基于PTP1B靶点和AMPK通路的卷柏中降糖活性成分研究

Insulin resistance is known to play a key role in the development of type 2 diabetes. The drugs currently used to improve insulin resistance are usually accompanied by side effects such as liver and kidney toxicity, cardiovascular side effects and so on. Therefore, it is important to develop new efficient and safe drug for treatment of type 2 diabetes. Selaginella tamariscina has good anti-hypoglycemic effect, however, most phytochemistical and pharmacological studies of S. tamariscina focus on amentoflavone. Many other potential active ingredients have not yet been discovered. Therefore, the strategy of chromatographic fractionation and purification monitored by activity will be applied to retro-screen more anti-hypoglycemic compositions from S. tamariscina in this project. Chemometric analysis was also performed in this project to study the possible synergies among the active ingredients. The isolates subsequently were evaluated for their abilities of anti-insulin resistance in vitro cell models (HepG2, 3T3-L1) and enzyme models (PTP1B, AMPK). Molecular biology techniques, such as immunofluorescence technique, RT-PCR test, western-blot test and so on, were further applied for mechanism study to clarify the anti-hypoglycemic material basis of S. tamariscina. The Research results of this project not only provide theoretical guidance for clinical application of S. tamariscina, but also lay a good foundation for development of new drugs for treatment of insulin resistance.

胰岛素抵抗是2型糖尿病发病的主要环节，目前用于改善胰岛素抵抗的药物多伴有肝肾毒性及心血管副作用等。因此，研发高效、安全的新型抗胰岛素抵抗药物研发是目前2型糖尿病治疗的重点之一。卷柏具有较好的降糖效果，但目前对卷柏降糖活性成分的研究主要集中于穗花杉双黄酮，它所含的其他多种潜在降糖活性成分的物质基础和作用机制尚未清楚。本项目拟以中药卷柏为研究对象，采用活性导向和色谱分离相结合的方法逆向筛选抗胰岛素抵抗活性成分，并引入化学计量学分析卷柏中多种活性成分之间可能的协同关系，通过体外细胞模型（HepG2、3T3-L1）和酶（PTP1B、AMPK）进行体外活性评价，采用免疫荧光染色法、RT-PCR法和Western-blot法等对活性单体进行作用机制研究，进一步阐明卷柏中降糖活性成分的药效物质基础。本项目的研究成果对卷柏在糖尿病治疗的临床应用具有重要的指导作用，也为抗胰岛素抵抗的药物研发提物研究基础

2型糖尿病是严重危害人类健康的代谢性疾病，胰岛素抵抗是2型糖尿病发病的主要环节,并贯穿于其发生、发展的整个过程。活血化瘀类中药往往具有较好的改善机体的代谢功能。药典中卷柏以卷柏或垫状卷柏的干燥全草为基源，是活血化瘀类中药之一，在民间有用于糖尿病的治疗。但目前对卷柏降糖活性成分的研究主要集中于穗花杉双黄酮，它所含的其他多种潜在降糖活性成分的物质基础和作用机制尚未清楚。本项目采用采用LC-MS导向以及多种色谱手段，对垫状卷柏的95%乙醇提取物进行了系统的化学成分分离。获得了22个化合物，结合波谱方法和化学沟通方法对它们的结构进行了鉴定，其中7个为新化合物，包括6个降碳木脂素和1个环肽。本项目首次鉴定了垫状卷柏中环肽的化学结构，丰富了卷柏属化学成分的结构多样性。进一步以3T3-L1细胞的葡萄糖摄取模型对相关化合物进行活性评价，发现从氯仿部位分离的两个降碳木脂素类化合物moellenoside C和pulvin A显著促进3T3-L1细胞对葡萄糖的摄取作用，显示了良好的剂量依赖性，而且没有明显的细胞毒性，具有较好的开发价值。本项目为从活血化瘀类中药中寻找开发天然降糖新药提供了思路和研究基础。