Cytotoxicity of extracellular histones on vascular endothelial cells and the underlying mechanisms in sepsis

Abstract

Objective To investigate the change of extracellular histone level as well as the mechanism of the cytotoxicity of extracellular histones on vascular endothelial cell in sepsis. Methods Septic children admitted to PICU in Shanghai Children′s Medical Center between January 2010 and December 2014 were included in the present study.According to the diagnostic criteria of sepsis, the patients were divided into the sepsis group(51 cases) and the severe sepsis group(79 cases), with healthy children as the control group(108 cases). Patients in the severe sepsis group were further divided into the survival group(45 cases) and the non-survival group(34 cases) based on 28-day mortality.The plasma concentration of extracellular histones in these children was determined and its correlation with the severity of sepsis was analyzed.Human umbilical vein endothelial cells(HUVECs) were incubated with calf thymus histone(CTH) at various concentrations(0, 50, 100, 200 and 300 μg/ml) or different time periods(200 μg/ml, 0, 5, 15, 30, 45 and 60 minutes). The treated cells were subject to flow cytometer to measure the cell survival rate and scanning/transmission electron microscopy to observe their morphological changes.Western blot was used to detect the expression of IκB and phosphor-p38/p38 in nuclear factor(NF)-κB and mitogen-activated protein kinase(MAPK)signaling pathways, while ELISA was used to determine the levels of tumor necrosis factor-α and interleukin-6. Results The levels of circulating histones in the septic children(2.29±1.00) and severe septic children(19.17±10.20) were significantly higher than that of healthy controls(0.23±0.26)(P<0.001), and the histone levels in the severe septic children were even higher(P<0.001). Among the children diagnosed as severe sepsis, the level of circulating histones in the non-survivors was significantly higher than that in the survivors(29.47±5.99 vs.10.94±2.68, P<0.001). The survival rate of HUVEC gradually decreased along with the increase of CTH concentration or the treatment period in vitro.Data from electron microscopy showed that CTH treatment could directly disrupt the plasma membrane of HUVEC.Histones could also activate NF-κB and MAPK pathways, leading to the release of large amount of tumor necrosis factor-α and interleukin-6. Conclusion The levels of extracellular histones in the septic children are correlated with the severity of sepsis.CTH can induce HUVEC death in a dose-and time-dependent manner.Extracellular histone-induced endothelial dysfunction may mediate the progression of sepsis and such cytotoxicity might be due to the destruction of endothelial cell membranes and activation of the NF-κB and MAPK signaling pathways. Key words: Sepsis; Extracellular histones; Endothelial cells; Multiple organ dysfunction syndrome; Inflammatory mediators