The Notch pathway attenuates burn-induced acute lung injury in rats by repressing reactive oxygen species

Background:Acute lung injury(ALI)is a common complication following severe burns.The underlying mechanisms of ALI are incompletely understood;thus,available treatments are not sufficient to repair the lung tissue after ALI.Methods:To investigate the relationship between the Notch pathway and burn-induced lung injury,we established a rat burn injury model by scalding and verified lung injury via lung injury evaluations,including hematoxylin and eosin(H&E)staining,lung injury scoring,bronchoalveolar lavage fluid and wet/dry ratio analyses,myeloperoxidase immunohistochemical staining and reac-tive oxygen species(ROS)accumulation analysis.To explore whether burn injury affects Notch1 expression,we detected the expression of Notch1 and Hes1 after burn injury.Then,we extracted pulmonary microvascular endothelial cells(PMVECs)and conducted Notch pathway inhibition and activation experiments,via aγ-secretase inhibitor(GSI)and OP9-DLL1 coculture,respectively,to verify the regulatory effect of the Notch pathway on ROS accumulation and apoptosis in burn-serum-stimulated PMVECs.To investigate the regulatory effect of the Notch pathway on ROS accumulation,we detected the expression of oxidative-stress-related molecules such as superoxide dismutase,nicotinamide adenine dinucleotide phosphate(NADPH)oxidase(NOX)2,NOX4 and cleaved caspase-3.NOX4-specific small interfering RNA(siRNA)and the inhibitor GKT137831 were used to verify the regulatory effect of the Notch pathway on ROS via NOX4.Results:We successfully established a burn model and revealed that lung injury,excessive ROS accumulation and an inflammatory response occurred.Notch1 detection showed that the expression of Notch1 was significantly increased after burn injury.In PMVECs challenged with burn serum,ROS and cell death were elevated.Moreover,when the Notch pathway was suppressed by GSI,ROS and cell apoptosis levels were significantly increased.Conversely,these parameters were reduced when the Notch pathway was activated by OP9-DLL1.Mechanistically,the inhibition of NOX4 by siRNA and GKT137831 showed that the Notch pathway reduced ROS production and cell apoptosis by downregulating the expression of NOX4 in PMVECs.Conclusions:The Notch pathway reduced ROS production and apoptosis by downregulating the expression of NOX4 in burn-stimulated PMVECs.The Notch-NOX4 pathway may be a novel therapeutic target to treat burn-induced ALI.