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.