Vitexin Reduces Mitochondrial Apoptosis Of Inner Ear Hair Cells Induced By Gentamicin By Regulating Epac1 Signaling Pathway

Objective:The purpose of this study was to investigate the protective mechanism of vitexin on gentamicin-induced hair cell damage in the inner ear of mice,and to provide new research ideas for the drug treatment of gentamicin ototoxicity.Methods:In the in vivo experiment,C57 mice were selected,and the mice were given a combination of gentamicin and furosemide by intraperitoneal injection for 10 days to establish a mouse model of drug ototoxicity.The hearing threshold changes of mice were detected by ABR test;the deletion of inner ear hair cells was detected by phalloidin staining;the mitochondrial damage in inner ear hair cells was detected by transmission electron microscopy（TEM）;the intracellular activity of HEI-OC1 was detected by DCFH-DA Oxygen（ROS）levels;JC-1 staining to detect mitochondrial membrane potential（MMP）in HEI-OC1 cells;flow cytometry to detect reactive oxygen species levels and apoptosis in HEI-OC1 cells;lentivirus transfection to detect related protein expressions;Western blot was used to detect the expression levels of Epac1,Rap1,Bax,Cleaved caspase-3,NOX4,Drp1,MFN2 and other proteins.Results:The results of the in vivo experiments showed that the hearing threshold（ABR）of the mice was significantly increased after the combined administration of gentamicin and furosemide,indicating that the hearing impairment of the mice was obvious;at the same time,the phalloidin staining results showed that the inner ear of the mice was Hair cells are severely absent.Pretreatment with vitexin can reduce the upward shift of the hearing threshold in mice,protect mice from gentamicin-induced hearing loss,and reduce the loss of inner ear hair cells in mice.The results of western blotting showed that after gentamicin treatment,the expression of Epac1 protein in the cochlea tissue of the mice in the model group was significantly up-regulated compared with the control group,which caused the activation of downstream Rap1;at the same time,the expression of the oxidative stress protein NOX4 was up-regulated,and the apoptosis protein The expression of CC3 increased,and the vitexin treatment group could inhibit the expression of Epacl protein and the activation of downstream Rapl in the cochlear tissue of mice,and reduce the expression levels of NOX4 and CC3,suggesting that vitexin could inhibit the oxidative damage of inner ear hair cells caused by gentamicin treatment and apoptosis.The results of transmission electron microscopy showed that after gentamicin-induced modeling,the outline of the mitochondrial outer membrane in the inner ear hair cells of mice was blurred,the inner mitochondrial cristae were severely broken,and some severely damaged mitochondria even appeared vacuolated.WB results showed that mitochondrial-related dynamics The expression of chemical protein MFN2 was down-regulated,and the expression of Drp1 was up-regulated;vitexin treatment group could reduce the gentamicin-induced damage of mitochondrial ultrastructure in mouse inner ear hair cells and protect mitochondrial function by down-regulating Epac1.In addition,the use of Epac1 agonist 8-CPT to up-regulate Epac1 further aggravated oxidative stress and mitochondrial damage in inner ear hair cells,thereby increasing gentamicin-induced injury of mouse inner ear hair cells,while Epac1 inhibitor ESI-09 Downregulation of Epac1 attenuated gentamicin-induced damage to mouse inner ear hair cells.The in vitro results showed that vitexin pretreatment could protect HEI-OC1 cell viability from gentamicin by CCK8 assay;The level of reactive oxygen species in OC1 cells increased,and Western blotting experiments showed that vitexin could inhibit the expression of NOX4,a protein related to oxidative stress in HEI-OC1 cells;JC1 staining showed that vitexin could significantly increase the expression of gentamicin Mitochondrial membrane potential of HEI-OC1 cells was decreased by gentamicin.WB results showed that the expression of mitochondria-related functional protein MFN2 in HEI-OC1 cells was significantly up-regulated,and the expression of Drpl was significantly down-regulated after vitexin pretreatment,and the apoptosis protein was inhibited.The expression of Bax and CC3 can reduce cell apoptosis.In addition,in vitro experiments,when HEI-OC1 cells were pretreated with the Epac1 agonist 8-CPT,the expressions of proteins Epacl and Rapl were significantly up-regulated,and the expressions of oxidative stress-related protein NOX4 and apoptosis protein CC3 were also up-regulated.,the expression of mitochondria-related dynamic protein MFN2 was down-regulated,and the expression of Drpl was up-regulated;DCFH-DA test results also showed that the level of intracellular reactive oxygen species was significantly increased after Epac1 agonist treatment;JC1 test results showed that after Epac1 agonist treatment,intracellular mitochondrial membrane The potential was significantly reduced;the results of flow cytometry showed that the number of HEI-OC1 cells apoptotic significantly increased after Epac1 agonist treatment.After pretreatment with Epac1 inhibitor ESI-09,the expressions of Epac1 and Rapl proteins were significantly down-regulated,while the expressions of oxidative stress-related proteins NOX4 and apoptosis protein CC3 were down-regulated,and the expression of mitochondria-related dynamic protein MFN2 was up-regulated,and Drpl The expression was down-regulated;the DCFH-DA test results also showed that the intracellular reactive oxygen species level was significantly reduced after Epac1 agonist treatment;the JC1 test results showed that after Epac1 agonist treatment,the intracellular mitochondrial membrane potential was significantly increased;the results of flow cytometry showed that,the apoptotic number of HEI-OC1 cells was significantly reduced after Epac1 agonist treatment.When the expression of Epac1 protein in HEI-OC1 cells was silenced by lentiviral transfection,it was confirmed that Epacl was involved in the gentamicin injury process.Conclusion:The results of this study suggest that vitexin can protect the hearing loss of mice by alleviating gentamicin-induced hair cell damage in the inner ear of mice.It protects the mitochondrial function of cells and inhibits apoptosis to play a protective role.