目的·研究不同月龄小鼠耳蜗中线粒体及烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide，NAD⁺）水平的变化，探索年龄相关性听力下降可能的机制。方法·分别选取1月龄、4月龄、8月龄及12月龄4组C57BL/6J雄性小鼠各10只，采用听性脑干反应（auditory brain response，ABR）和畸变产物耳声发射（distortion product otoacoustic emission，DPOAE）测定4组不同月龄小鼠听觉功能；通过实时荧光定量PCR（RT-qPCR）比较4组不同月龄小鼠耳蜗中线粒体能量代谢相关基因Ndufb5（NADH：ubiquinone oxidoreductase subunit B5）、Sdha（succinate dehydrogenase complex flavoprotein subunit A）、Sdhc（succinate dehydrogenase complex subunit C）和Atp5b（ATP synthase，H⁺ transporting mitochondrial F1 complex，beta subunit）的mRNA表达水平；通过免疫荧光染色分别观察1月龄和12月龄小鼠耳蜗毛细胞中的线粒体数量变化；通过透射电子显微镜（电镜）观察1月龄和12月龄小鼠耳蜗中内毛细胞、外毛细胞、螺旋神经节细胞及神经纤维中的线粒体形态结构；通过定量比色法检测并比较不同月龄小鼠耳蜗中的NAD⁺水平。结果·小鼠的ABR和DPOAE阈值呈现随月龄增长而升高的趋势，其中12月龄小鼠的ABR阈值在5.66~45.00 kHz的频率段较1月龄小鼠均显著升高（均P<0.01），DPOAE阈值在11.32~32.00 kHz的频率段较1月龄小鼠也显著升高（均P<0.01）。与1月龄小鼠相比，4、8、12月龄小鼠中4个线粒体功能相关基因（Ndufb5、Sdha、Sdhc和Atp5b）mRNA的表达水平呈现随月龄增长而下降的趋势，并在8月龄开始与1月龄的差异有统计学意义（均P<0.05）。免疫荧光染色结果显示12月龄小鼠耳蜗内毛细胞中的线粒体数量较1月龄明显减少。另外透射电镜在12月龄小鼠内毛细胞、螺旋神经节细胞及神经纤维中观察到空泡样变性的线粒体以及螺旋神经节细胞中出现较大的脂褐素。小鼠内耳NAD⁺水平呈现随月龄增长而下降的趋势，8月龄和12月龄小鼠NAD⁺水平较1月龄显著下降（均P<0.01）。结论·随月龄增长，C57BL/6J小鼠外周听觉关键细胞的线粒体功能下降、结构异常，耳蜗NAD⁺水平下降，这与小鼠听力阈值随着月龄增长而下降的表现一致。

Objective ·To investigate the changes of mitochondria and nicotinamide adenine dinucleotide (NAD⁺) levels in the cochleae of C57BL/6J mice at different ages, and explore potential mechanism of age-related hearing loss. Methods ·Forty C57BL/6J male mice aged 1, 4, 8, 12 months, respectively, were chosen and classified into 4 groups in terms of age (n=10). Auditory brain response (ABR) and distortion product otoacoustic emission (DPOAE) were conducted to detect the auditory function of mice at different ages; real-time quantitative PCR (RT-qPCR) was applied to compare the mRNA expression levels of the genes associated with mitochondrial energy metabolism in the cochleae of mice at different ages, including Ndufb5 (NADH: ubiquinone oxidoreductase subunit B5), Sdha (succinate dehydrogenase complex flavoprotein subunit A), Sdhc (succinate dehydrogenase complex subunit C), and Atp5b (ATP synthase, H⁺ transporting mitochondrial F1 complex, beta subunit); the changes of mitochondrial quantity in the cochlear hair cells from the mice aged 1 month and 12 months were observed by whole-mount immunofluorescence; the mitochondrial ultrastructure in the cochlear sensory epithelia including outer and inner hair cells, myelinate nerve fibers and spiral ganglion neurons of 1- and 12-month-old mice was observed by transmission electron microscope (TEM); NAD⁺ levels in the cochleae of mice at different ages were detected by quantitative colorimetry. Results ·The ABR thresholds of the 12-month-old mice were significantly elevated in comparison with those of the 1-month-old mice at the frequency range of 5.66?45.00 kHz (P<0.01); and the DPOAE thresholds of the 12-month-old mice were significantly elevated in comparison with those of the 1-month-old mice at the frequency range of 11.32?32.00 kHz (P<0.01). The expression levels of the genes related to mitochondrial functions including Ndufb5, Sdha, Sdhc, and Atp5b showed a downward trend with the age, which in the 8-month-old and 12-month-old mice were significantly different from those in the 1-month-old mice (P<0.05). Immunostaining showed that the number of mitochondria in cochlear inner hair cells of the 12-month-old mice was significantly lower than that of 1-month-old mice. It was observed by TEM that vacuolar degenerated mitochondria and larger lipofuscin existed in the inner hair cells, myelinate nerve fibers and spiral ganglion neurons of the 12-month-old mice. The NAD⁺ level showed a decreasing trend with age, declining significantly from 8 months of age, compared with the 1-month-old mice (P<0.01). Conclusion ·Mitochondrial dysfunction with abnormal structure and descending NAD⁺ levels in the cochleae is consistent with hearing function deterioration in C57BL/6J aging mice.