Enhancing Autophagy By Down-regulating GSK-3β Alleviates Cisplatin-induced Ototoxicity In Vivo And In Vitro

Part 1: Glycogen Synthetic Kinase 3(GSK-3β)inhibitor lithium chloride(LiCl)attenuates cisplatin-induced ear deafness in ratsObjective : To establish a rat model of cisplatin ear toxic deafness,and to explore the antagonistic effect of GSK-3β inhibitor LiCl on cisplatin ear toxic deafness and the changes of autophagy in outer hair cells.Method : Male SD rats(200-220g)were divided into four groups(Control group,Cisplatin group,LiCl group,Cisplatin + LiCl group)randomly.Cisplatin 4.6mg/kg was given daily by intraperitoneal injection in the first 3 days as Cisplatin group;besides the same dose of cisplatin,LiCl 100mg/kg was administered in Cisplatin + LiCl group.Auditory brainstem response(ABR)was measured before the start of the experiment and 72 hours after the last cisplatin administration.The difference in the survival rate of outer hair cells between groups was compared by counting the outer hair cells by basement membrane plaque.Immunofluorescence was used to compare the expression of glycogen synthase kinase 3βand autophagy-related proteins(LC3 Ⅱ and P62)in outer cochlear hair cells between each groups.Results : 1.The ABR response domain significantly higher in Cisplatin group,while the outer hair cells survival number significantly reduced compared with the control group.LiCl pretreatment reduced cisplatin ear toxic effects in rats(p<0.05).2.Compared with the control group,cisplatin group cochlear outer hair cells of the phosphorylated form of GSK-3β-Ser9 and the level of expression of autophagy were increased,and cisplatin + LiCl group this phenomenon is more obvious than the cisplatin group.Conclusions : 1.LiCl,a GSK-3β inhibitor,can reduce the elevation of ABR response domain induced by cisplatin in rats,increase the survival rate of rat cochlear outer hair cells under the action of cisplatin,and enhance autophagy in outer hair cells.2.GSK-3βand related autophagy may be involved in LiCl cisplatin antagonism toxicity deaf ears.Part 2: Autophagy in inhibiting GSK-3β antagonizing cisplatin-induced hair cells injury.Objective : To establish an in vitro model of cisplatin ototoxicity in HEI-OC1 cells,and to reduce GSK-3β expression in the above experimental model,and to reversely regulate autophagy.To explore whether autophagy inhibits GSK-3β against cisplatin-induced hair cell injury.Method : Adenovirus with GSK3β silencing sequence transfected HEI-OC1 cells to establish a stable GSK3β knockout HEI-OC1 cells(HEI-OC1 GSK3β-KO).Cells were divided into control group,cisplatin group,cisplatin + LiCl group,GSK3-KO group,cisplatin + GSK3-KO group,cisplatin + GSK3-KO + 3-MA group.CCK-8 method was used to detect the difference in cell survival rate between each groups,flow cytometry was used to detect the difference in the proportion of apoptosis between groups,transmission electron microscope was used to observe the differences in cell mitochondrial morphology and the number of autophagosomes,and western blot was used to detect autophagy And the expression of apoptosis-related proteins(LC3 Ⅱ,P62,bcl-2 and bax).Results : 1.Similar to the effects in animal experiments,the application of GSK3β inhibitor LiCl can increase the survival rate of HEI-OC1 cells under the action of cisplatin,reduce the proportion of apoptosis and the expression level of apoptotic proteins,enhance the expression of autophagy proteins and the number of phages,reduces the number of swollen vacuolar mitochondria in the cell.2.Knockout of GSK-3β in HEI-OC1 cells has similar cytoprotective effect as the use of GSK3β inhibitor LiCl;3.After the addition of autophagy inhibitor 3-MA,GSK-3β knockout and application of GSK3 β inhibitor The aforementioned cytoprotective effect of LiCl disappeared.Conclusions : 1.Inhibition of GSK-3β can alleviate HEI-OC1 injury induced by cisplatin in hair cell lines.2.The cytoprotective effect of inhibiting GSK-3β may be involved in inducing protective autophagy by reducing mitochondrial damage in cisplatin application.