Objective: To explore the role of the interaction between glycogen synthase kinase-3β (GSK-3β) and endoplasmic reticulum stress (ERS) in the high glucose (HG)-induced injury in human umbilical vein endothelial cells (HUVECs).
Methods: HUVECs treated with 40 mmol/L glucose for 24 h were examined for expression levels of GSK-3β, GRP78, CHOP and cleaved caspase-3 protein using Western blotting. The cell viability was examined using CCK-8 assay and cell apoptosis was detected with Hoechst 33258 nuclear staining and photofluorography. The intracellular level of reactive oxygen species (ROS) was measured with dichlorfluoresein staining and photofluorography. Mitochondrial membrane potential (MMP) was tested by rhodamine 123 (Rh123) staining and photofluorography.
Results: Treatment of HUVECs with 40 µmol/L glucose for 3-24 h activated GSK-3β in a time-dependent manner, leading to significantly down-regulated expression of phosphorylated (p)-GSK-3β (P<0.05). HG exposure of the cells for 1-24 h induced ERS, evidenced by time-dependently up-regulated expression of GRP78 and CHOP (P<0.05). LiCl, an inhibitor of GSK-3β, attenuated HG-induced ERS and significantly lowered the expression levels of GRP78 and CHOP (P<0.01). 4-PBA, an inhibitor of ERS, obviously ameliorated the activation of GSK-3β by HG as shown by the increase in p-GSK-3β expression level (P<0.01). HG exposure for 24 h induced obvious injuries in HUVECs, which exhibited decreased cell viability, increased cell apoptosis, increased expression of cleaved caspase-3 and ROS generation, and loss of MMP. Pretreatment of the cells with LiCl or 4-PBA for 60 min before HG exposure significantly lessened the cell injuries (P<0.01).
Conclusion: Interactions between GSK-3β and ERS occur in HUVECs exposed to HG and participate in HG-induced cell injuries.
目的: 糖原合酶激酶-3β（GSK-3β）与内质网应激（ERS）相互作用是否参与高糖引起的人脐静脉内皮细胞（HUVECs）损伤。
方法: 应用40 mmol/L葡萄糖作用HUVECs 24 h构建高糖血管内皮细胞损伤模型。应用Western blot法检测GSK-3β、糖调节蛋白78（GRP78）、CHOP和cleaved caspase-3蛋白的表达水平；CCK-8法检测细胞存活率；Hoechst 33258核染色荧光显微镜照相法测定细胞凋亡；DCFH-DA染色荧光显微镜照相法测定胞内活性氧（ROS）水平；罗丹明123（Rh123）染色荧光显微镜照相法测定线粒体膜电位（MMP）。
结果: 应用40 mmol/L葡萄糖处理HUVECs 3~24 h，磷酸化（p）-GSK-3β表达减少，与Con组比较，差异具有统计学意义（P < 0.05）；用高糖处理HUVECs 1~24 h，对促进GRP78和CHOP蛋白表达呈显著的时间依赖性，与Con组比较，差异具有统计学意义（P < 0.05）；氯化锂（LiCl，GSK-3β抑制剂）能减轻高糖引起的ERS，使GRP78和CHOP蛋白表达减少，与高糖组比较，差异具有统计学意义（P < 0.01）；4-苯基丁酸（4-PBA，ERS抑制剂）减弱高糖对GSK-3β的激活作用，使p-GSK-3β表达增多，与高糖组比较，差异具有统计学意义（P < 0.01）。高糖处理HUVECs 24 h能引起细胞存活率降低、凋亡细胞、cleaved caspase-3表达和ROS生成增多及MMP丢失等损伤；在高糖作用前，应用LiCl或4-PBA预处理60 min均减轻高糖引起的上述损伤，与高糖组分别比较，差异均具有统计学意义（P < 0.01）。
结论: 在高糖损伤的HUVECs，存在GSK-3β与ERS的相互作用并参与高糖对HUVECs的损伤。