Study On The Mechanism Of Cisplatin-induced Damage In Mouse Cochlear Hair Cells

Cisplatin is often used in the chemotherapy of various malignant tumors due to its strong anti-tumor effect,but its ototoxicity can lead to the occurrence of sensorineural hearing loss.So,the clinical application of this drug is greatly limited.Current studies suggest that cisplatin ototoxicity is caused by the death of cochlear hair cells.Although studies have been conducted,the exact mechanism by which cisplatin causes death in cochlear hair cells is still unclear.The endoplasmic reticulum is one of the important organelles of eukaryotes,which is involved in the important life activities of cells such as protein assembly,transport and storage of calcium ions.Stimulation of external harmful factors can result in to the destruction of the endoplasmic reticulum homeostasis and trigger endoplasmic reticulum(ER)stress.In this study,we aimed to investigate whether ER stress was involved in cisplatin-induced ototoxicity,and if so,to explore the possible mechanism underpinning such an action of cisplatin.Methods(1)The cell viability of HEI-OC1 cells under different treatment time and different concentrations was detected by CCK-8.(2)Western-blot was used to detect indicators of the ER stress of HEI-OC1 cells treated with cisplatin at different timesand concentrations.(3)Immunofluorescence technique was used to detect indicators of the ER stress of HEI-OC1 cells treated with cisplatin.(4)Flow cytometry was used to detect apoptosis of HEI-OC1 cells treated with or without cisplatin and the ER stress inhibitor.(5)Western-blot was used to detect indicators of the ER stress and the expression of cleaved-Caspase3 in HEI-OC1 cells treated with or without cisplatin and the ER stress inhibitor.(6)The expression indicators of ER stress and the expression of cleaved-Caspase3 in the hair cells of the cochlear basement membrane were detected by immunofluorescence.Results(1)Cisplatin can trigger HEI-OC1 cell damage and apoptosis:when HEI-OC1 cells were treated with cisplatin,the cell viability of HEI-OC1 cells decreased gradually,which was in a time-and concentration-dependent manner in vitro.Cisplatin induced cell injury via activation of apoptotic pathway in HEI-OC1 cells.(2)Cisplatin induced ER stress in HEI-OC1 cells:under cisplatin stimulation,expressions of indicators of ER stress,GRP78 and PERK were increasing,which exhibited in a time-and concentration-dependent manner;Mild endoplasmic reticulum stress can help cells resist harmful factors,and severe endoplasmic reticulum stress led to apoptosis(3)4-PBA inhibited ER stress and attenuated apoptosis in HEI-OC1 cells exposure to cisplatin:after using ER stress inhibitor 4-PBA,ER stress was inhibited,and the expressions of GRP78 and PERK were decreasing,whereas,the expression of cleaved-Caspase3 in HEI-OC1 cells was decreasing.(4)4-PBA inhibited ER stress and attenuated apoptosis in cochlea explants exposure to cisplatin:4-PBA can inhibit ER stress induced by cisplatin in cochlea explants in vitro.The expressions of GRP78 in cochlea explants and cleaved-Caspase3 were decreasing in hair cells in 4-PBA combination with cisplatin treatment group,the number of hair cells were increasing,and the morphology was better.Conclusion Cisplatin leads to auditory cells damage via activation of ER stress-mediated caspase-3 dependent apoptotic pathway in experiment in vitro.