The Effects And Mechanism Research Of Blebbistatin Inhibits Neomycin-induced Apoptosis In Hearing Hair Cells

Objective:In 2018,the WHO reported that there were about 466 million people with hearing impairment worldwide.Sensorineural hearing loss due to abnormal inner ear function accounts for about 90% of the patients.Aging,noise,and ototoxic drug induced hair cell(HC)loss are the major causes of sensorineural hearing loss.Aminoglycoside antibiotics are commonly used in the clinic,but these often have ototoxic side effects due to the accumulation of oxygen free radicals and the subsequent induction of HC apoptosis.In this study,the authors try to find a drug that can prevent aminoglycoside antibiotics’ damage in hair cells and explore its mechanism,so as to provide a feasible idea for the treatment of drug-induced deafness.Methods:In this study,the small-molecule drug Blebbistatin was selected as the research object,and the aminoglycoside antibiotic neomycin was selected for the experiment.The author first use hair cell like HEI-OC-1 cells and tissue culture of inner ear hair cells for cultivating models,joined the 0.5 mM neomycin as damage condition.Through the CCK-8 experiment,Live-Dead Cell Staining Kit and cell count methods explored whether Blebbistatin can improve the rate of cells survival after neomycin exposure.The author searched for the optimal protective concentration of Blebbistatin by experiment,and the concentration as a standard for the subsequent experiment.Similarly,in the HEI-OC-1 hair cells and the injury model of hair cells,flow cytometry analysis,qPCR detection and immunofluorescence staining were used to explore whether Blebbistatin can enhance the survival rate of cells induced by aminoglycoside antibiotics by inhibiting cell apoptosis.Then the authors used the same method to investigate the effects of Blebbistatin on the accumulation of mitochondrial membrane potential,reactive oxygen species and synaptic structure in the HEI-OC-1 hair cells and inner ear hair cell of mice after Blebbistatin and aminoglycoside antibiotics exposure.Results:Our results suggest after neomycin exposure,Blebbistatin can reduce the apoptosis induced by neomycin in both types of cells,and this effect is positively correlated with the concentration of Blebbistatin within a certain dose range.Blebbistatin can maintain the balance of oxidant and anti-oxidant gene expression and reduce the accumulation of ROS and thus maintain mitochondrial function and prevent apoptosis in HEI-OC-1 cells and cultured cochlear hearing cells after neomycin exposure.At the same time,Blebbistatin maintains the integrity of the synaptic structure between hair cells and spiral neurons,providing the necessary prerequisite for communication between hair cells and spiral neurons.Conclusion:In conclusion,our results suggest that Blebbistatin can maintain the balance of oxidant and anti-oxidant gene expression and reduce the accumulation of ROS and thus maintain mitochondrial function and prevent apoptosis in HEI-OC-1 cells and cultured cochlear hearing cells after neomycin exposure.These results suggest that Blebbistatin might have potential clinical application in preventing aminoglycoside-induced hearing cell loss and subsequent hearing loss,and we will further investigate its protective mechanism and clinical application in future studies.