The Role Of RIPK3-Dependent Necroptosis In Airway Epithelial Cell Injury

BackgroundsAcute lung injury(ALI) and acute respiratory distress syndrome(ARDS) is one of the most common emergency in the Department of respiration, which can be induced by sepsis.Bacterial lipopolysaccharide(LPS) is one of the main components of the cytoderm of gram negative bacilli.As the main pathogen of gram negative bacilli, it can cause strong immune response in vivo. Airway epithelial cells play an important role in initiation and maintenance inflammation of acute lung injury. Airway epithelial cells may damage 、proliferate even die in acute lung injury patients.Injury or death airway epithelial cells release a variety of cytokines and chemotactic factor activing immune cells, initiating inflammatory responses.Moreover the dysfunction of the airway epithelial cells barrier promotes foreign antigens directly contact with submucosa immune cells,which aggravates the inflammatory response. Necroptosis, programmed necrosis, is a newly discovered programmed cell death mode, which is regulated by RIPK1, RIPK3, MLKL and so on.Necroptosis is found to be a highly pro-inflammatory cell death and plays an important role in a variety of acute chronic inflammation.However,whether it is involved in inflammation of airway epithelial cells in acute lung injury has not been reported so far.Object:To explore the role of RIPK3-dependent necroptosis in inflammation of airway epithelial cells in acute lung injury.Methods:This experiment used bacterial lipopolysaccharide stimulating human airway epithelial cell line HBE,then detected necroptosis of airway epithelial cells by flow cytometric and tested the expression change of main signal molecule in necroptosis via western blot.Results:1. LPS induces death of airway epithelial cellsHBE cells were stimulated 24 h with LPS, the results showed that LPS increased the proportion of HBE death namely positive ratio of Annexin V+/PI+ and Annexin V+/PI-(23.90% + 1.749% vs 9.056% + 0.8710%, P < 0.05, n=9).2. LPS induces necroptosis in airway epithelial cells rather than apoptosis or pyroptosisHBE cells pre-treated with RIPK3 inhibitor dabrafenib(Da), and death rates of HBE cells was significantly reduced compared with that of directly added LPS stimulation(16.52% + 2.264% vs 23.90% + 1.749%, P < 0.05, n = 9); HBE cells anticipated caspase inhibitor z VAD fmk(Z) to inhibit apoptosis and pyroptosis, the death ratio of HBE cells did not significantly reduce(18.53% + 2.316% vs 22.29% + 1.890%, P > 0.05, n = 4).3. The role of RIPK3-MLKL signal pathway in LPS induced injury of airway epithelial cellsLPS upregrates the expression of p-MLKL,which is the activation of MLKL as the substrate of RIPK3; RIPK3 inhibitor dabrafenib partly reduced expression of p-MLKL.Conclusion:The experiment suggests that LPS can induce necroptosis of the airway epithelial cells.LPS promotes necroptosis pathway signal molecule MLKL to be active. RIPK3 inhibitors dabrafenib can partly alleviate necroptosis of airway epithelial cell caused by LPS.It reduces the activation of MLKL,which is the downstream signaling molecules of RIPK3.