Xu Huiyu, Tan Lijuan, Qu Qiaofang, Zhang Wutang
Department of Critical Care Medicine, Shanxi Cardiovascular Hospital, Taiyuan, Shanxi 030024, P.R. China.
Exp Ther Med. 2023 Jan 4;25(2):88. doi: 10.3892/etm.2023.11787. eCollection 2023 Feb.
Atherosclerosis chiefly results from inflammation as well as vascular endothelial cell dysfunction. Methylation levels of neuronally expressed developmentally downregulated 4 (NEDD4) were found to be fortified in atherosclerosis patients and NEDD4 deficiency enhanced vascular calcification. However, the exact function of NEDD4 in inflammation and vascular endothelial dysfunction remains to be elucidated. In the present study, CCK-8 assay was used to estimate cell viability. Reverse transcription-quantitative PCR was adopted to examine the expression of NEDD4, inflammation-associated enzymes and apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1). Western blotting was used to test NEDD4, endothelial nitric oxide synthase, inducible nitric oxide synthase and APEX1 protein levels. Cytotoxicity was detected by a lactate dehydrogenase (LDH) kit. Reactive oxygen species level was tested by a corresponding kit. Vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 contents were examined with ELISA. Cell adhesion assays evaluated the adhesion of endothelial cells. Co-immunoprecipitation assay was used to test the relationship between NEDD4 and APEX1. The data revealed that NEDD4 expression rapidly declined in oxidized low density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs). Following NEDD4 overexpression, the active damage, inflammatory release and endothelial cell dysfunction in ox-LDL-induced HUVECs were attenuated. After co-transfection of APEX1 interference plasmids and NEDD4 overexpression plasmids, cell damage, inflammatory release and endothelial cell dysfunction in ox-LDL-induced HUVECs were improved again. Taken together, NEDD4 attenuated ox-LDL-induced inflammation and endothelial dysfunction by regulating APEX1 expression.
动脉粥样硬化主要由炎症以及血管内皮细胞功能障碍引起。研究发现，神经元表达的发育下调蛋白4（NEDD4）在动脉粥样硬化患者中的甲基化水平升高，且NEDD4缺乏会加剧血管钙化。然而，NEDD4在炎症和血管内皮功能障碍中的具体作用仍有待阐明。在本研究中，采用CCK-8法评估细胞活力。采用逆转录定量PCR检测NEDD4、炎症相关酶和脱嘌呤/脱嘧啶内切脱氧核糖核酸酶1（APEX1）的表达。采用蛋白质免疫印迹法检测NEDD4、内皮型一氧化氮合酶、诱导型一氧化氮合酶和APEX1的蛋白水平。通过乳酸脱氢酶（LDH）试剂盒检测细胞毒性。采用相应试剂盒检测活性氧水平。用酶联免疫吸附测定法检测血管细胞黏附分子1和细胞间黏附分子1的含量。细胞黏附试验评估内皮细胞的黏附情况。采用免疫共沉淀试验检测NEDD4与APEX1之间的关系。数据显示，在氧化型低密度脂蛋白（ox-LDL）诱导的人脐静脉内皮细胞（HUVECs）中，NEDD4表达迅速下降。NEDD4过表达后，ox-LDL诱导的HUVECs中的活性损伤、炎症释放和内皮细胞功能障碍得到减轻。在共转染APEX1干扰质粒和NEDD4过表达质粒后，ox-LDL诱导的HUVECs中的细胞损伤、炎症释放和内皮细胞功能障碍再次得到改善。综上所述，NEDD4通过调节APEX1表达减轻ox-LDL诱导的炎症和内皮功能障碍。
