拮抗孕烷X受体的壮药黑老虎根抗NAFLD活性成分发现及作用机制研究

Zhuang medicine is an important part of medicinal resources in China, and the Radix Kadsurae Coccineae which is originated from Kadsura genus has been wildly used in popular. Mining ethical medicine resources and exploring the effective drug and action mechanism towards nonalcoholic fatty liver disease (NAFLD) has important social and economic benefits. Our previous research indicated that Radix Kadsurae Coccineae extraction can significantly ameliorate rats with NAFLD, and the containing constituent lignan gomisin E is a potent antagonist of human and rodent pregnane X receptor (PXR) which is an effective target towards NAFLD. Furtherly this project intends to use natural product separation and purification as well as spectral technology to track and prepare more bioactive dibenzocyclooctadiene lignan and other constituents from Radix Kadsurae Coccinea. We will adopt the DPX2 cell line which is derived from HepG2 cells with the stable transfection of human PXR and a luciferase reporter gene linked with a human PXR response element identified in the CYP3A4 gene promoter and rPXR cell lines to find potent and low toxic PXR antagonist with prospects of development. With in vitro hepatic steatosis cells induced by FFA and in vivo NAFLD model using C57BL/6J wild type and humanized PXR engineering mice on a C57BL/6J background treated with STZ+HFD, triglyceride metabolism and inflammatory signaling pathways were checked and drug action characteristic and mechanism were analyzed. This project can also provide scientific evidence for the rational development and use of Kadsuracoccinea (Lem.)A. C. Smith to prevent and treat NAFLD.

壮药是我国药材资源的重要组成部分, 其中的南五味子属植物黑老虎根在民间广泛应用。挖掘民族药物资源，探索抗非酒精性脂肪性肝病（NAFLD）的药物及作用机制具有重要的社会经济效益。我们前期研究工作表明：黑老虎根提取物对大鼠NAFLD具有明显改善作用，所含Gomisin E是NAFLD防治靶标—孕烷X受体（PXR）的强效拮抗剂，且对hPXR和mPXR无选择性。本项目拟进一步运用天然产物分离纯化、光谱结构鉴定技术从黑老虎根中活性追踪和分离制备新的联苯环辛烯类等成分，以稳定转染hPXR/rPXR和荧光素酶报告基因的DPX2/rPXR细胞系为工具发现强效低毒的新型PXR拮抗剂。采用FFA诱导的体外肝细胞脂肪变性和STZ+HFD诱导野生型与人源化PXR基因的C57BL/6J小鼠NAFLD模型，围绕甘油三酯代谢与炎症信号通路，探讨药物作用特点与机制, 为黑老虎的合理开发并应用于防治NAFLD提供科学依据。

黑老虎根来源于木兰科植物南五味子属厚叶五味子Kadsura coccinea (Lem.) A. G. Smith的干燥根，是壮医常用药，具有悠久的药用历史，且被《中华人民共和国药典》（1977年版）收载，既往药理学研究表明黑老虎根具有抗炎、抗氧化、抗肝纤维化作用、降低胆固醇、降酶、保护肝细胞、抑制血小板聚集及抗肿瘤、抗HIV作用等。我们前期工作发现孕烷X受体PXR能介导慢性肝损伤, 结合流行病学研究的提示, 从而提出PXR是NAFLD的生物靶点和PXR拮抗剂是治疗NAFLD有效手段。.运用天然产物分离纯化、LC-MS/MS、NMR等光谱结构鉴定技术结合稳转hPXR的DPX2细胞系等筛选和评价技术首次从黑老虎根中鉴定、发现其中的Gomisin E等联苯环辛二烯类木脂素和五环三萜酸类成分等强效hPXR/mPXR拮抗剂；采用了体外游离脂肪酸（FFA）诱导的HepaRG人肝细胞和BRL 3A大鼠肝细胞脂肪变性模型和整体动物模型，围绕甘油三酯代谢与炎症信号通路，探讨其作用特点与机制。结果表明黑老虎根中的拮抗PXR组分调节甘油三酯代谢作用可能与调控硬脂酰辅酶A去饱和酶、S14、脂素-1、脂肪酸合成酶、磷酸烯醇式丙酮酸羧激酶1、脂肪酸转位酶、长链游离脂肪酸延长酶、肉毒碱棕榈酰转移酶1α有关。对小样本PXR基因多态性与NAFLD易感性的初步关联研究表明 rs7643645与NAFLD显著关联，该位点的突变可致PXR自身表达的下调, 且也可导致脂肪肝的发生，其机制可能与上调醛酮还原酶1B10介导的乙酰辅酶A羧化酶有关。.获得与本项目有关的中国发明授权专利两项；截止目前发表科研论文5篇；获广东省科技进步奖一等奖一项。本项目所获授权的核心专利 “一种黑老虎提取物的制备方法及在治疗非酒精性脂肪肝中的应用”已转让给新药研发企业并在实施产业化。其它相关研究成果正在投稿。.本项目的完成对NAFLD的有效干预提供了潜在靶点，为合理综合开发利用黑老虎植物资源提供了科学依据，并进一步深化和丰富了“调肝降脂”的科学内涵，具有一定的科学、经济、学术和社会价值。