Background: Kanamycin (KM) is a commonly used antibacterial agent in clinical practice, but it can induce ototoxicity, leading to sensorineural hearing loss. Previous studies have reported that deletion of the vacuolar H⁺-ATPase B2 subunit (ATP6V1B2) results in hearing impairment and that the mitogen-activated protein kinase (MAPK) pathway exerts a protective effect on cochlear cells in mice. Therefore, this study aimed to elucidate the role of ATP6V1B2 in KM-induced cochlear hair cell injury in mice and to explore the specific mechanisms involved.

Methods: KM and siRNA/overexpression constructs were used in combination in vivo and in vitro to evaluate the protective effect of ATP6V1B2 on cochlear hair cell injury. Auditory function in individual mice was assessed using auditory brainstem response (ABR) testing. HEI-OC1 cell viability was measured using the Cell Counting Kit-8 (CCK-8). Apoptosis in HEI-OC1 cells was detected with flow cytometry. Intracellular reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorofluorescin diacetate (DCFH-DA) through flow cytometry. The expression of vital regulatory factors, apoptotic markers, inflammatory mediators, and MAPK pathway proteins in HEI-OC1 cells was evaluated by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis.

Results: Following KM induction, the ABR threshold increased, the number of viable cells decreased, apoptosis was promoted, ROS accumulation was enhanced, and inflammatory factor expression in HEI-OC1 cells was elevated (p < 0.05). Compared with the KM group, Ad-ATP6V1B2 reversed the effects of KM on the ABR threshold, cell viability, apoptosis, ROS accumulation, and inflammatory factor expression (p < 0.05). In contrast, si-ATP6V1B2 exacerbated the effects of KM on the ABR threshold, viable cell number, apoptosis, ROS accumulation, and expression of inflammatory factors (p < 0.05). Ad-ATP6V1B2 exerted its protective effects on injured HEI-OC1 cells by inhibiting the p38 MAPK pathway and activating the ERK MAPK pathway (p < 0.05).

Conclusion: Ad-ATP6V1B2 protects against KM-induced HEI-OC1 cell injury by inhibiting the p38/MAPK axis and activating the ERK1/2/MAPK pathway. ATP6V1B2 effectively reverses KM-mediated cellular damage by modulating the MAPK signaling pathway. These findings suggest ATP6V1B2 may represent a novel therapeutic target for preventing and treating KM-mediated HEI-OC1 cell injury and hearing impairment.