| Objective: Sepsis is one of the leading causes of death for patients in intensive care units(ICUs).The pathophysiological mechanism of sepsis is extremely complex,and there is still a lack of accurate and effective therapeutic drugs.Recent studies have found that histone is one of the important mediators of sepsis and can cause sepsis-like organ damage.Therefore,the drugs that can antagonize histones may become a new target for the treatment of sepsis.Different studies have reported that unfractionated heparin(UFH)can antagonize the toxic effects of histones and improve histone-induced organ damage.But such studies usually pretreat with UFH or co-injection with histones,which is inconsistent with the actual clinical situation.The purpose of this study is to confirm whether UFH can reduce histone-induced coagulation activation and thrombosis when histones have caused coagulation disorder already,thus providing a theoretical basis for the protect role of UFH in patients with sepsis.Methods: Survival assays: 20 males C57BL/6 mice aged 6-10 weeks were randomly divided into histone group and histone+UFH group(n=10 per group).The mice in the histone+UFH group were challenged with 75mg/kg histones followed by UFH(800U/kg)immediately and observed for 7days.The mice in the histone group was only challenged with 75mg/kg histones.In addition,24 males C57BL/6 mice aged 6-10 weeks were randomly divided into control group,histone group and histone+UFH group,with 8 mice in each group.The histone group and histone + UFH group were injected with 50 mg/kg histones through the tail vein,and the control group was injected equal volume of sterile saline.After 1 hour,the histone +UFH group was injected with 400U/kg UFH via the tail vein.The histone group and the control group were injected equal volume of sterile saline.After 4h of histones(sterile saline)injection,the lungs of the mice were harvested and the lung wet/dry weight ratio(W/D)and the pulmonary water contents were measured.The pathological changes in lung tissue were observed by hematoxylin and eosin(HE)staining under microscope,and the extent of lung injury was evaluated.The expression of von Willebrand factor(vWF)and fibrinogen were observed by immunohistochemistry.The quantitative real-time polymerase chain reaction(qRT-PCR)was used to determine the expression of fibrinogen mRNA and vWF mRNA in lung tissue.Observation of the morphology of pulmonary endothelial cells by transmission electron microscopy.Results: All mice died within 1h after injection 75mg/kg histones while the UFH treated mice survived for 7d.The lung W/D ratio and pulmonary water contents in the histone group were significantly higher than those in the control group(W/D ratio: 6.19±0.53 vs.4.54±0.25,pulmonary water contents: 82.59%±2.03% vs.78.52%±1.51%,both P<0.01).The lung W/D ratio and pulmonary water contents in the histone+UFH group were significantly lower than those in the histone group(W/D ratio: 4.84±0.35 vs.6.19±0.53,pulmonary water contents: 79.21%±1.48% vs.82.59%±2.03%,both P<0.01).Histological examination showed that the alveolar structure of the control group was intact,and the alveolar cavity was clean without exudation.In the histone group,the lungs were significantly damaged.The alveolar wall was thickened,infiltrated by inflammatory cells and focally alveolar hemorrhage,edema,associated with alveolar fibrin deposition and micro-thrombus formation.The lung histopathological score in the histone group was significantly higher than that in the control group(5.15±0.87 vs.0.18±0.17,P<0.01).All of the pathological changes were significantly alleviated in the histone+UFH group,and the histopathological score of the lung was significantly lower than that in the histone group(2.28±0.72 vs.5.15±0.87,P<0.01).Immunohistochemistry showed that the expression of vWF and fibrinogen in histone group was high,and the expression of vWF and fibrinogen in histone+UFH group was significantly reduced.The fibrinogen mRNA expression of lung tissue in the histone group was significantly higher than that in the control group(55.30±18.84 vs.1.11±0.45,P<0.01),and the expression of fibrinogen mRNA in the histone+UFH group was significantly lower than that in the histone group(26.50±9.97 vs.55.30±18.84,P<0.01).The vWF mRNA expression of lung tissue in the histone group was significantly higher than that in the control group(1.90±0.41 vs.1.08±0.65,P<0.05),and the expression of vWF mRNA in the histone+UFH group was significantly decreased after UFH treatment(1.23±0.49 vs.1.90±0.41,P<0.05).Transmission electron microscopy showed that the endothelial cells of the lung in the histone group were obviously swollen and exfoliated,and the thickness of the basement membrane was uneven,and the close connection was open.However,the endothelial cells in the histone+UFH group were basically normal in morphology.Conclusions: Histones can increase the expression of vWF and fibrinogen,thereby activating coagulation.UFH can effectively attenuate histone-induced coagulation activation and improve lung injury. |
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