达格列净改善高糖诱导的人脐静脉内皮细胞功能的机制研究

Mechanism of dapagliflozin improving the function of human umbilical endothelial cells induced by high glucose

目的:探讨达格列净对高糖诱导的人脐静脉内皮细胞(HUVECs)增殖、迁移、成管和凋亡的影响及其可能的分子机制。方法:将HUVECs分别采用5.5 mmol/L (对照)、25 mmol/L葡萄糖(高糖)、达格列净和高糖+达格列净干预细胞,用脂质体转染法将pcDNA、pcDNA-JAK2转入HUVECs,后用达格列净和(或)高糖处理。采用CCK8法检测细胞增殖活性,Transwell实验检测细胞迁移能力,血管形成实验检测细胞成管能力,流式细胞术检测细胞凋亡率,Western blot检测JAK2、p-JAK2、STAT3和p-STAT3蛋白表达情况。采用析因设计方差分析或单因素方差分析进行组间比较。结果:与对照比较,高糖干预细胞活力(0.37±0.05比0.69±0.07)、迁移数、管型形成数均降低,细胞凋亡率[(20.06±5.87)%比(1.95±0.56)%]、p-JAK2 (2.16±0.18比1.06±0.12)和p-STAT3均升高(P均< 0.05),达格列净干预可逆转高糖引起的上述变化。与pcDNA比较,pcDNA-JAK2的细胞活力(0.31±0.03比0.69± 0.10)、迁移数和管型形成数均降低,凋亡率[(20.59±2.81)%比(1.63±0.16)%]、p-JAK2(2.15±0.10比1.03±0.07)和p-STAT3蛋白表达均升高(P均< 0.05),过表达JAK2后高糖干预细胞活力、迁移数和管型形成数进一步降低,凋亡率、p-JAK2和p-STAT3蛋白表达进一步升高。与pcDNA-JAK2比较,pcDNA-JAK2+达格列净处理的细胞活力(0.65±0.06比0.31±0.03)、迁移数和管型形成数均升高,细胞凋亡率[(9.68±0.83)%比(20.59±2.81)%]、p-JAK2 (1.32±0.04比2.15±0.10)和p-STAT3蛋白的表达均降低(P均< 0.05)。与pcDNA-JAK2+高糖处理比较,pcDNA-JAK2+高糖+达格列净处理的HUVECs活力、体外迁移数量和管型形成数量升高,凋亡率、p-JAK2和p-STAT3蛋白表达水平均降低(P均< 0.05)。结论:达格列净促进高糖诱导的HUVECs增殖、迁移和成管能力的恢复,抑制细胞凋亡,其机制可能与抑制JAK2/STAT3信号通路有关。

Objective To investigate the effect of dapagliflozin on the function of human umbilical vein endothelial cells(HUVECs)induced by high glucose and its molecular mechanism.Methods HUVECs were treated with 5.5mmol/L glucose(ctrl),high glucose,high glucose+dapagliflozin.The cells were transfected with pcDNA or pcDNA-JAK2 by liposome-based transfection technology,and then treated with dapagliflozin and/or high glucose.Cell proliferation and apoptosis rate were detected by CCK8 and flow cytometry,respectively.CCK8,Transwell and Tube formation assay determined cell proliferation,migration and tube formation capacity.Proteins expression of JAK2/STAT3 was detected by western blotting.The differences between groups were compared by t-test and ANOVA compared the differences groups.Results Compared with the control,the high glucose-treated cells showed obviously lower cell proliferation(0.37±0.05 vs 0.69±0.07),migration and tube formation.However,the apoptosis rate(20.06±5.87 vs 1.95±0.56),p-JAK2(2.16±0.18 vs 1.06±0.12),and p-STAT3/STAT3 proteins expression were significantly higher,which can be reversed by dapagliflozin treatment.Compared with the pcDNA,the pcDNA-JAK2 significantly decreased-cell proliferation(0.31±0.03 vs 0.69±0.10),migration and tube formation capacity,while the apoptosis rate(20.59±2.81 vs 1.63±0.16),p-JAK2(2.15±0.10 vs 1.03±0.07)and p-STAT3 proteins expression were significantly increased,and the difference was statistically significant(all P<0.05).Overexpression of JAK2 further inhibited cell proliferation,migration and tube formation in high glucose-induced cells,but upregulated apoptosis rate,protein levels of p-JAK2 and p-STAT3(all P<0.05).Compared with the pcDNA-JAK2,the cell proliferation(0.65±0.06 vs 0.31±0.03),migration and tube formation capacity of pcDNA-JAK2+dapagliflozin-treated cells were significantly increased,while the apoptosis rate(9.68±0.83 vs 20.59±2.81),p-JAK2(1.32±0.04 vs 2.15±0.10)and p-STAT3 proteins expression were significantly decreased,and the difference was statistically significant(all P<0.05).Compared to JAK2+high glucose,the cell proliferation,migration and tube formation were increased in the cells treated with pcDNA+high glucose+dapagliflozin,wheras the apoptosis rate,p-JAK2 and p-STAT3 were decreased(all P<0.05).Conclusion Dapagliflozin restores diabetes-associated decline in cell proliferation,migration and tube formation of HUVECs,which may be related to the inhibition of JAK2/STAT3 signal path way.