过氧亚硝酸盐通过诱导己糖激酶1硝基化修饰促进血管内皮细胞凋亡

Peroxynitrite promotes apoptosis of vascular endothelial cells by inducing nitration modification of hexokinase-1

摘要:

目的]评估血管内皮细胞中特定蛋白质发生酪氨酸硝基化修饰的现象及其对线粒体介导的细胞凋亡的影响。 [方法]体外培养人脐静脉内皮细胞,分为对照组(二甲基亚砜处理)、3-吗啉磺胺(SIN-1)组和SIN-1＋5,0,5,0-四苯基卟啉铁(FeTPP)组,24 h后检测己糖激酶1(HK1)硝基化修饰水平、线粒体膜电位、活性氧生成以及内皮细胞增殖与凋亡水平。利用基因编辑技术构建HK1敲除人脐静脉内皮细胞系,检测其增殖与凋亡水平。 [结果]过氧亚硝酸盐生成剂SIN-1处理人脐静脉内皮细胞后,HK1蛋白质硝基化修饰水平显著升高(P＜0.01),活性氧生成显著增加,线粒体膜电位显著下降,内皮细胞增殖能力显著下降,内皮细胞凋亡水平显著增加(均P＜0.01)。过氧亚硝酸盐分解催化剂FeTPP能够逆转上述效应(P＜0.01)。此外,HK1基因敲除也展现出类似的抗氧化效果,内皮细胞的增殖能力显著降低,而凋亡水平则显著升高(P＜0.01)。 [结论]过氧亚硝酸盐能够诱导血管内皮细胞中HK1的硝基化修饰水平升高,这可能是通过促进线粒体活性氧的产生,进而加速内皮细胞凋亡的过程。

Abstract:

Aim To evaluate the tyrosine nitration modification of specific proteins in vascular endothelial cells and its impact on mitochondria-mediated apoptosis. Methods Human umbilical vein endothelial cells were cultured in vitro and divided into three groups:control group (treatment with dimethyl sulfoxide), 3-morphansulam (SIN-1) group, and SIN-1＋Fe(III) 5,0,5,0-(tetraphenyl)porphyrin (FeTPP) group. After 24 h, the levels of hexokinase 1 (HK1) nitration modification, mitochondrial membrane potential, reactive oxygen species (ROS) production, and endothelial cell proliferation and apoptosis were assessed. A human umbilical vein endothelial cell line knockout of HK1 was constructed using gene editing technology, and its proliferation and apoptosis levels were detected. Results After treatment of human umbilical vein endothelial cells with peroxynitrite generator SIN-1, the level of HK1 protein nitration modification significantly increased (P＜0.01), reactive oxygen species production significantly increased, mitochondrial membrane potential significantly decreased, endothelial cell proliferation ability significantly decreased, and endothelial cell apoptosis level significantly increased (all P＜0.01). Peroxynitrite decomposition catalyst FeTPP could reverse the above effect (P＜0.01). In addition, HK1 gene knockout also exhibited similar antioxidant effects, with a significant decrease in endothelial cell proliferation ability and a significant increase in apoptosis levels (P＜0.01). Conclusion Peroxynitrite can induce an increase in the level of nitration modification of HK1 in vascular endothelial cells, which may be achieved by promoting the production of mitochondrial reactive oxygen species, thereby accelerating the process of endothelial cell apoptosis.