Inhibitory Effect Of PEBP1 On Hyperoxia-exacerbated LPS-induced Alveolar Macrophage Pyroptosis

Acute Lung Injury（ALI）is one of the common complications in septic patients,and mechanical ventilation is the main treatment strategy for hypoxemia caused by acute lung injury.However,exposing lung tissue to hyperoxia can aggravate septic lung injury by accumulating intracellular reactive oxygen species（ROS）.The specific mechanism of hyperoxia aggravating septic lung injury and its prevention strategies have not been fully articulated.Therefore,this study was carried out by exploring the mechanisms by which hyperoxia aggravates septic lung injury through bioinformatics analysis and in vitro experiments,and to provide new targets for the prevention and treatment of hyperoxic lung injury in sepsis.PartⅠScreening for the sepsis-related molecule PEBP1Objective:Screening for the sepsis-associated molecule PEBP1.Methods:1.Download sepsis dataset GSE65682 and hyperoxia dataset GSE202458through GEO database,and obtain high-throughput sequencing data of the sepsis cell model from our desk group.2.Weighted gene overexpression network（WGCNA）analysis is performed on the dataset GSE65682 to obtain the most relevant modular signature genes for sepsis.3.The datasets GSE65682,GSE202458 and sequencing data are analyzed to screen for differential genes（DEGs）respectively.4.The above differential genes and the most relevant modular signature genes in WGCNA are analyzed using the Upset R package to obtain the intersection genes.5.Visualize the expression trend of PEBP1 in datasets GSE65682,GSE202458and sequencing data using box plots.6.q PCR is used to verify the expression trend of PEBP1 in an cell model of sepsis.Results:1.The most correlated module with sepsis was the yellow module with the highest correlation r=-0.57,P=1e-70.1912 yellow module genes were correlated with sepsis with cor=0.70,P=1e-200.2.A total of 376 DEGs were obtained from dataset GSE65682,including 155up-regulated genes and 221 down-regulated genes;1551 DEGs were obtained from Ctl vs LPS＿2h,including 655 up-regulated genes and 896 down-regulated genes,and4105 DEGs were obtained from Ctl vs LPS＿9h,including 820 up-regulated genes and3285 down-regulated genes.The dataset GSE202458 yielded a total of 1475 DEGs,including 762 up-regulated genes and 712 down-regulated genes.3.There were two intersecting genes obtained using the Upset R package,PEBP1and BIRC3,of which only PEBP1 showed consistent expression trends in the sepsis dataset and the hyperoxia dataset.4.PEBP1 was down-regulated in datasets GSE65682,GSE202458 and sequencing data.5.The q PCR results showed that the expression of PEBP1 m RNA was reduced in an in cell model of sepsis.Conclusion:The sepsis-associated molecule PEBP1 is screened and PEBP1 m RNA expression is decreased after LPS（1μg/ml）stimulation of NR8383 cells for 6h,12h,18h and 24h.PartⅡ Bioinformatics analysis of the relationship between PEBP1 and sepsisObjective:Validation of the correlation between PEBP1 and inflammatory response and immune cells of sepsis.Methods:1.The dataset GSE65682 is regrouped according to the high and low expression of PEBP1.2.Screen of the regrouped dataset for differential genes.3.The differential genes are subjected to GO/KEGG analysis.4.Perform GSEA/GSVA analysis on the regrouped data set.5.Gene expression matrix data are processed using CIBERSORT for immune cell infiltration assessment analysis and correlation analysis.6.Construct Protein-Protein Interaction（PPI）network,Random Forest（RF）model,and column line graph model.Results:1.GO/KEGG analysis showed that PEBP1 expression was closely associated with T cells,immunity,and inflammation.2.GSEA analysis showed that the major enrichment gene sets were also mostly associated with inflammation,oxidative stress,immunity,etc.3.GSVA analysis showed that the top six up-regulated gene sets（Top 6）and down-regulated gene sets（Bottom 6）with significant differences were also associated with inflammatory,immune,and glycolytic pathways.4.Immunocorrelation analysis showed that a total of 16 immune cells were significantly correlated with PEBP1,and the level of PEBP1 affected the immune activity of immune cells.5.The top five hub genes screened from the dataset regrouped according to PEBP1 expression can be used to predict the prevalence of sepsis.Conclusion:PEBP1 is strongly correlated with septic inflammation and PEBP1 levels affect the immune activity of immune cells.PartⅢ Overexpression of PEBP1 inhibits hyperoxia-exacerbated LPS-induced alveolar macrophage pyroptosisObjective:1.Exploring the mechanisms by which hyperoxia exacerbates lung injury in sepsis.2.Exploring the effect of PEBP1 on hyperoxia-exacerbated LPS-induced alveolar macrophage pyroptosis.Methods:1.A hyperoxic injury cell model of sepsis is constructed by culturing rat alveolar macrophage line NR8383 in a hyperoxic culture kit（37℃,95%O₂,5%CO₂）after pretreatment with LPS（1μg/ml）for 30min.2.Western Blot,q PCR and flow cytometry are used to clarify that hyperoxia exacerbates LPS-induced NR8383 cells pyroptosis.3.Using NLRP3 inhibitor MCC950 to clarify that hyperoxia exacerbates LPS-induced NR8383 cell injury by inducing pyroptosis.4.The NR8383 cell line of PEBP1 overexpression is constructed using lentiviral vector technology.5.Using Western Blot,q PCR and cell viability assays to clarify that overexpression of PEBP1 inhibits hyperoxia-aggravated LPS-induced NR8383 cells pyroptosis.Results:1.In a hyperoxic injury cell model of sepsis,the expression of PEBP1 m RNA was decreased at 6h,12h and 18h,while the ROS content was increased at all times.Ultimately 18h was chosen as the time point for the follow-up study.2.Compared with the LPS alone group or O₂alone group,the LPS+O₂group had higher intracellular ROS levels,significantly higher m RNA and protein of pyroptosis-related molecules NLRP3,GSDMD,Caspase-1,increased secretion of inflammatory factors（IL-18,IL-1β）,and decreased cellular activity.3.Compared with the LPS+O₂+DMSO group,the LPS+O₂+MCC950 group showed improved cell activity and decreased secretion of inflammatory factors（IL-18,IL-1β）;the m RNA and protein levels of the pyroptosis-related molecules NLRP3,GSDMD,and Caspase-1 were likewise significantly decreased.4.Compared with the LPS+O₂+OE-NC group,the m RNA and protein levels of the pyroptosis-related molecules NLRP3,GSDMD,and Caspase-1 were significantly decreased in the LPS+O₂+OE-PEBP1 group;the secretion of inflammatory factors（IL-18,IL-1β）was also significantly decreased;and cellular activity was increased.Conclusion:Hyperoxia induce pyroptosis to exacerbate LPS-induced NR8383 cell injury,whereas overexpression of PEBP1 inhibits hyperoxia-aggravated LPS-induced NR8383 cells pyroptosis.