Septic serum mediates inflammatory injury in human umbilical vein endothelial cells via reactive oxygen species, mitogen activated protein kinases and nuclear factor­κB.

ABSTRACT

Sepsis­induced blood vessel dysfunction is mainly caused by microvascular endothelial cell injury. However, the mechanism underlying sepsis­induced endothelial cell injury remains unclear. The present study hypothesized that sepsis­induced inflammatory injury of endothelial cells may be the first step of endothelial barrier dysfunction. Therefore, the present study aimed to uncover the mechanism underlying the inflammatory effects of sepsis. A rat model of cecal ligation and puncture­induced sepsis was established, and septic serum was collected. Subsequently, human umbilical vein endothelial cells (HUVECs) were treated with the isolated septic or normal serum. HUVEC viability was assessed using a Cell Count Kit­8 assay. Furthermore, transmission electron microscopy and reverse transcription­quantitative PCR (RT­qPCR) analysis were carried out to observe the cell morphology and determine the mRNA expression levels in septic serum­induced HUVECs. The protein expression levels were evaluated by western blot analysis, and the secretion of the inflammatory factors interleukin (IL)­1ß, IL­6 and tumor necrosis factor (TNF)­α was determined by ELISA. Additionally, reactive oxygen species (ROS) generation and nuclear factor (NF)­κB nuclear translocation were observed under a fluorescence microscope. The results of the present study demonstrated that HUVEC viability was significantly decreased following 12­ or 24­h treatment with septic serum. In addition, chromatin condensation, mitochondrial vacuolization and endoplasmic reticulum degranulation were observed following treatment with septic serum. Furthermore, the secretion levels of IL­1ß, IL­6 and TNF­α were increased in septic serum­stimulated HUVECs. Septic serum treatment also enhanced superoxide anion generation, promoted extracellular signal regulated kinase 1/2 (ERK1/2), N­terminal kinase (JNK) and p38 mitogen­activated protein kinase (p38) phosphorylation, and increased NF­κB levels in the nuclei of HUVECs. Finally, pre­treatment of HUVECs with the antioxidant N­acetylcysteine, the ERK1/2 inhibitor PD98059, the p38 inhibitor SB203580, the JNK inhibitor SP610025 or the NF­κB inhibitor pyrrolidine dithiocarbamate restored the septic serum­induced IL­1ß, IL­6 and TNF­α expression. In conclusion, the results of the current study suggested that the septic serum­induced endothelial cell injury may be mediated by increasing ROS generation, activation of mitogen­activated protein kinases and NF­κB translocation.