法尼醇X受体（FXR）抗肝细胞凋亡作用及其机制研究

Hepatocyte apoptosis is the common pathological feature of all kinds of liver diseases. Anti-apoptotic is regarded as one of the most effective ways to treat liver disorders. However, development of anti-apoptotic compounds as hepatoprotective drugs is still at its early stage. Few anti-apoptotic targets or strategies were reported. Farnesoid X receptor (FXR) is tightly coupled with the physiological function of liver. It is not clear whether FXR is benefit to hepatocyte apoptosis, although this nuclear receptor provides protective effect in verious kinds of liver diseases. We have demonstrated that over-expression of FXR in HepG2 cells exerted protective effect against ActD/TNFα-induced apoptosis. Thus, we assume that the FXR is benefit to hepatocyte apoptosis, this nuclear receptor exerts its anti-apoptotic effect through protein-protein interaction with an unknown protein. Inflammatory and fatty acid induced apoptosis models were enrolled in our project to validate the transcriptional activity independent anti-apoptotic effect of FXR. Furthermore, we intend to explore the apoptotic protein which binds to FXR and validate the binding domain using proteomic, Co-IP, and MST technologies. The role of this protein in the anti-apoptotic effect of FXR will also be explored. Besides, nonalcoholic steatohepatitis model will be enrolled to verify the hepatoprotective effect and possible side effects caused by FXR overexpression in livers. This project is expected to provide new strategies for the development of hepatoprotective medicines which targeted FXR.

肝细胞凋亡是各种肝病的共同核心病理进程，抗肝细胞凋亡被认为是肝病的治疗策略，但目前尚且缺乏针对肝细胞凋亡的有效治疗策略。法尼醇X受体（FXR）与肝脏生理功能密切相关，且FXR对不同原因导致的肝损伤均有保护作用，但这是否与其抗肝细胞凋亡作用有关尚不清楚。本项目前期发现FXR过表达可显著对抗ActD/TNFα诱导的肝细胞凋亡。基于此我们提出科学假说：FXR可对抗肝细胞凋亡，其通过与凋亡通路中的某一关键蛋白互作，进而阻断凋亡信号传导，发挥转录活性非依赖的抗凋亡作用。拟开展以下研究：在体内外多种肝细胞凋亡模型中，对比研究激动剂诱导的转录激活和腺病毒转染诱导的过表达的抗凋亡效应，明确其作用模式；通过蛋白组学、Co-IP、MST等技术发现并验证与FXR互作的凋亡蛋白及其结合位点，阐明FXR抗凋亡机制；并在脂肪肝炎模型中验证其药理作用及模式。通过本项目的研究，最终为靶向FXR的肝药研发提供新策略。

法尼醇X受体（FXR）是肝病治疗的重要靶标，然而其对肝细胞凋亡的作用尚不清楚。本项目围绕“FXR抗凋亡作用模式确证、作用机制探索及其在NASH治疗中的作用”展开系统研究。本项目按照立项计划执行，超额完成了各项研究内容：明确了肝脏FXR为抗凋亡分子，其抗凋亡作用模式为转录活性非依赖的方式，即FXR激动剂促进其转录激活后没有显著的抗凋亡作用，其抗凋亡作用主要依赖于其蛋白表达水平；其潜在机制在于FXR通过与caspase 8之前的蛋白-蛋白相互作用，阻断caspase 8向凋亡复合体的募集，进而阻断其自剪切活化和凋亡进程；FXR过表达可以显著对抗非酒精性脂肪肝及肝纤维化等多种肝病状态下凋亡，缓解肝病进展，且FXR过表达不会引起显著的胆汁酸和脂质代谢紊乱。上述研究表明，恢复FXR蛋白表达水平是治疗肝脏疾病的新策略。此外，我们还提出了FXR激动剂与凋亡抑制剂联用的策略来治疗肝纤维化，该策略具有更好的抗肝纤维化药效；此外，我们还建立了肠道限制性FXR激动剂的评价方式，合作得到具有生物学/药理学效应的候选分子Fex-3。超额完成了计划书约定的内容，达到预期目标。基于本项目的研究，发表SCI论文6篇，其中2篇影响因子大于5，另有1篇发表于本学科领域顶尖期刊Drug Metab Dispos上；获授权专利2项；基于本项目研究，培养博士研究生2名、硕士研究生3名。