The Role Of Autophagy In The Neomycin Ototoxicity

Aminoglycoside is a broad-spectrum antibiotic of mainly anti-aerobic gram bacteria,including Escherichia coli and Pseudomonas,in developing countries due to its low cost,low allergy,resistance less,still in the clinic for treatment of pulmonary tuberculosis,lung sac fibrosis,pediatric sepsis disease.Permanent irreversible hearing loss,vestibular dysfunction and renal injury are the major side effects.The ototoxicity is the main reason for limiting clinical application.Neomycin as a kind of aminoglycosides,with the most toxic and only ototoxicity and less likely to cause the characteristics of vestibular toxicity are mainly used in the treatment of hepatic encephalopathy and high cholesterol.Apoptosis induced by oxidative stress has been proved the main mechanism of ototoxicity of aminoglycoside antibiotic.Autophagy is widely concerned in recent years a cellular stress response,is a highly conserved protein degradation pathway,is the survival mechanism of the cells responding to a variety of harsh environments,almost occurred in all cells,in the inner ear,autophagy has been reported in the otolith development of mouse,otic vesicle early development of chicken and hearing loss of senescence accelerated mice plays an important role.However,there is no study on the autophagy mechanism in the normal postnatal and adult inner ear.Autophagy and oxidative stress and apoptosis were closely linked.Therefore,we hypothesized that autophagy may participate in the occurrence of the ototoxicity of aminoglycoside antibiotics,and may have a positive role in the protection and has an important correlation with concentration and time of the drug working.The purpose of the experiment is to establish ototoxicity model through C57BL/6J mouse cochlear basilar membrane and HEI-OC1 auditory cell lines injury by neomycin.The experiment was divided into the neomycin injury group and the control group,detection of LC3 by Western blot and observation of autophagy and autophagic lysosome through transmission electron microscope and cochlear Whole-mount and immunofluorescence staining to detect the LC3 expression,transfection with fluorescence of LC3 plasmid to detect of autophagy,and in different neomycin concentration and action time observation of the relationship between autophagy and apoptosis.The cochlear hair cell counting method and CCK8 experiment,PI-Annexin V apoptosis double staining flow cytometry assay were used to observe changes of the hair cell survival and apoptosis with the autophagy activation agent rapamycin which can up-regulate autophagy.The results verified the obvious existing autophagy and the development process inototoxicity model through Cellular and tissue levels.The level of autophagy was significantly related with the concentration and action time of the neomycin,a lot of autophagy appear when low concentration of neomycin injury.At this time,the activation autophagy will protect hair cell damage,reduce the apoptosis of hair cells,and high concentration of neomycin injury will cause substantial oxidative stress,leading to hair cells in a large number of apoptosis when autophagy has little effect.It can proved that the relationship between autophagy and apoptosis,indicating that autophagy is a cell intrinsic process,autophagy can reduce apoptosis,so as to reduce the damage of hair cells,which provide theoretical basis for the clinical prevention and treatment of aminoglycoside drug deafness and intervention time.