Protective Effect of ATP6V1B2 Against Kanamycin-Induced Cochlear Damage in Mice

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 <i>in vivo</i> and <i>in vitro</i> 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 (<i>p</i> < 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 (<i>p</i> < 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 (<i>p</i> < 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 (<i>p</i> < 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.