基于Nrf2/ARE信号系统的泽泻水提物降糖作用药效物质基础及作用机制研究

As a traditional medicine in China, Alisma orientale (AR) is an important part of many prescriptions and has been commonly used for treating a wide range of ailments related to hyperlipidaemia, diabetes as well as inflammation in clinical applications..This study aims to research the therapeutic material basis and mechanism in hypoglycemic activities of the water extracts of AR by Nrf2 / ARE signaling pathway. First, the chemical separation method was used to separate the small molecule and macromolecule compounds of the water extract of AR. The small molecular compounds were isolated, purified by a hydrophilic interaction chromatography (HILIC) coupled with mass spectrometric method (HILIC-MS) and further identified by NMR, HR-MS, IR, etc. The Sephadex gel chromatography with different molecular weights was developed for separating the macromolecular compounds such as polysaccharide, then identified the chemical units of polysaccharide, this study could improve the therapeutic material basis of the water extract of Alisma orientale; .Secondly, the glucose uptake method and the Nrf2 / ARE antioxidant system of fluorescent expression plasmid screening method was established. These High-throughput sequencing method were used to screen for the hypoglycemic conpounds and Nrf2/ARE target activator from the water extracts of AR, and the hypoglycemic through Nrf2 / ARE signaling system was further validated by siRNA method, which will clarify the real therapeutic material basis of the water extracts of AR in vitro; Third, The hypoglycemic mechanism of the active coumpounds of AR using IR mice and db/db mice, the research for the mechanism in vivo of water extract active components by Nrf2/ARE, NQO-1, HO-1 and other anti-oxidative system signaling pathway. Material foundation and hypoglycemic mechanisms of water extracts of AR can be elucidated by this study.

泽泻及其方剂临床上用于调脂降糖疗效确切，本研究拟通过Nrf2/ARE信号通路研究泽泻水提物的降糖药效物质及其作用机制。首先，采用化学分离方法对泽泻水提物进行小分子和大分子化合物两个部位的分离，对小分子部位采用HILIC模式的质谱引导分离纯化制备，鉴定其化学结构，对大分子化合物进行不同分子量大小的葡聚糖凝胶色谱分离，鉴定泽泻大分子的化学结构单元，明确泽泻水提物的化学物质基础；其次，构建细胞葡萄糖摄取和Nrf2/ARE抗氧化系统的荧光表达质粒筛选法，高通量筛选泽泻水提物中具有降糖作用和Nrf2/ARE靶点激活剂并进行肝细胞Nrf2/ARE信号系统及siRNA的验证，从体外水平阐明泽泻水提物的降糖物质基础；第三，对泽泻水提活性组分或者部位进行体内机制的研究，通过Nrf2/ARE、NQO-1、HO-1等抗氧化系统信号通路研究泽泻活性成分对IR小鼠的降糖机制，阐明泽泻水提物的药效物质和降糖机理。

本项目通过Nrf2靶点/胰岛素信号通路研究泽泻水提物的降糖药效物质及其作用机制。首先，采用化学分离方法对泽泻水提物进行小分子和大分子化合物两个部位的分离，对小分子部位采用质谱引导分离纯化制备，共分离鉴定15个化合物；其次，构建Nrf2/ARE抗氧化系统的荧光表达质粒筛选法，高通量筛选分离得到的15种化合物，结果表明16位羰基取代的泽泻三萜具有激活Nrf2靶点作用；同时也开展泽泻大分子多糖降糖作用的研究，在药效的基础上开展对大分子多糖的葡聚糖凝胶色谱分离，鉴定多糖化学结构单元，初步明确了泽泻水提物的化学物质基础；第三，开展泽泻水提物中微量三萜部位降糖的体内机制的研究，结果表明泽泻可能通过Nrf2靶点、Nfkb炎症信号通路和胰岛素信号磷酸化（AMPK、AKT、IRS-1等）发挥降糖作用。通过本项目研究初步阐明泽泻水提物中化学物质基础和降糖作用机制。