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OBJECTIVE: To investigate the induction and activation of heparinase by extracellular histones in acute respiratory distress syndrome(ARDS) induced by chlorine in mice.METHODS: The specific pathogen free adult male C57 BL/6 mice were randomly divided into control group, chlorine injured group, histone injured group, anti-histone antibody group and heparinase inhibitor group, with six mice in each group.The mice in the control group and histone injured group were exposed to clean air, and the mice in the other three groups were exposed to chlorine gas at a dose of 580.0 mg/m~3 for 30 minutes by systemic dynamic inhalation.Mice in the histone injured group were injected with 50 mg/kg body weight calf thymus histone by tail vein.One hour before exposure, mice in the anti-histone antibody group were pretreated with 20 mg/kg body weight anti-histone H4 antibody by tail vein injection, and mice in the heparinase inhibitor group were injected with 2 mg/kg body weight OGT2115(heparinase inhibitor). The other three groups were given equal volume of 0.9% sodium chloride solution by tail vein injection. After 24 hours of exposure, arterial blood was collected for blood gas analysis and the lung tissue was collected for histopathological examination. The protein level of heparinase in lung tissue were detected using enzyme-linked immunosorbent assay, and the activity of heparinase were detected by measuring the product of heparan degradation. The protein expression of pro-heparinase and active heparinase were detected by Western blotting.RESULTS: The dyspnea developed of mice in the chlorine injured group and histone injured group, diffuse inflammation occurred in lung tissue, the oxygenation index in arterial blood decreased(all P<0.05), and the protein level and activity of heparinase in lung tissue, as well as the relative expression of pro-heparinase and active heparinase were increased compared with the control group(all P<0.05). The dyspnea, hypoxemia and acute lung injury of mice in the anti-histone antibody group were alleviated, and the protein level of heparinase in lung tissue, as well as the relative expression levels of pro-heparinase and active heparinase were decreased(all P<0.05), compared with chlorine injury group and histone injury group.The dyspnea, hypoxemia and acute lung injury were alleviated in the heparinase inhibitor group, and the activity of heparinase and the relative expression of pro-heparinase in the lung tissue were decreased compared with the chlorine injury group(all P<0.05). CONCLUSION: During the occurrence and development of chlorine-induced ARDS in mice, extracellular histones aggravate lung injury by inducing the expression and activation of heparinase. Acute lung injury can be alleviated by inhibiting the expression and activation of heparinase.
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Objective@#To observe the changes of extracellular histones and pulmonary microvascular endothelial cells, and study the activating role of extracellular histones to pulmonary microvascular endothelial cells in the pathogenesis of acute respiratory distress syndrome (ARDS) .@*Methods@#The correlation of the severity of acute lung injury with extracellular histones and pulmonary endothelial damage was studied through mice model, and acute lung injury was produced by aspiration of different concentrations of hydrochloric acid (0.01、0.1、0.3 and 0.5 mol/L, 2 ml/kg). Tumor necrosis factor-α (TNF-α), soluble thrombomodulin (sTM) and lung pathological change were measured. The pro-inflammatory role of extracellular histones was tested by injecting calf thymus histones (CTH) or specific anti-H4 antibody through tail vein. The direct activating role of extracellular histones to pulmonary microvascular endothelial cells was studied through pulmonary endothelial model.@*Results@#The extracellular histones in plasma were increased obviously 6h after aspiration of different concentrations of hydrochloric acid in mice. A positive correlation was seen between extracellular histones and concentrations of aspirated hydrochloric acid (r=0.9180, P<0.05). The sTM in plasma also showed a positive correlation with concentrations of aspirated hydrochloric acid (r=0.8701, P<0.05). Merely administering CTH could not only increase TNF-α and sTM in plasma but also cause obvious lung injury, while specific anti-H4 antibody could relieve the inflammation and lung damage caused by CTH. Extracellular histones could directly damage pulmonary endothelial cells to release sTM in pulmonary endothelial model in vitro, while anti-H4 antibody could protect the endothelial cells.@*Conclusion@#Extracellular histones are the key endogenic inflammatory mediators during the pathogenesis of ARDS caused by aspiration of hydrochloric acid, which could promote inflammation by directly activating pulmonary endothelial cells.
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Objective To investigate the change of extracellular histone level as well as the mecha-nism of the cytotoxicity of extracellular histones on vascular endothelial cell in sepsis. Methods Septic chil-dren 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 con-centrations(0,50,100,200 and 300μg/ml) or different time periods(200μg/ml,0,5,15,30,45 and 60 mi-nutes) . The treated cells were subject to flow cytometer to measure the cell survival rate and scanning/trans-mission electron microscopy to observe their morphological changes. Western blot was used to detect the ex-pression 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 sep-tic 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 chil-dren 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 fac-tor-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. Extracellu-lar 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.
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Objective To investigate the effects of low dose of extracellular histone on endothelial cells in infectious diseases such as sepsis. Methods The endothelial cells were treated with 10 μg/mL recombinant human histone H3/H4 complex in replacement of calf thymus histones (CTH) for various periods of time, and the morphology changes and the viability of the endothelial cells were recorded. In addition, flow cytometry was applied to identify the characteristics of endothelial cells and enzyme-linked immunosorbnent assay (ELISA) was used to detect the extracellular histones level in endothelial cells culture. Results The low dose of CTH could continuously induce endothelial cells death, cell morphological changes and function loss, which was reproduced by 10 μg/mL recombinant histone H3/H4 complex. Results of histones quantitation showed that histone can cause a series of intracellular reactions in a short period of time. Conclusions It is showed that 10 μg/mL H3/H4 can induce the toxicity in infectious disease and this level of the dose is a lower than those used in previous studies and more close to the pathological conditions.
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Sepsis is a common desease with a high mortality in pediatric intensive care unit. The patho-genesis of sepsis remains unclear. Recent studies have found that extracellular histones play an important role in the incidence of sepsis. This review provides new ideas for the diagnosis and treatment of sepsis.
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Objective To investigate the changes of extracellular histones during the course of acute liver failure in mice as well as its therapeutic potential.Methods WT mice (C57BL/6) were randomly (random number) allocated to inducing acute liver failure by lethal doses of GalN/LPS injected i.p.Hepatic function,apoptosis of hepatocytes and histological indexes were measured at different intervals following GalN/LPS challenge.The levels of extracellular histones were determined by using ELISA and Western blot methods.Meanwhile,GalN/LPS-treated mice were administered with anti-histone H3 and antihistone H4 neutralized antibodies,respectively.Results Administration of GalN/LPS to mice caused acute liver failure,characterized by significant elevation of plasma ALT levels and massive hepatocyte apoptosis or necrosis.All mice died within 9-12 hours.The levels of nucleosomes and extracellular histones H3 and H4 were increased considerably in a time-dependent manner.The survival rates in GalN/LPS-treated mice were improved remarkably following administration of anti-histone H3 and H4 neutralized antibodies (P =0.037,P =0.025),likely due to the significant inhibition of TNF-production.Conclusions Extracellular histones are an important mediator implicated in the pathogenesis of acute liver failure.Anti-histones show promising potential in the treatment of acute liver failure,which deserves further investigation in the future.