Effect Of Endoplasmic Reticulum Stress On Apoptosis Of Mouse Cochlear Cell Induced By Cisplatin In Vitro

Objective To investigate the effect of endoplasmic reticulum stress(ERS) on apoptosis of mouse cochlear cell induced by cisplatin(CDDP) in vitro, and to explore the mechanism of CDDP-induced cochlear cell apoptosis, so as to provide a new idea for protecting against CDDP ototoxicity.Methods The cochlear basilar membranes from newborn Kunming mouse at 3 to 5 days were isolated and cultured in Dulbecco’s Modified Eagle Media: Nutrient Mixture F-12(DMEM/F12) containing 1% bovine serum albumin(BSA). The original medium was removed after 24 hours. 1. The basilar membranes were randomly divided into five groups. Control group was added into 2ml fresh medium without CDDP, other four groups were added into 2ml fresh medium containing different concentrations of CDDP(4μg/ml, 8μg/ml, 16μg/ml and 32μg/ml). The experiment was terminated after continually cultured for 24 hours. Fluorescein isothiocyanate(FITC)-labeled phalloidine staining was used to observe the morphological changes of cochlear hair cells. The missing of hair cell was detected by hair cell counts. Apoptosis of cochlear hair cell was detected by both tetramethylrhodamine isothiocyanate(TRITC)-labeled phalloidine staining and Hoechst 33258 staining, and was assessed by apoptotic hair cell counts. Western blot was carried out for detecting the expressions of glucose-regulated protein(GRP78) and cysteinyl aspartate specific proteinase 12(caspase-12) as ERS marker proteins in mouse cochlear cells. At the same time, Western blot was used to detect the expression of inositol-requiring enzyme 1(IRE1), c-Jun N-terminal kinase(JNK) and Bcl-2 associated X protein(Bax) in mouse cochlear cells. 2. The basilar membranes were randomly divided into four groups. Control group was added into 2ml fresh medium without CDDP. CDDP group was added into 2ml fresh medium containing 16μg/ml of CDDP. Tauroursodeoxycholic acid(TUDCA) group was added into 2ml fresh medium containing 500μg/ml of TUDCA. CDDP plus TUDCA group was first added into 2ml fresh medium containing 500μg/ml of TUDCA, and then added into 16 μg/ml of CDDP after 15 minutes, and cultured for 24 hours with other three groups. The experiment was terminated and Western blot was carried out for detecting GRP78 and caspase-12 expression in mouse cochlear hair cell.Results 1. FITC-labeled phalloidine staining showed green fluorescence. The staining of cochlear inner hair cell(IHC) and outer hair cell(OHC) were uniform, and the structure of cilia was intact and arranged orderly in control group. The results of hair cell counts showed that the percent of missing IHC and OHC were both lower than 1% in control group. While the cilia of cochlear hair cell were lodged, lost and arranged in disorder in CDDP group. When the concentration of CDDP was 4μg/ml, the percent of missing OHC was significantly increased as compared with control group(P<0.05), While the percent of missing IHC was not significantly changed(P>0.05). Moreover, with the concentration of CDDP was gradually increased, the percent of missing IHC and OHC were significantly increased, showing a clear dose-response relationship(P<0.01).2. The result of Hoechst 33258 staining showed that the nuclei of cochlear hair cells were light blue fluorescence, and the chromatin was well distributed in control group. The percent of apoptotic hair cells was lower than 5%. While in CDDP group, the nuclei of cochlear hair cells were shrunk with bright blue, and the chromatin was marginalized, agglutinated a block or broken to irregular pieces. With the concentration of CDDP was gradually increased, the broken nuclei increased significantly. The percent of apoptotic hair cells was significantly increased, showing a clear dose-response relationship(P<0.01). 3. The results of Western blot detection showed that there were weak expressions of GRP78 and caspase-12 in mouse cochleae in control group. While the expression levels of above proteins in CDDP groups were significantly elevated as compared with control group(P<0.01). Moreover, with the concentration of CDDP was gradually increased, the expressions of above proteins increased significantly, showing a clear dose-response relationship(P<0.01). After added into TUDCA, the expressions of GRP78 and caspase-12 was lower than those of in CDDP group(P<0.01). 4. The results of Western blot detection showed that there were weak expressions of IRE1, JNK1, JNK2 and Bax in mouse cochleae in control group. While the expressions of above proteins in CDDP groups were greater than those in control group(P<0.01). Moreover, with the concentration of CDDP was gradually increased, the expressions of above proteins increased significantly.Conclusions ERS-related proteins including GRP78, IRE1, JNK and caspase-12 were high expressions in mouse cochlear cells after CDDP intervention in vitro. Which suggesting ERS may be involved in the apoptosis of mouse cochlear cell induced by CDDP, and IRE1 pathway may be one of the pathways mediating mouse cochlear cells apoptosis by ERS in vitro.