Identification and validation of ferroptosis-related genes in lipopolysaccharide-induced acute lung injury

Emerging evidence reveals the important role of ferroptosis in the pathophysiological process of acute lung injury (ALI). We aimed to identify and validate the potential ferroptosis-related genes of ALI through bioinformatics analysis and experimental validation.Murine ALI model was established via intratracheal instillation with LPS and confirmed by H&E staining and transmission electronic microscopy (TEM). RNA sequencing (RNA-seq) was used to screen differentially expressed genes (DEGs) between control and ALI model mice. The potential differentially expressed ferroptosis-related genes of ALI were identified using the limma R package. Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, gene set enrichment analysis (GSEA), and protein-protein interactions (PPI) were applied for the differentially expressed ferroptosis-related genes. CIBERSORT tool was used to conduct immune cell infiltration analysis. Finally, protein expressions and RNA expression of ferroptosis DEGs were validated in vivo and in vitro by western blots and RT-qPCR.Among 5009 DEGs, a total of 86 differentially expressed ferroptosis-related genes (45 up-regulated genes and 41 down-regulated genes) were identified in the lungs between control and ALI. GSEA analysis showed that the genes enriched were mainly involved in response to molecule of bacterial origin and fatty acid metabolic process. The GO and KEGG enrichment analysis indicated that the top 40 ferroptosis DEGs were mainly enriched in reactive oxygen species metabolic process, HIF-1signaling pathway, lipid and atherosclerosis, and ferroptosis. The PPI results and Spearman correlation analysis suggested that these ferroptosis-related genes interacted with each other. Immune infiltration analysis confirmed that ferroptosis DEGs were closely related to immune response. Consistent with the RNA-seq data, the western blot and RT-qPCR unveiled increased mRNA expressions of Cxcl2, Il-6, Il-1β, and Tnfα, and protein expressions of FTH1, TLR4 as well as decreased ACSL3 in LPS-induced ALI. In vitro, the upregulated mRNA levels of CXCL2, IL-6, SLC2A1, FTH1, TNFAIP3, and downregulated NQO1 and CAV1 in LPS-stimulated BEAS-2B and A549 cells were verified.We identified 86 potential ferroptosis-related genes of LPS-induced ALI through RNA-seq. Several pivotal ferroptosis-related genes involved in lipid metabolism and iron metabolism were implicated in ALI. This study may be helpful to expand our understanding of ALI and provide some potential targets to counteract ferroptosis in ALI.