Wang Wenyu, Li Lin, Li Xia, Chen Jiaqi, Wang Rui, Yang Qi, Qu Yifan, Wang Changyuan, Fu Ting, Meng Qiang
Department of Clinical Pharmacology, College of Pharmacy, Dalian Medical University, Dalian, 116044, China.
Pharmacy Department of Affiliated Zhongshan Hospital of Dalian University, No. 6 Jiefang St. Dalian, 116001, China.
Free Radic Biol Med. 2025 Jun 22;238:152-168. doi: 10.1016/j.freeradbiomed.2025.06.039.
Cholestasis, a major driver of liver disease progression, is characterized by toxic bile acid accumulation due to impaired bile flow, potentially leading to hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. To date, the pathogenesis of cholestasis has remained incompletely understood. In the present study, we investigated the role of farnesoid X receptor (FXR) in modulating the NLR family CARD domain-containing protein 4 (NLRC4) inflammasome activity using in vitro (AML-12 hepatocytes) and in vivo (C57BL/6 mice) models of lithocholic acid (LCA)-induced cholestasis. LCA suppressed FXR expression, downregulated bile acid transporters (Bsep, Mrp2, Ntcp), and elevated serum biomarkers of liver injury. Through restored hepatic function by FXR lentiviral vectors, FXR overexpression reduced bile acid accumulation and mitigated inflammation and oxidative stress. In addition, FXR overexpression suppressed bile acid synthesis enzymes (Cyp7a1, Cyp8b1) by upregulating Shp and Fgf15, while enhancing detoxification through Ugt1a1 and Sult2a1. Interestingly, molecular docking analysis and Co-IP experiments demonstrated a direct interaction between FXR and NLRC4. Furthermore, FXR overexpression significantly inhibited NLRC4 inflammasome activation and decreased the expression of NLRC4, caspase-1, IL-1β, and IL-18, thereby attenuating inflammation and oxidative stress. Conversely, FXR knockdown reversed these effects. In addition, to delineate the contribution of NLRC4 inflammasome activation to IL-18 and IL-1β elevation, NLRC4-targeting siRNA-mediated knockdown and NLRC4-encoding plasmid-driven overexpression strategies were systematically employed. Furthermore, DCFH-DA staining was adopted to visualize reactive oxygen species (ROS). In conclusion, for the first time, we found that FXR overexpression alleviates LCA-induced cholestasis by regulating bile acid metabolism and inhibiting NLRC4 inflammasome activation, providing a novel therapeutic strategy for drug development targeting the FXR-NLRC4 axis.
胆汁淤积是肝病进展的主要驱动因素，其特征是胆汁流动受损导致毒性胆汁酸积累，可能导致肝纤维化、肝硬化和肝细胞癌。迄今为止，胆汁淤积的发病机制仍未完全明确。在本研究中，我们使用体外（AML-12肝细胞）和体内（C57BL/6小鼠）模型，研究了法尼醇X受体（FXR）在调节含NLR家族CARD结构域蛋白4（NLRC4）炎性小体活性中对石胆酸（LCA）诱导的胆汁淤积的作用。LCA抑制FXR表达，下调胆汁酸转运体（Bsep、Mrp2、Ntcp），并升高肝损伤血清生物标志物。通过FXR慢病毒载体恢复肝功能，FXR过表达减少了胆汁酸积累，并减轻了炎症和氧化应激。此外，FXR过表达通过上调Shp和Fgf15抑制胆汁酸合成酶（Cyp7a1、Cyp8b1），同时通过Ugt1a1和Sult2a1增强解毒作用。有趣的是，分子对接分析和免疫共沉淀实验证明FXR与NLRC4之间存在直接相互作用。此外，FXR过表达显著抑制NLRC4炎性小体激活，并降低NLRC4、半胱天冬酶-1、白细胞介素-1β和白细胞介素-18的表达，从而减轻炎症和氧化应激。相反，FXR敲低逆转了这些作用。此外，为了阐明NLRC4炎性小体激活对白细胞介素-18和白细胞介素-1β升高的作用，系统采用了靶向NLRC4的小干扰RNA介导的敲低和NLRC4编码质粒驱动的过表达策略。此外，采用DCFH-DA染色来可视化活性氧（ROS）。总之，我们首次发现FXR过表达通过调节胆汁酸代谢和抑制NLRC4炎性小体激活来减轻LCA诱导的胆汁淤积，为靶向FXR-NLRC4轴的药物开发提供了一种新的治疗策略。
