背景:法尼醇X受体(FXR)是胆汁酸的重要受体,在胆汁淤积性肝病的治疗中起着重要作用。除了传统的基于胆汁酸的甾体激动剂,合成生物碱是最常见的非甾体FXR激动剂。SarmentolH是一种从SedumsarkentosumBunge获得的去甲倍半萜,和体外筛选实验表明,在先前的研究中,它可能与FXR途径的调节有关。
目的:研究沙门特醇H对胆汁淤积的治疗作用,并确定沙门特醇H是否直接靶向FXR减轻胆汁淤积。此外,本研究旨在通过定点诱变的方法探索沙门特醇H与FXR结合的关键氨基酸残基。
方法:建立肝内胆汁淤积小鼠模型,研究沙门托醇H对胆汁淤积的治疗作用。体外实验,包括Co-Ip和FXR-EcRE-Luc测定,进行评估是否通过招募受体辅激活因子SRC1来激活FXR。CETSA,SIP,达人,和ITC用于确定沙门托醇H与FXR蛋白的结合。通过分子对接和定点诱变分析了沙门托醇H与FXR结合的关键氨基酸残基。最后,我们对野生型和Fxr-/-小鼠进行了体内实验,以进一步验证沙门托醇H的抗胆汁淤积作用。
结果:沙门托醇H对ANIT诱导的胆汁淤积小鼠的病理状况有明显的改善作用。体外实验表明,它能够通过募集SRC1激活FXR并调节下游信号通路。目标验证实验表明,沙门托醇H具有作为配体(KD=2.55μmol/L)与FXR结合的能力,并增强了其空间结构的稳定性。此外,定点诱变显示THR292和TYR365是沙门托醇H和FXR的关键结合位点。此外,Fxr基因敲除导致ANIT诱导的胆汁淤积性肝损伤程度明显高于野生型胆汁淤积小鼠,而且在Fxr-/-胆汁淤积小鼠中,沙门托醇H改善胆汁淤积或对FXR下游基因的调节作用也消失了。
结论:沙美托醇H是FXR激动剂。这是第一个研究表明它对胆汁淤积小鼠有显著的治疗作用,并且可以直接与FXR结合并通过招募辅激活剂SRC1来激活它。
BACKGROUND: Farnesoid X receptor (FXR) is a vital receptor for bile acids and plays an important role in the treatment of cholestatic liver disease. In addition to traditional bile acid-based steroidal agonists, synthetic alkaloids are the most commonly reported non-steroidal FXR agonists. Sarmentol H is a nor-sesquiterpenoid obtained from Sedum sarmentosum Bunge, and in vitro screening experiments have shown that it might be related to the regulation of the FXR pathway in a previous study.
OBJECTIVE: To investigate the therapeutic effects of sarmentol H on cholestasis and to determine whether sarmentol H directly targets FXR to mitigate cholestasis. Furthermore, this study aimed to explore the key amino acid residues involved in the binding of sarmentol H to FXR through site-directed mutagenesis.
METHODS: An intrahepatic cholestasis mouse model was established to investigate the therapeutic effects of sarmentol H on cholestasis. In vitro experiments, including Co-Ip and FXR-EcRE-Luc assays, were performed to assess whether sarmentol H activates FXR by recruiting the receptor coactivator SRC1. CETSA, SIP, DARTS, and ITC were used to determine the binding of sarmentol H to FXR protein. The key amino acid residues for sarmentol H binding to FXR were analyzed by molecular docking and site-directed mutagenesis. Finally, we conducted in vivo experiments on wild-type and Fxr-/- mice to further validate the anticholestatic target of sarmentol H.
RESULTS: Sarmentol H had significant ameliorative effects on the pathological conditions of cholestatic mice induced with ANIT. In vitro experiments suggested that it is capable of activating FXR and regulating downstream signaling pathways by recruiting SRC1. The target validation experiments showed that sarmentol H had the ability to bind to FXR as a ligand (KD = 2.55 μmol/L) and enhance the stability of its spatial structure. Moreover, site-directed mutagenesis revealed that THR292 and TYR365 were key binding sites for sarmentol H and FXR. Furthermore, knockout of the Fxr gene resulted in a significantly higher degree of ANIT-induced cholestatic liver injury than that in wild-type cholestatic mice, and the amelioration of cholestasis or regulatory effects on FXR downstream genes by sarmentol H also disappeared in Fxr-/- cholestatic mice.
CONCLUSIONS: Sarmentol H is an FXR agonist. This is the first study to show that it exerts a significant therapeutic effect on cholestatic mice, and can directly bind to FXR and activate it by recruiting the coactivator SRC1.