PLoS One PMID:26295804 (2015) 

NaHS Protects Cochlear Hair Cells from Gentamicin-Induced Ototoxicity by Inhibiting the Mitochondrial Apoptosis Pathway



Aminoglycoside antibiotics such as gentamicin could cause ototoxicity in mammalians, by inducing oxidative stress and apoptosis in sensory hair cells of the cochlea. Sodium hydrosulfide (NaHS) is reported to alleviate oxidative stress and apoptosis, but its role in protecting aminoglycoside-induced hearing loss is unclear. In this study, we investigated the anti-oxidant and anti-apoptosis effect of NaHS in in vitro cultured House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and isolated mouse cochlea. Results from cultured HEI-OC1 cells and cochlea consistently indicated that NaHS exhibited protective effects from gentamicin-induced ototoxicity, evident by maintained cell viability, hair cell number and cochlear morphology, reduced reactive oxygen species production and mitochondrial depolarization, as well as apoptosis activation of the intrinsic pathway. Moreover, in the isolated cochlear culture, NaHS was also demonstrated to protect the explant from gentamicin-induced mechanotransduction loss. Our study using multiple in vitro models revealed for the first time, the potential of NaHS as a therapeutic agent in protecting against aminoglycoside-induced hearing loss.House Ear Institute-Organ of Corti 1 (HEI-OC1) cell line was derived from the cochlea of the Immorto-mouse and characterized by Kalinec et al [21]. Cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) at 33°C in a humidified incubator with 5% CO2.The Atoh1/GFP transgenic mice carrying GFP under the control of Atoh1 enhancer [22] were purchased from Cyagen, Shanghai. All procedures and experiments involving animals in this study were performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. The study protocol was approved by the Animal Ethics Committee at Shengjing Hospital Affiliated to China Medical University. The IACUC committee members at Institute of Shengjing Hospital Affiliated to China Medical University approved this study. All efforts were made to minimize the number of animals used and their suffering. The animals were anesthetized using an inhalant anesthetic and sacrificed by decapitation.In order to establish an in vitro gentamicin ototoxicity model, we first exposed the HEI-OC1 cells to increasing concentrations of gentamicin in the culture medium (0, 1, 2.5 and 5 mM). As shown in Fig 1A, gentamicin treatment markedly reduced the number of cells in the culture in a dose-dependent manner. Cell viability was also significantly reduced by gentamicin exposure, as analyzed by MTT assay (Fig 1B), in a similar dose-dependent fashion. This result suggested that the use of gentamicin was able to exert toxicity to HEI-OC1 cells in our culturing conditions. Next, in order to test whether NaHS was able to protect HEI-OC1 cells from this gentamicin-induced toxicity, cells in the presence of 2.5 mM gentamicin were co-treated with 0, 10, 50 and 250 μM of NaHS respectively. We observed an obvious increase in cell numbers of NaHS-treated cultures compared to culture treated with gentamicin alone (Fig 1C). Using MTT assay, we confirmed that cell viability in the presence of NaHS was indeed significantly higher than in the absence of NaHS, in a dose-dependent manner to NaHS concentration (Fig 1D). Of note, NaHS alone (0 to 250 μM) does not affect the viability of HEI-OC1 cells in these experiments (S1 Fig). Therefore this result clearly indicated that NaHS was able to protect viability of HEI-OC1 cells upon gentamicin toxicity.HEI-OC1 cells were treated with 0, 10, 50, 250, 500 μM of NaHS for 48 hours and subject to viability measurement by MTT assay. Values were represented as the mean ± SEM from three independent experiments. n.s. not significant vs control. * P < 0.05 vs control.(TIF)
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