The Role Of Unfolded Protein Responses In Murine Cisplatin-induced Ototoxicity

Part ? The alteration of unfolded protein responses in cochlear cells in a cisplatin-induced ototoxicity rat modelObjective: To observe the alteration of unfolded protein responses(UPR)and analyze its significance in a cisplatin-induced ototoxicity rat model.Methods: Male SD rats were received cisplatin intraperitoneal injection of 4.6mg/kg/d for 3 consecutive days.Auditory brainstem response(ABR)was used to test the threshold shifts before and after cisplatin injection.The cochlea samples of the rats were collected after the final ABR test.TUNEL test and western blot were applied to test cell apoptosis in rats' cochleae.The difference in expression level of the key members in the UPR between the control and the cisplatin-treated group was compared by immunofluorescence.Results: Cisplatin elevated the rats' thresholds and induced apoptosis in rats' cochlear cells(p < 0.05),especially in outer hair cells,marginal cells of the stria vascularis and cells in the Reissner's membranes.Significant eleviation in the expression level of GRP78,CHOP,cleaved-caspase-9 and cleaved-caspase12 were seen in the cochlear tissues of cisplatin-treated rats(p < 0.05).In addition,the reduction in the expression level of total ubiquitinated protein in the cochlear tissues of cisplatin-treated rats was also observed.Conclusions: 1.Cisplatin elevated the rats' thresholds and induced apoptosis in rats' cochlear cells.2.Mitochondria-dependent intrinsic apoptosis signal may play an important role in cisplatin-induced apoptosis in cochlear cells,and the cisplatin-enhanced UPR in the rats' cochlear cells may be one of the reasons to the activation of the apoptosis signal.3.The disturbance of protein ubiquitylation by cisplatin may also be a possible reason to the disruption of proteostasis and the activation of UPR.Part ? Tauroursodeoxycholic acid(TUDCA)alleviates cisplatin-induced ototoxicity by attenuating unfolded protein responsesObjective: To study the alleviation effect of TUDCA on cisplatin-induced ototoxicity in vitro and in vivo.Methods: In vitro study: HEI-OC1 cells were divided into four groups: the control group,the TUDCA group,the cisplatin group and the cisplatin + TUDCA group.CCK-8 method was used to measure the difference in cell viability between the four groups.Flow cytometry(FCM)was applied to test the difference in apoptosis between the four groups.The alteration in cell ultrastructures was observed by transmission electron microscope(TEM).The difference in the expression level of GRP78 and CHOP was tested by western blot.In vivo study: Male SD rats were divided into four groups as it was in vitro study.ABR was used to test the threshold shifts of the rats in the four groups.The number of outer hair cells(OHCs)in the same area of the basal turn was counted.The expression level of GRP78 and CHOP in the organ of Corrti(OC)and in the OHCs was tested by immunofluorescence.Results: In vitro study: Cisplatin reduced the cell viability and induced apoptosis in HEI-OC1 cell line(p < 0.05).The dilation of endoplasmic reticulum and the swell and vacuollation of the mitochondria were observed in the cisplatin-treated cells under TEM,and the combined application of TUDCA and cisplatin partially alleviated the cytoxicity effects of cisplatin.Compared to the cisplatin group,the expression level of GRP78 and CHOP in the cisplatin + TUDCA group was reduced(p < 0.05).In vivo study: Compared to the control group,TUDCA alone did not affect the thresholds of the rats(p > 0.05).The threshold shifts of the rats in the cisplatin + TUDCA group were smaller than it in the cisplatin group,and the co-administration of TUDCA reduced the percentage of OHC loss induced by cisplatin(p < 0.05).The co-administration of TUDCA also reversed the cisplatin induced elevated expression of GRP78 and CHOP in the OHCs(p < 0.05).Conclusions: TUDCA itself does not have significant ototoxicity,and it can partially alleviate cisplatin-induced ototoxicity possibly by ameliorating the UPR and its related apoptosis.