Abstract
[Background and Purpose] Age-related hearing loss (HL), which is caused by age-related disorders in inner ear tissues, has been a social problem because there is no clinical medicine for HL and HL in middle age is the most severe risk factor for dementia. Recent studies have reported that oxidative stress is deeply involved in disorders of inner ear cells and that suppressive effect on oxidative stress in the inner ear may delay the progression of age-related HL. In our study, we established an age-related HL in vitro model that hydrogen peroxide was treated with HEI-OC1 cells, a mouse cochlear cell line, and evaluated its effects on the autophagy mechanism.
[Methods] HEI-OC1 cells were cultured at 33℃ and 10% CO2. The expression of hair cell, supporting cell, and Spiral ganglion cell marker genes (Myo7a, Gjb2, Bdnf) in HEI-OC1 cells was analyzed by qPCR. HEI-OC1 cells treated with hydrogen peroxide at concentrations of 0.5 to 2 mM were measured LC3 protein levels and lysosomal activity, key markers of autophagy. In addition, 5 mM N-acetyl-L-cysteine (NAC), which has antioxidant effect, was applied HEI-OC1 cells with oxidative stress and was measured level of LC3 and lysosomal activity.
[Results] HEI-OC1 cells expressed specific inner ear marker genes such as Myo7a, Gjb2, and Bdnf. Hydrogen peroxide increased the level of LC3 protein of HEI-OC1 cells and decreased lysosomal activity of them in a dose dependent manner. Moreover, NAC ameliorated these impairments caused by oxidative stress significantly.
[Conclusion] HEI-OC1 cells were a undifferentiated inner ear cell line expressing various specific marker genes. We established an age-related HL in vitro model by exposure to hydrogen peroxide to them and an assay system using autophagy lysosomes pathway. The impairment of autophagy lysosome pathway by oxidative stress was ameliorated in treatment with NAC. Our data suggested that this system is used for screening drugs having strong antioxidant effects.
