基于“肠-肝轴”-脂质代谢信号通路的苗药赶黄草干预非酒精性脂肪肝药效物质和作用机制研究

Penthorum chinense Pursh. (P. chinense), which is a kind of Miao medicine, has been proven effectively in clinic to protect liver function. Based on the metabolic characteristics of the major constituents of P. chinense in vivo and the pathogenesis of nonalcoholic fatty liver disease NAFLD, we have hypothesized that the mechanisms of the anti-nonalcoholic fatty liver disease (anti-NAFLD) of P. chinense may be through the regulation of the intestinal microecology by working on the multi-targets in the lipid metabolism signaling pathways, which are associated with insulin resistance. In this work, the active constituents of P. chinense of anti-NAFLD were screened in silico through pharmacophore modelling and molecular docking and further validated both in cellular and animal models. Using 16SrRNA high-throughput sequencing techniques, the interactions of active constituents and intestinal microbiota were detected by analyzing the changes of gut microbiota in mice and the transformation of the active constituents incubated in flora in vivo and in vitro. By using NAFLD disease models, the mechanisms of anti-NAFLD of P.chinense were illustrated through multiple studies, including the analysis of the contents of endotoxin, inflammatory factors and the expression of key proteins related to lipid metabolism signaling pathways. As a result, we have identified the active constituents of P. chinense involved in anti-NAFLD and illustrated the mechanisms of anti-NAFLD of P.chinense via the regulation of the intestinal microecology by working on the multi-targets in the lipid metabolism signaling pathways. This work may provide the theoretical basis for the further expanding the application of P. chinense.

苗药赶黄草的保肝作用临床疗效确切，在前期研究基础上，根据其主要成分的体内药物代谢特点和非酒精性脂肪肝病（NAFLD）的发病机制，提出其作用机制可能是通过对肠道微生态的调节与胰岛素抵抗相关联的脂质代谢信号通路的多靶点协同干预NAFLD。本项目采用分子对接计算机虚拟筛选技术，细胞与整体动物模型相结合，对赶黄草中干预NAFLD的活性成分进行筛选与验证。以肠道菌群为切入点，采用16SrRNA高通量测序技术通过表征小鼠肠道菌群的结构差异以及肠道菌群对活性成分的转化，探讨赶黄草中活性成分与肠道菌群的相互作用。通过血浆中内毒素的含量变化、相关炎症因子的检测、脂质代谢信号通路关键蛋白的表达，从肠道菌群、肠-肝轴、脂质代谢信号通路的多靶点及肠道菌群代谢产物转化等方面，阐明赶黄草干预NAFLD的药效物质基础和作用机制，为扩大赶黄草的应用和寻找新的保肝降脂药物奠定理论基础。

非酒精性脂肪肝病（Nonalcoholic fatty liver disease，NAFLD），也被称为代谢相关性脂肪肝病（Metabolic associated fatty liver disease, MAFLD），已成为世界范围内慢性肝病的首要病因，发病率高达25%。NAFLD是代谢综合征在肝脏的表现，发病机制涉及脂质代谢障碍、炎症反应、胰岛素抵抗等。NAFLD尚无有效的治疗药物，现阶段临床使用的有降脂药物、胰岛素增敏剂、抗氧化剂等，但效果不佳、副作用明显。近年来，肠道菌群及相关代谢物（如LPS、短链脂肪酸、胆汁酸等）被证实与NAFLD的发生发展密切相关，为NAFLD的治疗提供了新的研究思路。民族药赶黄草防治NAFLD的药效明确，但是作用机制尚不清楚。课题组前期研究发现，赶黄草中二氢黄酮苷乔松素-7-O-β-D-葡萄糖苷（PCBG）是其保肝作用的主要成分，因其生物利用度低推测其与肠道菌群存在相互作用。本课题首先开展赶黄草黄酮苷类成分的有效组分富集和分离鉴定；其次采用高脂饮食诱导的NAFLD小鼠模型和HepG2细胞模型验证了赶黄草浸膏及其组分通过降低肝脏脂质代谢发挥药效；采用网络药理学、分子对接和FFA诱导脂质沉积的L02细胞模型相结合的方法，预测赶黄草中二氢黄酮类成分抗NAFLD的作用通路及靶点并进行验证，提示胆汁酸代谢途径的相关靶点可能是其发挥作用的关键。在明确药效的基础上，选取代表性活性成分PCBG，采用靶向代谢组学技术和粪菌移植方法，从“肠道菌群-菌群相关代谢物-胆汁酸代谢通路”的角度阐明其作用机制，为赶黄草的临床应用提供科学依据,也为民族药研究提供参考。