基于蛋白质组学与代谢组学联用技术探讨桑叶治疗2型糖尿病作用机制及信号元件交互作用

The pathogenesis of diabetes is complex and diverse. It is of great significance to comprehensively understand the pathogenesis and the crosstalk with different signal pathways. Morus L. is the commonly known traditional Chinese Medicine used to treat T2DM, however, its hypoglycemic mechanism varies due to different ingredients. Previous studies have shown that Morus L. has the function of hypoglycemia, improves insulin resistance (IR) in relation to insulin (Ins) pathway and immune inflammatory pathways, but it is still unknown whether there exist other pathways to play a role in treating diabetes with Morus L., and whether there exist network and crosstalk among different signal elements and pathways..It is prepared to combine proteomics and metabolomics to analyze the pathology of T2DM and the mechanism of treating T2DM using Morus L. from the upstream protein and downstream small metabolites in a systematic way. PPI experiment is carried out for validation, and then the tools like Cytoscape and online String network analysis are used to find out the specific differences in protein and terminal metabolites, focusing on the crosstalk with the protein of Ins and inflammatory signaling pathways with Co-IP、GST-pull down technology, which aims to establish the networked mechanism of pathology and pharmacology in treating T2DM and improving IR with Morus L.. The project is expected to discover the special markers of T2DM, and to complete the 3D and networked mechanism of hypoglycemia, hoping to provide the theoretical basis and experimental means for the early detection and prevention of T2DM and new ideas for the modernization of TCM’s mechanism.

糖尿病发病机制复杂多样，全面认识其病理机制及其不同信号通路间交互作用对防治糖尿病意义重大。桑叶是治疗2型糖尿病（T2DM）常用中药，其降糖机制多样。本团队先期研究表明，桑叶降糖作用与胰岛素通路及免疫性炎症通路有关，但桑叶是否通过其它通路发挥作用，各信号通路不同元件间有否网络化交互作用尚不明确。.本课题采用蛋白质组学联合代谢组学技术，基于string、KEGG数据库从上游蛋白质和下游小分子代谢产物水平，利用Cytoscape分析工具构建桑叶治疗T2DM网络化作用机制，并进行蛋白验证实验，Co-IP、GST-pull down技术确定蛋白间交互作用，重点观察Ins与炎症信号通路蛋白、代谢产物变化及交互作用，以建立T2DM及桑叶降糖网络化病理、药效学作用机制。本项目有望发现T2DM特异性标记物，完善桑叶降糖立体网络化作用机制，为实现T2DM早期发现及中药现代化研究提供新的思路与方法。

项目根据既定的实验方案和研究计划，经过四年的研究，利用蛋白质组学与代谢组学联用等技术揭示了桑叶治疗2型糖尿病（T2DM）作用机制及信号元件交互作用。（1）提取分离桑叶总多糖和总黄酮两个有效部位，对其进行定性定量研究，同时采用传统工艺制备桑叶总水提物，并对三者进行降糖药效比较，证明桑叶降糖最佳药效部位为桑叶总水提物；（2）利用蛋白质组学与代谢组学技术，筛选出了T2DM形成不同阶段模型大鼠肝脏、血、尿中的差异代谢物及肌肉和肝脏中的差异蛋白，明确了T2DM形成相关特异性差异蛋白与差异代谢物；（3）在桑叶降糖药效学基础上，采用蛋白质组学与代谢组学联用技术，明确桑叶降糖、改善IR作用相关特异性差异蛋白与差异代谢物；（4）利用Cytoscape网络分析工具及在线String、KEGG数据库对特异性差异蛋白及差异代谢物进行交互作用分析，并用Western blot技术对差异蛋白进行验证。本项目完善了T2DM网络化病理机制及桑叶改善IR网络化作用机理，为中药现代化研究提供了基于蛋白质组学与代谢组学的现代化技术平台。