基于“肠道菌群-代谢”特异性调控的壮药山绿茶多组分配伍防治NAFLD作用机制研究

The therapeutic benefits of Ilex hainanensis Merr., a kind of Zhuang medicine, towards nonalcoholic fatty liver disease (NAFLD) have been well-defined. The total triterpenoid saponins from I. hainanensis have been proved to decrease adiposity, insulin resistance and markers of inflammation by modulating gut microbiota in high-fat fed-induced NAFLD mice. The relative abundance of bacteria droped a hint that I. hainanensis might have metabolic regulating effect. However, the underlying mechanisms haven’t been revealed. We aim to address these issues based on the concept of holism and compatibility of traditional Chinese medicine. The pharmacodynamics of the polysaccharide, polyphenols, total saponins and their compounds were investigated through the indices including body weight, the level of blood lipids, glucose homeostasis and the histopathology of the liver and so on. The specificity of the active fractions and the synergitic effect of the compounds was analyzed through the gut microbiota composistion combined with the abundance of correlated bacteria. The underlying mechanisms were illustrated by combining pathways (the metabolism of bile acid, lipid, SCFAs and chronic inflammation, gut permeability) with the changes in the gut microbiota. The rationality of component compatibility than single component will be revealed from the various of microbiota-metabolic-pharmacodynamics modulation factors. The findings obtained are expected to expand the scope of clinical application of I. hainanensis, provide promising scientific clues for the principles of “compatibility to promote the effect” and enhance the innovative research of drug discovery from ethno medicine.

壮药山绿茶防治非酒精性脂肪肝（NAFLD）药效明确，前期研究证明山绿茶皂苷调节肠道菌群抑制炎症反应发挥药效，相关菌丰度分析提示山绿茶可能对体内多种代谢反应具有调控作用，机制尚不清楚。本项目以传统医学的“整体观”与“配伍”理论为指导，采用高脂饲料诱导小鼠NAFLD模型，从体脂、血脂、糖稳态及肝组织病变等药效学指标，评价山绿茶总皂苷、总多酚、总多糖部位及组合物药效；从菌群整体结构、NAFLD相关菌丰度变化等指标，评价各组分在结构调整中的特异性及配伍组分的协同性；将胆汁酸代谢、脂代谢、SCFAs、LPS-TLR炎症通路、肠屏障等方面与药效指标、菌群结构变化结合，揭示山绿茶组分配伍治疗NAFLD的作用机制。本研究以肠道菌群整体调控策略为基础，建立“菌群-代谢-药效”关联的复杂组分作用机制研究方法，为山绿茶资源的开发利用准备理论依据，对诠释组分配伍协同增效的科学内涵及民族药创新研究具有重要意义。

本课题采用高脂饲料诱导的非酒精性脂肪肝（NAFLD）小鼠为模型，应用代谢组学和16S rRNA 基因测序等技术，从药效、代谢调控、肠道菌群、相关蛋白表达，阐明了山绿茶各有效部位及其组合物对NAFLD的保护作用及各有效部位的贡献，为阐明山绿茶干预NAFLD作用机制、成分配伍规律和临床应用提供了参考。.药效学研究显示，山绿茶各有效部位和组合物均能够改善高脂饲料诱导的NAFLD小鼠肝脂肪变性，血脂异常，胰岛素抵抗及肠道菌群紊乱导致的肠屏障的破坏和肠道炎症。.采用LC-MS/MS靶向代谢组学技术，研究山绿茶各有效部位对NAFLD小鼠胆汁酸代谢的作用。结果表明山绿茶总皂苷组小鼠肝脏结合型胆汁酸水平显著降低，肝脏胆汁酸蓄积减少；小鼠粪便中胆汁酸含量升高，促进胆汁酸的排泄。.应用 16S rRNA 基因测序和代谢组学技术，研究NAFLD小鼠肠道菌群和宿主代谢相关性，并考察山绿茶各有效部位及组合物对肠道菌群紊乱的调控作用。结果表明，NAFLD小鼠肠道菌群结构和代谢表型均发生显著改变，并伴有肠屏障破坏和肠道炎症的发生，而山绿茶各有效部位及组合物对肠道菌群紊乱具有一定调控作用。山绿茶总皂苷给药后能够显著提高肠道菌群中胆盐水解酶（BSH）相关菌的丰度，并提高BSH活力，从而增加胆汁酸去结合后排泄。胆汁酸池变化通过肝肠循环作用于胆汁酸-FXR轴，促进胆汁酸替代途径相关合成酶、肝脏外排转运体BSEP的基因表达，降低重吸收转运体NTCP的表达，促进胆汁酸生成的同时避免了NAFLD小鼠肝脏的胆汁酸蓄积。高脂饲料诱导的肠道菌群紊乱随后引发肠屏障受损，肠源性脂多糖（LPS）通过门静脉入血，在肝脏激活TLR通路，直接导致炎症反应的发生。山绿茶各给药组及组合物均能够一定程度上调整菌群结构，修复肠屏障，减少肠源性毒性成分入血。山绿茶各有效部位均能够一定程度改善NAFLD小鼠肠道菌群紊乱及内源代谢，且与组合物存在差异，提示通过调控肠道菌群干预NAFLD存在多种途径，但如何实现肠道菌群与代谢的最佳调控效果仍需要探索药效以外的评价标准，从而获得更加科学、合理的组分配伍方案。