VPAC2介导高稳定性PACAP衍生物MHDBAY促胰岛素功能的作用及机制研究

The design of type Ⅱ diabetes drug is usually based on insulin resistance, but long-term use of these drugs may lead to damage of pancreatic β-cell function and insulin dysfunction. Pituitary adenylate cyclase-activating polypeptide(PACAP) and its derivatives are important human neural polypeptides, which can promote insulin function, improve insulin resistance, protect beta cells and so on through VPAC2 receptor-mediated effects, and has no risk of hypoglycemia. Many VPAC2-specific agonists such as RMBAY, have the defects of poor stability and low bioavailability. The recombinant PACAP derivative MHDBAY with high stability was developed by our team in early study. The recombinant MHDBAY has good glucose-dependent hypoglycemic effect. Compared to other VPAC2-specific agonists such as RMBAY, the half-life of MHDBAY was increased by more than 23 times, up to 11.5 hours, and its effective dose of the animal experiments is 5μg/kg without toxic side effects. In this study, VPAC2-CHO cells, normal pancreatic β-cells, β-cells of insulin resistance, normal 3T3-L1 cells, 3T3-L1 cells of insulin resistance, normal rats and mice and type Ⅱ diabetic rat or mice model will be used to systematically research the effects that VPAC2 mediated MHDBAY to promote β-cell proliferation, gene transcription and expression of insulin and insulin signal transduction and so on. So that the biological role of MHDBAY and its molecular mechanism of promoting insulin function are revealed, and the experimental basis of MHDBAY for its pharmaceutical research and development is provided.

Ⅱ型糖尿病药物多是针对胰岛素抵抗进行设计，但长期使用会导致胰岛β细胞功能损伤和胰岛素功能障碍。垂体腺苷酸环化酶激活肽（PACAP）及其衍生物是人体重要的神经多肽，通过VPAC2受体介导具有促胰岛素功能、改善胰岛素抵抗和保护β细胞等作用，且无低血糖症风险。课题组针对VPAC2特异激动剂RMBAY等，存在生物利用度低、稳定性差等问题，研制了高稳定性重组PACAP衍生物MHDBAY，其具有良好的葡萄糖依赖性降血糖作用。与RMBAY等比较，其半衰期提高23倍以上，达11.5小时，动物实验有效剂量为5μg/kg，无毒副作用。本研究利用VPAC2-CHO细胞、正常和胰岛素抵抗的β-细胞及3T3-L1脂肪细胞、正常和Ⅱ型糖尿病模型鼠等，系统研究VPAC2介导MHDBAY促β细胞增殖、促胰岛素基因转录和表达、促胰岛素信号转导等作用，阐明其促胰岛素功能的生物学作用及其分子机制，也为其药用研发提供实验基础。

本课题是在课题组前期研究的基础上，利用结构优化的基因重组多肽MHDBAY、VPAC2-CHO细胞、正常和胰岛素抵抗的β-细胞或3T3-L1脂肪细胞、正常和Ⅱ型糖尿病模型鼠等，深入研究了VPAC2介导MHDBAY促β细胞增殖、促胰岛素基因转录和表达、促胰岛素信号转导等生物学作用及其分子机制。实验结果表明：重组肽MHDBAY具有较高的稳定性和良好的缓释效应，重组肽MHDBAY与HSA的结合可有效延长其半衰期；重组肽MHDBAY具有对VPAC2受体的高激活活性以及特异选择性，MHDBAY与HSA具有较高的亲和力，从而有效延伸了MHDBAY的体内半衰期，MHDBAY在实验鼠体内的半衰期可达12.19 h；MHDBAY可促进胰岛β细胞的增殖，有效改善胰岛素抵抗的3T3-L1 细胞模型的胰岛素抵抗程度，显著促进3T3-L1 脂肪细胞的葡萄糖摄取利用，MHDBAY 可显著促进胰岛素的合成和分泌，并可有效促进胰岛素与其受体的结合，提高胰岛素的敏感性；MHDBAY 可通过激活VPAC2受体有效调节胰岛素信号传导通路，通过有效调节胰岛素信号传导通路关键蛋白的表达及磷酸化水平以及提高葡萄糖转运蛋白GLUT4的表达和膜转位水平，从而有效促进葡萄糖的摄取利用，降低血糖水平。同时，动物学实验表明，连续用药MHDBAY 可有效控制db/db实验鼠的饮食、饮水量和体重，有效提高其急性糖耐量水平和胰岛素分泌。初步的相关病理学研究表明，重组肽MHDBAY对实验动物无明显的毒副作用。总之，本研究表明新型重组肽MHDBAY具有良好的抗Ⅱ型糖尿病作用且安全性较高，并阐明了其促胰岛素功能的生物学作用及其分子机制，也为其药用研发提供实验基础。