注射用重组艾塞那肽-人血清白蛋白融合蛋白E2HSA对小鼠胰岛β细胞功能的保护作用

Protective effects of the injectable recombinant protein of exendin-4 and human serum albumin,E2HSA,on mouse islet β cell function

注射用艾塞那肽-人血清白蛋白融合蛋白(injectable recombinant protein of exendin-4 and human serum albumin,E2HSA)是我国自主研发的一种长效胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)受体激动剂,目前处于临床研究阶段。本研究在细胞和整体动物水平研究E2HSA对胰岛β细胞的保护作用。在细胞水平,采用小鼠胰岛素瘤细胞株NIT-1考察E2HSA对细胞活力和增殖的影响;采用水溶性胆固醇诱导NIT-1细胞损伤,考察E2HSA对细胞活力和凋亡的影响,并研究其相关作用机制。在整体动物水平,采用四氧嘧啶高血糖小鼠考察长期注射E2HSA对血糖、血胰岛素和胰岛内胰岛素含量的影响。动物实验操作和福利均遵循中国医学科学院、北京协和医学院药物研究所实验动物伦理与动物福利委员会的规定。结果显示,E2HSA可明显增加NIT-1细胞的活力和掺入的溴脱氧尿嘧啶核苷(bromodeoxyuridine,BrdU);同时显著增加水溶性胆固醇诱导NIT-1细胞损伤后的细胞活力,减少其凋亡发生率,并增加胞内胰腺-十二指肠同源框1(pancreatic duodenal homeobox-1,PDX-1)和蛋白激酶B(protein kinase B,PKB)蛋白的表达。E2HSA长期注射可明显降低四氧嘧啶小鼠的空腹血糖和非禁食血糖,增加其血清和胰岛内的胰岛素含量。综上,E2HSA可以促进NIT-1细胞增殖,抑制水溶性胆固醇诱导的细胞损伤和凋亡,同时增加四氧嘧啶高血糖小鼠血清和胰岛内的胰岛素水平,提示E2HSA可改善小鼠胰岛β细胞功能。

The injectable recombinant protein of exendin-4 and human serum albumin(HSA),E2 HSA,is a long-acting glucagon like-peptide-1(GLP-1)receptor agonist,which is in clinical research stage now.This study aimed to evaluate the protective effects of E2 HSA on mouse isletβcell function in vitro and in vivo.In vitro,the mouse insulinoma cells NIT-1 were used to assay the effects of E2 HSA on cell viability and proliferation.Besides,the water soluble cholesterol was adopted to induce cell injury,and then the effects of E2 HSA on cell viability and apoptosis,as well as the mechanism,were studied.In vivo,the alloxan-induced hyperglycemic mice were repeatedly administered with E2 HSA by subcutaneous injection,and the blood glucose,serum insulin,and content of insulin in islets were measured.All animal experiments were carried out with approval of the Experimental Animal Welfare Ethics Committee of the Institute of Materia Medica(Chinese Academy of Medical Sciences and Peking Union Medical College).The results showed that E2 HSA significantly increased the viability of NIT-1 cells and the amount of bromodeoxyuridine(BrdU)incorporated in cells.Besides,the water soluble cholesterol significantly decreased the cell viability and induced apoptosis of NIT-1 cells,but E2 HSA significantly reversed the injury of NIT-1 cells.Nevertheless,E2 HSA significantly increased the expression of pancreatic duodenal homeobox-1(PDX-1)and protein kinase B(PKB)of NIT-1 cells after injured by water soluble cholesterol.Furthermore,repeated injections of E2 HSA significantly reduced the fasting and non-fasting blood glucose,increased the serum insulin level and raised the insulin content inβcells of alloxan-induced hyperglycemic mice.In conclusion,E2 HSA could promote proliferation of NIT-1 cells,inhibit the water soluble cholesterol induced injury and apoptosis,and increase the insulin content in serum and islets of alloxan-induced hyperglycemic mice,suggesting the protective effects on pancreatic isletβcell function.