Dry eye disease (DED) is a multifactorial ocular condition marked by tear film instability, inflammation, and oxidative stress. Current therapies, including artificial tears and immunosuppressants, offer limited long-term benefit and potential side effects. Natural flavonoids such as luteolin possess anti-inflammatory and antioxidant properties, yet their therapeutic potential in DED remains underexplored.

This study aimed to evaluate the therapeutic efficacy and mechanisms of luteolin in experimental DED.

Flavonoids were initially screened using lipopolysaccharide-stimulated RAW264.7 macrophages. A benzalkonium chloride-induced DED mouse model was treated with topical luteolin or cyclosporine A. Ocular surface evaluations included tear secretion, corneal fluorescein staining, and goblet cell density. Inflammatory and oxidative markers were examined via histology, immunofluorescence, and qPCR. RNA sequencing and network pharmacology were used to identify target pathways. In vitro studies with RAW264.7 cells and hyperosmolarity-stressed human corneal epithelial cells further validated mechanism-specific effects. Target engagement was further validated using pathway-specific pharmacological inhibitors. Ocular safety and pharmacokinetics were also assessed.

Luteolin showed superior anti-inflammatory activity among tested flavonoids. It improved tear production, reduced epithelial damage, and preserved goblet cells. Mechanistically, it modulated macrophage-driven inflammation through JAK-STAT signaling and mitigated oxidative epithelial damage by suppressing the MAPK pathway. Transcriptomic and pharmacological analyses confirmed its dual immunomodulatory and cytoprotective roles. Combined STAT1/3 and MAPK inhibition mimicked its therapeutic effects. Topical luteolin was well-tolerated and achieved corneal bioavailability.

Luteolin provides a dual protective effect in DED by targeting inflammation and oxidative stress, supporting its development as a safe and effective disease-modifying therapy.