Amphiregulin Protects Against TNF-?-induced Alveolar Epithelial Cell Death Via EGFR Signaling Pathway

Background: Acute respiratory distress syndrome(ARDS)is a devastating respiratory disease with very high mortality of 35-46%.Until now,the clinical treatment of ARDS is still limited.And the research on the pathogenesis and treatment of ARDS has always been the emphases and difficulties of critical care medicine.The damage of alveolar epithelial cells is the pathologic basis of ARDS.When ARDS happens,lung tissue highly expresses tumor necrosis factor-alpha(TNF-?),activating TNF-?-induced cell death signaling pathway,injuring alveolar epithelial cells.Amphiregulin(Areg)is the member of epidermal growth factor receptor(EGFR),which can promote cell proliferation and inhibit apoptosis.It is reported that Areg can reduce the acute lung injury,but the underlining mechanisms are poorly understood.We hypothesized that Areg can protect against TNF-?-induced alveolar epithelial cell death via activating EGFR signaling pathway and inhibiting TNF-?-induced cell death signaling pathway.Objective: To investigate the protective effect and the underlying mechanism of Areg against TNF-?-induced alveolar epithelial cell death.Methods: There are two parts in the experiments.Experiments in vitro were conducted on the murine lung epithelial cell line(MLE12 cells).MLE12 cells were randomly divided into control group,Areg group,PBS+TNF-? group,Areg+TNF-? group,siRNA-Scramble group and siRNA-EGFR group.Cells in control group were treated with PBS(the same volume as Areg).Cells in Areg group were treated with Areg(50 ng/m L).Cells in PBS+TNF-? group were treated with PBS(the same volume of Areg),and 30 minutes later,they were treated with TNF-?(100 ng/m L).Cells in Areg+LPS group were treated with Areg(50 ng/m L),and 30 minutes later,they were treated with TNF-?(100 ng/m L).Mixing the Opti-MEM with scramble siRNA and the Opti-MEM with Lipofectamine 3000,incubating 10 to 15 minutes.Cells in Scramble-siRNA group were treated with the mixture,culturing 4-6 hours,then replacing the medium to complete medium containing 10% fetal bovine serum.Continuing cultivating 24 hours,then cells were treated with Areg(50 ng/m L),30 minutes later,treated with TNF-?(100 ng/m L).Cells in si-EGFR group were treated with mixture containing EGFR-siRNA instead of scramble siRNA,and the remaining steps are the same as above.TUNEL immunofluorescence,CCK-8 and LDH release of each group were compared.The activation of EGFR signaling pathway and TNF-?-induced cell death signaling pathway were examined by western blotting.Experiments in vivo were conducted on male C57BL/6 mice of 6 to 8 weeks old,weighing 20 to 25 g.Mice were randomly divided into control group,Areg group,PBS+LPS group and Areg+LPS group.Mice in the control group were injected intraperitoneally with phosphate-buffered saline(PBS,the same volume as Areg).Mice in Areg group were injected intraperitoneally with Areg(5 ?g per mouse).Mice in PBS+LPS group were injected intraperitoneally with PBS(the same volume as Areg),and 30 minutes later,they were treated intratracheally with lipopolysaccharide(LPS,3 mg/kg).Mice in Areg+LPS group were injected intraperitoneally with Areg(5 ?g per mouse),and 30 minutes later,they were treated intratracheally with LPS(3 mg/kg).Lung injury score,level of protein,Ig M,neutrophils,IL-6,TNF-? in bronchial alveolar lavage fluid of each group were compared.The activation of EGFR signaling pathway and TNF-?-induced cell death signaling pathway were examined by western blotting.Results: Areg significantly alleviated LPS induced pulmonary histopathological injury,protected alveolar capillary barrier,relieved inflammatory response and greatly reduced TNF-?-induced alveolar epithelial cells apoptosis.Areg promoted the phosphorylation of EGFR and AKT,inhibiting the activation of Caspase 3,Caspase 8 and Caspase 9.Knockdown of EGFR inhibited the phosphorylation level of EGFR and AKT,promoted the activation of Caspase 3,Caspase 8,Caspase 9,and eliminated the protective effect of Areg to alveolar epithelial cells.Conclusion: Areg protects against TNF-?-induced alveolar epithelial cell death via promoting EGFR-signaling pathway and inhibiting TNF-?-induced cell death signaling pathway.