Effects Of Connexin43on Seawater Drowning Induced Pulmonary Edema And Its Mechanism

Background:Seawater drowning is the common cause of accidental death. Duringdrowning, the alveolar-capillary membrane is damaged, resulting in disruptionof barrier function and accumulation of fluid and protein in the alveolar space.Therefore, pulmonary edema induced by seawater drowning (PE-SWD) ischaracteristic of lung injury induced by seawater aspiration. PE-SWD ischaracterized by the increased of pulmonary permeability, hyoxemia anddyspnea. And in severe cases, PE-SWD can lead to seawater respiratorydistress syndrome (SW-RDS), which is one of the acute respiratory distresssyndrome (ARDS). In recent years more and more researches are focus onPE-SWD/SW-RDS, however, the pathogenesis is not well established. Becauseof the absence specific and effective treatment, PE-SWD/SW-RDS is still at ahigh mortality.Connexion43(Cx43) is the major connexin in alveolar epithelium and capillary endothelium. Cx43does not only form transmembrane channels, whichprovide direct intercellular exchange of information and direct cytoplasmiccontinuity between adjacent cells, but also plays an important role forpulmonary development, differentiation, and cell apoptosis. The expression ofCx43in the lung may be altered in lung injury such as radiation-inducedpulmonary fibrosis, lipopolysaccharide or bleomycin-induced lung injury, andeven lung cancer. What’s more, it is reported that Cx43is associated withvascular permeability. However, there is little evidence regarding the role ofCx43in PE-SWD/SW-RDS. The aim of this study was to investigate the role ofCx43in PE-SWD/SW-RDS and its mechanism.Objective:⑴To investigate the role of Cx43in pulmonary permeability, both in vivoand in vitro, following exposure to seawater.⑵To discover the potential mechanism of Cx43on PE-SWD/SW-RDS.Method:PartⅠAnimal experiment24rats were randomly divided into4groups: pre-seawater aspiration group(0h), and1,4, and8h post-seawater aspiration groups (1h,4h,8h). PE-SWDmodels were reproduced by drip seawater into trachea. The lung tissuewet-to-dry weight ratio (W/D ratio), protein concentration in bronchoalveolarlavage fluid (BALF), Evans blue leak index (ELI), in order to investigate thechanges in pulmonary barrier function. Cx43expression was assessed byRT-PCR and Western Blot.Cell experimentCultured A549cells were randomly divided into2groups: control group (Control) and seawater exposure group (SW). For SW group, cells were exposedto25%seawater for4h. Cx43expression was assessed by RT-PCR and WesternBlot. Co-cultured monolayer A549cells permeability was assessed toinvestigate the altered barrier function by FITC-dextran.PartⅡInhibitors of mitogen-activated protein kinase signalling pathways wereused to explore the mechanism by which Cx43altered pulmonary barrierfunction following seawater aspiration1. A549cells were divided into2groups: control group (Control) andseawater exposure group (SW), Western Blot was performed to investigate therole of seawater on MAPKs.2. Alveolar typeⅡcells were isolated from normal rats, and divided intocontrol group (Control), seawater exposure group (SW), PD98059group (PD),SP600125group (SP), SB203580group (SB). For SW group, cells wereexposed to25%seawater for4h. For PD, SP, SB groups, the cells wererespectively pre-treated with MAPK inhibitors by incubation with PD98059, SP600125(μM), or SB203508(50μM) for30min. The expression of Cx43wasassessed by FCM after staining with fluorescein-conjugated (FITC) goatanti-mouse IgG.3. A549cells were divided into6groups: control group (Control), seawaterexposure group (SW), PD98059group (PD), SP600125group (SP), SB203580group (SB) and ASODN group (ASODN). The cell treatments were followed theabove description. For ASODN group, freshly diluted ASODN (30μM) wasthen applied to the cell culture for48h as a100μL/mL liposomal DOTAPpreparation. Cx43expression was assessed by Western Blot. Co-culturedmonolayer A549cells permeability was assessed to investigate the altered barrier function by FITC-dextran. Pearson’s correlation was used to assess thecorrelation between Cx43expression and the permeability of the cellmonolayers.Results:The lung tissue W/D ratio, protein concentration in BALF, ELI andpermeability of cell monolayers were increased following seawater aspiration.Seawater aspiration in rats, exposure of both alveolar type II (ATII) cells andA549cells to seawater resulted in the up-regulation of Cx43expression.Up-regulation of Cx43by incubation with inhibitors of extracellularsignal-regulated kinase (ERK)1/2and c-Jun N-terminal kinase (JNK) increasedthe permeability of cell monolayers, whereas down-regulation of Cx43with anantisense oligonucleotide (ASODN) decreased the permeability. Cx43expression was positively correlated with pulmonary permeability.Conclusion:⑴Seawater aspiration induced up-regulation of Cx43, resulting inincreased of pulmonary permeability and disruption of pulmonary barrierfunction. And the expression of Cx43was associated with pulmonarypermeability.⑵Seawater induced the increased Cx43expression through ERK1/2andJNK MAPK signalling pathways.