Change And Mechanism Of Alveolar Epithelial Glycocalyx And Tight Junction In ARDS

Background:Acute respiratory distress syndrome(ARDS)is an acute and diffuse lung injury caused by various intrapulmonary and/or extrapulmonary factors.It is characterized by pulmonary vascular endothelial and alveolar epithelial cell damage,increased pulmonary vascular permeability,alveolar permeability,and pulmonary edema.ARDS is a common acute and severe disease with a mortality rate up to 46%.At present,mechanical ventilation is the main treatment for ARDS.Effective drug therapy are still lacking.A large amount of edema fluid escaping from the airway and the chest imaging findings of "white lung" are the typical clinical manifestations of severe pulmonary edema in patients with severe ARDS.ARDS is usually divided into endogenous and exogenous pulmonary ARDS,according to different initial factors.The main inductive factors of pulmonary exogenous ARDS include severe non-pulmonary infection in the abdominal cavity,skin and soft tissue,acute pancreatitis,major extrathoracic trauma,and car accidents.The first damaged cells are usually the pulmonary vascular endothelial cells.In endogenous pulmonary ARDS,such as S ARS,H1N1 influenza,COVID-19 infection,aspiration,drowning,lung contusion,etc.alveolar epithelial cells are the primary target cells to be damaged.The destruction of the epithelial barrier is key to the occurrence and development of ARDS.Studies have shown that injury of endothelial cells alone is not sufficient to induce pulmonary edema.In recent years,increasing attention has been paid to the destruction of alveolar epithelial cells.In particular,for ARDS that is caused by intrapulmonary factors,the structure and function of the alveolar epithelial barrier are key factors.Therefore,an in-depth study into the mechanism of alveolar epithelial barrier injury may provide a breakthrough in the treatment of ARDS.The alveolar epithelial barrier is mainly composed of type I and type II epithelial cells,intercellular juncture,active substances on the cell surface,and the glycocalyx of the alveolar epithelial cells.Alveolar epithelial cells have a sugar-rich external epithelial cell surface layer consisting of negatively-charged polysaccharides and a protein complex of mainly core protein and side chains.The side chain structure mainly consists of heparan sulphate(heparan sulfate,HS),hyaluronic acid,chondroitin sulfate,etc.Of these,the HS content is the highest,accounting for more than 50%of the side chain structure.HS is attached to the sugar packets,contributes to the integrity,and plays an important function role.It is an important component of the alveolar epithelial barrier.Heparinase(HPA)is the only mammalian endoglucuronidase that specifically cleaves the glycoside chain of HS into small fragments and regulates the HS.Heparinase III,a specific type of HS glucuronic acid bacteria enzyme can degrade many sugar packets and side chains.Non-anticoagulant heparin(N-desulfated/re-N-acetylated heparin,NAH)is a competitive antagonist of HPA.At present,most available studies have focused on the glycocalyx of the endothelial cells,while the glycocalyx of alveolar epithelial cells has been neglected.In recent years,an animal model of pulmonary endogenous ARDS has shown that alveolar epithelial cells glycocalyx plays an important role in maintaining lung parenchymal structure and promoting cell signal transduction,and thus is an important barrier to prevent host infection.As an important part of the alveolar barrier,the tight junction between the alveolar epithelium and the saccharide envelope is an important contribution to pulmonary edema in ARDS.Damage to the glycocalyx and tight junctions of the alveolar epithelial cells is an important target of ARDS,especially endogenous ARDS.Therefore,further studies on the damage of the glycocalyx and tight junctions of the alveolar epithelial cells during the development of ARDS and its potential mechanism will provide a new target for clinical drug treatment of ARDS.This study aimed to investigate the glycocalyx and tight junctions of the alveolar epithelial cells.The study will include four parts(as shown below).The specific technical route is shown in the figure A(page 15).PART 1:Effect of LPS on the glycocalyx side chain HS in alveolar epithelial cellsObjective:Increased permeability caused by the destruction of the alveolar barrier is an important factor in the development of pulmonary edema in ARDS.In recent years,it has been found that the epithelial glycocalyx is an important component of the alveolar barrier,and its damage may be involved in the formation of pulmonary edema in ARDS.The purpose of this study was to observe changes in the glycocalyx side chain HS in the alveolar epithelium in LPS-ARDS.Methods:C57BL/6 mice were used to establish a mouse model of ARDS by intratracheal infusion of LPS.The mice were randomly divided into the normal control and LPS groups.In the in vitro xperiment,human alveolar epithelial cells(A549)were divided into normal control and LPS groups.The degree of lung injury was assessed by observing the histopathological changes of the lung tissue.The change in alveolar permeability was assessed by measuring the wet/dry ratio(W/D)of lung tissue.Immunofluorescence was used to analyze the changes in the glycocalyx HS in mouse alveolar epithelial cells and human alveolar epithelial cells.Results:Compared with the control group,the pathology of lung tissue in the LPS group showed increased inflammatory cell infiltration,thickened alveolar septum,increased W/D,increased alveolar exudation,and formation of pulmonary edema.Immunofluorescence results showed that the expression of HS in mouse alveolar epithelial cells and human alveolar epithelial cells in the LPS groups was lower than that in the normal groups(P<0.05).Conclusion:LPS reduced the expression of the glycocalyx side chain HS in ARDS alveolar epithelial cells.PART 2:Effects of HPA on the structure and function of the glycocalyx of alveolar epithelial cells in LPS-induced ARDSObjective:HPA is an HS-specific regulatory enzyme and non-anticoagulant heparin.N-desulfated/re-N-acetylated heparin(NAH)is a competitive antagonist of HPA.The main objective of this study was to investigate the possible mechanism of reduced epithelial HS expression in LPS-induced ARDS.Methods:C57BL/6 mice and human alveolar epithelial cells were used in the research.The mouse ARDS model was established by intratracheal infusion of LPS.The human alveolar epithelial cells(A549)were stimulated by LPS to establish a cell injury model.For both the in vivo and in vitro experiments the mice and human alveolar epithelial cells were divided into the normal control group,LPS group,heparinase Ⅲ(exogenous add HPA)group,the LPS+NAH competitive antagonist(HPA)group,the inactivated heparinase Ⅲ group,and the NAH group.The degree of lung injury was assessed by observing the histopathological change in the lung tissue.The change in alveolar permeability was assessed by measuring the wet/dry ratio(W/D)of lung tissue.The levels of HS in the bronchoalveolar lavage fluid(BALF),blood,BALF/blood,and cell supernatants were determined by ELISA to assess the degree of degradation of the glycocalyx of alveolar epithelial cells.Immunofluorescence was used to analyze the changes in the glycocalyx HS in epithelial cells.Ultrastructural changes in the epithelial cells of the glycocalyx in mice were observed by transmission electron microscopy.Results:HE staining of the in vivo LPS and heparinase Ⅲ groups mice HE staining showed increased inflammatory cell infiltration,increased alveolar exudate,thickening of the alveolar interval,and increased W/D.At the same time,the HPA inhibitor NAH pretreatment reduced thepathological changes and W/D.The ELISA results showed that the HPA content in the cell supernatant of the LPS group was significantly increased.In the LPS and heparinase Ⅲ groups,the BALF supernatant fluid loss of the HS content in serum and BALF was higher than that in the normal group,and the NAH pretreatment group showed an improvement in the LPS group(P<0.05).Immunofluorescence showed that the LPS and heparinase Ⅲ groups of mice alveolar epithelial cells and the HS group of human alveolar epithelial cells were lower than those in the normal group,and the NAH pretreatment group showed greater improvement than in the LPS group(P<0.05).Transmission electron microscopy(TEM)showed that the alveolar epithelial cells in the normal group were continuous,while the alveolar epithelial cells were partially or completely shed after LPS stimulation.The alveolar epithelial cells in the NAH preconditioning group were also somewhat relieved.Conclusion:The degradation of the glycocalyx side chain HS in LPS-induced ARDS is related to an increase of the specific regulatory enzyme HPA,and the competitive antagonist of HPA,NAH,that can inhibit the degradation of HS by HPA.Moreover,NAH can reduce the damage of LPS to HS,protect the integrity of alveolar epithelial cells and improve pulmonary edema.PART 3:Changes of LPS on tight junction of the alveolar epithelial cells in ARDSObjective:Alveolar epithelial glycocalyx and tight junction constitute the alveolar barrier.Its destruction is an important pathophysiological mechanism of pulmonary edema in ARDS.The results of the first and second parts of this study showed that LPS could lead to the destruction of glycocaly in the alveolar epithelial cells of ARDS,which was associated with the formation of pulmonary edema.However,the disruption of the tight junctions of epithelial cells is also closely associated with the development of pulmonary edema.The purpose of this study was to observe the changes of tight juncture of alveolar epithelial cells in ARDS induced by LPS.To further explore the mechanism of lung injury caused by LPS.Methods:C57BL/6 mice and human lung epithelial cells(A549)were used in the research.The mouse ARDS model was established by intratracheal infusion of LPS.The human alveolar epithelial cells were stimulated by LPS to establish a cell injury model.Experiments in vivo and in vitro were divided into the normal control group,LPS group,the heparinase III group,LPS+NAH group,heparinase Ⅲ group,and NAH group.The changes in tight junction proteins(occludin,ZO-1,and claudin 4)in the epithelial cells were analyzed by immunofluorescence and western blot.The ultrastructure of tight junctions of the mouse alveolar epithelial cells was observed using transmission electron microscopy.Results:Immunofluorescence analysis showed that the expression levels of the tight junction proteins occludin,ZO-1,and claudin 4 in mice in the LPS group and heparinseⅢ groups were lower than those in the normal group.After LPS and heparinse Ⅲstimulation of alveolar epithelial cells,immunofluorescence and western blot showed that occludin,ZO-1,and claudin 4 expression levels were lower than those in the normal group.The damage to tight junction proteins in the NAH pretreatment group was reduced.The heparinase Ⅲand NAH groups showed no difference between the normal group.Transmission electron microscopy showed that the tight junctions of alveolar epithelial cells were intact in the normal group,while cracks between cells were widened and the structure of tight junction proteins was destroyed after LPS stimulation.Damage to tight junctions of the alveolar epithelial cells was improved in the NAH pretreatment group.Conclusion:(1)LPS can destroy tight junction proteins(occludin,ZO-1,and claudin 4)in alveolar epithelial cells,leading to the destruction of the alveolar barrier.(2)Heparinse Ⅲ can damage the alveolar epithelial cells glycocalyx HS and can also cause the tight junction protein(occludin,ZO-1,and claudin 4)of epithelial cell damage increase,cause alveolar permeability increase and the destruction of the epithelial barrier.(3)NAH,a competitive antagonist of heparinase,can reduce the degradationof glycocalyx HS in alveolar epithelial cells and the damage of tight junction proteins between epithelial cells,thus improving the alveolar barrier.PART 4:Explore the correlation between glycocalyx side chain HS and tight junction damageObjective:Previous studies have found that exogenous HPA can destroy the glycocalyx HS of alveolar epithelial cells,and can also cause damage to tight junction proteins of the epithelial cells(occludin,ZO-1,and claudin 4)to increase,leading to an increase in alveolar permeability.The HPA competitive antagonist NAH can reduce the degradation of the glycocalyx HS of the alveolar epithelial cells,the close connection between epithelial cell protein damage and improve the alveolar barrier.HPA is an HS-specific regulatory enzyme.HPA does not act directly on tight junctions,and the damage of tight junctions increases after HPA degrades the glycocalyx side chain HS,suggesting a potential correlation between tight junctions and the glycocalyx side chain HS.The purpose of this study was to further investigate the possible mechanisms of HPA-HS and tight junction injury.Methods:(1)To investigate the relationship between HPA-HS and ZO-1 damage,human alveolar epithelial cells(A549)were stimulated with endogenous recombinant HPA(2 U/mL)for 12h to establish a cell injury model.The cells were divided into the normal and recombinant HPA groups.Immunofluorescence was used to observe the changes in the HS-expression in cells and to analyze the changes of HS in the glycocalyx side chain.The expression of the tight junction protein ZO-1 was observed using cellular immunofluorescence and western blot.The expression of tight junction proteins(ZO-1 mRNA)was observed using real-time quantitative PCR.(2)To investigate whether the effect of recombinant HPA on tight junction proteins is related to the STAT3 signaling pathway.Human alveolar epithelial cells were divided into normal control,recombinant HPA,recombinant HPA+STAT3-IN-1(STAT3 inhibitors),and STAT3-IN-1 groups.The differences in the expression of p-STAT3 in each group were detected by western blot.Results:(1)Immunofluorescence analysis showed that recombinant HPA decreased the expression of glycocalyx HS in cells(P<0.05).At the same time,cell immunofluorescence analysis and western blot showed that the expression of tight junction protein ZO-1 in the recombinant HPA group was disrupted compared with that in the normal group(P<0.05).The ZO-1 mRNA in human alveolar epithelial cells stimulated by the recombinant HPA group was also significantly decreased(P<0.001).(2)Western blot results showed that there was no significant difference in p-STAT3 expression the control,HPA,HPA+STAT3-IN-1,and STAT3-IN-1 groups.Conclusion:(1)Recombinant HPA can degrade alveolar epithelial cells,HS,and destroy the tight junction protein ZO-1 of the alveolar epithelial cells.Degradation of the glycocalyx side chain HS inhibits the expression of tight junction ZO-1 proteins in alveolar epithelial cells,and the mechanism is related to the reduction of ZO-1 mRNA synthesis.(2)The STAT3 signaling pathway does not contribute to the effect of recombinant HPA-HS on tight junction proteins of epithelial cells.In conclusion,LPS can increase the HPA of alveolar epithelial cells,destroy the glycocalyx HS,and impair the glycocalyx structure and function,thus participating in the formation of pulmonary edema of ARDS.In the process of LPS lung injury,the structure and function of tight junction proteins are also damaged,which together with the damage of glycocalyx are involved in the formation of pulmonary edema in ARDS,which provides a new way for ARDS drug treatment and application.The relationship between LPS-HPA-HS and tight junction injury needs further study.