In vivo and in vitro evaluation of the protective effects of osthole against ototoxicity using the zebrafish model and HEI-OC1 cell line.

Osthole, a coumarin derivative with potent antioxidant and anti-inflammatory properties, has demonstrated promising therapeutic potential in protecting against ototoxicity. This study investigated the protective effects of osthole through both in vitro and in vivo experimental models. A high-content screening of 1505 natural compounds in HEI-OC1 cells identified osthole as the most effective compound in alleviating gentamicin-induced cellular damage. Our results indicate that osthole confers protection by restoring autophagic flux and reducing the accumulation of reactive oxygen species (ROS). In HEI-OC1 cells, cell viability was significantly improved following co-treatment with gentamicin and osthole. Western blot analysis revealed that osthole modulates key signaling pathways involved in cell survival and autophagy. Furthermore, LysoTracker staining in zebrafish larvae confirmed that osthole preserved autophagic activity compromised by gentamicin exposure. In vivo experiments using wild-type and Tg(Brn3c:EGFP) zebrafish lines assessed neuromast hair cell survival in the lateral line system. Compared with the gentamicin-only group, the osthole co-treated group exhibited increased hair cell counts, a reduced number of TUNEL-positive apoptotic cells, decreased ROS levels, and enhanced autophagy. These outcomes collectively demonstrate the potential protective effects of osthole against gentamicin-induced ototoxicity in both cellular and zebrafish models. Taken together, these findings highlight osthole as a promising candidate for therapeutic development against aminoglycoside-induced hearing loss, offering a multi-targeted mechanism involving oxidative stress reduction, autophagy restoration, and inhibition of apoptosis.