不同月龄C57BL/6J小鼠耳蜗线粒体和NAD+水平比较

doi:10.3969/j.issn.1674-8115.2022.08.002

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (8): 980-986.doi: 10.3969/j.issn.1674-8115.2022.08.002

不同月龄C57BL/6J小鼠耳蜗线粒体和NAD⁺水平比较

冯宝怡¹^,²^,³(), 董庭婷⁴, 郑晓飞¹^,²^,³, 陶永¹^,²^,³, 吴皓¹^,²^,³()

^1.上海交通大学医学院附属第九人民医院耳鼻咽喉头颈外科，上海 200011
^2.上海交通大学医学院耳科学研究所，上海 200092
^3.上海市耳鼻疾病转化医学重点实验室，上海 200092
^4.上海交通大学医学院附属第九人民医院样本库，上海 200092

收稿日期:2022-01-27 接受日期:2022-06-05 出版日期:2022-08-12 发布日期:2022-08-12
通讯作者: 吴皓 E-mail:cathrine_hyi@163.com;wuhao@shsmu.edu.cn
作者简介:冯宝怡（1996—），女，硕士生；电子信箱：cathrine_hyi@163.com。
基金资助:
国家自然科学基金重点项目(81730028);上海市教育委员会高峰高原学科建设计划(2019821);上海市高水平地方高校创新团队(SSMU-ZLCX20180601);上海交通大学医学院附属第九人民医院基础研究助推计划资助(JYZZ096)

Comparison of mitochondria and NAD⁺ level in the murine cochleae of C57BL/6J mice at different ages

FENG Baoyi¹^,²^,³(), DONG Tingting⁴, ZHENG Xiaofei¹^,²^,³, TAO Yong¹^,²^,³, WU Hao¹^,²^,³()

^1.Department of Otolaryngology-Head and Neck Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
^2.Ear Institute, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
^3.Shanghai Key Laboratory of Translation Medicine on Ear and Nose Disease, Shanghai 200092, China
^4.Biobank of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China

Received:2022-01-27 Accepted:2022-06-05 Online:2022-08-12 Published:2022-08-12
Contact: WU Hao E-mail:cathrine_hyi@163.com;wuhao@shsmu.edu.cn
Supported by:
National Natural Science Foundation of China(81730028);Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support(2019821);Innovative Research Team of High-level Local Universities in Shanghai(SSMU-ZLCX20180601);Fundamental Research Program Funding of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine(JYZZ096)

摘要/Abstract

摘要：

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。

Abstract:

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.

Key words: aged-related hearing loss, hearing loss, mitochondria, aging, nicotinamide adenine dinucleotide (NAD⁺)

中图分类号:

R764.3

冯宝怡, 董庭婷, 郑晓飞, 陶永, 吴皓. 不同月龄C57BL/6J小鼠耳蜗线粒体和NAD⁺水平比较[J]. 上海交通大学学报（医学版）, 2022, 42(8): 980-986.

FENG Baoyi, DONG Tingting, ZHENG Xiaofei, TAO Yong, WU Hao. Comparison of mitochondria and NAD⁺ level in the murine cochleae of C57BL/6J mice at different ages[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(8): 980-986.

图/表 5

表1 实时荧光定量PCR引物序列

Tab 1 Primer sequences for real-time qPCR

Name	Sequence（5'→3'）
Ndufb5-Forward	CTTCGAACTTCCTGCTCCTT
Ndufb5-Reverse	GGCCCTGAAAAGAACTACG
Sdha-Forward	GGAACACTCCAAAAACAGACCT
Sdha-Reverse	CCACCACTGGGTATTGAGTAGAA
Sdhc-Forward	GCTGCGTTCTTGCTGAGACA
Sdhc-Reverse	ATCTCCTCCTTAGCTGTGGTT
Atp5b-Forward	GGTTCATCCTGCCAGAGACTA
Atp5b-Reverse	AATCCCTCATCGAACTGGACG
Rpl19-Forward	ACCTGGATGAGAAGGATGAG
Rpl19-Reverse	ACCTTCAGGTACAGGCTGTG

图1 不同月龄C57BL/6J小鼠听力比较 (n=5)Note： A. ABR thresholds from 5.66 kHz to 45.00 kHz. B. DPOAE thresholds from 5.66 kHz to 32.00 kHz. ①P=0.002, ②P=0.001, ③P=0.000, ④P=0.034, ⑤P=0.007, ⑥P=0.014, compared with 1 month.

Fig 1 Comparison of auditory function in C57BL/6J mice at different ages (n=5)

图2 不同月龄C57BL/6J小鼠耳蜗线粒体功能相关基因和蛋白表达水平的比较Note： A. mRNA expression levels of Ndufb5, Sdha, Sdhc, and Atp5b in the cochleae of mice at ages of 1, 4, 8 and 12 months. ①P=0.042, ②P=0.009, ③P=0.006, ④P=0.000, ⑤P=0.011, ⑥P=0.002. B. Representative confocal images with HSP60 (green) and parvalbumin (red) stained at the middle turn of the cochleae of mice aged 1 month and 12 months.

Fig 2 Comparison of expression levels of mitochondrial function-related genes and proteins in the cochleae of C57BL/6J mice at different ages

图3 1月龄和12月龄C57BL/6J小鼠耳蜗中不同细胞的线粒体超微结构对比Note： Mitochondrial ultrastructure in outer hair cells (OHC), inner hair cells (IHC), myelinated nerve fibers and spiral ganglion neurons (SGN) in the cochleae of mice aged 1 month and 12 months. The rectangles indicate mitochondria, and the structures in the rectangles are enlarged and displayed on the right; the arrow indicates lipofuscin.

Fig 3 Comparison of mitochondrial ultrastructure in different cells in the cochleae of mice aged 1 month and 12 months

图4 不同月龄C57BL/6J小鼠耳蜗NAD+ 水平比较Note： ①P=0.002, ②P=0.000.

Fig 4 Comparison of NAD+ levels in the cochleae of mice at different ages

参考文献 24

1	CHADHA S, KAMENOV K, CIEZA A. The world report on hearing, 2021[J]. Bull World Health Organ, 2021, 99(4): 242-242A.
2	JAFARI Z, KOLB B E, MOHAJERANI M H. Age-related hearing loss and tinnitus, dementia risk, and auditory amplification outcomes[J]. Ageing Res Rev, 2019, 56: 100963.
3	KEITHLEY E M. Pathology and mechanisms of cochlear aging[J]. J Neurosci Res, 2020, 98(9): 1674-1684.
4	FUJIMOTO C, YAMASOBA T. Mitochondria-targeted antioxidants for treatment of hearing loss: a systematic review[J]. Antioxidants (Basel), 2019, 8(4): 109.
5	COVARRUBIAS A J, PERRONE R, GROZIO A, et al. NAD⁺ metabolism and its roles in cellular processes during ageing[J]. Nat Rev Mol Cell Biol, 2021, 22(2): 119-141.
6	WU P Z, O'MALLEY J T, DE GRUTTOLA V, et al. Age-related hearing loss is dominated by damage to inner ear sensory cells, not the cellular battery that powers them[J]. J Neurosci, 2020, 40(33): 6357-6366.
7	LYU A R, KIM T H, PARK S J, et al. Mitochondrial damage and necroptosis in aging cochlea[J]. Int J Mol Sci, 2020, 21(7): 2505.
8	HEQUEMBOURG S, LIBERMAN M C. Spiral ligament pathology: a major aspect of age-related cochlear degeneration in C57BL/6 mice[J]. J Assoc Res Otolaryngol, 2001, 2(2): 118-129.
9	TAN W J T, THORNE P R, VLAJKOVIC S M. Characterisation of cochlear inflammation in mice following acute and chronic noise exposure[J]. Histochem Cell Biol, 2016, 146(2): 219-230.
10	DRÖSE S, BRANDT U. Molecular mechanisms of superoxide production by the mitochondrial respiratory chain[J]. Adv Exp Med Biol, 2012, 748: 145-169.
11	LIN M T, BEAL M F. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases[J]. Nature, 2006, 443(7113): 787-795.
12	RENTON J P, XU N Y, CLARK J J, et al. Interaction of neurotrophin signaling with Bcl-2 localized to the mitochondria and endoplasmic reticulum on spiral ganglion neuron survival and neurite growth[J]. J Neurosci Res, 2010, 88(10): 2239-2251.
13	WHITE K, KIM M J, HAN C, et al. Loss of IDH2 accelerates age-related hearing loss in male mice[J]. Sci Rep, 2018, 8(1): 5039.
14	SONG X, CHEN Z Z, JIA R Y, et al. Transcriptomics and proteomic studies reveal acaricidal mechanism of octadecanoic acid-3,4 - tetrahydrofuran diester against Sarcoptes scabiei var. cuniculi[J]. Sci Rep, 2017, 7: 45479.
15	GO Y M, SUTLIFF R L, CHANDLER J D, et al. Low-dose cadmium causes metabolic and genetic dysregulation associated with fatty liver disease in mice[J]. Toxicol Sci, 2015, 147(2): 524-534.
16	QUINTANA-CABRERA R, MEHROTRA A, RIGONI G, et al. Who and how in the regulation of mitochondrial cristae shape and function[J]. Biochem Biophys Res Commun, 2018, 500(1): 94-101.
17	LAUTRUP S, SINCLAIR D A, MATTSON M P, et al. NAD⁺ in brain aging and neurodegenerative disorders[J]. Cell Metab, 2019, 30(4): 630-655.
18	FANG E F, KASSAHUN H, CROTEAU D L, et al. NAD⁺ replenishment improves lifespan and healthspan in ataxia telangiectasia models via mitophagy and DNA repair[J]. Cell Metab, 2016, 24(4): 566-581.
19	LIU D, PITTA M, JIANG H Y, et al. Nicotinamide forestalls pathology and cognitive decline in Alzheimer mice: evidence for improved neuronal bioenergetics and autophagy procession[J]. Neurobiol Aging, 2013, 34(6): 1564-1580.
20	LEE C F, CHAVEZ J D, GARCIA-MENENDEZ L, et al. Normalization of NAD⁺ redox balance as a therapy for heart failure[J]. Circulation, 2016, 134(12): 883-894.
21	MUKHERJEE S, CHELLAPPA K, MOFFITT A, et al. Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration[J]. Hepatology, 2017, 65(2): 616-630.
22	BROWN K D, MAQSOOD S, HUANG J Y, et al. Activation of SIRT3 by the NAD⁺ precursor nicotinamide riboside protects from noise-induced hearing loss[J]. Cell Metab, 2014, 20(6): 1059-1068.
23	KIM H J, OH G S, SHEN A, et al. Augmentation of NAD⁺ by NQO1 attenuates cisplatin-mediated hearing impairment[J]. Cell Death Dis, 2014, 5(6): e1292.
24	KIM H J, CAO W, OH G S, et al. Augmentation of cellular NAD⁺ by NQO1 enzymatic action improves age-related hearing impairment[J]. Aging Cell, 2019, 18(5): e13016.

[1]	林祎嘉, 程丽珍, 苗雅. 线粒体自噬异常在阿尔茨海默病中的作用及机制研究综述[J]. 上海交通大学学报（医学版）, 2022, 42(3): 387-392.
[2]	党国栋, 洪新宇, 蔡美琴. 烟酰胺单核苷酸对衰老小鼠代谢的干预作用[J]. 上海交通大学学报(医学版), 2022, 42(2): 158-165.
[3]	熊雷, 易茜, 许明芳, 陈健. MRPL12在肺腺癌中的表达和预后分析[J]. 上海交通大学学报(医学版), 2021, 41(8): 1033-1040.
[4]	袁笑, 田野野, 薛峥. 6-OHDA诱导的帕金森病小鼠表现出以p16^Ink4a上调和星形胶质细胞衰老为特征的衰老表型[J]. 上海交通大学学报(医学版), 2021, 41(7): 876-883.
[5]	韦亚忠, 薛晓梅, 何斌. 活性氧介导心肌缺血再灌注损伤的研究进展[J]. 上海交通大学学报(医学版), 2021, 41(6): 826-829.
[6]	蔡明琪, 陈焱, 林开斌, 黄冬. 生长分化因子11在心血管疾病中的作用[J]. 上海交通大学学报(医学版), 2021, 41(6): 834-838.
[7]	代华杰, 禤立平, 项家丽, 林泓, 赵志云, 王天歌, 李勉, 徐瑜, 陆洁莉, 王卫庆, 毕宇芳, 徐敏. 上海市社区中老年人群健康的血管衰老和心血管疾病患病风险的相关性研究[J]. 上海交通大学学报(医学版), 2021, 41(2): 223-227.
[8]	陈巍，韩峥，邹艳丽，黄莎莎，田霞. 溃疡性结肠炎小鼠血清miR-23a-3p和miR-27a-3p的表达水平及其作用的研究[J]. 上海交通大学学报(医学版), 2020, 40(8): 1069-1074.
[9]	杨硕瑶1, 2，戚紫怡3，向军4. 线粒体Lon蛋白酶及其相关疾病的研究进展[J]. 上海交通大学学报(医学版), 2020, 40(5): 683-687.
[10]	贾欢1, 2，陈颖1, 2，张治华1, 2，李静洁3，李蕴1, 2，陈见清1, 2，李孛1, 2，谭皓月1, 2，汪照炎1, 2，吴皓1, 2. 人工听觉脑干植入在先天性耳聋低龄儿童中的应用探索[J]. 上海交通大学学报(医学版), 2020, 40(10): 1324-1329.
[11]	菅朝慧，包玉倩. 自噬与非酒精性脂肪性肝病的研究进展[J]. 上海交通大学学报（医学版）, 2018, 38(6): 690-.
[12]	刘阳春，叶海峰，李小燕，胡川，黄健,郑月慧. 卵巢生殖干细胞巢功能的影响因素和调控机制[J]. 上海交通大学学报（医学版）, 2018, 38(6): 708-.
[13]	徐辰祺 1，闵璐琳 1，陈晓欢 3,陆任华 1,朱铭力 1,谢可炜 1,钱盈盈 1,郭相江 2,倪兆慧 1,阿依加肯·卡司木马力 3#，顾乐怡. 终末期肾病患者骨骼肌中丙酮酸脱氢酶活性与丙酮酸脱氢酶激酶 4 的表达[J]. 上海交通大学学报（医学版）, 2018, 38(3): 305-.
[14]	韩可琪，阮昕，贺明. SIRT2在肝癌发生发展中的作用[J]. 上海交通大学学报（医学版）, 2018, 38(10): 1247-.
[15]	郭竹英,徐芒华,赫玮,王世婷 . 线粒体通透性转换孔道在脂多糖致 H9c2心肌细胞凋亡中的作用[J]. 上海交通大学学报（医学版）, 2017, 37(7): 942-.

不同月龄C57BL/6J小鼠耳蜗线粒体和NAD⁺水平比较

Comparison of mitochondria and NAD⁺ level in the murine cochleae of C57BL/6J mice at different ages

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 24

相关文章 15

编辑推荐

Metrics

不同月龄C57BL/6J小鼠耳蜗线粒体和NAD+水平比较

Comparison of mitochondria and NAD+ level in the murine cochleae of C57BL/6J mice at different ages

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 24

相关文章 15

编辑推荐

Metrics

不同月龄C57BL/6J小鼠耳蜗线粒体和NAD⁺水平比较

Comparison of mitochondria and NAD⁺ level in the murine cochleae of C57BL/6J mice at different ages