基于肠道稳态调节的壮药山绿茶防治非酒精性脂肪肝作用机制研究

Non-alcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver disease. Our previous study found that triterpenes and their saponins of Zhuang medicine Ilex hainanensis can significantly improve NAFLD. Triterpenes of Ilex hainanensis can take effect in target organ for their good absorption, while mechanism of saponins against NAFLD havn’t been illuminated for their low oral bioavailability. Intestinal microflora dysbiosis plays an important role on the procession of NAFLD. Our preliminary experiments show that: Ilex hainanensis saponins were transformed into aglycones by intestinal bacteria which revealed significant antibacterial activity, and can reduce serum endotoxin in NAFLD mice. The results showed that Ilex hainanensis saponins can prevent and treat NAFLD by regulating the intestinal homeostasis. Ilex hainanensis triterpenes and their saponins can control NAFLD by repairing intestinal microflora. The improvation of intestinal microflora will be studied by bacteria structure analysis, bacteria metabolic product profile as well as intestinal barrier function after oral triterpenes and saponins from Ilex hainanensiscan。The metabolic function of intestinal bacteria will also be studied on analysis of triterpene saponins and their bacteria metabolic products from intestinal contents. Our study willl illuminate the mechanism against NAFLD of Ilex hainanensis triterpene saponins based on gut homeostasis, and elucidate the contradiction between pharmacokinetics and pharmacodynamic. It will provide evidence for prevention of NAFLD by Ilex hainanensis triterpene saponins from multi-target and give reference for drug research on saponins regulating intestinal microflora.

非酒精性脂肪肝（NALFD）已成为慢性肝病的首要病因。前期研究表明：壮药山绿茶皂苷及其苷元具有显著的防治NAFLD作用，其中三萜苷元吸收良好，能够直达靶器官起效；但皂苷口服利用度低，其作用机制尚不明确。肠道微生态失衡在NAFLD发展过程起重要作用。初步实验表明：山绿茶皂苷经肠道代谢为三萜苷元，具有显著抑菌活性，并能降低血清内毒素，提示其能通过肠道稳态调节防治NAFLD。本课题通过肠道菌群结构、NAFLD相关菌群代谢产物变化，结合肠屏障功能研究山绿茶三萜皂苷对NAFLD小鼠肠道微生态的修复作用；通过皂苷肠道代谢产物分析探讨肠道菌群结构变化对皂苷代谢转化功能的影响。课题的完成，通过山绿茶三萜皂苷的肠道稳态调节作用阐明其防治NAFLD的作用机制，揭示其“药代-药效”相矛盾的真正原因，为山绿茶多靶点防治NAFLD提供理论依据，为皂苷类药物用于肠道稳态调节的研发提供参考。

非酒精性脂肪肝（NALFD）已成为慢性肝病的首要病因。研究显示，肠道微生态失衡在NALFD的发展过程中扮演重要的角色。山绿茶皂苷具有显著的防治NAFLD作用，但其口服利用度低，作用机制尚不明确。本课题拟通过“肠-肝轴”，研究山绿茶皂苷防治NAFLD的作用机制。. 首先，建立山绿茶富集工艺，得到高纯度山绿茶总皂苷（含量>90%）；并对主要三萜、皂苷类成分进行分离纯化，共分离得到22个三萜成分，其中新化合物2个；以山绿茶中主要6种三萜、皂苷类成分HPLC-ELSD含量测定方法，用于该类药材及提取物的质量控制。. 建立UPLC-MS/MS法定性、定量分析灌胃山绿茶总皂苷后正常及NAFLD小鼠血浆原型及代谢产物含量。结果显示，与正常小鼠相比，NAFLD小鼠体内皂苷的吸收减少，苷元的吸收增加，可能与NAFLD小鼠菌群与肝药酶改变有关；为山绿茶皂苷作用机制探讨提供依据。. 采用高脂饲料诱导小鼠 NAFLD 模型，通过体重、肝重、脂肪积聚、转氨酶、血脂、肝组织切片综合评价山绿茶皂苷及冬青皂苷A1防治小鼠 NAFLD 的作用。结果显示，低给药组均可显著降低小鼠的体重、肝重、脂肪积聚及血脂、转氨酶含量，肝组织形态得到明显改善，且具有较好的剂量依赖性。.采用PA刺激内皮细胞建立血管内膜功能紊乱的细胞模型，Western Blot检测内皮细胞中IRE1α、PERK、LKB1、AMPK、ACC的磷酸化表达。结果显示，冬青素A可以增强LKB1依赖性的AMPK的活性，降低IRE1α和PERK的磷酸化作用，从而抑制ER stress。. 采用试剂盒检测血胰岛素、LPS、IL-6的含量，qRT-PCR检测肝组织中TNF-α、IL-1β、IL-6、TLR2的mRNA表达，western blot检测近端结肠组织occludin的蛋白表达。采用Illumina Miseq PE300高通量测序平合对小鼠肠道菌进行16S rDNA测序，并对测序数据进行生物信息学分析。结果显示，山绿茶皂苷可以增加肠道中有益微生物的丰度，同时抑制有害微生物的过度增值，作用于LPS-TLR2通路，改善胰岛素抵抗、代谢性代谢性内毒素血症、炎症和肠屏障功能。. 课题的完成，揭示山绿茶皂苷通过肠道稳态调节防治NAFLD的作用机制，为山绿茶多靶点防治NAFLD提供理论依据。