NETs Inhibit GPX4 And Promote Pyroptosis Of Alveolar Macrophages Exacerbating Sepsis-Associated Lung Injury

Objective:In vivo and in vitro models of lung injury caused by sepsis,the molecular mechanism of NETs regulating oxidative stress in alveolar macrophages and inducing pyroptosis was investigated.Methods:1、The sepsis model of mice was constructed by cecal puncture and ligation technology.Hematoxylin-Eosin(HE)staining was used to detect the severity of lung injury in Sham group,CLP group and CLP+DnaseΙ group.The protein expressions of NLRP3,GSDMD,Caspase-1,GPX4 and Cit H3 in the lung tissues of the above groups were detected by Western Blot,and the expressions of IL-1β and TNF-α in bronchoalveolar lavage fluid were detected by enzyme-linked immunosorbent assay(ELISA).The expression of GPX4 m RNA in lung tissues was detected by qRT-PCR.2、Peripheral blood neutrophils of rats were extracted with kits and NETs were induced,and identified by immunofluorescence.3、The rat alveolar macrophage(AM)cells were grouped into CON,LPS,LPS+NETs,LPS+NETs+DnaseΙ group.Western Blot and qRT-PCR detected the expression levels of NLRP3,GSDMD and Caspase-1 in pairs after NETs stimulation in AM cells.The changes of reactive oxygen species(ROS)were detected by the kit and fluorescence technology,the content of MDA was detected by the kit,the content of IL-1β and IL-18 in the supernatant of cells was detected by ELISA,and the cell viability was detected by CCK-8.4、The experimental groups were CON,LPS,LPS+NETs and LPS+NETs+NAC groups.Western Blot was used to detect the contents of NLRP3,GSDMD and Caspase-1 in AM cells after the ROS scavenger NAC was used to regulate reactive oxygen species.The contents of IL-1β and IL-18 in the supernatant were detected by ELISA,the cell viability was detected by CCK-8,and the changes of reactive oxygen species(ROS)were detected by the kit and fluorescence technology.5、Plasmid was extracted,GPX4-overexpressing lentivirus was packaged,and GPX4-overexpressing AM cell metastases were infected and screened.6、The experimental groups were CON,LPS,LPS+NETs+NC-GPX4 and LPS+NETs+OE-GPX4 groups.Western Blot and qRT-PCR were used to detect the expression levels of NLRP3,GSDMD and Caspase-1 in AM cells stimulated by NETs.The changes of reactive oxygen species(ROS)were detected by the kit and fluorescence technology,the content of MDA was detected by the kit,the content of IL-1β and IL-18 in the supernatant of cells was detected by ELISA,and the cell viability was detected by CCK-8.Results:1、Pyroptosis related proteins,inflammatory factors and NETs signature protein Cit H3 in lung tissue of sepsis mice were correlated with the degree of lung injury,and the expression level of GPX4 gene and protein was negatively correlated with the degree of lung injury.Inhibition of NETs could reduce the level of scorch death in lung tissue of sepsis mice,while the expression level of GPX4 was increased.2、Neutrophils were successfully extracted from rat blood and NETs were induced by cell staining and immunofluorescence.3、In vitro experiments,NETs will amplify the LPS-induced pyroptosis level of AM cells and affect the expression of GPX4 gene and protein,resulting in decreased cell viability and increased oxidative stress level.The pyroptosis level and oxidative stress level of cells are improved after NETs is inhibited by DnaseⅠ.4、Compared with the control group,after the removal of reactive oxygen species by NAC,NETs stimulated AM cells again,and pyroptosis level decreased and cell viability increased.5、The successful construction of GPX4 overexpressed stable strains was verified by fluorescence technique,qRT-PCR and Western Blot.6、Compared with the control group,NETs stimulated GPX4 overexpression group,pyroptosis level,reactive oxygen species and MDA content were significantly reduced,and cell activity was improved.Conclusion:1、During lung injury in sepsis,NETs promote oxidative stress in alveolar macrophages and induce pyroptosis.2、NETs induce pyroptosis of alveolar macrophages by inhibiting GPX4 and aggravate lung injury in sepsis.