The Study On The Mechanism Underlying The Streptomycin-induced Cochlear Hair Cell Damage

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PARP-1 modulated AIF translocation is involved in streptomycin-inducing cochlear hair cell deathObjective:The hair cell loss induced by aminoglycosides(AGs)is considered as an important issue in the sensorineural hearing loss.Ototoxic drug,represented by the streptomycin(SM),is applied as an important part in the clinical medicine.The definite mechanism underlying the drugs-induced hair cell(HC)loss remains unclear.Researchers previously classified the AGs-induced HC loss into apoptosis and placed Caspases(a family of cys proteases),as a central role in carrying out cell death,but recently the newly discovered Caspase-independent cell death pathways were also detected in HC death.In this study,we wanted to establish two SM-induced HC loss models and to explore the role of apoptosis-inducing factor(AIF),an important cell death mediator located in mitochondria,in the models,so as to decipher the mechanisms of SM ototoxicity and to explore new targets for the intervention of SM-induced deafness.Methods:The two cochlear HC loss models were established by organic culture of basilar membranes of neonatal rats cochleae and continuous culture of HEI-OC1 cells.The SM sulfate and the PARP-1 inhibitor—3-AB were applied into the models to induce the injury and protection of HCs.Immunofluorescence staining of myosin ?a protein was used to detect the number of remaining HCs in Organ of Corti after 24h treatment of SM and to confirm the best treatment concentration of drugs in the following experiments.MTT assay was applied to explore the relative viability of HEI-OC1 cell under varying concentration of SM,and to confirm the working concentration in the subsequent experiments.The protective effect of 3-AB(1 mM)on SM treated HEI-OC1 cells was also examined by MTT assay.Immunostainings were used to explore the translocation of AIF and activation of PARP-1.The isolation of mitochondria and Western-blot was applied to quantify the change of AIF protein in mitochondria and cytosol.The expressions of PARP-1 mRNA and protein were achieved by qRT-PCR and Western-blot respectively.Results:Immunostaning of Myosin ?a showed that Organ of Corti exhibited HC loss,disordered arrangement,and contraction of basilar membrane at different degree depending on the variation of SM concentration.Under the same drug concentration,the loss of auditory sensory cell progressed from the basal turn to the apex.No difference was detected in the relative remaining ratio of inner and outer HCs.MTT assay revealed a dose-dependent cell loss manner in HEI-OC1 cells treated with SM and survival ratios were significantly reduced with increase of SM concentration.After 24h treatment of 2mg/ml SM,the nuclei of remaining HCs of basilar membrane showed both atrophic and swollen appearance.In the control group,AIF distributed in the cell body and no AIF was detected in the nuclear region.However,the AIF staining was strengthened in most HCs and aggregated in the nuclei after drug exposure.SM(5mg/ml)treated HEI-OC1 cell also showed the co-staining of AIF and nuclei.Western-blot assay showed that the expression of AIF protein in mitochondria was decreased,whereas increased in cytosol.No obvious PARP-1 expression was detected in HC of the control group by immunofluorescence staining.However,a strengthened staining of PARP-1 was exhibited in nuclear region of the SM treated cells.qRT-PCR and Western-blot showed that the expression of mRNA and protein of PARP-1 in HEI-OC1 cells increased significantly.MTT assay showed a partial alleviation from 3-AB treatment in SM induced HEI-OClcell loss.The tendency of AIF alteration in mitochondria and cytosol was also reversed by 3-AB.Conclusions:SM could induce dose-and location-dependent cochlear HC death in vitro.AIF might be translocated from mitochondria to nucleus and cytosol within SM-treated hair cells.The translocation of AIF might be modulated by PARP-1.Part ? Changes of inner hair cell ribbon synapses in a hearing loss model of rat induced by co-administration of streptomycin and furosemideObjective:The researches concerning the ototoxicity of AGs were mostly focused on the mechanism about how drugs undermined the cochlear hair cell and spiral ganglion neurons(SGNs).The alteration of ribbon synapses,the first relay part of auditory pathway,in the acute hearing loss has not been reported.The cochlear ribbon synapse is the specialized synaptic structure located between inner hair cell and SGNs,playing an important role in the rapid,sustainable and accurate acoustic signal transduction.Ribbon synapse was viewed as the most vulnerable component to the external stimuli in the hearing pathway.We want to build an acute hearing loss model of rat by the co-administration of furosemide and SM and to decipher the alteration of ribbon synapses after the drugs treatment.Methods:The acute hearing loss model was established in rats by intraperitoneal injection with furosemide(200 mg/kg)and intramuscular injection with SM sulfate(200mg/kg)at the same time.Auditory brainstem response(ABR)was employed to assess the hearing thresholds of rats under drugs.The immunofluorescence stainings of CtBP2,Glu R2&3,Myosin ?a and Tuj-1 were used to identify the changes of pre-and post-synaptic parts of ribbon synapses,hair cell and SGNs respectively.Results:ABR showed that the hearing loss at all tested frequencies in the rats 1 d after the drugs exposure,and no restoration was detected until 1 month.The immunostaining of CtBP2 revealed the significant decrease in the number of ribbon synapses,which exacerbate from the apex to the basal turn,worse than the loss of inner hair cells.Part of the remaining synapses showed swelling appearance and moved location,which was consistent with the loss of synapses.The abruption of pre-and post-synaptic part marked by the CtBP2 and Glu R2&3 was also detected 10 d after drugs exposure.SGNs were intact 10 d after drugs treatment,whereas the cell body and neural fibers of SGNs significantly decreased 1 month later.Conclusion:The co-administration of furosemide and SM could induce an irreversible hearing loss at all frequency.The destruction of ribbon synapses induced by drugs might play an important role in the early hearing loss.