The Role Of Connexin Hemichannels In Acute Lung Injury

Background:Acute lung Injury(ALI)and its more severe form,Acute Respiratory Distress Syndrome(ARDS),refer to the excessive inflammatory response in the lung tissue after the body is hit by infection,trauma and sepsis,etc.,which is characterized by massive inflammatory cell infiltration in the lung tissue and increased pulmonary vascular permeability.The mechanism of the disease is complex and the fatality rate is high,which has become a focus of clinical research.ARDS is characterized by intractable hypoxemia,presenting as acute progressive dyspnea.Currently,the combination of drug and non-drug therapy has become a recognized direction for ARDS treatment.But so far it appears to only improve short-term clinical effects,but not long-term survival rate.Therefore,it is still necessary to continue to explore the pathogenesis of ARDS and develop related drugs.High Mobility Group Box 1(HMGB1),initially identified as a nuclear DNA binding protein,is essential for transcriptional regulation and gene expression.In addition,a large number of clinical and experimental research have shown that extracellular HMGB1 is closely related to the pathogenesis of ALI/ARDS.However,the regulatory mechanism of HMGB1 release is still not fully elucidated.There are about 20 members in the connexin(Cx)family,which form not only the gap junction between cells,but also the hemichannel to communicate the internal and external environment of cells.These channels may play important roles in a variety of physiological and pathological processes.However,due to the lack of means to selectively interfere with gap junction or hemichannel,the function of hemichannel has not been clearly established.EL1P is a broad-spectrum selective hemichannel blocker.Previous studies have shown that EL1P can effectively inhibit the permeability(broad-spectrum)of macrophages(expressing Cx43 and Cx37)but does not interfere with the Cx43 phosphorylation of NIH3T3 fibroblasts and the formation and function of gap junctions(selective-hemichannel).It was also found that EL1P can selectively inhibit the secretion of HMGB1 from macrophages induced by LPS,indicating that hemichannels are important regulator of HMGB1 release.Because the amino acid sequence of EL1P is a relatively conserved region in different Cxs,EL1P is probably to inhibit permeability of hemichannels formed by other Cxs.We have to futher determine the property of EL1P as a hemichannel blocker.Due to the important role of extracellular HMGB1 in the pathogenesis of ALI/ARDS,the hemichannel is likely to be involved in the pathogenesis of inflammatory diseases such as ALI/ARDS by regulating HMGB1 release.Objective:1.To determine the property of EL1P as a broad-spectrum hemichannel blocker2.To research the role of Cx hemichannels in ALI with EL1P.Methods:1.Establishment of a hemichannel blocker.Based on the highly homologous amino acid sequence in the first extracellular ring of the gap junction protein family,we designed a Cx mimic peptide(EL1P)of which the amino acid sequence is ENVCYD.By detecting the effect of EL1P on the hemichannels of HUVEC,HepG2 cells and Cx43-deficient mouse cortical astrocytes,and its property as a wide-spectrum selective hemichannel blocker was determined.2.Human cord vein endothelial cells(HUVEC)were used to detect the level of HMGB1 in the medium after LPS stimulation by EL1P,so as to determine the effect of EL1P on the release of pro-inflammatory factor HMGB1 in these cells.3.To detect the effect of EL1P on the hemichannel permeability of lung cells in vivo.lung injury was induced by ip of bacterial endotoxin twenty hours later,saline(control group)or EL1P(EL1P group)was administrated through ip,followed by a hemichannel tracer Lucifer yellow,15 minutes later the lung was pulm-cardiacly perfused,fixed and cut into sections,which were subsequently stained with Lucifer yellow and DAPI fluorescence,signal ratio was used to determine by Image J software.4.The role of hemichannel in acute lung injury.Acute lung injury was prepared by injecting LPS into the trachea.Intraperitoneal injection of saline(control group)or EL1P was performed 2 and 20 h after lung injury.Subsequent observation or detection: mouse mortality,HMGB1,IgM,ALT,AST and LDH levels in branchoalveolar lavage fluid,lung tissue Cx37,Cx40,Cx43 hemichannel protein levels,alveolar leukocyte infiltration and other indicators.5.Student's t-test was used for statistical analysis of experimental data.The p values of the following experimental results were all less than 0.05.Results:Under culture conditions,EL1P can significantly inhibit the hemichannel permeability of PMVEC,HepG2 cells,and Cx43-deficient mouse cortical astrocytes.Because these cells express different Cxs,these results suggest that EL1P is a broad-spectrum hemichannel blocker.In vitro,EL1P inhibited the release of HMGB1 from HUVEC.In vivo,EL1P did not significantly affect the levels of Cx37,Cx40,Cx43,but reduced lung cell hemichannel permeability.The survival rate of mice with acute lung injury was significantly increased by delayed injection of EL1P.The level of Leukocyte infiltration in ALI lung was reduced.The levels of HMGB1,ALT,AST,LDH and IgM in branchoalveolar lavage fluid of mice in the EL1P group were lower than those in the control group.Conclusions:Hemichannels play an important role in the process of acute lung injury.EL1P is a broad-spectrum hemichannel blocker,which can protect lung tissue by blocking the hemichannel mediated HMGB1,and may have the potential to treat acute lung injury and acute respiratory distress syndrome.