Experimental Study Of FoxM1 In Age-related Hearing Loss In Mice

objectiveAge-related hearing loss(ARHL)is the most common cause of hearing loss in the elderly.Hearing loss not only affects the quality of life of ARHL patients,but also causes social isolation,depression,anxiety,cognitive impairment and other symptoms,thus increasing the social and economic burden.Therefore,early detection,intervention and delay of the occurrence and development of ARHL are of great significance to the elderly population.Forkhead box protein M1(FoxM1)as an important regulator of oxidative stress response,delays cell senescence by regulating autophagy and DNA damage.At the same time,FoxM1 is also involved in the development of the inner ear of mice.However,the role of FoxM1 in the occurrence and development of ARHL is not clear and needs to be further studied.The purpose of this study is to clarify the role of FoxM1 in ARHL and its possible mechanism,and to provide theoretical basis for clinical prevention and treatment of ARHL.The purpose of the study is divided into the following two parts.Part one: to clarify the relationship between the decrease of FoxM1 expression and ARHL in aging cochlea.Part two: to explore the effect of FoxM1 on the senescence of hair cells(HCs)and its mechanism.Methods1.In vivo experiment to clarify the relationship between FoxM1 and ARHL2-month-old and 12-month-old male C57BL/6 mice were selected and 12 in each group.After auditory brainstem evoked potential Auditory brain response(ABR)was used to detect the auditory function of the mice,the mice were killed and the bilateral cochleas were collected.The localization and expression of FoxM1,8-hydroxy-2 deoxyguanosine(8-OHdG),microtubule associated protein-1 light chain 3(LC3)and p-p53 in mice cochlea were detected by immunofluorescence,and the expression of Selected autophagy effector protein Beclin1 and autophagy-related gene 5(ATG5)in mice cochlea was detected by Western blot.2.To verify the effect and mechanism of FoxM1 on senescence of HCs in vitro.(1)In order to investigate whether the endogenous oxidizer 4 hydroxy nonenal(4HNE)can induce senescence of mice cochlear hair cell like HEIOC1 cells,the cells were treated with different concentrations of 4-HNE(0 μ M,5 μ M,10 μ M,20 μ M)for 5 h,and cultured in DMEM high glucose medium with 10% fetal bovine serum for 48 h.The cell viability was detected by CCK-8method.The senescence of HEI-OC1 cells was observed by β-galactosidase staining,and the expression of senescence-related proteins p21 and p16 was detected by Western blot to determine the conditions of senescence induced by 4-HNE.Western blot was used to observe the changes of FoxM1 protein level in HEI-OC1 cells after senescence induced by different concentrations of4-HNE.(2)To detect the role of FoxM1 in the senescence of HCs cells.First,FoxM1 inhibitor forkhead domain inhibitor 6(FDI-6)was given to reduce the level of FoxM1 in HEI-OC1 cells.After HEI-OC1 cells were treated with different concentrations of FDI-6(0 μ M,5 μ M,10 μ M,20 μ M)for 24 hours,the cell viability was detected by CCK-8,the senescence of cells was observed by flow cytometry(C12FDG fluorescence staining),and the expression of FoxM1,p21,p16,Beclin1,ATG5,LC3,p53,p-p53 protein was detected by Western blot.At the same time,the expression of p53 binding protein 1(53BP1)was detected by immunofluorescence.(3)in order to further elucidate the mechanism of FoxM1 in the senescence of HCs cells,the cells were pretreated with N-acetyl-L-cysteine(NAC)for 1 h,and then treated with 4-HNE(20 μ M for 5 h followed by 48 h culture)or FDI-6(10 μ M for 24 h).After drug treatment,the expression of Beclin1,LC3,p53 and p-p53 protein was detected by Western blot,Autophagy flow detection of Autophagosome Lysosome fusion,and the expression of 53BP1 was observed by immunofluorescence staining.Results1.Localization and expression of FoxM1 in mice cochlea.The expression of autophagy-related proteins LC3 and Beclin1 in cochlear cells of 12-month-old mice decreased,while the levels of DNA oxidative damage markers 8-OHdG and DNA damage protein p-p53 increased significantly,and bound to the ABR threshold of each frequency.It is suggested that the hearing loss of 12-month-old mice is closely related to the decrease of autophagy and DNA damage of cochlear cells.At the same time,it was observed that the expression of FoxM1 in Cody organ,Spiral Ganglion(SG)and Stria Vascularis(SV)of 12-month-old mice cochlea decreased significantly,and mainly concentrated in outer hair cells(OHCs).It is inferred that FoxM1 may be mainly involved in the amplification of sound signal by OHCs,suggesting that ARHL may be related to the decreased expression of FoxM1.2.Inhibition of the effect of FoxM1 on senescence of HCsafter treatment with 4-HNE,the senescence positive markers of β-galactosidase in HEI-OC1 cells increased,and the levels of aging markers p21 and p16 increased significantly.HCs aging model in vitro was established successfully.After 4-HNE treatment,the expression level of FoxM1 decreased.It is suggested that the endogenous oxide 4-HNE may induce the aging of HEIOC1 cells by inhibiting FoxM1.After treatment with FDI-6,the level of FoxM1 in HEI-OC1 cells decreased significantly.The inhibitory effect of FDI-6 on FoxM1 was confirmed.After FDI-6 treatment,obvious cell senescence was observed,and the expression level of senescence-related proteins increased significantly.3.Mechanism of inhibiting FoxM1 promoting HCs senescenceFDI-6 treatment can significantly reduce the level of autophagy in HEI-OC1 cells and induce DNA damage.It is suggested that inhibition of FoxM1 can cause autophagy disorder and DNA damage in HEI-OC1 cells.4-HNE and FDI-6 activated p53 phosphorylation,resulting in autophagy imbalance and DNA damage,while NAC treatment reduced the level of p-p53,impaired autophagy and alleviated DNA damage in HEI-OC1 cells.It is suggested that inhibition of FoxM1 can induce p53 phosphorylation and senescence of HEI-OC1 cells,resulting in autophagy disorder and DNA damage.ConclusionsAging induces a decrease in the level of FoxM1 in mice cochlear cells and activates p53 phosphorylation,which in turn leads to autophagy and DNA damage in cochlear cells,promotes cell senescence and causes ARHL.