Cinchonine And Cinchonidine Alleviate Cisplatin-induced Ototoxicity By Regulating PI3K-AKT Signaling

ObjectiveCisplatin is a widely used chemotherapy drug for the treatment of many types of cancer,but its side effects,such as ototoxicity,limit its use.Cisplatin ototoxicity side effects mainly manifest as hearing loss and tinnitus,which seriously affect the quality of life of patients.There are very few drugs available for the treatment of cisplatin-induced hearing loss and the therapeutic effect is unclear,so it is important to find effective anti-cisplatin ototoxic drugs.Cisplatin damages inner ear cells,such as auditory hair cells,mainly by inducing apoptosis,which involves multiple signaling pathways and cytokines,and the PI3K/AKT pathway is one of the most important pathways.Therefore,the aim of this study is to investigate the natural compounds that can protect the auditory hair cells and the hearing after PIK3 CA binding and treatment with cisplatin,and to screen the natural compounds with the tightest binding and the best protective effect,namely,cinchonine and its isomer Cinchonidine,and to investigate the possible mechanism of hearing protection with this natural compound as the focus of the study.possible mechanisms for hearing protection and identify new potential targets for auditory protection.MethodsIn this study,we screened the tightly bound products,cinchonine and Cinchonidine,from a library of natural compounds by molecular docking and molecular dynamics simulations using PIK3 CA as the target.In vitro P2 neonatal mouse Corti organelles were cultured and treated with cisplatin and cinchonine(and Cinchonidine)in vitro as a single addition and combined treatment.Morphology and number of hair cells in Corti organoids were assessed by immunofluorescence staining with Parvalbumin and Myosin 7a;apoptosis of hair cells was quantified by Cleaved Caspase-3 and TUNEL immunofluorescence;intra-mitochondrial reactive oxygen levels in hair cells in Corti organoids were assessed by Mito SOX Red staining;TUJ-1 staining was used to observe The morphology and number of spiral neurons and nerve fibers in the cochlea of newborn mice were observed by TUJ-1 staining.In HEI-OC1 cells,the protective effects of Cinchonine and Cinchonidine on HEI-OC1 cells were observed and assessed under cisplatin injury.Cell viability was detected by cell counting kit-8(CCK8);changes in reactive oxygen species in mitochondria in HEI-OC1 cells were assessed by Mito SOX Red staining,and differences in mitochondrial membrane potential levels were assessed by TMRM staining,and mitochondria-related changes were characterized and quantified by immunofluorescence and flow cytometry,respectively;apoptosis-related proteins were detected by protein immunoblotting.The expression of Cleaved Caspase 3 was detected by protein immunoblotting to verify the regulation of related proteins on PI3K/AKT pathway,including PI3K、p-PI3K、AKT、p-AKT proteins.Finally,cell viability was measured by cell viability assay kits for the co-treatment of PI3 K inhibitor(LY294002)or AKT inhibitor(MK-2206)with cisplatin and cinchonine or cinchonidine.Results1,Cinchonine and Cinchonidine can interact with PIK3 CA protein and form stable compounds.2,Cinchonine and Cinchonidine pretreatment combined with cisplatin treatment reduced the reduction in the number of hair cells in the Corti organ of the neonatal mouse cochlea caused by cisplatin.3,Pre-treatment of Cinchonine or Cinchonidine then treated with cisplatin can reduce the apoptosis of hair cells and ROS accumulation in the Corti apparatus of neonatal mice in vitro compared with the cisplatin group.4,Pre-treatment of Cinchonine and Cinchonidine before cisplatin compared with cisplatin only group can reduce the decrease of HEI-OC1 cell viability,and mitochondrial dysfunction and ROS accumulation caused by cisplatin.5,Cinchonine and Cinchonidine inhibited cisplatin-induced neuronal damage and nerve fiber disruption and shortening in the cochlear spiral ganglion of neonatal mice in vitro.6,Pre-treatment of Cinchonine and Cinchonidine before cisplatin compared with cisplatin only group reduced the expression of cisplatin-induced apoptosis-related proteins through PI3K/AKT signaling pathway.7,The cytoprotective effect of Cinchonine or Cinchonidine on cisplatin-injured HEI-OC1 cells was blocked after co-treatment with PI3 K inhibitor(LY294002)or AKT inhibitor(MK-2206).ConclusionsWe used neonatal mouse cochlear Corti organ and HEI-OC1 cells as the models and confirmed that Cinchonine and Cinchonidine can reduce cisplatin-induced apoptosis and mitochondrial oxidative stress in neonatal mouse Corti organ and HEI-OC1 cells in vitro via PI3K/AKT pathway,thus reducing cisplatin-induced ototoxicity and ultimately achieving protection of auditory hair cells,which also suggests that it can be used as potential therapeutic agents for the treatment of cisplatin-induced hearing loss.