Experimental Study Of Carbonic Anhydrase 8 Inhibiting Cisplatin-induced Ototoxicity

ObjectiveTo study the role of carbonic anhydrase Ⅷ（CAⅧ）in cisplatin-induced ototoxicity,so as to provide a basis for the clinical prevention and treatment of cisplatin ototoxicity.Methods1.In vivo experiment40 BALB/c mice aged 6 weeks were randomly divided into 4 groups:control group,cisplatin group,cisplatin + puerarin group and puerarin group.Cisplatin group received cisplatin（4.5mg/kg/d）intraperitoneally for 5 days.Puerarin（200mg/kg/d）was injected intraperitoneally for 9 days in cisplatin +puerarin group,and cisplatin（4.5 mg/kg/d）was injected intraperitoneally for 5days on the 10 th day.Puerarin group was injected with puerarin（200mg/kg/d）for 14 days.The control group was intraperitoneally injected with the same amount of normal saline for 5 days.Auditory brainstem response（ABR）was used to detect the auditory function of mice.Immunofluorescence staining and western blot were used to detect the localization of CAⅧ and inositol 1,4,5-trisphosphate receptor type 1（IP₃R1）in mouse cochlea.2.In vitro experimentThe House Ear Institute-Organ of Corti 1（HEI-OC1）mouse cochlear hair cell line was cultured in 33℃,10% CO₂ incubator.HEI-OC1 cells were divided into 4 groups: control group,cisplatin group,cisplatin + puerarin group and puerarin group.The control group was given normal culture medium,the cisplatin group was given culture medium containing 20μM cisplatin,the cisplatin + puerarin group was given culture medium containing 20μM cisplatin and 20μM puerarin,the puerarin group was given culture medium containing20μM puerarin.CCK-8 assay was used to detect cell viability.Western blot and immunofluorescence staining were used to detect the expression of CAⅧ and other proteins in cochlear hair cells.Mag-Fluo-4,AM Ca²⁺ fluorescent probes were used to detect the changes of Ca²⁺ level in endoplasmic reticulum of HEIOC1 cells.Rhod-2,AM Ca²⁺ fluorescent probes were used to detect the changes of Ca²⁺ level in mitochondria of HEI-OC1 cells.Results1.The expression of CAⅧ in normal mouse cochleaImmunofluorescence results showed that CAⅧ protein was expressed in the spiral ganglion and cochlear hair cells of mouse cochlea,but less in other parts,suggesting that CAⅧ protein may play a role in the generation of hearing.2.The expression of CAⅧ in the cochlea of cisplatin-induced mice.Western blot analysis showed that the expression of CA Ⅷ protein was detected in HEI-OC1 cells.Compared with the control group,The decrease in CAⅧ.protein was more obvious under the action of 20μM cisplatin.ABR test showed that the ABR threshold of cisplatin group was significantly higher than that of control group at 8k Hz,12 k Hz,24 k Hz and 32 k Hz.The ABR threshold of cisplatin + puerarin group was significantly lower than that of cisplatin group（P <0.05）.There was no significant difference in ABR threshold between puerarin group and control group.The results showed that puerarin had obvious protective effect on ototoxicity induced by cisplatin.The results of Western blot and immunofluorescence showed that the expression of CAⅧ protein was significantly decreased and the expression of IP3R1 protein was significantly increased in the cisplatin group compared with the control group.Compared with cisplatin group,the expression of CA Ⅷprotein in mouse cochlea and HEI-OC1 cells was significantly increased and the expression of IP3R1 protein was significantly decreased in cisplatin +puerarin group.IP3R1 is a Ca²⁺ efflux channel in endoplasmic reticulum.These results suggest that CAⅧ may play a role in cisplatin ototoxicity by regulating IP3R1-mediated Ca²⁺ channels.3.CAⅧ inhibits cisplatin-induced apoptosis of cochlear cellsThe results of cytoactive staining showed that the content of Ca²⁺ in endoplasmic reticulum of HEI-OC1 cells was significantly decreased and the content of Ca²⁺ in mitochondria was significantly increased in cisplatin group compared with control group.Compared with cisplatin group,the Ca²⁺ content in endoplasmic reticulum of HEI-OC1 cells was significantly increased and the Ca²⁺ content in mitochondria was significantly decreased in cisplatin + puerarin group.The results suggest that the decrease of CAⅧ protein content can lead to the decrease of Ca²⁺ in endoplasmic reticulum and the increase of Ca²⁺ in mitochondria,which mediate the ototoxicity of cisplatin.Puerarin can alleviate the ototoxicity of cisplatin by reducing mitochondrial calcium overload.The results of Western blot and immunofluorescence showed that the apoptosis level of HEI-OC1 cells in cisplatin group was significantly higher than that in control group.Compared with cisplatin group,the apoptosis level of cisplatin + puerarin group was significantly decreased.The results showed that puerarin could reduce the level of apoptosis induced by cisplatin.The CCK-8 assay showed that puerarin at 20μM had more protective effect on the cell viability of HEI-OC1 cells than cisplatin.ConclusionsCAⅧ was expressed in normal mouse cochlea and HEI-OC1 cells.After cisplatin treatment,the expression of CAⅧ decreased significantly,while the expression of IP3R1 increased,which caused mitochondrial calcium overload and finally led to cochlear cell apoptosis,suggesting that CAⅧ inhibits the ototoxicity of cisplatin...