, Volume 22, Issue 11, pp 1419–1430 | Cite as

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

  • Zhixin Cao
  • Qianqian Yang
  • Haiyan Yin
  • Qi Qi
  • Hongrui Li
  • Gaoying Sun
  • Hongliang Wang
  • Wenwen Liu
  • Jianfeng Li
Original Paper


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.


Hair cells Peroxynitrite Apoptosis Hearing loss Immunofluorescence 



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).


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Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Zhixin Cao
    • 1
    • 2
  • Qianqian Yang
    • 2
    • 3
  • Haiyan Yin
    • 2
    • 3
  • Qi Qi
    • 2
    • 3
  • Hongrui Li
    • 2
    • 3
  • Gaoying Sun
    • 2
    • 3
  • Hongliang Wang
    • 4
  • Wenwen Liu
    • 2
    • 3
  • Jianfeng Li
    • 2
    • 3
  1. 1.Department of PathologyShandong Provincial Hospital Affiliated to Shandong UniversityJinanPeople’s Republic of China
  2. 2.Department of Otolaryngology-Head and Neck SurgeryShandong Provincial Hospital Affiliated to Shandong UniversityJinanPeople’s Republic of China
  3. 3.Shandong Provincial Key Laboratory of OtologyJinanPeople’s Republic of China
  4. 4.Laboratory of Physical and Chemical AnalysisShandong Academy of Occupational Health and Occupational Medicine, Shandong Academy of Medical SciencesJinanPeople’s Republic of China

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