自噬抑制2-DG诱导内皮细胞凋亡

Autophagy inhibits endothelial cell apoptosis induced by 2-DG

目的探讨2-脱氧.D-葡萄糖(2-DG)诱导人脐静脉血管内皮细胞发生凋亡与自噬及两者之间的关系。方法传代培养人脐静脉血管内皮细胞,在常氧实验条件下,细胞被分为对照组,不同浓度的2-DG组、2-DG联合甘露糖组、阳性对照组(衣霉素组),利用MTS比色法检测2-DG对人脐静脉血管内皮细胞增殖活性的影响;利用Western blot免疫印记技术,检测细胞内质网应激相关蛋白-葡萄糖调节蛋白78(GRP78)的表达水平,以反映处理后的细胞内质网应激的水平变化;以Annexin V-PI流式细胞术检测人脐静脉血管内皮细胞凋亡。为了检测2.DG对细胞自噬的影响,引入自噬抑制剂3-MA,采用Western blot免疫印记技术,检测自噬相关蛋白Beclin 1和Atg 5的表达水平,以反映处理后的细胞自噬水平变化。结果 2-DG组细胞增殖活力明显降低,与对照组相比具有统计学意义,且该抑制作用呈剂量依赖性;2.DG联合甘露糖组增殖活力有所增加,与单独2-DG组相比具有统计学意义;2-DG组凋亡率明显增加,与对照组相比具有统计学意义,2.DG联合甘露糖组细胞凋亡减少,与单独2.DG组相比具有统计学意义;2-DG组GRP78蛋白水平增加,与对照组相比具有统计学意义;加入甘露糖后,GRP78蛋白水平减少,与单独2-DG组相比具有统计学意义;2-DG组Beclin1和Atg5蛋白表达均有所增加,与对照组相比具有统计学意义;3-MA预处理细胞抑制自噬后,2-DG诱导的细胞凋亡增多,Beclin1和Atg5蛋白表达也有所减少,与2-DG组相比具有统计学意义。结论 2-DG显著抑制人脐静脉血管内皮细胞增殖,诱导人脐静脉血管内皮细胞凋亡及自噬;内质网应激可能是介导2-DG诱导细胞凋亡的相关作用机制;2-DG诱导的细胞自噬则能抑制细胞凋亡。

Objective To explore the autophagy and apoptosis of human umbilical vein endothelial cells （HUVECs） induced by 2-deoxidation-D-glucose （2-DG） and the relationship between them. Methods HUVECs cultured under normoxia conditions were divided into control group, 2-DG group, 2-DG combined with mannose group and positive control group （ tunicamycin group）. The effects of the proliferation activity of 2-DG on HUVECs were evaluated by MTS assay at 24 hours after culture. The expression level of the Endoplasmic Reticulum Stress （ERS）-related protein, glucose regulated protein 78 （RP78）, was detected by Western blot to check the changes of ERS after treatment. The level of HUVECs apoptosis was detected by flow cytometry ; the autophagy inhibitor 3-MA was introduced into this experiment so as to detect the effects of 2-DG on cell autophagy, then the expression levels of Beclinl and Atg5 were detected by Western blot technique to reflect the changes of autophagy after treatment. Results Compared with the control group, the cell proliferation activity of the 2-DG group was significantly decreased, and this inhibition was dose-dependent. Proliferation activity of the 2-DG joint mannose group increased compared with the 2-DG group, with a significant difference. The apoptosis rate of the 2-DG group increased significantly compared with the control group with a significant difference, and the apoptosis rate of the 2-DG joint mannose group decreased significantly compared with the 2-DG group. The GRP78 protein level of the 2-DG group increased, compared with the control group with a significant difference. After adding mannose, the GRP78 protein level reduced significantly, compared with the 2-DG group. Beclinl and Atg5 protein levels of the 2-DG group increased, as compared with the control group, with a significant difference. After 3-MA pretreatment, the 2-DG-induced apoptosis rate increased, Beclinl and Atg5 protein expression levels also decreased significantly, compared with the 2-DG group. Conclusion 2-DG significantly inhibited the proliferation of HUVECs and induced HUVECs apoptosis and autophagy. ERS may mediate the cell apoptosis effect of 2-DG. However, autophage may reduce the apoptosis.