| Background: Severe burn is a disease with high mortality.Shock that occurs in the early stage after severe burn is the main cause of death,and it is also the focus and difficulty of treatment.However,the pathological mechanism of shock in the early stage of severe burns have not been fully elucidated.It has been reported that heparin binding protein(HBP)and myeloperoxidase(MPO)secreted by neutrophils may be involved in vascular leakage and glycocalyx injury,respectively,but their mechanism of action in severe burns is unknown.Objective: To study the synergistic effect and specific mechanism of HBP and MPO produced by degranulation of neutrophils on shock in the early stage of severe burns,and provide new ideas for the treatment of shock after severe burns.Methods: This study is divided into three parts: clinical observation,in vitro experiment and animal experiment.In the clinical observation part,ELISA was used to detect the expressions of plasma granule proteins(HBP,MPO,MMP9,NE)and glycocalyx injury-related molecules(HA,HS,SDC-1)in patients with early severe burns.Agarose chemotaxis assay was used to detect neutrophil chemotaxis.The expression of ROS and apoptosis of neutrophils were detected by flow cytometry.The number of neutrophils was detected by automatic hemocytometer.Quantitative proteomics was used to detect the difference of plasma protein expression between healthy volunteers and severely burned patients and cluster analysis was performed.In the in vitro experiment,the immune cells and proteins that cause the increase of vascular endothelial permeability were screened by the permeability test of single-layer endothelial cells.Caspase-3 and other apoptosis related molecules were detected by RTPCR after r HBP stimulated HMEC-1 cells.The expression of CD35,CD63,ROS and HOCl in neutrophils were detected by immunofluorescence.Flow cytometry was used to detect the expression of CD44 in HMEC-1 cells treated with different recombinant proteins and inhibitors.The expression of intracellular domain(ICD)and extracellular domain(ECD)of CD44 molecule in HMEC-1 cells under different stimulation conditions such as HOCl was detected by immunofluorescence.RT-PCR was used to detect the expression of CD44 m RNA in HMEC-1 cells after r MPO stimulation.In the animal experiment,20% TBSA third degree burn mice were prepared by scalding in boiling water at 100 ℃ for 10 s,and edema of lung,spleen and other major organs in the burn group and sham group was observed by HE staining.Evans blue staining and wet dry ratio method were used to detect vascular leakage and pulmonary edema in 20% TBSA burn-model-mice and sham injured mice.The in vivo imaging model of vascular leakage in mice was established by tail vein injection of fluorescently labeled dextran.r HBP and / or aprotinin were injected into tail vein to observe the vascular leakage of mice.30% TBSA third degree burn rats were prepared by boiling water at 100 ℃ for 13 s.The effects of low molecular weight heparin(LMWH)and methylprednisolone(MP)on rat vascular glycocalyx were detected by transmission electron microscopy,and the expression of HA,HS and SDC-1 in rat plasma was detected by ELISA.Neutrophils were labeled with fluorescent antibodies and HOCL was injected into the tail vein of mice.Laser confocal microscopy was used to observe the expression of CD44 molecule in vascular endothelial cells,neutrophil adhesion and vascular leakage in mice.r HBP and r MPO were used in combination to observe the vascular leakage by in vivo vascular imaging in mice.MPO inhibitor was used to intervene mice in burn group and sham group,and the thickness of glycocalyx was observed by transmission electron microscope.Pulmonary vascular leakage was detected by Evans blue staining after the intervention of HBP and MPO combined inhibitor in the burn group and sham group.Results: The clinical observation data showed that the percentage of neutrophils in the severely burned group increased(84.1 ± 5.3%,58 ± 3.7%,P < 0.0001),the chemotactic distance shortened(1161.8 ± 266.1μm,2103.5 ± 171.7μm,P < 0.0001),the percentage of apoptosis decreased(19.9 ± 11.1%,60.3 ± 7.8%,P < 0.0001),and the expression of cytosolic ROS decreased(636 ± 204.8.8a.U.,1376.2 ± 121.8a.U.,P < 0.0001)compared with the healthy control group.The expression of neutrophil related granule protein HBP(539.1 ± 206.5ng/ m L,63.7 ± 14ng/ m L,P < 0.0001),MPO(622.4± 380.3ng/ m L,350.3 ± 20.2ng/ m L,P < 0.05),NE(302.9 ± 157.9ng/ m L,49.7 ± 13ng/ m L,P < 0.0001),MMP-9(518.1 ± 197.1ng/ m L,298.9 ± 25.5ng/ m L,P < 0.001)in the severe-burned group was significantly higher than that in the healthy control group.The expression of glycocalyx injury related molecules HA(20.27 ± 2.99ng/ m L,5.41 ± 1.19ng/ m L,P < 0.0001),HS(91.6 ± 17.4ng/ m L,17.68 ± 3.63ng/ m L,P < 0.0001)and SDC-1(11.98 ± 2.58ng/ m L,5.07 ± 0.81ng/ m L,P < 0.0001)were significantly increased in the severely burned group compared with the healthy control group.Quantitative proteomic analysis showed that the expression of CD44 and ICAM-1 molecules in peripheral blood of patients with severe burns in the early stage was significantly higher than that of healthy volunteers(P < 0.001,P < 0.0001).The in vitro data showed that the permeability of HMEC-1 monolayer cells increased after the neutrophils of healthy volunteers were co incubated with HMEC-1 compared with the untreated control group(7.67 ± 0.66ng/ m L,4.97 ± 0.31 ng/m L,P < 0.01).After the neutrophils of severely burned patients were co incubated with HMEC-1,the permeability of HMEC-1 monolayer cells increased significantly compared with the untreated control group(14.17 ± 1.65ng/m L,7.67 ± 0.66ng/m L,P < 0.0001).Compared with the control group,the permeability of HMEC-1 monolayer cells in r HBP group was significantly increased(15.8 ± 1.64ng/m L,4.69 ± 0.77ng/m L,P < 0.001).The permeability of HMEC-1 monolayer cells in r HBP + aprotinin group(9.32 ± 2.56ng/ m L)was significantly decreased compared with r HBP group(15.8 ± 1.64ng/m L,P < 0.05).There was no significant difference between r HBP + aprotinin group and the control group(P = 0.072).Quantitative proteomics showed that the expression of CD44 and ICAM-1 molecules in peripheral blood increased significantly in the early stage of severe burns(P < 0.001,P < 0.0001).Immunofluorescence data showed that transient thermal stimulation could induce the increase of the expression of CD35 and CD63 on the neutrophil membrane and the expression of ROS and HOCl in the cytoplasm.Compared with the control group,the expression of CD44 in the HOCl intervention group was significantly decreased(12.33 ± 0.79%,98 ± 1.15%,P < 0.0001).Immunofluorescence data showed that HOCl induced the shedding of the extracellular domain of CD44 molecule in HMEC-1 cells,but had no significant effect on the intracellular domain of CD44 molecule.After HMEC-1 cells were treated with r MPO,the expression of CD44 m RNA in HMEC-1 cells was significantly decreased at 4h,8h,24 h and 48 h compared with the control group(P < 0.05,P < 0.01,P < 0.01,P < 0.001).The data of animal experiments showed that the HE staining of the lungs of the mice in the burn group was significantly larger than that in the sham injury group.In vivo vascular imaging data showed that r HBP could significantly induce vascular leakage in mice,and the induction of r HBP could be significantly inhibited by aprotinin.The results of Evans blue staining showed that the concentration of Evans blue in the lungs of the r HBP group(124.46 ± 6.01μg/m L)was significantly higher than that of the control group(64.61 ± 2.16μg/m L)(P < 0.0001).The r HBP+20% TBSA group(158.64 ± 20.6μg/m L)was significantly higher than the control group(64.61 ± 2.16μg/m L)(P < 0.0001).The pulmonary Evans blue concentration in the r HBP+20% TBSA group(158.64 ± 20.6μg/m L)was significantly higher than that in the 20% TBSA group(114.4 ± 13.06μg/m L)or the r HBP group(124.46 ± 6.01μg/m L)(P < 0.0001,P < 0.001).Compared with the r HBP+20% TBSA group(158.64 ± 20.6μg/m L),the r HBP+aprotinin+20% TBSA group(98.37 ± 7.76μg/m L)significantly decreased the concentration of Evans blue in the lungs(P < 0.0001).There was no significant difference in aprotinin+20% TBSA group(78.08 ± 2.14μg/m L)compared with 20% TBSA group(114.4 ± 13.06μg/m L)(P = 0.0614).The results of transmission electron microscopy showed that LMWH and MP could antagonize the injury of rat vascular glycocalyx caused by burn injury.The data of in vivo vascular imaging and scanning electron microscopy in mice showed that HOCl downregulated the expression of CD44 on the surface of vascular endothelial cells in mice,induced increased adhesion of neutrophils to the vascular wall,and aggravated vascular leakage after burn;Using r HBP or r MPO separately can induce vascular leakage in vivo in mice,and the induction effect of HBP is more significant than that of MPO.Combined use of r HBP and r MPO can induce vascular leakage in mice faster and more significantly.The transmission electron microscope data also showed that the use of the MPO inhibitor AZD5904 could significantly antagonize the injury of blood vessel glycocalyx in mice after burn.At 4h after burn,there was no significant difference in glycocalyx thickness between the AZD5904 + burn group(250.3 ± 23.1nm)and the burn group(221.7 ± 8.5nm)(P = 0.8005);At 24 h after injury,the glycocalyx was significantly thicker in the AZD5904 + burn group(228.3 ± 14.5nm)than in the burn group(75.7 ± 16.3nm)(P < 0.001),and there was no significant difference in the thickness of the glycocalyx between the AZD5904 + burn group(228.3 ± 14.5nm)and the sham group(325.3 ± 21.5nm)(P = 0.0551).The combined use of HBP inhibitor aprotinin and MPO inhibitor AZD5904 intervened burn mice.Evans blue staining data showed that the content of Evans blue in the lungs of burn r HBP group(158.93 ± 15.96μg/m L)was significantly higher than that of sham group(57.96 ± 3.28μg/m L)(P < 0.0001),and the content of Evans blue in the lungs of burn + r HBP + combined-inhibitor group(106.47 ± 10.02μg/m L)was significantly lower than that of burn r HBP group(158.93 ± 15.96μg/m L)(P < 0.001),but it was significantly higher than that in the sham group(57.96 ± 3.28μg/m L)(P < 0.001),and there was no significant difference in the content of Evans blue in the lung between the sham group(57.96 ± 3.28μg/m L)and the sham + combined-inhibitor group(70.91 ± 7.9μg/m L)(P = 0.1058).The combined use of HBP inhibitor aprotinin and MPO inhibitor AZD5904 intervened burn mice.Evans blue staining data showed that the content of Evans blue in the lungs of burn r HBP group(158.93 ± 15.96μg/m L)was significantly higher than that of sham group(57.96 ± 3.28μg/m L)(P < 0.0001),and the content of Evans blue in the lungs of burn + r HBP + combined-inhibitor group(106.47 ± 10.02μg/ m L)was significantly lower than that of burn r HBP group(158.93 ± 15.96μg/ m L)(P < 0.001),but it was significantly higher than that in the sham group(57.96 ± 3.28μg/ m L)(P < 0.001),and there was no significant difference in the content of Evans blue in the lung between the sham group(57.96 ± 3.28μg/ m L)and the sham + combined-inhibitor group(70.91 ± 7.9μg/ m L)(P = 0.1058).Conclusions: This study found that HBP and MPO secreted by neutrophil degranulation can synergistically induce vascular leakage in the early stage of severe burns.HBP acts on vascular endothelial cells and significantly increases vascular endothelial permeability.The MPO catalyzed product HOCl induces the shedding of CD44 molecules on vascular endothelial cells and then damages the glycocalyx.After glycocalyx injury,the adhesion between neutrophils and vascular endothelial cells increases,which further triggers neutrophils to release HBP and continuously increases vascular endothelial permeability,and the two act synergistically to induce vascular leakage.This study reveals the specific mechanism of HBP and MPO synergistically inducing vascular leakage in the early stage of severe burns,which will provide a new idea for the treatment of shock in the early stage of severe burns. |
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