基于肠道微生态学结合代谢组学的黄连解毒汤改善2型糖尿病胰岛素抵抗的药效物质基础及其作用机制研究

Huang-lian-jie-du decoction is a very famous and classic decoction which has been used to treat damp-heat syndrome patient for more than one thousand years. Until now, a number of studies have demonstrated that it can obviously improve the insulin resistance in type 2 diabetes mellitus. As insulin resistance in type 2 diabetes mellitus was one of the core pathogenesis, which was closely relevant to gut microbiota. However, the mechanism of Huang-lian-jie-du decoction improving insulin resistance in type 2 diabetes mellitus based on gut microbiota was poorly understood. Preliminary study in our lab has shown that the change of gut microbiota composition was a key issue for the Huang-lian-jie-du decoction improving insulin resistance in type 2 diabetes mellitus. Therefore, the mechanism and therapeutic material basis research of Huang-lian-jie-du decoction improving insulin resistance in type 2 diabetes mellitus will be studied in this project, which will be performed by the combination of 'metabonomics' with 'intestinal microecology' based on the previous research results through the breakthrough point of 'imbalance' or 'balance' of gut microflora. Molecular biology technologies such as polymerase chain reaction-denatured gradient gel electrophoresis (PCR-DGGE) that bases on the 16S rRNA gene, real time polymerase chain reaction (RT-PCR), and metabonomics methods which based on modern analysis instrument will be performed to analysis urine, feces, blood and tissue samples collected from rats. Samples were collected before and after insulin resistance in type 2 diabetes mellitus modeling, before and after Huang-lian-jie-du decoction intervention in nomal and model rats. Through metabonomics and gut microbiota fingerprint data was analysed using SPSS, SIMCA-P+, Matlab, and Cytoscape, the metabolites and functional bacteria which are covariation and correlation each other will be found. Then, the correlative metabolites and functional bacteria will be identified by the high resolution mass spectrometry, cloning and sequencing technology, respectively. The biological basis which relate to relationship of the intestinal microecology 'imbalance' or 'balance' with Huang-lian-jie-du decoction improving insulin resistance in type 2 diabetes mellitus will be well understood. In addition, an evaluation system, 'Huang-lian-jie-du decoction / intestinal microecology and metabonomics / improving insulin resistance in type 2 diabetes mellitus' will be set up. With the use of the evaluation system, the mechanism and therapeutic material basis research of Huang-lian-jie-du decoction improving insulin resistance in type 2 diabetes mellitus will be carried out. At the end, metabonomics in combination with intestinal microecology will provide a new platform for studying the the mechanism about traditional Chinese medicine used to remedy obesity, type 2 diabetes mellitus, and other metabolic diseases which are very closely related to gut microbiota.

胰岛素抵抗（IR）是2型糖尿病（T2DM）的核心机制之一。黄连解毒汤能明显改善T2DM-IR。虽然T2DM-IR与肠道菌群关系密切，但鲜有从肠道菌群角度来探讨黄连解毒汤改善T2DM-IR机制的报道。本课题组前期研究发现肠道菌群结构变化是黄连解毒汤改善T2DM-IR的重点环节。因此本项目拟进一步从肠道菌群切入，基于肠道微生态学和代谢组学，利用PCR-DGGE等分子生物学技术和现代分析仪器的代谢组学方法分析T2DM-IR模型大鼠造模前后及黄连解毒汤干预前后的血、尿、粪等的肠道菌群谱和代谢组学谱，结合多变量分析，发现鉴定并定量与改善T2DM-IR关联的代谢物与功能菌，构建"黄连解毒汤-肠道菌群-T2DM-IR"研究体系，研究其改善T2DM-IR与肠道微生态失衡、平衡间关系，阐明其改善T2DM-IR的药效物质基础及机制，并为研究中医药治疗与肠道菌群相关的肥胖、T2DM等代谢性疾病的机制提供新思路。

胰岛素抵抗（IR）是2型糖尿病（T2DM）的核心机制之一。黄连解毒汤能明显改善T2DM-IR 。虽然T2DM-IR与肠道菌群关系密切，但鲜有从肠道菌群角度来探讨黄连解毒汤改善T2DMIR机制的报道。本课题组前期研究发现肠道菌群结构变化是黄连解毒汤改善T2DM-IR的重点环节。因此本项目进一步从肠道菌群切入，基于肠道微生态学和代谢组学，利用分析T2DM-IR模型大鼠造模前后及黄连解毒汤干预前后的血、尿、粪等的肠道菌群谱和代谢组学谱，结合多变量分析，发现：黄连解毒汤对2型糖尿病模型大鼠干预治疗后，具有降血糖、抗炎抗氧化等治疗作用，2型糖尿病大鼠主要被扰乱的代谢通路包括氨基酸代谢、三羧酸循环、牛磺酸代谢等。黄连解毒汤对2型糖尿病大鼠扰乱的代谢轮廓具有回调作用。模型组大鼠厚壁菌门水平上升而拟杆菌门下降，经过黄连解毒汤干预后，菌群结构发生回调。与模型组相比，黄连解毒汤给药组中，阿克曼菌属（Akkermansia）、萨特氏菌属（Sutterella）、布劳特氏菌属（Blautia）和副杆状菌属（Parabacteroides）等有益菌含量均显著上升；而葡萄球菌属（Staphylococcus）、气球菌属（Aerococcus）和费克蓝姆菌属（Facklamia）等有害菌丰度显著下降。结合代谢组学和生物信息学两方面数据分析，2型糖尿病会对机体的代谢物和肠道菌群产生影响，从而影响正常的生理功能。黄连解毒汤能够不同程度的逆转上述的改变，从而改善2型糖尿病和胰岛素抵抗。发现鉴定并定量与改善T2DM-IR关联的代谢物与功能菌，构建"黄连解毒汤-肠道菌群-T2DM-IR" 研究体系，研究其改善T2DM-IR与肠道微生态失衡、平衡间关系，阐明其改善T2DM-IR的药效物质基础及机制，并为研究中医药治疗与肠道菌群相关的肥胖、T2DM等代谢性疾病的机 制提供新思路。