Peroxynitrite induces apoptosis of mouse cochlear hair cells via a Caspase-independent pathway in vitro.
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.
['Cao|Zhixin|Z|', 'Yang|Qianqian|Q|', 'Yin|Haiyan|H|', 'Qi|Qi|Q|', 'Li|Hongrui|H|', 'Sun|Gaoying|G|', 'Wang|Hongliang|H|', 'Liu|Wenwen|W|', 'Li|Jianfeng|J|']
D000818:Animals D000831:Animals, Newborn D017209:Apoptosis D051033:Apoptosis Inducing Factor D053477:Apoptotic Protease-Activating Factor 1 D053148:Caspase 3 D053181:Caspase 8 D053453:Caspase 9 D002470:Cell Survival D003051:Cochlea D005786:Gene Expression Regulation D051379:Mice D008810:Mice, Inbred C57BL D016877:Oxidants D030421:Peroxynitrous Acid D019253:Proto-Oncogene Proteins c-bcl-2 D015398:Signal Transduction D046509:Tissue Culture Techniques D051028:bcl-2-Associated X Protein
Peroxynitrite Apoptosis Hair cells Immunofluorescence Hearing loss