Ursolic acid ameliorates ocular surface dysfunction in dry eye via targeting EGFR/RAS/RAF/MAP2K1/MAPK1 pathway

Dry eye (DE), a multifactorial ocular surface disease, is predominantly characterized by inflammation as a central pathological factor. Ursolic acid (UA), a pentacyclic triterpenoid with well-documented anti-inflammatory properties, was evaluated in this study for its therapeutic effects on ocular surface dysfunction associated with DE and its underlying mechanisms. A hyperosmotic stress model (500 mOsM) using human corneal epithelial cells (HCEs) and an animal model of DE was established to assess UA's protective effects on both cellular and organismal levels. Comprehensive assessments, including phenol-red cotton tests and slit-lamp examinations, were performed to evaluate ocular surface damage in the DE mouse model. Potential UArelated targets and their relevance to DE pathology were identified through database mining. Protein-protein interaction (PPI) network construction and pathway enrichment analysis using the Metascape platform highlighted core targets and signaling pathways. Molecular docking simulations using AutoDock and PyMOL further elucidated the interaction modes between UA and its targets. To validate the molecular mechanisms underlying UA's therapeutic effects, integrative analyses were conducted using singlecell sequencing data from the Single Cell Portal and RNA sequencing of tissue samples. The results demonstrated that UA eye drops significantly preserved ocular surface functional units and alleviated DE symptoms, through modulation of the epidermal growth factor receptor (EGFR)/rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase kinase 1 (MAP2K1)/mitogen-activated protein kinase 1 (MAPK1) signaling pathway, as supported by network pharmacological analysis. Single-cell sequencing localized the distribution of key pathway proteins to the anterior ocular segment, particularly the cornea. In vivo experiments confirmed the therapeutic efficacy of UA eye drops via the EGFR/RAS/RAF/MAP2K1/MAPK1 pathway. Collectively, these findings underscore the potential of UA eye drops as a promising therapeutic approach for managing ocular surface disorders in DE.