Wogonoside alleviates hyperosmotic stress-induced inflammation and apoptosis in human corneal epithelial cells via PI3K/AKT signaling

Background: Dry eye is a multifactorial ocular surface disorder in which tear hyperosmolarity acts as a major stressor that promotes inflammatory injury and Apoptosis in corneal epithelial cells. Wogonoside (WGS), a flavonoid glycoside derived from Scutellaria baicalensis, has reported anti-inflammatory and cytoprotective properties; however, its role and mechanism in dry eye-related epithelial injury remain insufficiently defined.

Methods: Network pharmacology was used to identify putative targets shared by WGS and dry eye, followed by pathway enrichment analysis. Molecular docking, molecular dynamics simulation, and cellular thermal shift assay (CETSA) were further used to evaluate the interaction between WGS and Akt1. Experimental validation was performed in transformed human corneal epithelial cells (HCE-T cells) exposed to hyperosmotic medium (500 mOsm). Cell viability, proliferation, Apoptosis, inflammatory mediator levels, and PI3K/Akt signaling-related changes were assessed. To interrogate pathway involvement, cells were additionally treated with the Akt Activator SC79 (10 μM) or the Akt Inhibitor MK-2206 (2.5 μM).

Results: A total of 203 overlapping targets were identified between WGS-related and dry eye-related target sets. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment highlighted PI3K/Akt signaling as a potentially relevant pathway, and Akt1 was prioritized as a candidate target. Molecular docking and molecular dynamics simulation supported a stable interaction between WGS and Akt1, while CETSA provided additional evidence of Akt1 target engagement in cells. In hyperosmotic HCE-T cells, WGS improved cell viability and proliferative capacity, attenuated Apoptosis, and reduced interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and matrix metalloproteinase-9 (MMP-9) levels. These effects were accompanied by restoration of PI3K/Akt signaling. Mechanistically, MK-2206 partially attenuated the protective effects of WGS, whereas SC79 produced a similar protective profile, supporting a PI3K/AKT-dependent component.

Conclusion: Wogonoside alleviates hyperosmotic stress-induced inflammatory injury and Apoptosis in HCE-T cells. Integrated evidence from network pharmacology, molecular docking, molecular dynamics simulation, CETSA, and in vitro validation supports Akt1 as a candidate target of WGS. The protective effects of WGS were closely associated with restoration of PI3K/Akt signaling, suggesting that WGS may be a promising candidate for hyperosmotic stress-related corneal epithelial injury and merits further validation in appropriate animal models of dry eye.