谷氧还蛋白1在高糖诱导的血管内皮细胞凋亡中的作用及其机制

Mechanism underlying the protective effects of glutaredoxin-1 against high glucose-induced apoptosis of umbilical vein endothelial cells

目的:观察谷氧还蛋白1(glutaredoxin1,Grx1)在高糖诱导的血管内皮细胞凋亡中的作用及机制.方法:在高糖环境中培养人脐静脉内皮细胞,制备人脐静脉内皮细胞损伤模型.细胞分为正常对照组、损伤组、Grx1预保护组.倒置显微镜下观察细胞形态.MTT比色法检测细胞活力以初步观察Grx1对细胞损伤的作用;采用Annexin VFITC/PI双染法,流式细胞仪检测Grx1对人脐静脉内皮细胞凋亡的影响;利用Western blot法分析Grx1对Akt、JNK蛋白水平表达的影响.结果:Grx1预保护组与损伤组比较,细胞状态明显改善.与高糖损伤组比较,Grx1预保护组的细胞存活率显著提高(78%±3% vs 59%±2%,P<0.05),早期凋亡率(0.2360%±0.0156% vs 0.4156%±0.0374%,P<0.05)和晚期凋亡率(0.2433%±0.0278% vs 0.3689%±0.0083%,P<0.05)均明显降低.与正常对照组相比,高糖组p-JNK相对含量明显增多(0.64±0.07 vs 0.48±0.03,P<0.05),而高糖组p-Akt水平较正常对照组显著降低(1.29±0.035 vs 0.69±0.11,P<0.01).同时,hGrx1保护后p-JNK蛋白水平较高糖组显著降低(0.39±0.05 vs 0.64±0.07,P<0.05);而p-Akt蛋白水平较高糖组显著增高(0.69±0.11 vs 1.07±0.13,P<0.01).结论:Grx1可通过抑制JNK激活及激活Akt通路来拮抗高糖诱导的内皮细胞凋亡.

AIM： To investigate the mechanism underlying the protective effects of glutaredoxin-1 （Grxl） against high glucose-induced apoptosis of umbilical vein endothelial cells. METHODS： The apoptosis of cultured human umbilical vein endothelial cells （HUVECs） was induced under high glucose conditions.HUVECs were then divided into three groups, namely, control group, high glucose group and Grxl plus high glucose group. The cells were observed under an inverted light microscope to examine their morphological changes. The proliferation of cells was measured by MTT assay. The influence of Grxl on the apoptosis of HUVECs was determined by flow cytometry with annexin V-FITC/PI double staining. The expression levels of p-JNK and p-Akt proteins were evaluated by Western blot. RESULTS： Compared to the high glucose group, cells in the Grxl plus high glucose group showed a significant improvement in morphology and growth state, a remarkable increase in viability （59% ± 2% vs 78% ± 3%, P 〈 0.05）, as well as an obvious decrease in early （0.4156% ± 0.0374% vs 0.2360% ± 0.0156%, P 〈 0.05） and late （0.3689% ± 0.0083% vs 0.2433% ± 0.0278%, P 〈 0.05） apoptotic rates. Compared to the normal control group, the expression level of p-JNK protein in cells in the high glucose group significantly increased （0.48±0.03 vs 0.64 ± 0.07, P 〈 0.05） while that of p-Akt protein significantly decreased （0.69 ± 0.11 vs 1.29 ± 0.035, P 〈 0.01）. After pretreatment with hGrxl, the expression level of p-JNK protein decreased （0.64 ± 0.07 vs 0.39 ±0.05, P 〈 0.05） while that of p-Akt protein increased （0.69 ± 0.11 vs 1.07 ± 0.13, P 〈 0.01）. CONCLUSION： Grxl is able to antagonize high glucose-induced apoptosis of human umbilicus vein endothelial cells through inhibition of JNK and activation of the Akt signaling pathway.