Protective Effect And Mechanism Of Icariin Against Gentamicin-induced Ototoxicity

Objective: According to WHO data,the world ' s hearing-impaired population is about 460 million,of which drug deafness accounts for a certain proportion.Aminoglycoside antibiotics are cheap and have broad spectrum antibacterial properties,which are currently one of the most widely used antibiotic in the world.However,in the course of clinical treatment,the incidence of ototoxicity is very high(3.2% ? 47%).Aminoglycosides can enter auditory hair cells,and then induce the production and accumulation of reactive oxygen species(ROS).The excessive production and excessive deposition of ROS destroy the normal balance,leading to the oxidation of DNA,lipids,proteins and other important cellular components,and ultimately leading to cell death.Human hair cells are not renewable,so the hearing damage caused by aminoglycoside antibiotics is permanent and irreversible.This has caused a heavy burden on the lives of hundreds of millions of people around the world,not only to bear the dysfunction,but also to have a social and emotional imbalance,causing a huge economic burden to the society.Several strategies have been reported to overcome the irreversible loss of hair cells induced by aminoglycosides in mammals,among which antioxidant is an important part.In recent years,Icariin(ICA),the main active ingredient of traditional herb Epimedium,has been widely concerned because of its antioxidant,anti-inflammatory and anti-apoptotic properties.ICA has been shown to activate SIRT3,which is closely related to oxidative stress and plays a key role in ROS removal,and has been proved to be closely related to the protective effect of oxidative stress injury in inner ear hair cells.In some disease models,AMPK is the upstream of SIRT3.AMPK has been confirmed to be involved in the pathophysiological process of aminoglycoside-induced ototoxicity,and phosphorylated AMPK is down-regulated in gentamicin-treated hair cells.The activation of AMPK-SIRT3 signaling pathway contributes to the improvement of mitochondrial function,thereby reducing the progression of disease.However,the pharmacological effect of Icariin on gentamicin induced ototoxicity in hair cells deserves our attention.Whether icariin can activate AMPK-SIRT3 signaling pathway in gentamicin induced ototoxicity is still unknown.The aim of this study was to investigate the protective effect of Icariin on gentamicin induced ototoxicity and the role of AMPK-SIRT3 signaling pathway in the protection of Icariin on gentamicin induced ototoxicity.Methods: 1.The ototoxicity model induced by aminoglycoside antibiotics was established in HEI-OC1 cells and isolated cochlea using gentamicin.2.The pharmacological effects of icariin on gentamicin-induced ototoxicity were determined by cell viability assay and immunofluorescence assay.3.The effect of icariin on gentamicin-induced oxidative stress injury in HEI-OC1 and cochlear tissues was detected by flow cytometry labeled with DCFH-DA probe and fluorescence detection.4.The effects of icariin on gentamicin-induced apoptosis of HEI-OC1 cells were detected by quantitative RT-PCR analysis,Td T-mediated d UTP Nick-End Labeling(TUNEL)assay and Western blotting.5.Western blot,cell viability test,DCFH-DA probe fluorescence test,and TUNEL staining were performed on HEI-OC1 cultured in groups.Immunofluorescence test was performed on cochlear tissue cultured in groups to detect the mechanism of AMPK and SIRT3 in the protection of icariin against ototoxicity.Results: 1.Gentamicin treatment can reduce the survival rate of hair cells in HEI-OC1 cells and cochlear tissues in vitro.The survival rate of hair cells in HEI-OC1 cells and cochlear tissues in vitro was significantly increased by icariin pretreatment.2.Gentamycin treatment can increase the apoptosis of HEI-OC1 cells,icariin pretreatment can inhibit the apoptosis of HEI-OC1 cells induced by gentamicin.3.Gentamycin treatment can increase ROS in HEI-OC1 cells and cochlear tissues in vitro,icariin pretreatment can reduce the production of ROS in HEI-OC1 cells and cochlear tissues in vitro induced by gentamicin.4.Icariin exerts ototoxicity protection by activating AMPK-SIRT3 signaling pathway to inhibit gentamicin-induced apoptosis and oxidative stress.Conclusion : Gentamicin can induce ROS production,activate mitochondrial apoptosis,damage HEI-OC1 and cochlear hair cells.Icariin can prevent gentamicin ototoxicity by anti ROS and anti-apoptosis.AMPK-SIRT3 signaling pathway plays a key role in the protection of icariin against gentamicin induced ototoxicity.This study provides a theoretical basis for the development of icariin as a preventive drug for gentamicin ototoxicity.