Exosome-Associated Gene Network and the Role of SPP1 in Herpes Stromal Keratitis and the Therapeutic Modulation by Ursolic Acid.

PURPOSE: This study aimed to explore the exosome-related gene network in herpes stromal keratitis (HSK) and to identify key molecular drivers, focusing on the role of secreted phosphoprotein 1 (SPP1) and investigating the therapeutic potential of ursolic acid (UA).

METHODS

Transcriptomic RNA sequencing was performed on corneal tissues from herpes simplex virus type 1 (HSV-1)-infected mice. Bioinformatics analyses included identification of differentially expressed genes (DEGs), immune cell infiltration assessment, exosome-related gene network construction, functional enrichment analyses, and compound-gene network creation. Immune infiltration findings were further validated using single-cell RNA sequencing and flow cytometry. Key findings were validated in a mouse HSK model using quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry. The therapeutic effect and mechanism of UA were investigated through subconjunctival administration, with subsequent evaluation of corneal lesions, angiogenesis, fibrosis, and key signaling pathways.

RESULTS

Through analysis, 222 DEGs associated with exosomes were identified in HSK, and the exosome-related gene network revealed that the primary functional cluster was centered on the positive regulation of cell adhesion. Among the genes in the primary functional cluster, SPP1 emerged as a hub gene, demonstrating significant upregulation that correlated with clinical progression. SPP1 expression exhibited a shift from corneal epithelial cells under normal conditions to infiltrating immune cells, particularly neutrophils and monocytes, during HSK. In a mouse model, treatment with UA significantly reduced corneal opacity, vessel in-growth, and inflammatory cell infiltration. Mechanistically, UA downregulated SPP1 expression and subsequently inhibited the activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway.

CONCLUSIONS

These findings provide new insights into the exosome-related gene network and the mechanisms of HSK, while identifying UA as a promising candidate for therapeutic development.