The Protective Effect Of Unfractionated Heparin On The Intestinal Microcirculation Dysfunction Induced By Extracellular Histone In Sepsis

Objective: Sepsis is systemic inflammatory response caused by infection,and it is a life-threatening organ dysfunction caused by dysregulated host response to infection.Microcirculation is composed of arteriole,venule and capillary.It is an important place to maintain homeostasis.The normal microcirculation function is an important intermediate link for the host to maintain homeostasis and to control pathogen.The abnormal function of microcirculation is the pathophysiological manifestation and also the initiating factors of the development of sepsis.Endothelial cells are the main structural cells of microcirculation.Endothelial cells are at the interface between inflammation and coagulation in sepsis,and are important structures for regulating immune thrombus.Endothelial dysfunction is an important factor of microcirculation dysfunction.Intestinal blood perfusion is first sacrificed in sepsis and shock,prone to have microcirculation dysfunction,will further cause intestinal barrier damage,and not only leads to intestinal dysfunction,but also aggravates sepsis and remote organ function dysfunction.Histone is a structural protein that forms chromatin in eukaryotic cells and is an alkaline protein with positive charge.The extracellular histone is derived from the release of nucleosome resulting from intracellular chromatin depolymerization.The concentration of extracellular histone can be significantly increased when inflammation and injury occur.Extracellular histone is a kind of damage related model molecule(DAMPs),which can cause extensive cell damage,especially endothelial cell injury,induce leukocyte recruitment and adhesion,activate Toll like receptors in immune cells,activate intracellular signaling pathways,cause inflammatory factors release,activate platelets and cause coagulation disorders.These effects suggest that the role of extracellular histone may be an important intermediate link in the pathogenesis of microcirculation dysfunction when inflammation and injury occur.Unfractioned heparin(UFH)is an anticoagulant drug commonly used in clinic.As a biological molecule with strong negative charge,heparin can bind to positive charged histone and reduce the toxicity and inflammation mediated by histone.This study aimed to establish rat sepsis model by intravenous injection of LPS,and to investigate the effect of UFH on the intestinal microcirculation and the content of extracellular histone in sepsis;Another rat model was established by intravenous injection of histone,then the effects of histone stimulation to intestinal microcirculation and endothelial cells were observed,and the protective effects of heparin were also observed;Human intestinal microvascular endothelial cells(HIMEC)were stimulated by histone,the protective effect of UFH on histone damaged HIMEC,and the effect of heparin on v WF which is a endothelial cell damage and coagulation activation marker was studied.Whether unfractioned heparin can reduce HIMEC damage and coagulation activation by mechanism of inhibiting the calcium ion inflow caused by histone was also explored.Methods: 1.In vivo experiment: The protect effect of unfractioned heparin on the extracellular histone and intestinal microcirculation dysfunction in sepsis rats.24 male Wistar rats were randomly divided into 3 groups,which were the control group,the LPS group and the LPS+UFH group,and each group had 8 rats.The LPS group model was established by bolus injection of 10mg/kg LPS intravenously,and the UFH group was given 100IU/kg*h heparin solution for continuous intravenous infusion.The changes of heart rate and blood pressure in rats within 6 hours were observed continuously.After opening the abdomen,the microcirculation of the ileum was observed by IDF.Microcirculation of the mucosa in the rats was observed at 6 hours in the experiment.Microcirculation of the serous layer of intestine was observed on 0,2,4 and 6 hours in the experiment,respectively.The microcirculation in the intestinal mucosa was observed in 6 hours.Euthanasia was performed by injection of excess pentobarbital at 6 hours,and the plasma specimens were collected.The changes of total histone in rat plasma were measured by ELISA,plasma H3 and H4 histone were measured by Western blot.2.In vivo experiment: The effect of extracellular histone on intestinal microcirculation and endothelial cell injury in rats and the protective effect of unfractioned heparin.24 male Wistar rats were randomly divided into three groups,which were the control group,the histone group and the histone+ UFH group,and each group had 8 rats.The model of histone group was established by bolus injection of 30mg/kg calf thymus histones intravenously.The histone+ UFH group was treated with 100IU/kg*h heparin solution after histone infusion.The changes of heart rate and blood pressure in rats within 6 hours were observed continuously.After opening the abdomen,the microcirculation of the ileum was observed by IDF,and microcirculation of the intestinal serous layer was observed at 0,2,4 and 6 hours.Microcirculation of the mucosa was observed at 6 hours in the experiment after the ileum was opened.Euthanasia was performed by injection of excess pentobarbital at 6 hours,and the samples of plasma and ileum were collected.Plasma s TM and v WF contents were measured by ELISA and the ultrastructural changes of the ileum microvascular endothelial cells were observed by transmission electron microscope.3.In vitro experiment: The protective effect and mechanism of unfractioned heparin on human intestinal microvascular endothelial cell injury induced by extracellular histone.The human intestinal microvascular endothelial cells(HIMEC)were selected and divided into 4 groups: the control group,the histone group,the histone +UFH group and the UFH group.10IU/ml UFH pretreatment was added before the stimulus was added,and HIMEC was stimulated by the addition of PBS dissolving calf thymus histone.The effect of different concentration of histone stimulation on the cell viability of HIMEC was measured by MTT.PI staining was used to determine the survival rate of HIMEC by flow cytometry and fluorescence microscopy.The changes of v WF content in HIMEC cytoplasm were observed by immunofluorescence,and the concentration of v WF in the supernatant of HIMEC cells was determined by ELISA.Cytosolic free calcium concentration in calcium free and calcium containing buffers was measured by Fura 2-AM fluorescence staining.Results: 1.(1)The heart rate and blood pressure of the rats in LPS group was increased,and the rate of heart rate and blood pressure in the rats with sepsis was not improved by the treatment of UFH.(2)The intestinal serous layer and mucous layer of the LPS group rats had obvious microcirculation disorder.Compared with the the control group,serous microcirculation of LPS group's TVD,PVD,PPV and MFI of 2,4,6 hours and TVD of 2 hours were significantly different.Microcirculation of mucous layer was analyzed for 6 hours.The TVD,PVD and PPV in LPS group was statistically different from that of the control group.(3)Heparin therapy can improve the intestinal serous and mucous layer microcirculation disorder in septic rats.At 6 hours in the LPS+ UFH group,serous layer TVD,PVD,MFI,and TVD,PVD,PPV and MFI in the mucous layer were statistically different from the LPS group.(4)The plasma total histone,H3 and H4 histone levels in LPS group increased,and the total plasma histone,H3 and H4 histone levels in the LPS+ UFH group decreased.2.(1)There was no significant change in heart rate and blood pressure in the histone group and histone + UFH group.(2)There were obvious microcirculation changes in the intestine serous layer and mucous layer of the histone group.Compared with the control group,PVD and MFI of the serosa layer decreased significantly at the 2 hours time point after the infusion of histone,and the PVD,PPV and MFI decreased significantly at 4 hours.There was a significant difference in TVD,PVD,PPV and MFI of mucous layer at 6 hours.(3)Heparin therapy could improve the microcirculation disturbance of intestinal serous and mucosa layer in rats stimulated by histone.6 hours TVD,PPV,4,6 hours PVD of The serous layer in histone+ UFH group had statistical difference compared with the histone group.The TVD,PVD,PPV and MFI of the mucous layer in histone+ UFH group were significantly different from those of the histone groups at 6 hours.(4)The levels of plasma s TM and v WF in rats were significantly increased by histone infusion,and UFH treatment could significantly reduce the s TM and v WF in plasma.(5)The ultrastructural damage of the ileum microvascular endothelial cells in the histone group was seriously damaged,and the heparin therapy could reduce the ultrastructural injury of the ileum microvascular endothelial cells.3.(1)Viability of HIMEC cells decreased with the increase of the concentration of the histone stimulation.(2)Histone stimulation led to a decrease in the survival rate of HIMEC,and heparin preconditioning could inhibit the HIMEC death caused by histone stimulation.(3)Histone stimulation led to the v WF exocytosis of HIMEC.The content of v WF in the cytoplasm was reduced,and the concentration of v WF in the supernatant of cell culture was increased.Heparin pretreatment could significantly inhibit the HIMEC release v WF during the histone stimulation.(4)Histone stimulation can lead to influx of extracellular calcium in HIMEC cells,and concentration preconditioning can inhibit the intracellular calcium influx caused by histone stimulation.Conclusion:(1)Unfractionated heparin alleviates intestinal microcirculation dysfunction and reduce the plasma level of extracellular histone in sepsis.(2)Histone stimulation cause intestinal microcirculation dysfunction and endothelial cell injury in rats.Unfractionated heparin improves the intestinal microcirculation,reduces endothelial cell injury induced by histone stimulation.(3)Histone stimulation can cause HIMEC cell damage and v WF release through influx of extracellular calcium ions.Unfractionated heparin can antagonize the extracellular calcium influx caused by histone stimulation,reduce the damage of HIMEC cells and the v WF release induced by histone stimulation.(4)Unfractionated heparin may prevent the intestinal microcirculation dysfunction in sepsis by antagonizing endothelial cell injury caused by extracellular histone.