Exendin-4对人脐静脉内皮细胞内质网应激损伤的拮抗作用

Effect of exendin-4 on human umbilical vein endothelial cells with endoplasmic reticulum stress

目的研究exendin-4(Ex-4)对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)内质网应激相关凋亡的拮抗作用。方法 RPMI1640培养基培养HUVECs,待细胞长到培养瓶底部80%左右后,分4组干预细胞,每组8瓶,共32瓶。实验分组:①正常对照组(n=8):不做任何处理;②Ex-4组(n=8):(Ex-4,8μg/mL,45 min);③EPBS组(n=8):(EPBS,100μL/mL,30 min);④双药组(n=8):Ex-4预处理(8μg/mL,45 min),后加EPBS(100μL/mL,30 min)。免疫荧光法检测确认HUVECs上存在胰高血糖素样肽-1受体(glucagon-like peptide-1 receptor,GLP-1R);内源性过氧化氢酶抑制物(endogenous peroxidase blocking solution;EPBS)诱导HUVECs的凋亡,流式细胞仪检测细胞凋亡率变化;加入Ex-4预处理后,观察EPBS诱导HUVECs凋亡率的变化。Western blot检测细胞Calnexin、(C/EBP homologous protein,CHOP)和Bax表达情况,探讨内质网应激相关凋亡的机制。结果①HUVECs上有GLP-1受体表达;②与正常对照组相比,EPBS组HUVECs的凋亡率显著增加[(52.27±3.66)%vs(7.25±0.62)%,P<0.01)];与EPBS组比较,Ex-4的预处理可明显降低EPBS诱导的HUVECs凋亡[(37.77±2.86)%vs(52.27±3.66)%,P<0.01)]。与正常对照组相比,EPBS能增加Calnexin[(1.76±0.16)vs(1.00±0.05),P<0.01]、CHOP[(4.58±0.32)vs(1.00±0.12),P<0.01]和Bax[(2.25±0.10)vs(1.00±0.06),P<0.01]的表达;与EPBS组比较,Ex-4的预处理可显著降低Calnexin[(0.42±0.05)vs(1.76±0.16),P<0.01]、CHOP[(0.64±0.10)vs(1.65±0.12),P<0.01]和Bax[(1.48±0.08)vs(2.25±0.10),P<0.01)]的表达。结论 Ex-4对EPBS诱导的HUVECs凋亡有明显抑制作用,其抑制作用可能与EX-4减少内质网应激相关蛋白Calnexin、CHOP和Bax相关。

Objective To study the antagonistic effect of exendin-4 （Ex-4） on human umbilical vein endothelial cells （HUVECs） with endoplasmic reticulum （ER） stress-induced cell apoptosis. Methods HUVECs were cultured with RPMI 1640 medium. When the HUVECs grew to around 80% at the bottom of culture flasks, the cells were divided into 4 groups （n=8） including： a control group （without any treatment）, an Ex-4 group （Ex-4, 8 μg/ml for 45 min）, an endogenous peroxidase blocking solution （EPBS） group （EPBS, 100 μL/mL for 30 min）, and an Ex-4＋EPBS group [Ex-4 pretreatment （8 μg/mL for 45 min） and EPBS treatment （100 μL/mL for 30 min）]. The glucagon-like peptide-1 receptor （GLP-1R） was verified by immunofluorescence method. The cell apoptosis was detected by flow cytometry. The expression of calnexin, C/EBP homologous protein （CHOP） and Bax were detected by Western blotting. Results GLP-1R existed in HUVECs. EPBS could significantly increase the apoptosis of HUVECs compared with the control group [（52.27±3.66）% vs （7.25±0.62）%, P〈0.01）]. The rate of cell apoptosis in the Ex-4＋EPBS group was significantly lower than that in the EPBS group [（37.77±2.86）% vs （52.27±3.66）%, P〈0.01）]. Compared with the control group, EPBS group could increase the expression of calnexin [（1.76±0.16） vs （1.00±0.05）, P〈0.01], CHOP [（4.58±0.32）vs. （1.00±0.12）, P〈0.01] and Bax [（2.25±0.10） vs （1.00±0.06）, P〈0.01]. Compared with the EPBS group, Ex-4 group could significantly reduce the expression of calnexin [（0.42±0.05） vs （1.76±0.16）, P〈0.01], CHOP [（0.64±0.10）vs（1.65±0.12）, P〈0.01] and Bax [（1.48±0.08） vs （2.25±0.10）, P〈0.01）]. Conclusion Ex-4 can inhibit EPBS-induced apoptosis in HUVECs through down-regulating the expression of ER stress-related proteins such as calnexin, CHOP and Bax.