基于GLUT4靶点的壮药“勾瓢更”抗Ⅱ型糖尿病药效物质基础及作用机制研究

Diabetes is a chronic metabolic disease that severely affects human health, and GLUT4 is a potential new target for diabetes treatment. “Gou Piao Geng” is a kind of characteristics of zhuang medicine commonly used in the treatment of diabetes mellitus, which mainly including Gymnema sylvestre (Retz.) Schult. and Gymnema tingens Sprenge. the studies home and abroad and our previous works were all shown that the main chemical compositions of G. sylvestre. such as triterpenoid saponins and C 21 Steroidal Glycosides possessed obvious antidiabetic activity in vitro, which are related to GLUT4 target. But what is Action Mechanisms and the effective substances of them? Are there a similar chemical compositions contained in G. tingens. that play the same pharmacological activity like G. sylvestre ? That all questions need to be solved. Combining with screening for GLUT4 transmembrane and glucose uptake in vitro, this project made a comprehensive evaluation of "Gou Piao Geng" anti-type II using mice with high-fat diet and STZ-induced diabetes and KK-Ay mouse model with spontaneous type II diabetes. With the separation of the chemical constituents of the active extract by chromatography, and the study of structure-activity relationship of the active compounds by computer-aided drug design techniques, the similarities and differences between the anti-type 2 diabetes activity and the active ingredients of the two herbs were evaluated from the molecular-cell-total animal level, to provide scientific basis for whether G. tingens. This project can be used as medicinal herbs origin of "Gou Piao Geng", and provide theoretical guidance for discovering novel GLUT4 target normal phase modulators through clarifying the "Gou Piao Geng" pharmacodynamics basis and mechanism of action.

糖尿病是严重影响人类健康的慢性代谢性疾病，GLUT4是潜在的糖尿病治疗新靶点。“勾瓢更”是治疗糖尿病的特色壮药，来源包括匙羹藤和习用品大叶匙羹藤。国内外研究和前期工作表明匙羹藤中三萜皂苷和C21甾苷有良好的体外抗Ⅱ型糖尿病活性，是作用于GLUT4靶点的活性化合物，但其作用机制和物质基础如何？大叶匙羹藤是否具有类似化学成分和活性？是亟需回答的科学问题。本项目结合促GLUT4转膜和葡萄糖摄取的体外活性筛选，用高脂饲料和STZ诱导的糖尿病小鼠和自发性Ⅱ型糖尿病KK-Ay小鼠模型来评价“勾瓢更”的抗Ⅱ型糖尿病活性；利用色谱法分离其中化学成分，用计算机辅助药物设计技术研究活性化合物的构效关系，从分子-细胞-整体动物水平上评判两种药材抗Ⅱ型糖尿病活性和活性成分的异同，为大叶匙羹藤能否作为“勾瓢更”基原提供科学依据；阐明“勾瓢更”药效物质基础和作用机制，为发现新颖的GLUT4靶点正相调节剂提供理论指导。

项目主要研究工作包括（1） 建立促GLUT4转膜和促L6肌肉细胞葡萄糖摄取活性筛选方法，明确匙羮藤和大叶匙羮藤活性提取物和部位。（2）从匙羹藤中分离鉴定61个化合物，含17个C21甾苷新化合物；从大叶匙羹藤中分离鉴定73个化合物，含15个新化合物（11个C21甾体，4个三萜皂苷）。（3）对单体化合物进行基于GLUT4靶点的体外抗糖尿病活性筛选，发现活性化合物35个，先导化合物 3 个，均为C21甾体和三萜类成分。（4）采用 SYBYL-x 软件中的分子对接技术对活性化合物与靶蛋白的结合方式进行研究，分析体外抗糖尿病活性成分与 GLUT4 的构效关系，发现大部分C21甾体和三萜皂苷与靶蛋白GLUT4 之间结合能力较强，具有抑制GLUT4转运蛋白的潜在作用。（5）采用色谱法富集活性化合物部位，建立高脂饲料和STZ诱导的Ⅱ型糖尿病小鼠模型，以空腹血糖（FBG）、糖耐量（OGTT）、胰岛素耐量（ITT）、胰岛敏感性指数（ISI）等为观察指标，结合组织形态学观察，综合评价其体内活性，发现大叶匙羹藤活性部位对实验小鼠呈现明显的毒性作用；匙羹藤活性部位高剂量组具有显著抗糖尿病活性，疗效优于阳性对照二甲双胍。采用免疫印迹技术检测化合物sylvepregoside A和tigensides B对糖尿病小鼠体内与调控通路相关的关键调控因子表达水平以及关键性翻译后修饰的影响，发现其作用机制与PI3K-AKT、AMPK等信号通路与调控GLUT4的基因表达与细胞膜转位相关。. 研究表明匙羮藤和大叶匙羮藤都有抗Ⅱ型糖尿病活性，成分类型相似，但毒性和主要药效物质C21甾体和三萜皂苷结构存在明显差异。研究结果初步厘清壮药“勾瓢更”的药用基原，并为两种药材质量评价提供参考；扩展了匙羹藤属植物的化学成分库，丰富和深化了对萝藦科植物中C21甾体结构多样性的认识；将为发现新颖的针对GLUT4靶点的活性化合物提供依据；为C21甾体和三萜皂苷类抗糖尿病构效关系和作用机制深入研究奠定基础。