Fig 1.
NaHS protects viability of HEI-OC1 cells upon gentamicin toxicity.
(A and B) HEI-OC1 cells were cultured as described in materials and methods. Gentamicin (GM) was added to the culture medium to a concentration of 0 (control), 1, 2.5 and 5 mM respectively incubated for 48 hours. Representative culture images (A) and viability measured by MTT assay (B) of HEI-OC1 cell culture after gentamicin (GM) treatment were shown. (C and D) HEI-OC1 cells in the presence of 2.5 mM gentamicin were co-treated with 0, 10, 50 and 250 μM of NaHS respectively for 48 hours. Representative culture images (C) and viability (B) were shown. Scale bar 100 μm. Values were represented as the mean ± SEM from three independent experiments. * P < 0.05 vs control.
Fig 2.
NaHS inhibits gentamicin-induced ROS production, mitochondrial depolarization and apoptosis.
(A and B) HEI-OC1 cells in the presence of 2.5 mM gentamicin were co-treated with 0, 50 and 250 μM of NaHS respectively for 48 hours. Representative DCFH-DA (green) staining fluorescence images (A) and quantification of fluorescent intensity (B) were shown. (C) Mitochondrial depolarization was measured in HEI-OC1 cells as treated in (A). (D and E) Hoechst (blue)/PI (red) staining (D) and percentage of PI-positive apoptotic cells (E) in HEI-OC1 culture treated as in (A) were shown. Scale bar 100 μm. Values were represented as the mean ± SEM from three independent experiments. * P < 0.05 vs control. n.s. not significant vs control. # P < 0.05 vs 0 and 50 μM NaHS treatment.
Fig 3.
NaHS reverses expressions of gentamicin-induced intrinsic apoptotic pathway factors in HEI-OC1 cell culture.
(A to C) mRNA levels of Bax (A), Bcl-2 (B) and Caspase-3 (C) were examined with RT-PCR in HEI-OC1 cells treated as in Fig 2. Values were represented as the mean ± SEM from three independent experiments, normalized to GAPDH mRNA levels. * P < 0.05 vs control. n.s. not significant vs control. # P < 0.05 vs 0 and 50 μM NaHS treatment. (D) Protein levels of Bax, Bcl-2 and Caspase-3 were examined with western blot analysis in HEI-OC1 cells treated as in Fig 2.
Fig 4.
NaHS inhibits gentamicin-induced ototoxicity in mouse cochlear explant.
Mouse cochlear explant was isolated and cultured as described in materials and methods. After 24 hours cultured in the incubator, cochlear explant were divided into 3 treatment groups: PBS (control), 80 μM gentamicin (GM) and 80 μM gentamicin + 250 μM NaHS (GM+NaHS) for 24 hours. (A) Hair cells from mid apex segment of cochlear showing outer hair cells (OHC) and inner hair cells (IHC) labeled with Atoh1/GFP (green). Scale bar 25 μm. (B) Scanning electron micrographs of the mid apex segment from cochlear explant as treated in (A). (C) Quantification of hair cells in three cochlear explant groups. Values were represented as the mean ± SEM from three independent experiments. * P < 0.05 vs control. n.s. not significant vs control. # P < 0.05 vs GM.
Fig 5.
NaHS reverses gentamicin-induced apoptotic gene expressions in cochlear explant.
(A to C) mRNA levels of Bax (A), Bcl-2 (B) and Caspase-3 (C) were examined with RT-PCR in cochlear explant treated as in Fig 4. Values were represented as the mean ± SEM from three independent experiments, normalized to GAPDH mRNA levels. * P < 0.05 vs control. n.s. not significant vs control. # P < 0.05 vs GM. (D) Protein levels of Bax, Bcl-2 and Caspase-3 were examined with western blot analysis in cochlear explant treated as in Fig 4.
Fig 6.
NaHS protects mechanotransduction activity of cochlear explant from gentamicin-induced ototoxicity.
Mechanotransduction currents were recorded at −75 mV as described in materials and methods, from the mid apex segment of cochlear explant bathed in 1.3 mM Ca2+, treated as in Fig 4. Stimulus protocol and scale bar applies to all current families.