机译
自噬障碍作为对乙酰氨基酚诱导的耳毒性的关键特征
摘要:a HEI-OC1 cells were treated with APAP (2, 4, 10, 25, 50 mM) for 24 h. The cell viability was detected by CCK-8 assay. *P < 0.05 vs. control cells (Con). b The IC50 of APAP treatment was 19.24 mM at 24 h. IC50 with the curve fit line and the IC50 value are illustrated. c The cell viability was also determined by the crystal violet assay. HEI-OC1 cells were treated with APAP (10, 20 mM) or vehicle (1% DMSO with the volume equal to that used in 20 mM APAP) for 24 h. The cells were then stained with crystal violet, and morphological alterations were observed under a light microscope. Scale bar = 0.86 mm. d The quantitative bar graph shows the relative density of cells from the crystal violet assay. *P < 0.05 vs. control cells. e HEI-OC1 cells were treated with 1% DMSO or APAP (10, 20 mM). The percentage of apoptotic cells was determined by flow cytometry using annexin V and PI labeling. The flow cytometric plots are representative of four experiments; mean ± SEM are shown in the bar plots (f), AnxV+/PI− (green bar; early apoptotic; lower right quadrant), AnxV+/PI+ (red bar; late apoptotic; upper right quadrants), AnxV−/PI− (white bar; live cells; lower left quadrant); and AnxV−/PI+ (gray bar; dead cells; upper left quadrants) are shown. g Quantification of the proportion of apoptotic cells (early and late apoptotic cells) after APAP treatment from the flow cytometric data. *P < 0.05 vs. control cells. h HEI-OC1 cells were treated with 20 mM APAP for a designated period (6, 12, or 24 h) or vehicle (1% DMSO) for 24 h. The protein expression of cleaved caspase-3 and Bcl-xl was detected by western blotting. The right two panels show the results of densitometric analysis. *P < 0.05 vs. control cells. i The cochlear explants treated with vehicle (0.5% DMSO) and 5 mM or 10 mM APAP for 24 h were stained for F-actin with phalloidin (green fluorescence). The images are representatives of four individual preparations. Confocal images were taken from the middle turn of the cochlea. Scale bar = 10 μm. OHC1, OHC2, and OHC3 represent the first, second, and third row of outer HCs, respectively; IHC inner HCs. The mean percentages of OHC loss are shown on the bar graph. Data are presented as the mean ± SEM (n = 3 mice per group). *P < 0.05 vs. control group. j The cochlear explants treated with vehicle (0.5% DMSO) or 10 mM APAP for 24 h. Apoptotic HCs were detected with a caspase 3 (CASP3) staining assay. Apoptotic cells were seen as caspase3-positive cells in the middle turns of cochlear explants from the APAP-treated groups. Scale bar = 5 μm. The right panel shows the comparison of the numbers of positive cells between the treated and control groups. *P < 0.05 vs. control group.