Skip to main content
SpringerLink
Log in

Peroxynitrite induces apoptosis of mouse cochlear hair cells via a Caspase-independent pathway in vitro

  • Original Paper
  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

Peroxynitrite (ONOO−) is a potent and versatile oxidant implicated in a number of pathophysiological processes. The present study was designed to investigate the effect of ONOO− on the cultured cochlear hair cells (HCs) of C57BL/6 mice in vitro as well as the possible mechanism underlying the action of such an oxidative stress. The in vitro primary cultured cochlear HCs were subjected to different concentrations of ONOO−, then, the cell survival and morphological changes were examined by immunofluorescence and transmission electron microscopy (TEM), the apoptosis was determined by Terminal deoxynucleotidyl transferase dUNT nick end labeling (TUNEL) assay, the mRNA expressions of Caspase-3, Caspase-8, Caspase-9, Apaf1, Bcl-2, and Bax were analyzed by RT-PCR, and the protein expressions of Caspase-3 and AIF were assessed by immunofluorescence. This work demonstrated that direct exposure of primary cultured cochlear HCs to ONOO− could result in a base-to-apex gradient injury of HCs in a concentration-dependent manner. Furthermore, ONOO− led to much more losses of outer hair cells than inner hair cells mainly through the induction of apoptosis of HCs as evidenced by TEM and TUNEL assays. The mRNA expressions of Caspase-8, Caspase-9, Apaf1, and Bax were increased and, meanwhile, the mRNA expression of Bcl-2 was decreased in response to ONOO− treatment. Of interesting, the expression of Caspase-3 had no significant change, whereas, the expression alteration of AIF was observed. These results suggested that ONOO− can effectively damage the survival of cochlear HCs via triggering the apoptotic pathway. The findings from this work suggest that ONOO−-induced apoptosis is mediated, at least in part, via a Caspase-independent pathway in cochlear HCs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Rios N, Piacenza L, Trujillo M, Martínez A, Demicheli V, Prolo C, Álvarez MN, López GV, Radi R (2016) Sensitive detection and estimation of cell-derived peroxynitrite fluxes using fluorescein-boronate. Free Radic Biol Med 101:284–295

    Article  CAS  PubMed  Google Scholar 

  2. Labbé D, Bloch W, Schick B, Michel O (2016) Hearing impairment, cochlear morphology, and peroxynitrite (ONOO−) formation in adult and aging NOS II knockout mice. Acta Otolaryngo 10:991–998

    Article  Google Scholar 

  3. Degendorfer G, Chuang CY, Kawasaki H, Hammer A, Malle E, Yamakura F, Davies MJ (2016) Peroxynitrite-mediated oxidation of plasma fibronectin. Free Radic Biol Med 16:30304–30305

    Google Scholar 

  4. Liu X, Gao RW, Li M, Si CF, He YP, Wang M, Yang Y, Zheng QY, Wang CY (2016) The ROS derived mitochondrial respirstion not from NADPH oxidase plays key role in Celastrol against angiotensin II-mediated HepG2 cell proliferation. Apoptosis 11:1315–1326

    Article  Google Scholar 

  5. Terashita T, Saito S, Nabeka H, Hato N, Wakisaka H, Shimokawa T, Kobayashi N, Gyo K, Matsuda S (2013) Prosaposin-derived peptide alleviates ischaemia-induced hearing loss. Acta Otolaryngol 5:462–468

    Article  Google Scholar 

  6. Wu WJ, Sha SH, McLaren JD, Kawamoto K, Raphael Y, SchachtJ (2001) Aminoglycoside ototoxicity in adult CBA, C57BL and BALB mice and the Sprague-Dawley rat. Hear Res 158:165–178

    Article  CAS  PubMed  Google Scholar 

  7. Klein M, Koedel U, Kastenbauer S, Pfister HW (2008) Nitrogen and oxygen molecules in meningitis-associated labyrinthitis and hearing impairment. Infection 36:2–14

    Article  CAS  PubMed  Google Scholar 

  8. Poirrier AL, Pincemail J, Van Den Ackerveken P, Lefebvre PP, Malgrange B (2010) Oxidative stress in the cochlea: an update. Curr Med Chem 17:3591–3604

    Article  CAS  PubMed  Google Scholar 

  9. Han C, Someya S (2013) Mouse models of age-related mitochondrial neurosensory hearing loss. Mol Cell Neurosci 55:95–100

    Article  CAS  PubMed  Google Scholar 

  10. Lee JE, Nakagawa T, Kim TS, Endo T, Shiga A, Iguchi F, Lee SH, Ito J (2004) Role of reactive radicals in degeneration of the auditory system of mice following cisplatin treatment. Acta Otolaryngol 124:1131–1135

    Article  CAS  PubMed  Google Scholar 

  11. Liu W, Fan Z, Han Y, Lu S, Zhang D, Bai X, Xu W, Li J, Wang H (2011) Curcumin attenuates peroxynitrite-induced neurotoxicity in spiral ganglion neurons. Neurotoxicology 1:150–157

    Article  Google Scholar 

  12. Liu W, Fan Z, Han Y, Zhang D, Li J, Wang H (2012) Intranuclear localization of apoptosis-inducing factor and endonuclease G involves in peroxynitrite-induced apoptosis of spiral ganglion neurons. Neurol Res 10:915–922

    Article  Google Scholar 

  13. Ding D, Roth J, Salvi R (2011) Manganese is toxic to spiral ganglion neurons and HCs in vitro. Neurotoxicology 2:233–241

    Article  Google Scholar 

  14. Li P, Ding D, Salvi R, Roth JA (2015) Cobalt-induced ototoxicity in rat postnatal cochlear organotypic cultures. Neurotox Res 28:209–221

    Article  CAS  PubMed  Google Scholar 

  15. Pacher P, Beckman JS, Liaudet L (2007) Nitric oxide and peroxynitrite: in health and disease. Physiol Rev 1:315–424

    Article  Google Scholar 

  16. Kim SJ, Ho Hur J, Park C, Kim HJ, Oh GS, Lee JN, Yoo SJ, Choe SK, So HS, Lim DJ, Moon SK, Park R (2015) Bucillamine prevents cisplatin-induced ototoxicity through induction of glutathione and antioxidant genes. Exp Mol Med 47:e142

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Radi R (2013) Peroxynitrite, a stealthy biological oxidant. J Biol Chem 37:26464–26472

    Article  Google Scholar 

  18. Szabó C, Ischiropoulos H, Radi R (2007) Peroxynitrite: biochemistry, pathology and development of therapeutics. Nat Rev Drug Discov 8:662–680

    Article  Google Scholar 

  19. Nguyen MC, Park JT, Jeon YG, Jeon BH, Hoe KL, Kim YM, Lim HK, Ryoo S (2016) Arginase inhibition restores peroxynitrite-induced endothelial dysfunction via l-arginine-dependent endothelial nitric oxide synthase phosphorylation. Yonsei Med J 6:1329–1338

    Article  Google Scholar 

  20. Selimoglu E (2007) Aminoglycoside-induced ototoxicity. Curr Pharm Des 13:119–126

    Article  CAS  PubMed  Google Scholar 

  21. Ding D, He J, Allman BL, Yu D, Jiang H, Seigel GM, Salvi RJ (2011) Cisplatin ototoxicity in rat cochlear organotypic cultures. Hear Res 282:196–203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Jamesdaniel S, Coling D, Hinduja S, Ding D, Li J, Cassidy L, Seigel GM, Qu J, Salvi R (2012) Cisplatin-induced ototoxicity is mediated by nitroxidative modification of cochlear proteins characterized by nitration of Lmo4. J Biol Chem 287:18674–18686

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Furness DN (2015) Molecular basis of hair cell loss. Cell Tissue Res 1:387–399

    Article  Google Scholar 

  24. Infante EB, Channer GA, Telischi FF, Gupta C, Dinh JT, Vu L, Eshraghi AA, Van De Water TR (2012) Mannitol protects HCs against tumor necrosis factor α-induced loss. Otol Neurotol 9:1656–1663

    Article  Google Scholar 

  25. Tadros SF, D’Souza M, Zhu X, Frisina RD (2008) Apoptosis-related genes change their expression with age and hearing loss in the mouse cochlea. Apoptosis 13:1303–1321

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Pan N, Jahan I, Kersigo J, Duncan JS, Kopecky B, Fritzsch B (2012) A novel Atoh1 “self-terminating” mouse model reveals the necessity of proper Atoh1 level and duration for hair cell differentiation and viability. PLoS ONE 7:e30358

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Han W, Shi X, Nuttall AL (2006) AIF and endoG translocation in noise exposure induced hair cell death. Hear Res 211:85–95

    Article  CAS  PubMed  Google Scholar 

  28. Zhang D, Fan Z, Han Y, Bai X, Liu W, Lu S, Wang M, Xu L, Luo J, Li J, Wang H (2012) Apoptosis-inducing factor is involved in gentamicin-induced vestibular hair cell death. ORL J Otorhinolaryngol Relat Spec 1:1–5

    Article  CAS  Google Scholar 

  29. Ding ZJ, Chen X, Tang XX, Wang X, Song YL, Chen XD, Wang J, Wang RF, Mi WJ, Chen FQ, Qiu JH (2015) Apoptosis-inducing factor and calpain upregulation in glutamate-induced injury of rat spiral ganglion neurons. Mol Med Rep 2:1685–1692

    Article  Google Scholar 

  30. Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot E, Costantini P, Loeffler M, Larochette N, Goodlett DR, Aebersold R, Siderovski DP, Penninger JM, Kroemer G (1999) Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397:441–446

    Article  CAS  PubMed  Google Scholar 

  31. Cande C, Cecconi F, Dessen P, Kroemer G (2002) Apoptosis-inducing factor (AIF): key to the conserved caspase-independent pathways of cell death. J Cell Sci 115:4727–4734

    Article  CAS  PubMed  Google Scholar 

  32. Sevrioukova IF (2011) Apoptosis-inducing factor: structure, function, and redox regulation. Antioxid Redox Signal 14: 2545–2579

    Article  Google Scholar 

  33. Higgins GC, Devenish RJ, Beart PM, Nagley P (2012) Transitory phases of autophagic death and programmed necrosis during superoxide-induced neuronal cell death. Free Radic Biol Med 10:1960–1967

    Article  Google Scholar 

  34. Song Y, Fan Z, Bai X, Liu W, Han Y, Xu L, Wang M, Li J, Zheng Q, Zhang D, Wang H (2016) PARP-1-modulated AIF translocation is involved in streptomycin-induced cochlear hair cell death. Acta Otolaryngol 6:545–550

    Article  Google Scholar 

  35. Lu H, Wang X, Sun W, Hu Y, Gong S (2010) New insights into glutamate ototoxicity in cochlear hair cells and spiral ganglion neurons. Acta Otolaryngol 130:1316–1323

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by Shandong Province Key Research and Development Program Project (No. 2016GSF201087), the National Natural Science Foundation of China (No. 81570918), the Shandong Provincial Natural Science Foundation, China (ZR2014HQ076), the Key Project of Shandong Provincial Programs for Research, and Development (2015GSF118153).

Author information

Authors and Affiliations

  1. Department of Pathology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, 250021, People’s Republic of China

    Zhixin Cao

  2. Department of Otolaryngology-Head and Neck Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, 250021, People’s Republic of China

    Zhixin Cao, Qianqian Yang, Haiyan Yin, Qi Qi, Hongrui Li, Gaoying Sun, Wenwen Liu & Jianfeng Li

  3. Shandong Provincial Key Laboratory of Otology, Jinan, 250021, People’s Republic of China

    Qianqian Yang, Haiyan Yin, Qi Qi, Hongrui Li, Gaoying Sun, Wenwen Liu & Jianfeng Li

  4. Laboratory of Physical and Chemical Analysis, Shandong Academy of Occupational Health and Occupational Medicine, Shandong Academy of Medical Sciences, Jinan, 250062, People’s Republic of China

    Hongliang Wang

Authors
  1. Zhixin Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qianqian Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Haiyan Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qi Qi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hongrui Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gaoying Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hongliang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Wenwen Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jianfeng Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Wenwen Liu or Jianfeng Li.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cao, Z., Yang, Q., Yin, H. et al. Peroxynitrite induces apoptosis of mouse cochlear hair cells via a Caspase-independent pathway in vitro. Apoptosis 22, 1419–1430 (2017). https://doi.org/10.1007/s10495-017-1417-8

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-017-1417-8

Keywords

Search

Navigation