背景:胆汁淤积性肝病(CLD)包括各种胆汁形成障碍,这通常会导致肝脏中胆汁酸(BA)的慢性暴露,并导致肝毒性和进行性肝胆损伤。Wedelactone(7-甲氧基-5,11,12-三羟基-香豆,WED),天然活性化合物来自于EclipseHerba,已报道具有保护肝脏的有价值的生物活性。然而,WED对胆汁淤积性肝损伤(CLI)的影响仍未被研究.
目的:本研究旨在阐明WED对α-萘基异硫氰酸盐(ANIT)诱导的CLI小鼠的保护作用,并探讨其潜在的药理机制。
方法:在ANIT诱导的CLI小鼠中评估WED的抗胆汁淤积和肝保护作用。非靶向代谢组学研究结合分子通路分析(IPA)探讨WED的关键机制。分析肝肠循环中的BA代谢谱,以评估WED在调节BA代谢中的作用。此外,分子动力学(MD)模拟和细胞热转移测定(CETSA)用于模拟和验证WED对法呢素X受体(FXR)的靶向激活。FXR在WED促进BA运输中的核心作用,在WT和FXR敲除小鼠或肝细胞中进一步评估了BA积累诱导的肝毒性。
结果:WED剂量依赖性地减轻ANIT诱导的小鼠胆汁淤积和肝损伤,同时抑制核因子-κB/核因子-红细胞2相关因子2(NF-κB/NRF2)的信号通路,以减轻炎症和氧化应激。在代谢物水平,WED改善了CLI小鼠的代谢紊乱,重点是BA的代谢,花生四烯酸,和甘油磷脂,这与BA监管过程密切相关,炎症,和氧化损伤。WED靶向激活的FXR,然后转录它的目标基因,包括胆盐出口泵(BSEP)和BA转运机,随后增加BA的运输,以恢复BA受损的肝肠循环。同时,WED通过NF-κB/NRF2信号通路减轻肝脏BA积累并保护肝脏免受BA诱导的损伤。此外,FXR缺乏在体外和体内抑制WED的保护作用。
结论:WED调节BA代谢,减轻胆汁淤积性肝损害。它通过调整BA运输保护肝脏,并通过FXR-胆汁酸-NF-κB/NRF2轴减轻BA积累相关的肝毒性。我们的研究提供了新的见解,即WED可能是胆汁淤积性肝病的有希望的策略。
BACKGROUND: Cholestatic liver diseases (CLD) comprise a variety of disorders of bile formation, which causes chronic exposure to bile acid (BA) in the liver generally and results in hepatotoxicity and progressive hepatobiliary injury.
Wedelolactone (7-methoxy-5, 11, 12-trihydroxy-coumestan, WED), the natural active compound derived from Ecliptae Herba, has been reported with valuable bioactivity for liver protection. Nevertheless, the effect of WED on cholestatic liver injury (CLI) remains unexplored.
OBJECTIVE: The present study aims to elucidate the protective effect of WED on Alpha-naphthylisothiocyanate (ANIT)-induced CLI mice, and to investigate its potential pharmacological mechanism.
METHODS: The anit-cholestatic and hepatoprotective effects of WED were evaluated in ANIT-induced CLI mice. Non-targeted metabolomics study combined with ingenuity pathway analysis (IPA) was used to explore the key mechanism of WED. The BA metabolic profile in enterohepatic circulation was analyzed to evaluate the effect of WED in regulating BA metabolism. Furthermore, molecular dynamics (MD) simulation and cellular thermal shift assay (CETSA) were used to simulate and verify the targeting activation of WED on the Farnesoid X receptor (FXR). The core role of FXR in WED promoting BA transportation, and alleviating BA accumulation-induced hepatotoxicity was further evaluated in WT and FXR knockout mice or hepatocytes.
RESULTS: WED dose-dependently alleviated ANIT-induced cholestasis and liver injury in mice, and simultaneously suppressed the signaling pathway of nuclear factor-kappa B/nuclear factor-erythroid 2-related factor 2 (NF-κB/NRF2) to relieve inflammation and oxidative stress. At the metabolite level, WED improved the metabolic disorder in CLI mice focusing on the metabolism of BA, arachidonic acid, and glycerophospholipid, that closely related to the process of BA regulation, inflammation, and oxidative damage. WED targeting activated FXR, which then transcribed its target genes, including the bile salt export pump (BSEP) and the BA transporter, and subsequently increased BA transportation to restore the damaged enterohepatic circulation of BA. Meanwhile, WED alleviated hepatic BA accumulation and protected the liver from BA-induced damage via NF-κB/NRF2 signaling pathway. Furthermore, FXR deficiency suppressed the protective effect of WED in vitro and in vivo.
CONCLUSIONS: WED regulated BA metabolism and alleviated hepatic damage in cholestasis. It protected the liver according to adjusted BA transportation and relieved BA accumulation-related hepatotoxicity via FXR-bile acid-NF-κB/NRF2 axis. Our study provides novel insights that WED might be a promising strategy for cholestatic liver disease.