Bioinformatics Analysis Identifies Potential Ferroptosis Key Genes in the Pathogenesis of Pulmonary Fibrosis

Objective: Ferroptosis has an important role in developing pulmonary fibrosis. The present project aimed to identify and validate the potential ferroptosis-related genes in pulmonary fibrosis by bioinformatics analyses and experiments. Methods: First, the pulmonary fibrosis tissue sequencing data were obtained from Gene Expression Omnibus (GEO) and FerrDb databases. Bioinformatics methods were used to analyze the differentially expressed genes (DEGs) between the normal control group and the pulmonary fibrosis group and extract ferroptosis-related DEGs. Hub genes were screened by enrichment analysis, protein-protein interaction (PPI) analysis, and random forest algorithm. Finally, mouse pulmonary fibrosis model was made for performing an exercise intervention and the hub genes' expression was verified through qRT-PCR. Results: 13 up-regulated genes and 7 down-regulated genes were identified as ferroptosis-related DEGs by comparing 103 lung tissues with idiopathic pulmonary fibrosis (IPF) and 103 normal lung tissues. PPI results indicated the interactions among these ferroptosis-related genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway enrichment and Genome-Ontology (GO) enrichment analyses showed that these ferroptosis-related genes involved in the organic anion transport, response to hypoxia, response to decrease oxygen level, HIF-1 signaling pathway, renal cell carcinoma, and arachidonic acid metabolism signaling pathway. The confirmed genes using PPI analysis and random forest algorithm included CAV1, NOS2, GDF15, HNF4A, and CDKN2A. qRT-PCR of the fibrotic lung tissues from the mouse model showed that the mRNA levels of NOS2 and GDF15 were up-regulated, while CAV1 and CDKN2A were down-regulated. Also, treadmill training led to an increased expression of CAV1 and CDKN2A and a decrease in the expression of NOS2 and GDF15. Conclusion: Using bioinformatics analysis, 20 potential genes were identified to be associated with ferroptosis in pulmonary fibrosis. CAV1, NOS2, GDF15, and CDKN2A were demonstrated to be influencing the development of pulmonary fibrosis by regulating ferroptosis. These findings suggested that, as an aerobic exercise treatment, treadmill training reduced ferroptosis in the pulmonary fibrosis tissues, and thus, reduces inflammation in the lungs. Aerobic exercise training initiate concomitantly with induction of pulmonary fibrosis reduces ferroptosis in lung. These results may develop our knowledge about pulmonary fibrosis and may contribute to its treatment.