Identification of ferroptosis-related genes in dry eye disease and their effects on inflammatory response and immune infiltration.

BACKGROUND

Dry Eye Disease (DED) is an ocular surface disease characterized by reduced tear secretion, abnormal fluid dynamics, and decreased tear quality, leading to instability of the tear film. It is accompanied by eye discomfort, visual impairment, and ocular surface tissue damage. DED is not only associated with the death of corneal epithelial cells but also involves damage to multiple ocular surface tissues, including the meibomian glands, lacrimal glands, conjunctival cells, and goblet cells. Ferroptosis plays a role in corneal epithelial cell death and keratopathy, but its underlying mechanisms remain unclear and require further investigation.

METHODS

This study utilized the GSE208297 dataset (from the Gene Expression Omnibus, GEO) to analyze the gene expression profiles of Mus musculus with DED. Five untreated DED samples and five normal control samples were selected for differential gene expression analysis to identify ferroptosis-related differentially expressed genes (FRDEGs). Based on these FRDEGs, comparative gene expression analysis, functional enrichment analysis, and small-molecule interaction network analysis were conducted. Additionally, gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) were performed, and immune infiltration analysis was conducted. A rat model of DED was induced using benzalkonium chloride (Benzalkonium chloride, 0.2 % solution, 5 µl, twice daily for 15 days). The tear secretion was assessed by the Schirmer I test, and structural changes in corneal tissue were observed by hematoxylin and eosin (HE) staining. Mitochondrial changes were examined by transmission electron microscopy (TEM), and ferroptosis was confirmed using malondialdehyde (MDA) and glutathione (GSH) kits. RT-PCR was used for the validation of gene expression analysis, confirming the mRNA expression changes of 8 key genes in the corneal tissue of DED rats.

RESULTS

Differential gene expression analysis identified 136 genes with abnormal expression. After intersecting these genes with 833 known ferroptosis-related genes, 13 FRDEGs were identified. These genes were primarily enriched in functions related to muscle tissue development, regulation of white blood cell differentiation, positive regulation of apoptosis signaling pathways, and inflammatory response regulation, as well as KEGG pathways such as MAPK, TNF, and IL-17 signaling. Immune cell infiltration analysis revealed significant differences in immune cell infiltration between DED and control groups, with CD8 T cells, macrophages, and NK cells showing correlations with the expression of FRDEGs. In the small-molecule interaction network analysis, eight key genes (ARNTL, ATF3, CCL5, JUN, KRT14, MYC, NR4A1, and TNF) exhibited strong interaction relationships with other small molecules. RT-PCR verification confirmed significant mRNA expression changes of these eight key genes in the corneal tissue of DED rats. MDA and GSH experiments further confirmed the ferroptosis phenotype in the corneas of DED rats, and TEM and HE staining showed corneal epithelial damage and mitochondrial cristae reduction.

CONCLUSIONS

Ferroptosis plays a key role in corneal cell death and keratopathy, amplifying inflammatory responses and contributing to the pathogenesis of DED. This study provides new molecular targets for therapeutic interventions in DED and reveals the potential mechanisms of ferroptosis in immune responses.