基于药物蛋白质组学与多靶点超滤筛选研究匙羹藤干预餐后高血糖的作用机制与活性物质基础

Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia that seriously threatens human health. Postprandial hyperglycemia is a major feature of DM patients. By inhibiting the activity of digestive enzymes, such as the α-glycosidase, in the small intestine, the decomposition of carbohydrates can be reversibly inhibited, and the absorption of glucose can be delayed, which is of great significance for preventing the abnormal postprandial blood glucose. The traditional Chinese Medicine of Gymnema sylvestre has good therapeutic effects for the treatment of DM. However, the mechanisms of action and bioactive components of its effect on DM by reducing the postprandial hyperglycemia remain elusive. This study aims to develop a new multi-target approach for studying the overall anti-diabetes mechanisms of action and the associated bioactive components of G. sylvestre. Firstly, we propose to investigate the overall protein biomarkers between the DM models treated with and without the Gymnema sylvestre extract using DiART labeling proteomics. Secondly, we plan to establish the multi-target ultrafiltration mass spectrometry (UF-LC/MS) to fast fish out the active components in G. sylvestre. Thirdly, by revealing the interactions between active components and protein targets, we propose to build the possible target protein signaling pathways of G. sylvestre for the treatment of DM, and finally elucidate its overall mechanisms of action and associated bioactive components. This study embodies the characteristics of "holistic" of traditional Chinese Medicine. The research results are expected to provide strong technical support for studying the overall mechanisms of action and bioactive components of traditional Chinese Medicine for the treatment of DM.

糖尿病（DM）是一种严重威胁人类健康的以高血糖为特征的代谢紊乱疾病，餐后高血糖是DM患者的重要病理特征。通过可逆地抑制小肠中影响碳水化合物消化吸收的α-糖苷酶等关键酶，延缓小肠对葡萄糖的吸收，对抑制餐后血糖升高具有重要意义。中药匙羹藤治疗DM疗效显著，但目前对其通过干预餐后高血糖发挥治疗DM的作用机制和活性物质基础仍不清楚。本项目以系统生物学的整体研究思路为指导，先采用已知的降糖药物靶标进行多靶点筛选，然后应用DiART蛋白质组学方法探索匙羹藤治疗DM的新的关键靶点蛋白，并采用多个单靶点和多靶点组合超滤筛选匙羹藤中的活性成分；通过揭示匙羹藤中不同活性成分与不同关键靶点蛋白间的相互作用关系，构建匙羹藤干预餐后高血糖的靶点蛋白网络，阐明匙羹藤治疗DM的整体作用机制及活性物质基础。本项目研究方法体现了中药作用“整体性”的特点，研究结果将为中药治疗DM整体作用机制的研究提供有力的技术支持。

匙羹藤（Gymnema sylvestre (Retz.) Schult., GS）传统上用于防治糖尿病（DM），应用历史悠久，现代研究也证实其降血糖的作用。我国GS资源丰富，治疗DM具有巨大的开发潜能。食物中碳水化合物从被消化水解、到被吸收入血形成血糖，涉及多步复杂过程的分工和协作。研究表明，肠道菌群稳态失调伴随着机体的生理代谢紊乱，如诱发体内代谢性内毒素血症、胆汁酸代谢紊乱和短链脂肪酸（SCFA）生成减少，导致机体慢性低度炎症和胰岛素抵抗，是DM发生的重要病理过程。因此，调节肠道菌群成为了防治DM的潜在靶点和新方向。当前GS以茶饮和汤剂为主要途径面向T2DM患者，目前尚少有GS通过调节肠道菌群防治T2DM的机制研究，极大地限制了GS降糖药物的开发和应用。本项目从肠道菌群及其代谢产物SCFAs出发，通过对T2DM模型大鼠肠道菌群及SCFAs的检测，探讨基于肠道微生物的GS治疗T2DM的作用研究，为临床上更好地应用GS治疗T2DM提供科学数据。. 本项目采用“高脂饲料诱导+链脲佐菌素（STZ）腹腔注射”方法造T2DM大鼠模型，并灌胃给药30天，通过对肠道微生物和SCFAs的检测，探讨GS通过肠道菌群途径降血糖的作用机制。研究结果表明，在给予GS药物干预30天后，与模型组比较，GS各给药组大鼠空腹血糖、TC、TG和LDL-C水平降低，HDL-C水平升高，体重下降趋势明显（p<0.05，p<0.01）；肠道中Bacteroides、Lactobacillus、Clostridium属丰度和Firmicutes/Bacteroidetes比值均有所增加，乙酸、丙酸、正丁酸及总SCFAs含量均有所升高（p<0.05，p<0.01）；KEGG信号通路分析表明，GS显著影响了包括glucose 6-phosphate和glucose糖酵解等多个碳水化合物代谢过程。. 本研究结果表明，除了抑制肠道碳水化合物消化酶活性外，GS降低T2DM大鼠血糖、血脂与其有效调节肠道菌群、增加SCFAs含量有关，这可能是GS防治T2DM的新机制，也是本项目研究成果的创新点。因此，本项目的研究成果不仅有助于理解中药GS治疗DM整体作用机制及其物质基础，还对研发GS相关中药新药具有重要的参考价值，应用前景广阔。