Damage To Hair Cells Treated With Low-dose Cisplatin And Low-dose D-galactose In Different Order And Its Possible Mechanism

Objective: 1.To explore the appropriate drug concentration for D-galactose(D-gal)and cisplatin(CP)to act on the organ of corti explants without causing hair cells death.2.To explore whether the low concentration of D-galactose or cisplatin increases the sensitivity of organ of corti explants to drugs.3.To explore the possible mechanism of the result that the treatment of low-concentration cisplatin first followed by low-concentration Dgalactose caused hair cells loss in organ of corti explants,while the treatment with lowconcentration D-galactose first followed by cisplatin of low concerntration did not.Methods: 1.Select 4-5 days postnatal day C57 mice,dissect the basilar membrane from the cochlea and place them in medium containing 1u M,2u M,4u M CP and 40mg/ml,50mg/ml,60mg/ml and 70mg/ml D-gal respectively for 48 h while the control group was cultured in normal medium for 48 h.The stereocilia of auditory hair cells were labeled with phalloidin.The differences in the survival numbers of inner hair cells and outer hair cells of the same length of basilar membrane in the apical,middle and basal turn were compared between groups.2.The organ of corti explants were divided into control group,CP group,D-gal group,CP+D-gal group and D-gal+CP group.The CP+D-gal group was treated with CP at a concentration of 1u M for the first 48 h,and then changed to medium containing D-gal at a concentration of 40mg/ml for the second 48 h,while the D-gal+CP group was treated with D-gal at a concentration of 40mg/ml for the first 48 h,and changed to 1u M CP for the second 48 h,the CP group was only treated with CP of the same concentration for 48 h,the D-gal group was only treated with D-gal of the same concentration for 48 h and the control group was only replaced with normal medium.The auditory hair cells were labeled with phalloidin and the difference in the number of living hair cells between groups was compared.Trizol method was used to extract RNA from organ of corti explants and real-time quantitative PCR was used to measure the transcription level of Bcl2 and Bax in each group.The m RNA expression levels of Bcl-2and Bax,as well as Bcl-2/Bax ratio values were compared between groups.The apoptosis level of organ of corti explants was detected by TUNEL method and compared between groups.The mitochondrial ROS levels of organ of corti explants in each group were detected by Mito SOX RED kit and compared between groups.3.The organ of corti explants were divided into control group,cisplatin group and D-galactose group.The cisplatin group was treated with CP of 1u M concentration for 48 h and the D-galactose group was treated with D-galactose of 40mg/ml concentration for 48 h,while the control group was only replaced with normal medium.Real-time quantitative PCR was used to detect the difference in the transcription levels of HSP70 among the groups while western blot and immunofluorescence was used to detect the difference in protein expression level and the distribution of HSP70 among the groups.4.The organ of corti explants were divided into control group D-gal+CP group,inhibitor group,D-gal+dmso+CP group,inhibitor+CP,D-gal+inhibitor and D-gal+inhibitor+CP group.The auditory hair cells were labeled by phalloidin and the difference in the number of living hair cells between groups was compared.Results: 1.Compared with the control group,both 4 u M CP and 70mg/ml D-gal had a certain damage effect on the outer hair cells in the apical,middle and basal turn of organ of corti explants.Besides,D-gal also caused loss of inner hair cells.D-gal at the concentration of 60mg/ml only caused the loss of outer hair cells in the basal turn of organ of corti explants.2.Compared with the control group,the outer hair cells of the apical,middle and basal turn of the organ of corti explants in the CP+D-gal group were significantly damaged,while no obvious loss was observed in the auditory hair cells in the D-gal+CP group.3.Compared with the control group,the number of ribbon synapses in the D-gal group,the CP group,the CP+D-gal group and the D-gal+CP group decreased to different degrees.4.Compared to the control group,the levels of Bax m RNA expression in D-galactose group and cisplatin+D-galactose group were elevated.The m RNA expression levels of Bcl-2 in D-galactose+cisplatin group significantly decreased.Compared with the control group,the Bcl2/Bax ratio of the cisplatin group did not change significantly,while the Bcl-2/Bax ratio of D-galactose group,cisplatin+Dgalactose and D-galactose+cisplatin group decreased compared with the control group.5.TUNEL positive staining cells number in the cisplatin group,D-galactose group,Dgalactose+cisplatin group and cisplatin+D-galactose group increased compared with the control group.6.Compared with the control group,the level of HSP70 m RNA and protein did not change significantly in cisplatin group which was upregulated in Dgalactose group 7.Compared to the control group,the numbers of auditory hair cells did not change significantly in D-gal+CP group,inhibitor group and D-gal+dmso+CP group,which deacreased in inhibitor+CP,D-gal+inhibitor and D-gal+inhibitor+CP group in the basal turn.Moreover,the basal turn outer hair cells in D-gal+inhibitor+CP group were less than the ones in inhibitor+CP,D-gal+inhibitor group.The above differences are all statistically significant(p < 0.05).Conclusion:1.Cisplatin and D-galactose can cause loss of hair cells of organ of corti explants.2.Organ of corti explants treated with cisplatin at low concentration are more susceptible to damage by D-galactose,while low-concentration D-galactose treatment does not make them more susceptible to cisplatin damage;3.Treatment with low concentration of cisplatin and low concentration of D-galactose,respectively and cotreated,causes damage to ribbon synapses of inner hair cells in organ of corti explants.