二精方中枸杞-黄精抗П型糖尿病量效关系及作用机制研究

Er Jing Fang, also known as Goji Pill, is a traditional prescription composed by Goji and Huang Jing. The previous study in our research group has revealed that the Er Jing Fang polysaccharides has hypoglycemic and hypolipidemic effects on type 2 diabetic mouse. Based on our previous study, we believethat different proportions of Goji and Huangjing can perform specific dose-effect relationship and interactions. In additional, the hypoglycemic mechanism on regulating insulin production and levels in inflammatory signaling pathway will be further explored in the current project. Therefore, we intend to preform the proportion optimization and study the material foundation of Er Jing Fang in hypoglycemic activity by using spectrum-activity relationship. Then, high performance liquid chromatography (HPLC) and animal experiment will be performed to study the interaction of Goji and Huangjing in Er Jing Fang based on the perspective of chemical-pharmacokinetic-pharmacodynamic study. Finally, focused on the hypoglycemic effect, we will investigate the effects of material foundation of Goji and Huangjing on insulin production and the inflammatory signaling pathway both in vitro and in vivo, and we will identify the potential mechanism of Er Jing Fang in the treatment of diabetes by observing the relative protein expression, such as IRS-1、PI3K、IKKα、IKKβ、NF-κB、Akt、P65、IKBα with Western blot method. This study could reveal the deep meaning of Er Jing Fang compositions, and provide ideas and methods to understand the scientific cognition of traditional prescription.

二精方是由枸杞和黄精组成的经典方，前期课题组研究显示二精方多糖具有改善实验性П型糖尿病小鼠糖代谢和脂代谢的作用。在此基础上，提出不同的枸杞-黄精配比具有特定的量效关系及相互作用的性质和特点，并进一步探讨其调节胰岛和炎症信号相关通路分子水平的抗П型糖尿病可能机理。因此，本项目拟开展（1）运用谱效关系研究，阐述二精方中枸杞-黄精抗糖尿病活性的最佳配比以及抗糖尿病活性的物质基础；（2）采用高效液相色谱法结合动物实验，从化学-药代-药效三维方向研究二精方中枸杞-黄精相互作用；（3）以降糖作用为着眼点，从整体动物、细胞、分子三个水平考察枸杞-黄精物质基础对胰岛和炎症信号相关通路分子水平的影响，采用蛋白质印记定量分析IRS-1、PI3K、IKKα、IKKβ、NF-κB等蛋白表达，探讨二精方降糖活性的可能机制。本研究可深入揭示二精方组方的内涵，为深化对疗效确切的传统方剂的科学认知提供思路和探索方法。

二精方是由枸杞和黄精组成的经典名方，前期课题组研究显示二精方具有改善П型糖尿病（T2DM）小鼠糖代谢和脂代谢的作用。基于此，提出不同的枸杞-黄精配比具有特定的量效关系及相互作用的性质和特点，并进一步探讨其抗T2DM的机理。项目从以下四个方面开展了研究：（1）运用谱效关系，明确二精方抗T2DM活性的最佳配比为1：1，抗T2DM活性物质主要包括总多糖及黄酮类成分；（2）采用HPLC及LC-Q/TOF-MS，从化学-药代揭示了二精方中枸杞-黄精相互作用的科学内涵，鉴定出枸杞、黄精体外化学成分分别有53个和31个，其中入血成分有45个，5个来源于黄精，40个来源于枸杞。测定betaine，beta-sitosterol和chlorogenic acid在大鼠体内24h的药代动力学；（3）通过网络药理学预测二精方治疗T2DM的潜在靶点及作用机理与PI3K/AKT信号通路有关，从动物-细胞-分子三个水平验证二精方对相关通路分子水平的影响，通过体外胰岛素抵抗细胞模型实验，RT-PCR及蛋白免疫印迹法测定基因及蛋白表达，上调IRS1、PI3K p85、AKT1 mRNA的表达，抑制NF-κBp65、GSK3β、Myc mRNA的表达同时，IRS1、PI3K p85、AKT1蛋白表达水平增加，p-IRS1、p-PI3K p85和p-AKT的表达均上调。动物体内实验表明，二精方通过调节炎症反应和缓解氧化应激指标治疗T2DM，蛋白免疫印迹实验结果与体外蛋白表达结果一致，确定二精方降糖活性的可能机制与IRS1/PI3K/AKT通路有关；（4）优化了二精方颗粒制备工艺，筛选出适合枸杞-黄精组方颗粒的制粒工艺。本研究揭示二精方治疗T2DM的科学内涵，为传统方剂的科学认知提供思路和方法。项目已完成了预期的实验内容，发表期刊论文4篇，发明专利2项，培养硕士研究生4名。