Abstract:

Objective To identify the core fatty acid metabolism-related genes （FAMRGs） in idiopathic pulmonary fibrosis （IPF） tissue using bioinformatics methods， and to experimentally verify whether these genes are significantly differentially expressed in IPF tissue and normal lung tissue. Methods Two IPF gene chip datasets， including the GSE47460 dataset and the GSE150910 dataset， were downloaded from the Gene Expression Omnibus （GEO） database； FAMRGs were screened from the Human Gene Comprehensive Database （GeneCards） database； the GEO2R tool was used to screen the differentially expressed genes （DEGs） between IPF tissue and normal lung tissue in GSE47460 dataset； Venn diagram was drawn between the DEGs and FAMRGs to obtain the differentially expressed FAMRGs. Xiantao Academic Tool was used to perform functional enrichment analysis on the above genes； the STRING database was used for analysis and Cytoscape software was used to establish a protein-protein interaction （PPI） network and screen the key FAMRGs； the GSE150910 dataset was used to verify the expression levels of the above genes， and receiver operating characteristic （ROC） curves were plotted to screen the core FAMRGs. The CIBERSORT database was used to analyze the relationship between the core FAMRGs and immune cell infiltration in IPF tissue. Eighteen healthy male SD rats were selected and randomly divided into control group， IPF group， and pirfenidone group， with 6 rats in each group. The rats in IPF group and pirfenidone group were intratracheally injected with bleomycin to establish the pulmonary fibrosis model， and the rats in control group were intratracheally injected with an equal volume of normal saline. After successful modeling， the rats in control group and IPF group were given 10 mL·kg^-1·d^-1 normal saline by gavage， and the rats in pirfenidone group were given 10 mL·kg^-1·d^-1 pirfenidone suspension by gavage； after 14 d of drug intervention， the rats were sacrificed and lung tissue was taken. Masson staining was used to observe the degree of fibrosis in lung tissue of the rats in the three groups； Western blotting method was used to detect the expression of FAMRGs proteins in lung tissue of the rats in three groups. Results A total of 182 differentially expressed FAMRGs were screened. The Gene Ontology （GO） analysis results showed that the above FAMRGs were mainly involved in functions such as hormone regulation， response to peptide substances， signaling receptor agonist activity， and cytokine activity. The Kyoto Encyclopedia of Genes and Genomes （KEGG） analysis results showed that the above FAMRGs were mainly enriched in signaling pathways such as interleukin-17 （IL-17） signaling pathway， advanced glycation end products-receptor for advanced glycation end products （AGE-RAGE） signaling pathway， and hypoxia-inducible factor-1（HIF-1） signaling pathway. Ten key FAMRGs were identified from the PPI network. Through validation with the GSE150910 dataset， matrix metalloproteinase 3 （MMP3）， secreted phosphoprotein 1 （SPP1）， and insulin-like growth factor 1 （IGF1） were determined as the core FAMRGs in IPF tissue， and their expressions were significantly higher compared with normal lung tissue （P<0.001）. The immune infiltration analysis results showed that in IPF tissue， the expressions of plasma cells， regulatory T cells， M0 macrophages， and resting mast cells were significantly up-regulated （P<0.05）， while the expressions of resting CD4 memory T cells， resting natural killer （NK） cells， monocytes， eosinophils， and neutrophils were significantly down-regulated （P<0.05）； the Spearman correlation analysis results showed that in IPF tissue， the expression level of SPP1 was positively correlated with the expression level of M0 macrophages （P<0.001）， and negatively correlated with the expression level of monocytes （P<0.001）. The Masson staining results showed that compared with control group， the collagen fiber deposition in lung tissue of the rats in IPF group was significantly increased； compared with IPF group， collagen fiber deposition in lung tissue of the rats in pirfenidone group was significantly decreased. The Western blotting results showed that compared with control group， the expression levels of MMP3， SPP1， and IGF1 proteins in lung tissue of the rats in IPF group were significantly increased （P<0.05）； compared with IPF group， the expression levels of MMP3， SPP1， and IGF1 proteins in lung tissue of the rats in pirfenidone group were significantly decreased （P<0.05）. Conclusion MMP3， SPP1， and IGF1 are the FAMRGs in IPF tissue and can serve as potential targets for future IPF treatment.

Key words: Idiopathic pulmonary fibrosis, Fatty acid metabolism, Bioinformatics, Immune infiltration, Pirfenidone