Oxidative stress plays a critical role in the pathogenesis of hearing loss, and 2,3,4′,5-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) exerts antioxidant effects by inhibiting reactive oxygen species (ROS) generation. With the aim of developing new therapeutic strategies for oxidative stress, this study investigated the protective mechanism of THSG in vitro using a normal mouse cochlear cell line (UB/OC-2). The THSG and ascorbic acid have similar free radical scavenger capacities. H₂O₂, but not THSG, reduced the UB/OC-2 cell viability. Moreover, H₂O₂ might induce apoptosis and autophagy by inducing morphological changes, as visualized by microscopy. As evidenced by Western blot analysis and monodansylcadaverine (MDC) staining, THSG might decrease H₂O₂-induced autophagy. According to a Western blotting analysis and Annexin V/PI and JC-1 staining, THSG might protect cells from H₂O₂-induced apoptosis and stabilize the mitochondrial membrane potential. Furthermore, THSG enhanced the translocation of nucleus factor erythroid 2-related factor 2 (Nrf2) into the nucleus and increased the mRNA and protein expression of antioxidant/detoxifying enzymes under H₂O₂-induced oxidative stress conditions. Collectively, our findings demonstrate that THSG, as a scavenging agent, can directly attenuate free radicals and upregulate antioxidant/detoxifying enzymes to protect against oxidative damage and show that THSG protects UB/OC-2 cells from H₂O₂-induced autophagy and apoptosis in vitro.

Cisplatin is a widely used standard chemotherapy for various cancers. However, cisplatin treatment is associated with severe ototoxicity. Fucoidan is a complex sulfated polysaccharide mainly derived from brown seaweeds, and it shows multiple bioactivities such as antimicrobial, anti-inflammatory, anticancer, and antioxidant activities. Despite evidence of the antioxidant effects of fucoidan, research on its otoprotective effects remains limited. Therefore, the present study investigated the otoprotective effects of fucoidan in vitro using the mouse cochlear cell line UB/OC-2 to develop new strategies to attenuate cisplatin-induced ototoxicity. We quantified the cell membrane potential and analyzed regulators and cascade proteins in the apoptotic pathway. Mouse cochlear UB/OC-2 cells were pre-treated with fucoidan before cisplatin exposure. The effects on cochlear hair cell viability, mitochondrial function, and apoptosis-related proteins were determined via flow cytometry, Western blot analysis, and fluorescence staining. Fucoidan treatment reduced cisplatin-induced intracellular reactive oxygen species production, stabilized mitochondrial membrane potential, inhibited mitochondrial dysfunction, and successfully protected hair cells from apoptosis. Furthermore, fucoidan exerted antioxidant effects against oxidative stress by regulating the Nrf2 pathway. Therefore, we suggest that fucoidan may represent a potential therapeutic agent for developing a new otoprotective strategy.

Excessive levels of reactive oxygen species (ROS) lead to mitochondrial damage and apoptotic cell death in gentamicin-induced ototoxicity. 2,3,4’,5-Tetrahydroxystilbene-2-O-β-d-glucoside (THSG), a bioactive constituent, isolated from Polygonum multiflorum Thunb., exhibits numerous biological benefits in treating aging-related diseases by suppressing oxidative damage. However, its protective effect on gentamicin-induced ototoxicity remains unexplored. Therefore, here, we aimed to investigate the otoprotective effect of THSG on gentamicin-induced apoptosis in mouse cochlear UB/OC-2 cells. We evaluated the effect of gentamicin and THSG on the ROS level, superoxide dismutase (SOD) activity, mitochondrial membrane potential, nuclear condensation, and lactate dehydrogenase (LDH) release, and the expression of apoptosis-related proteins was assessed to understand the molecular mechanisms underlying its preventive effects. The findings demonstrated that gentamicin increased ROS generation, LDH release, and promoted apoptotic cell death in UB/OC-2 cells. However, THSG treatment reversed these effects by suppressing ROS production and downregulating the mitochondrial-dependent apoptotic pathway. Additionally, it increased the SOD activity, decreased the expression of apoptosis-related proteins, alleviated the levels of the apoptotic cells, and impaired cytotoxicity. To the best of our knowledge, this is the first study to demonstrate that THSG could be a potential therapeutic option to attenuate gentamicin-induced ototoxicity.

Aminoglycoside antibiotics are critical in clinical use for treating severe infections, but they can occasionally cause irreversible sensorineural hearing loss. To establish a rational pathway for otoprotectant discovery, we provide an integrated, three-tier methodology—comprising cell-model selection, transcriptomic analysis, and a gentamicin–Texas Red (GTTR) uptake assay—to guide the development of otoprotective strategies. We first utilized two murine auditory cell lines—UB/OC-2 and HEI-OC1. We focused on TMC1 and OCT2 and further explored the underlying mechanisms of ototoxicity. UB/OC-2 exhibited a higher sensitivity to gentamicin, which correlated with elevated OCT2 expression confirmed via RT-PCR and Western blot. Transcriptomic analysis revealed upregulation of PI3K-Akt, calcium, and GPCR-related stress pathways in gentamicin-treated HEI-OC1 cells. Protein-level analysis further confirmed that gentamicin suppressed phosphorylated Akt while upregulating ER stress markers (GRP78, CHOP) and apoptotic proteins (cleaved caspase 3, PARP). Co-treatment with PI3K inhibitors (LY294002, wortmannin) further suppressed Akt phosphorylation, supporting the role of PI3K-Akt signaling in auditory cells. To visualize drug entry, we used GTTR to evaluate its applicability as a fluorescence-based uptake assay in these cell lines, which were previously employed mainly in cochlear explants. Sodium thiosulfate (STS) and N-acetylcysteine (NAC) significantly decreased GTTR uptake, suggesting a protective effect against gentamicin-induced hair cell damage. In conclusion, our findings showed a complex ototoxic cascade involving OCT2- and TMC1-mediated drug uptake, calcium imbalance, ER stress, and disruption of PI3K-Akt survival signaling. We believe that UB/OC-2 cells serve as a practical in vitro model for mechanistic investigations and screening of otoprotective compounds. Additionally, GTTR may be a simple, effective method for evaluating protective interventions in auditory cell lines. Overall, this study provides molecular-level insights into aminoglycoside-induced ototoxicity and introduces a platform for protective strategies.