基于肠道微生态学与代谢组学的葛根-丹参配伍改善T2DM胰岛素抵抗药效物质基础及机理研究

The connection between gut microbiota and type 2 diabetes mellitus (T2DM) and its role in the pathogenesis of insulin resistance (IR) and/or T2DM are increasingly recognized. The compatibility of Radix Puerariae and Salvia Miltiorrhiza has long been used in the treatment of T2DM, which accompanied with Qi stagnation and Blood stasis in Chinese Medicine system. However, the benificial effects of the compatibility of Radix Puerariae and Salvia Miltiorrhiza are mainly based on empirical experiences, and its active components and machanism have never been investigated. Our previous study has shown that the compatibility of Radix Puerariae and Salvia Miltiorrhiza could obviously attenuate metabolic disturbances such as IR and chronic low-grade inflammation, and also adjust the structural change of urine metabolic spectrum and gut microbiota, which suggested that its effects may be associated with the role of gut microbiota. Based on previous research results, through the breakthrough point of the modulation of gut microbiota and metabolic network, the active components form the compatibility of Radix Puerariae and Salvia Miltiorrhiza and their machanism will be research in this project. First, different fractions was made from Radix Puerariae and Salvia Mitiorrhiza, and the influences on metabolic disturbances and gut microbiota structure will be investigated in C57BL/6J fed with high fat food. Second, the technology of denaturing gradient gel electrophoresis based on the 16S rDNA molecular ecology will be used to get the intestinal micro ecology characteristics treat with the different fractions, and UPLC-MS/MS will be used to achieve the data of the urine and blood metabolomics. Through pricipal components analysis, partial least squares discrimination analysis, and pattern recogition, the functional bacteria and the metabolites which correlated with the efficacy of active components will be found. Then, the effects of active components on the intestinal barrier function and mucous membrane integrity will be studied to clarify overall biological mechanism. At the end, this project will be developed as one of the modern researchs of traditional Chinese Cedicine and provide theoretical basis for effective prevention and treatment with T2DM.

肠道菌群与胰岛素抵抗（IR）及2型糖尿病（T2DM）的发生发展密切相关。葛根-丹参配伍常用于治疗气滞血瘀型T2DM，疗效确切，但二者配伍增效的药效物质和机理尚不明确。前期研究发现葛根-丹参配伍可改善IR、慢性低度炎症等异常，并改变尿液代谢谱及肠道菌群结构，提示其可能通过调节肠道菌群而发挥改善IR效应。本项目拟以干预肠道菌群和代谢网络为切入点，探讨葛根、丹参不同组分及组分配伍对代谢异常、肠道菌群紊乱等影响，明确二者配伍改善IR的药效物质；采用PCR-DGGE分子生态学技术及LC-MS/MS代谢组学技术，对葛根-丹参配伍干预前、中、后的菌群结构变化规律和代谢组学特征进行研究，并利用模式识别及偏最小二乘回归法分析，寻找相关联及共变化的功能菌、代谢物及影响通路，同时结合肠道黏膜及屏障功能等关联研究，阐明葛根-丹参配伍改善IR的整体效应机制，为中药复方的现代研究和T2DM的有效防治提供理论依据。

本课题建立了葛根-丹参药对水煎液及丹参菲醌类组分、葛根异黄酮类组分、丹参酚酸类组分等不同组分的制备方法，对各组分进行HPLC-QTOF-MS定性分析，并结合IR小鼠模型对上述不同组分及配伍组合进行药理效应评价，确定丹参酚酸类组分和葛根异黄酮类组分为葛根-丹参药对抗IR效应的主要药效组分。建立GC-MS及UPLC-MS/MS代谢组学分析方法，以尿液、血浆为生物样本，分析各有效组分或组分配伍对IR小鼠模型干预前、中、后的内源性代谢组学特征的影响，结合改善糖脂代谢、全身炎症等药效数据，确定6个氨基酸类及6个炎症消退介质类主要差异生物标记物；结合PCR-DGGE技术初步确定与生物标记物相关联及共变化的2个可能功能菌。在此基础上，进一步结合体内外实验模型，确证丹酚酸B对2型糖尿病大鼠模型具有改善糖脂代谢、胰岛素抵抗、氧化应激等作用，可显著改善高脂高糖诱导C57BL/6J肥胖小鼠的胰岛素抵抗及高血脂状态，其作用与调节PPARγ、C/EBPα、GATA-2、GATA-3等脂肪代谢关键蛋白有关；还可显著改善db/db糖尿病小鼠模型糖脂代谢水平，其作用与调节糖异生、AMPK通路等有关。