渥曼青霉素对高糖培养小鼠胰岛β细胞去分化的影响

Effect of wortmannin on dedifferentiation of mouseβcells induced by high concentration of glucose

目的探讨渥曼青霉素对高糖培养小鼠胰岛β细胞去分化的影响及其作用机制。方法高糖培养小鼠胰岛β细胞后,采用蛋白质印迹法检测细胞内磷酸化叉头转录因子1(p-FoxO1)表达水平,确定高糖诱导细胞FoxO1磷酸化最佳时间。小鼠胰岛β细胞根据培养条件分为4组:(1)常规糖浓度组:用含25mmol/L葡萄糖浓度的DMEM培养液培养;(2)高张浓度组:用含25mmol/L葡萄糖浓度和35mmol/L甘露醇的DMEM培养液培养;(3)高糖浓度组:用含60mmol/L葡萄糖浓度的DMEM培养液培养;(4)高糖浓度+渥曼青霉素干预组:用含60mmol/L葡萄糖浓度和50nmol/L渥曼青霉素的DMEM培养液培养。培养12h后采用蛋白质印迹法检测各组小鼠胰岛β细胞p-FoxO1、FoxO1蛋白表达水平;培养72h后采用蛋白质印迹法和实时荧光定量聚合酶链式反应法检测各组小鼠胰岛β细胞神经元素3(ngn3)、胰岛十二指肠同源盒-1(PDX1)蛋白和mRNA表达水平。结果在一定时间范围内,高糖能刺激FoxO1蛋白磷酸化;高糖培养小鼠胰岛β细胞后细胞ngn3蛋白和mRNA表达水平均增加,PDX1蛋白和mRNA表达水平均减少。渥曼青霉素同步干预后,FoxO1磷酸化减少,细胞ngn3蛋白和mRNA表达水平均减少,PDX1蛋白和mRNA表达水平均增加。结论渥曼青霉素能抑制高糖诱导的小鼠胰岛β细胞去分化,渥曼青霉素可能通过抑制FoxO1的磷酸化而产生该抑制作用的。

Objective To investigate the effects of wortmannin on the dedifferentiation of mouseβcells(MIN-6)incubated in high concentration of glucose and its mechanism.Methods The expression of phosphorylated forkhead box O-1(p-FoxO1)in mouse isletβcells was detected by Western blot after cultured with high concentration of glucose,and the phosphorylation time of FoxO1 was explored.Mouseβcells were divided in to four groups:(1)Normal glucose concentration group:cultured with DMEM medium containing 25mmol/L glucose;(2)high tension concentration group:cultured with DMEM medium containing 25mmol/L glucose and 35mmol/L mannitol;(3)high glucose concentration group:cultured with DMEM medium containing 60mmol/L glucose;(4)high glucose concentration+wortmannin intervention group:cultured with DMEM medium containing 60mmol/L glucose and 50nmol/L wortmannin.The protein expression of p-FoxO1 and FoxO1 was detected by Western blot 12h after culture,and the protein and mRNA expression of neuroelement3(ngn3),pancreatic duodenal homeobox-1(PDX1)were detected by Western blot and real-time fluorescence quantitative RT-PCR 72h after culture.Results The phosphorylation of FoxO1 was activitied by high glucose.The expression of ngn3 protein and mRNA increased,PDX1 protein and mRNA decreased in mouseβcells after cultured with high glucose.After wortmannin intervention the phosphorylation of FoxO1 decreased,the expression of ngn3 protein and mRNA decreased,and the expression of PDX1 mRNA and protein increased.Conclusion Wortmannin can inhibit high glucose-induced dedifferentiation of mouseβcells and it may be associated with the reduced phosphorylation of FoxO1.