Mechanisms of Erlong Zuoci Wan alleviating damage to cochlear spiral ganglion neurons by upregulation of SIRT1 in mice with presbycusis

WANG Yirong; YAN Xirui; OUYANG Zhaoming; LI Jingquan; YAN Li; ZHANG Xinlei; LIU Qing; SHI Jianrong; CHU Min; DONG Yang

doi:10.16305/j.1007-1334.2023.2303073

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Mechanisms of Erlong Zuoci Wan alleviating damage to cochlear spiral ganglion neurons by upregulation of SIRT1 in mice with presbycusis

更新时间：2023-08-05

- Mechanisms of Erlong Zuoci Wan alleviating damage to cochlear spiral ganglion neurons by upregulation of SIRT1 in mice with presbycusis
- Shanghai Journal of Traditional Chinese Medicine Vol. 57, Issue 8, Pages: 81-88(2023)
- 作者机构：
  
  1.上海中医药大学中西医结合学院（上海 201203）
  2.上海中医药大学中医学院（上海 201203）
- 作者简介：
- 基金信息：
- DOI：10.16305/j.1007-1334.2023.2303073
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王艺蓉,阎希芮,欧阳朝明,等.耳聋左慈丸上调SIRT1减轻老年性聋小鼠耳蜗螺旋神经节细胞损伤的作用机制研究[J].上海中医药杂志,2023,57(8):81-88.

WANG Yirong,YAN Xirui,OUYANG Zhaoming,et al.Mechanisms of Erlong Zuoci Wan alleviating damage to cochlear spiral ganglion neurons by upregulation of SIRT1 in mice with presbycusis[J].Shanghai Journal of Traditional Chinese Medicine,2023,57(8):81-88.
王艺蓉,阎希芮,欧阳朝明,等.耳聋左慈丸上调SIRT1减轻老年性聋小鼠耳蜗螺旋神经节细胞损伤的作用机制研究[J].上海中医药杂志,2023,57(8):81-88. DOI： 10.16305/j.1007-1334.2023.2303073.

WANG Yirong,YAN Xirui,OUYANG Zhaoming,et al.Mechanisms of Erlong Zuoci Wan alleviating damage to cochlear spiral ganglion neurons by upregulation of SIRT1 in mice with presbycusis[J].Shanghai Journal of Traditional Chinese Medicine,2023,57(8):81-88. DOI： 10.16305/j.1007-1334.2023.2303073.

摘要

目的,2,探讨耳聋左慈丸对老年性聋小鼠耳蜗螺旋神经节细胞损伤的保护作用及其机制。,方法,2,①将27只C57BL/6J小鼠随机分为3组，即对照组、老年性聋组和耳聋左慈丸组，每组9只。耳聋左慈丸组小鼠从3月龄开始给予含有中药的饲料喂养至9月龄，老年性聋组同时喂养普通饲料；对照组采用2月龄小鼠。听性脑干反应（ABR）法检测小鼠听阈值的变化；苏木精-伊红（HE）染色法观察小鼠耳蜗螺旋神经节细胞形态学变化；免疫荧光染色法观察小鼠耳蜗螺旋神经节细胞微管相关轻链蛋白3（LC3）、选择性自噬接头蛋白（p62）和沉默信息调节因子1（SIRT1）蛋白的表达；转移酶介导的三磷酸脱氧鸟苷-生物素刻痕末端标记（TUNEL）法检测小鼠耳蜗螺旋神经节细胞的凋亡情况。②体外实验采用C57BL/6J小鼠耳蜗基底膜离体培养模型，分为空白对照组、过氧化氢（H,2,O,2,）模型组（0.3 mmol·L,-1,）和耳聋左慈丸低、中、高剂量组（5 μg·mL,-1,、10 μg·mL,-1,和50 μg·mL,-1,）。神经丝蛋白200（NF200）免疫荧光染色法观察耳蜗基底膜螺旋神经节细胞形态变化；Western blot法检测SIRT1、LC3-Ⅱ、p62、切割的半胱天蛋白酶-3（cleaved Caspase-3）、半胱天蛋白酶-3（Caspase-3）的蛋白表达。,结果,2,①与对照组比较，老年性聋组小鼠（9月龄）听阈值显著升高（,P,<,0.05），耳蜗底回螺旋神经节细胞数量显著下降（,P,<,0.05），SIRT1蛋白表达明显下降（,P,<,0.05），自噬相关蛋白LC3-Ⅱ和p62表达升高（,P,<,0.05），TUNEL阳性细胞数表达增多（,P,<,0.05）。耳聋左慈丸可明显降低老年性聋小鼠的听阈值（,P,<,0.05），显著增加耳蜗底回螺旋神经节细胞的数量（,P,<,0.05）。与老年性聋组比较，耳聋左慈丸组SIRT1蛋白表达显著增加（,P,<,0.05），自噬相关蛋白LC3-Ⅱ和p62表达降低（,P,<,0.05），TUNEL阳性细胞数显著减少（,P,<,0.05）。②体外实验发现，与空白对照组干预措施比较，H,2,O,2,可致螺旋神经节细胞出现核碎裂或胞体皱缩，听神经纤维紊乱，SIRT1蛋白表达显著下降（,P,<,0.05），LC3-Ⅱ和p62的蛋白表达及cleaved Caspase-3/Caspase-3比值上升（,P,<,0.05）。与H,2,O,2,模型组干预措施比较，耳聋左慈丸可显著降低H,2,O,2,所致小鼠耳蜗基底膜螺旋神经节细胞的损伤与丢失，减少听神经纤维紊乱，耳聋左慈丸中、高剂量组SIRT1蛋白表达显著增加（,P,<,0.05），LC3-Ⅱ、p62蛋白表达及cleaved Caspase-3/Caspase-3比值明显下调（,P,<,0.05）。,结论,2,耳聋左慈丸可减轻老年性聋小鼠螺旋神经节细胞的退化，改善自噬，抑制凋亡，其机制可能与上调SIRT1蛋白表达有关。

Abstract

Objective,2,To study the protective effects of Erlong Zuoci Wan （ELZC） on cochlear spiral ganglion neurons （SGNs） in mice with presbycusis and explore its mechanisms.,Methods,2,①Twenty-seven C57BL/6J mice were randomly divided into three groups： control group， ARHL group and ELZC group， with nine mice in each group. The 2-month-old C57BL/6J mice were taken as control， and the 3-month-old C57BL/6J mice were randomly divided into two groups that were fed diets containing Erlong Zuoci Wan （ELZC group） or standard diets （presbycusis group） for 6 months. Auditory brainstem response （ABR） was employed to test changes in hearing threshold. HE staining was used to evaluate the morphologic changes in mouse cochlear spiral ganglion neurons. The expression levels of LC3， p62 and SIRT1 in SGNs were detected by immunofluorescence. TUNEL assay was used to detect the apoptosis of SGNs. ②In the ,ex vivo, study， cochlear basement membrane in C57BL/6J mice was used as an ,ex vivo ,culture model， which were divided into 5 groups： blank control group， H,2,O,2, model group （0.3 mmol L,-1,） and ELZC groups with low， middle and high concentrations of ELZC （5 μg·mL,-1,， 10 μg·mL,-1,， 50 μg·mL,-1,）. The morphological changes in SGNs were assessed by NF200 immunohistochemistry. Western blot was employed to analyze the protein expressions of SIRT1， LC3-Ⅱ， p62， cleaved Caspase-3， and Caspase-3.,Results,2,①The mice （9 months old） in presbycusis group had significantly higher hearing threshold （,P,<,0.05）， significantly fewer SGNs in cochlea （,P,<,0.05）， significantly lower expression of SIRT1 protein （,P,<,0.05）， higher expressions of autophagy related proteins LC3-Ⅱ and p62 （,P,<,0.05）， and more TUNEL positive cells （,P,<,0.05） than mice in control group. ELZC could significantly lower hearing threshold （,P,<,0.05） and significantly improve SGNs survival （,P,<,0.05） in mice with age-related hearing loss. The mice in ELZC group showed significantly higher SIRT1 protein expression （,P,<,0.05）， lower LC3-Ⅱ and p62 expressions （,P,<,0.05）， and significantly fewer TUNEL-positive cells （,P,<,0.05） than those in presbycusis group. ②In the ,ex vivo, study， exposure to H,2,O,2, led to significant damage， shrinkage， and condensation of SGNs. After H,2,O,2, exposure， SIRT1 protein expression was significantly lower （,P,<,0.05）， and the protein expression levels of LC3-Ⅱ and p62 and the ratio of cleaved Caspase-3/Caspase-3 were higher than the condition in the blank control group ,（P,<,0.05）. SGNs damage and loss were alleviated， accompanied by the improvement of nerve fiber thinning and fragmentation in,ELZC group compared with the condition in H,2,O,2, model group. Besides， 10 μg·mL,-1 ,and,50 μg·mL,-1 ,ELZC treatment significantly increased SIRT1 expression ,（P,<,0.05）， and significantly downregulated the protein expressions of LC3-Ⅱ and p62 and the ratio of cleaved Caspase-3/Caspase-3 （,P,<,0.05）.,Conclusion,2,ELZC can reduce degeneration， improve autophagy and inhibit apoptosis of SGNs in presbycusis mice， and the mechanisms may be related to upregulation of SIRT1 protein expression.

关键词

耳聋经典名方作用机制自噬凋亡模型小鼠中药研究

Keywords

deafnessclassical famous prescriptionsmechanism of actionautophagyapoptosismodel mousetraditional Chinese herbal medicine research

references

KEITHLEY E M， CANTO C， ZHENG Q Y， et al. Cu/Zn superoxide dismutase and age-related hearing loss［J］. Hear Res， 2005， 209（1-2）： 76-85.

WANG J， PUEL J L. Presbycusis： An update on cochlear mechanisms and therapies［J］. J Clin Med， 2020， 9（1）： 218-239.

ZIA A， SAHEBDEL F， FARKHONDEH T， et al. A review study on the modulation of SIRT1 expression by miRNAs in aging and age-associated diseases［J］. Int J Biol Macromol，2021， 188： 52-61.

XU C Y， WANG L， FOZOUNI P， et al. SIRT1 is downregulated by autophagy in senescence and ageing［J］. Nat Cell Biol， 2020， 22（10）： 1170-1179.

XIONG H， CHEN S， LAI L， et al. Modulation of miR-34a/SIRT1 signaling protects cochlear hair cells against oxidative stress and delays age-related hearing loss through coordinated regulation of mitophagy and mitochondrial biogenesis［J］. Neurobiol Aging， 2019， 79： 30-42.

国家药典委员会.中华人民共和国药典（2020年版）：一部［M］. 北京：中国医药科技出版社，2020： 865-866.

LIU Q， LI N， YANG Y， et al. Prediction of the molecular mechanisms underlying Erlong Zuoci treatment of age-related hearing loss via network pharmacology-based analyses combined with experimental validation［J］. Front Pharmacol， 2021， 12： 719267.

DONG Y， GUO C R， DING Y， et al. Effects of Erlong Zuoci decoction on the age-related hearing loss in C57BL/6J mice［J］. J Ethnopharmacol， 2016， 181： 59-65.

BENKAFADAR N， FRANCOIS F， AFFORTIT C， et al. ROS-induced activation of DNA damage responses drives senescence-like state in postmitotic cochlear cells： implication for hearing preservation［J］. Mol Neurobiol， 2019， 56（8）： 5950-5969.

RIVAS-CHACÓN L D M， MARTÍNEZ-RODRÍGUEZ S， MADRID-GARCÍA R， et al. Role of oxidative stress in the senescence pattern of auditory cells in age-related hearing loss［J］. Antioxidants （Basel）， 2021， 10（9）： 1497.

DONG Y， DING D， JIANG H， et al. Ototoxicity of paclitaxel in rat cochlear organotypic cultures［J］. Toxicol Appl Pharmacol， 2014， 280（3）： 526-533.

董杨，施建蓉. 中医肾主耳理论的现代生物学研究进展与思路［J］. 中西医结合学报，2012， 10（2）： 128-134.

宋海燕，董杨，王静，等. 耳聋左慈丸防治庆大霉素诱发大鼠肾耳毒性实验研究［J］. 上海中医药杂志，2014， 48（5）： 101-103.

韩晓蕾. 针灸联合耳聋左慈丸水煎剂治疗肾虚耳鸣85例临床观察［J］. 中国民族民间医药，2019， 28（2）： 91-92.

王翔，谭业农，谢柳，等. 耳聋左慈丸加味联合声治疗对肾精亏虚型耳鸣及抗氧化作用的观察［J］. 中国实验方剂学杂志，2018， 24（23）： 189-193.

GUPTA R， AMBASTA R K， PRAVIR K. Autophagy and apoptosis cascade： which is more prominent in neuronal death？［J］. Cell Mol Life Sci， 2021， 78（24）： 8001-8047.

SOMEYA S， XU J， KONDO K， et al. Age-related hearing loss in C57BL/6J mice is mediated by Bak-dependent mitochondrial apoptosis［J］. Pro Natl Acad Sci U S A， 2009， 106（46）： 19432-19437.

WU J， YE J， KONG W， et al. Programmed cell death pathways in hearing loss： A review of apoptosis， autophagy and programmed necrosis［J］. Cell Prolif， 2020， 53（11）： e12915.

SHA S H， CHEN F Q， SCHACHT J. Activation of cell death pathways in the inner ear of the aging CBA/J mouse［J］. Hear Res， 2009， 254（1-2）： 92-99.

BALCHIN D， HAYER-HARTL M， HARTL F U. In vivo aspects of protein folding and quality control［J］. Science， 2016， 353（6294）： aac4354.

MENARDO J， TANG Y， LADRECH S， et al. Oxidative stress， inflammation， and autophagic stress as the key mechanisms of premature age-related hearing loss in SAMP8 mouse Cochlea［J］. Antioxid Redox Signal， 2012， 16（3）： 263-274.

LIU H， LI F， LI X， et al. Rapamycin ameliorates age-related hearing loss in C57BL/6J mice by enhancing autophagy in the SGNs［J］. Neurosci Lett， 2022， 772： 136493.

BROSEL S， LAUB C， AVERDAM A， et al. Molecular aging of the mammalian vestibular system［J］. Ageing Res Rev， 2016， 26： 72-80.

PANG J， XIONG H， OU Y， et al. SIRT1 protects cochlear hair cell and delays age-related hearing loss via autophagy［J］. Neurobiol Aging， 2019， 80： 127-137.

SALAM S A， MOSTAFA F， ALNAMSHAN M M， et al. Thymoquinone ameliorates age-related hearing loss in C57BL/6J mice by modulating Sirt1 activity and Bak1 expression［J］. Biomed Pharmacother， 2021， 143： 112149.

SONG M K， KIM Y J， KIM S H， et al. Environmental enrichment modulates silent information regulator 1 （SIRT1） activity to attenuate central presbycusis in a rat model of normal aging［J］. Exp Gerontol， 2021， 155： 111552.

LUO G， JIAN Z， ZHU Y， et al. Sirt1 promotes autophagy and inhibits apoptosis to protect cardiomyocytes from hypoxic stress［J］. Int J Mol Med， 2019， 43（5）： 2033-2043.

杨焘，宋厚盼，陈哲，等. 基于SIRT1/AMPK信号通路探讨茵陈蒿汤治疗急性乙醇性肝损伤的效应及机制［J］. 中药药理与临床，2022， 38（1）： 36-40.

GUO Y M， XU A M， WANG Y. SIRT1 in endothelial cells as a novel target for the prevention of early vascular aging［J］. J Cardiovasc Pharm， 2016， 67（6）： 465-473.

LI X， WANG Y， XIONG Y， et al. Galangin induces autophagy via deacetylation of LC3 by SIRT1 in HepG2 cells［J］. Sci Rep， 2016， 6： 30496.

YAMAKUCHI M， LOWENSTEIN C J. MiR-34， SIRT1 and p53： the feedback loop［J］. Cell Cycle， 2009， 8（5）： 712-715.

XIONG H， PANG J， YANG H， et al. Activation of miR-34a/SIRT1/p53 signaling contributes to cochlear hair cell apoptosis： implications for age-related hearing loss［J］. Neurobiol Aging， 2015， 36（4）： 1692-1701.

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