Modulatory Effects Of Fas/Fasl-mediated Pathway On Seawater-induced Apoptosis In Alveolar Epithelial Cells

Background:Drowning is one of the most common causes for human accidental deathsand public health problems in the world. Seawater drowning occurs frequently inagricultural and industrial production, military action and everyday life at sea.During drowning, seawater can block airway and lead to drowning death due toapnea. Seawater into pulmonary alveolar can induce pulmonary edema ofseawater drowning (PE-SWD), seawater drowning-induced acute lung injury(SW-ALI）and further deteriorate into seawater drowning-induced respiratorydistress syndrome (SW-RDS).Seawater can directly damage alveolar epithelial cells (AECs) and then leadto hypoxia and acidosis in SW-ALI. AEC is of critical importance for thepathogenesis and progression as well as resolution of SW-ALI. Previous studieshave showed that Fas/FasL pathway plays a major role in the induction of AECapoptosis during acute lung injury (ALI). Previously, our experiments haveconfirmed that seawater could result in apoptosis in rat lung tissue cells. However,little is known about whether seawater can induce AEC apoptosis via Fas/FasLpathway. Objective:To investigate the effects of seawater on AEC apoptosis and to explorewhether Fas/FasL pathway for its possible contribution to apoptosis in AECthrough establishing rat animal models of SW-ALI and seawater inducing A549cells.Methods:1. Animal experiment:48rats were randomly divided into4groups: Anormal control group，1h seawater group，2h seawater group and4h seawatergroup. After the rats being anesthetised, seawater (4ml/kg body weight) wasinstilled into both lungs with a steady speed in4min except the normal controlgroup. The rats were sacrificed at the indicated time points after seawatertreatment. Lung wet-to-dry ratio (W/D), the protein in bronchoalveolar lavagefluid (BALF) and the leak index of Evans blue (ELI) were measured.Histological sections of rat lungs were stained with haematoxylin-eosin.Expression of Fas and FasL was assessed by immunohistochemistry in lungtissue from the rats4h after seawater intratracheal instillation. Fas, FasL, cleavedcaspase-8and cleaved caspase-3were analysed by western blot analysis.2. Cell experiment: Seawater was added into medium to obtain a finalconcentration of10%,20%,40%and60%(0.1ml,0.2ml,0.4ml and0.6mlseawater per1ml total volume) for6h or40%for1h,2h,4h and6h to inducehuman lung alveolar epithelial (A549) cell apoptosis to examine the effects ofseawater on AEC apoptosis. To further explore the relationship between FasL,caspase-8activation and seawater-induced apoptosis, we chose the40%concentration for6h for our further experiments in A549cells, which werepreincubated in the presence of10μg/ml anti-FasL neutralising antibody NOK-2or20μΜ caspase-8inhibitor Z-IETD-FMK for60min before exposure to seawater. The cells undergoing apoptosis were assayed by Annexin V-FITC andPI staining and flow cytometry. Fas, FasL, cleaved caspase-8and cleavedcaspase-3were analysed by western blot analysis.Results:1. Animal experiment: After seawater instillation, lung W/D ratios, theBALF protein levels and ELI elevated significantly, which showed that seawaterinduced typical ALI associated with histopathological results. The expression ofFas, FasL, cleaved caspase-8and cleaved caspase-3was up-regulated in the ratlungs after the instillation of seawater compared with the normal control group.2. Cell experiment: Treatment with seawater induced a marked increase inthe apoptosis of A549cells in a concentration-and time-dependent manner.Up-regulation in Fas, FasL, cleaved caspase-8and cleaved caspase-3in A549cells was detected by immunoblotting analysis. The incubation of A549cells inthe presence of NOK-2or Z-IETD-FMK resulted in a decrease ofseawater-induced cell apoptosis. NOK-2inhibited Fas and FasL interaction, andreduced the cleavage of caspase-8and caspase-3, but had no effect on Fasexpression. Z-IETD-FMK blocked caspase-8and caspase-3activation and didnot inhibit Fas and FasL expression.Conclusion:Seawater treatment triggered AEC apoptosis, and AEC apoptosis mediatedby the Fas/FasL pathway could play an important role in seawater-induced ALI.This finding may provide some experiment evidences for treating SW-ALI fromthe perspective of apoptosis.