。以期为口服难吸收柴胡作用机理的认识开启新视角。

NAFLD不仅是全球重要的公共健康问题之一，也逐渐成为我国愈来愈突出的慢性肝病问题，但由于其发病机制的复杂性和治疗目标的多元化导致临床尚缺乏公认且效果理想的药物。中医学家提出NAFLD的病机遵肝郁为始、痰湿致病的过程，在痰湿论治之中应注重肝气郁结的始发病机。临床中疏肝要药-----柴胡是防治NAFLD的常用中药，广泛用于肝损伤，肝炎和肝纤维化，然而柴胡皂苷类成分的口服生物利用度差，PK-PD不相关而难以全面解释柴胡发挥药效的作用机制。本研究首先通过检测胆汁酸肠肝循环相关转运体和代谢酶表达水平以及反应胆汁酸重吸收障碍的血清和粪便指标证实柴胡通过抑制ASBT阻碍胆汁酸的肠肝循环；其次利用靶向胆汁酸代谢组学和双荧光素酶报告基因法探明柴胡通过抑制ASBT反馈调节胆固醇-胆汁酸代谢改变肝脏胆汁酸代谢轮廓，并通过高表达FXR-HepG2细胞模型进一步证实肝脏胆汁酸组成变化对于FXR的调控作用；最后在亲和超滤色谱-UPLC-Q-TOF-MS/MS筛选潜在活性成分的基础上利用pSR-CMV-GFP-ASBT-HEK293细胞模型揭示柴胡防治NAFLD的药效物质基础。以期诠释“肠-肝对话”模式下柴胡抑制ASBT而发挥肝脏的保护作用。本课题的顺利实施不仅为口服难吸收中药治疗NAFLD的作用机理开启了新视角，也为"以肠道为靶位"的中医药治疗策略提供依据。