FXR/NF-κB负反馈环调控肝细胞凋亡与水飞蓟宾双靶干预机制

FXR is a potential therapeutic target for various liver diseases; however, the FXR target drug development remains a great challenge due to multiple and difficult-to-predict undesirable side effects by FXR activation. On the basis of strong preliminary experiment results, we hypothesize that the dual target interference of FXR and NF-κB both at a weak level would be promising to complementing a selective FXR activation at the damaged liver while sparing the normal FXR regulated physiological functions. We intend to confirm this hypothesis, by focusing on the role and mechanisms of FXR/NF-κB feedback loop on regulating apoptotic hepatocytes death, in the MCD diet induced NASH and thioacetamide induced chronic hepatic fibrosis mice models, and TNFα/MCD challenged primary mice and human hepatocytes and Hepg2 cell models. To provide strong evidence on determining the role and mechanism of FXR/NF-κB feedback loop in regulating hepatocytes apoptosis, various chemical interferences and genetic handling strategies will be applied to specifically interfering FXR or NF-κB alone or both at both strong and weak level. With these models and experimental strategies, we intend to determine how FXR/NF-κB feedback loop regulates the external and internal apoptotic pathways of hepatocytes, which is the key pathological process in various liver diseases and represent a pivotal link between inflammation and fibrosis/cirrhosis. Then, the effect and involved mechanisms of silybin, a natural hepatoprotective agent, in regulating the FXR/NF-κB feedback loop and the apoptotic pathways of hepatocytes will be determined. The characteristics of silybin in targeting both FXR and NF-κB at a relatively weak level but confers a strong hepatoprotective effects via a feed-forward mechanism will be especially focused. Afterwards, silybin can be used as a chemical probe to determine the differential effects in regulating both the physiological and pathological bile acids catabolism and the carbohydrate and lipid metabolism, between dual target interference of FXR and NF-κB at a weak level and the strong activation of FXR by the typical agonists. This proposed research will provide novel insights into the understanding of hepatoprotective role of FXR and will open a new idea to the FXR target new drug development.

FXR是肝病治疗的潜在靶标，但由于广泛的和难以预期的副作用，靶向FXR的肝保护药物研发一直未能突破。本项目基于良好的实验基础及炎症/代谢交互负调控的特点，提出通过"弱效应双靶干预FXR/NF-κB负反馈通路"以避免或降低FXR激动对正常生理功能影响的科学假说。拟在NASH及慢性肝纤维化小鼠模型、原代肝细胞与Hepg2细胞炎症损伤模型，利用多种化学干预及基因操纵技术特异性干预FXR/NF-κB，研究FXR/NF-κB负反馈通路调控内外源性肝细胞凋亡通路的作用与分子机制，比较单独干预及协同干预、强干预与弱干预对肝细胞凋亡的影响；研究水飞蓟宾调控FXR/NF-κB负反馈通路的作用特点与分子机制，揭示其潜在作用靶标；比较研究水飞蓟宾与经典FXR强激动剂对正常与肝损时胆汁酸合成代谢通路及糖脂代谢的差异作用。项目研究对于深入理解FXR的肝保护机理和推动靶向FXR的创新药物研发具有重要理论和实际意义。

FXR 是肝病治疗的潜在靶标，但由于广泛的和难以预期的副作用，靶向 FXR 的肝保护药物研发一直未能突破。本项目基于良好的实验基础及炎症/代谢交互负调控的特点，提出通过“弱效应双靶干预 FXR/NF-κB负反馈通路”以避免或降低 FXR 激动对正常生理功能影响的科学假说，并拟以水飞蓟宾为模型药物来研究FXR/NF-κB负反馈通路对肝脏疾病的干预保护作用。在项目研究中，我们首先系统研究了水飞蓟宾对生理、病理状态下核受体-代谢酶系统的调控作用，明确了水飞蓟宾对FXR等核受体的调控作用以及病理状态下FXR等核受体表达和转录活性的改变；在此基础上，系统研究了FXR/NF-κB负反馈环在脂肪肝、急性肝衰竭等肝病中的失调，并通过基因操作、化学干预等手段揭示了该负反馈环对肝脏疾病的调控/保护作用；并在此基础上揭示了基于核受体调控的水飞蓟宾保肝作用分子机制，发现水飞蓟宾对肝细胞损伤的保护作用以及对损伤状态下脂肪酸胆汁酸等内源性物质失调的纠正作用依赖于核受体，初步阐释了水飞蓟宾的保肝分子机制。另外，代谢组学研究发现与水飞蓟宾相比，经典FXR激动剂对糖酵解通路具有显著调控作用，这是FXR促进细胞增殖的分子机制之一。在以上研究的基础上，我们对项目研究进行了拓展：考察了另外一种保肝药物甘草酸对核受体-代谢酶体系的调控作用以及基于代谢的保肝机制研究；考察了核受体/NF-κB对结肠炎的调控作用以及基于此的人参皂苷作用机制研究。本项目初步揭示了水飞蓟宾和甘草酸的保肝作用分子机制，另外项目研究对于深入理解 FXR 的肝保护机理和推动靶向 FXR等核受体的创新药物研发具有重要理论和实际意义。