| ObjectiveHearing impairment was the fourth leading cause of disability in the world in 2021.According to estimates by the World Health Organization,approximately 1.5 billion people worldwide suffer from hearing impairment,placing a heavy burden on society and the economy.Currently,cisplatin(cis-diamminedichloroplatinum(Ⅱ))is widely used as a first-line tumor chemotherapy drug;however,its use may cause many adverse side effects,such as ototoxicity.Some reports have shown that hearing loss caused by cisplatin is mainly caused by the induction of oxidative stress in cells,and other reports also suggest that inflammation may be a triggering event,which can lead to inner ear cell death through endoplasmic reticulum(ER)stress,autophagy,necrosis,and intrinsic apoptosis.However,the exact mechanism underlying cisplatin-induced ototoxicity remains unclear.The transcription factor c-Myb is encoded by Myb,and the normal expression of Myb is extremely important for cell survival.The deletion of both alleles of this gene causes embryo death during embryonic development.The c-Myb protein is implicated in the control of proliferation and differentiation of both normal and transformed cells.Studies of the inner ear have reported that c-Myb is expressed within the chicken ear placode and may play a role in inner ear development.We previously confirmed that c-Myb is expressed in mouse cochlear hair cells(HCs),downregulated in neomycin-damaged HCs,and has a protective role in neomycin-damaged HCs;however,the role of c-Myb and its regulatory mechanism in cochlear HCs remain unclear and requires further research.Therefore,the purpose of this study was to explore the role of c-Myb in cisplatin-induced cochlear HC damage,investigate whether the upregulation of c-Myb in cochlear HCs in vitro and in vivo would change their sensitivity to cisplatin and its underlying molecular regulatory mechanisms,and provide a new potential target for the prevention and treatment of cisplatin ototoxicity.Methods1.C57BL/6 mice were intraperitoneally injected with cisplatin(3 mg/kg)from postnatal day(P)30 once daily for seven consecutive days.Auditory brainstem potentials(ABR)were used to detect hearing function in the mice,and immunofluorescence staining was performed.Laser confocal scanning microscopy and cell counting were used to observe and detect damage to cochlear HCs,and western blotting was performed to detect changes in c-Myb expression levels in HCs.2.An adeno-associated viral recombinant vector for c-Myb overexpression(AAV-iemNeonGreen-C-Myb-HA,AAV-c-Myb)was constructed.The infection efficiency of the virus was first defined,and the cochlear HCs of P3 mice were primary cultured in vitro.After different titers(2×109,4×109,7×109,and 2×1010 genome-containing particles[GCs])of the empty vector(AAV-ie)were infected for 60 h,the efficiency of different titers of virus infecting cochlear HCs was determined using immunofluorescence staining and cell counting.The most appropriate titer of the virus was selected according to the infection efficiency,and changes in c-Myb expression levels in cochlear HCs were detected using western blotting.3.The cultured HCs of mice were infected with 2×1010 GCs of AAV-c-Myb or the empty vector AAV-ie and co-treated with 30 μM cisplatin for 48 h.Immunofluorescence staining and cell counting were performed to compare the number of inner hair cells(IHCs)and outer hair cells(OHCs)in the apical,middle,and basal turns of the cochlea in all groups.TUNEL assay and cleaved-caspase 3 staining were used to detect apoptosis in cultured HCs,and the numbers of TUNEL/myosin 7a and cleaved caspase 3/myosin 7a double-positive cells in the cochlear bases of all groups were counted and compared.Mito-SOX staining was performed to detect changes in Mito-SOX Red fluorescence intensity in HCs,and the number of Mito-SOX/myosin 7a double-positive cells in the cochlear bases of all groups was counted and compared.4.The cochlear HCs of P16 mice were infected in vivo by injecting 1×1010 GCs empty vector AAV-ie through a round window membrane(RWM)for 14 days,and ABRs were recorded to evaluate the hearing function of the mice.Immunofluorescence staining was performed to observe the efficiency of infecting cochlear HCs with different titers of AAV-ie.The appropriate viral titer was selected,AAV-c-Myb of the selected titer was injected through the RWM,cochlear HCs of P16 C57BL/6 mice were infected in vivo for 14 days,and western blotting was carried out to detect the relative change in c-Myb expression levels in cochlear HCs.5.The cochlear HCs of P16 mice were infected in vivo by injecting 1×1010 AAV-cMyb or empty vector AAV-ie through the RWM for 14 days and then injected intraperitoneally with cisplatin(3 mg/kg)for 7 consecutive days.ABR tests were performed to compare hearing function,and immunofluorescence staining and cell counting were performed to determine the number of IHCs and OHCs lost in the apex,middle,and base of the cochlea.The number of C-terminal binding protein 2(Ctbp2,inner hair cell ribbon synapse presynaptic marker)puncta per IHC were counted in all groups.TUNEL staining was performed to compare the numbers of TUNEL-positive IHCs and OHCs in all groups(TUNEL/myosin 7a double-positive cells).6.c-Myb flox/flox mice were crossed with Prestin-CreER mice,double-positive mice(Prestin;c-Myb-cKO)were screened and intraperitoneally injected with tamoxifen(3 mg/40 g)from P10 once a day for three consecutive days.ABR tests were performed at P30 to evaluate the hearing function of the mice,and immunofluorescence staining and cell counting were performed to compare the changes in c-Myb expression in cochlear HCs and the number of HCs in the two groups.At P30,Prestin;c-Myb-cKO mice were injected daily with cisplatin(3 mg/kg)for seven consecutive days,and ABR tests were performed to compare hearing function.Immunofluorescence staining,cell counting,and TUNEL assays were performed to compare the number of IHCs and OHCs lost and the number of TUNEL-positive IHCs and OHCs in all groups.7.The cultured HCs of P3 mice were pre-treated with 2×1010 GCs of AAV-c-Myb or empty vector AAV-ie for 12 h and co-treated with cisplatin(30 μM)or cisplatin(30μM)+LY294002 for 48 h.Western blotting was used to detect changes in the PAkt/Akt signalling pathway in all groups,and immunofluorescence staining and cell counting were performed to compare the changes in the number of cochlear HCs lost in each group.Results1.After cisplatin treatment,the hearing threshold shifts of all frequencies(4-32kHz)of the mice in the cisplatin group were significantly higher than those in the control group,and those at 24 and 32 kHz were significantly increased.Compared with the control group,HC loss in the cochlear basal turn was significantly increased,and the c-Myb expression level in the cochlear HCs was significantly decreased in the cisplatin group.2.Immunostaining and cell counting results showed that after the HCs were infected with 2×1010 GCs AAV-ie in vitro,approximately 83.250±0.944%,80.470±2.976%,and 76.600±2.804%of the HCs were in the apical,middle,and basal turns,respectively.Western blotting showed that after AAV-c-Myb-infected cochlear HCs were incubated in vitro for 60 h,the c-Myb expression level in the AAV-c-Mybinfected group was significantly higher than that in the control group.3.Immunofluorescence staining and cell counting results showed that after cultured HCs were treated with AAV-c-Myb or AAV-ie and cisplatin,the numbers of IHCs and OHCs decreased in the cochlear apical,middle,and basal turns and the numbers of TUNEL/myosin7a,cleaved caspase3/myosin7a,and Mito-SOX/myosin 7a doublepositive IHCs and OHCs in the cisplatin+AAV-c-Myb group were significantly lower than those in the cisplatin+AAV-ie group.4.After mice were injected with 1×1010 AAV-ie GCs through the RWM,ABR tests showed no change in the hearing function of mice in the AAV-ie infection group.The infection efficiency of HCs in the cochlear apex,middle,and base was 94.587±1.381%,93.150±1.370%,and 86.320±2.228%,respectively.The number of IHCs and OHCs in the cochlear apical,middle,and basal turns did not differ significantly between the AAV-ie and control groups.In vivo,when mice were injected with 1×1010GCs AAVc-Myb through the RWM,the expression level of c-Myb in the AAV-c-Myb infection group was significantly higher than that in the control group.5.After mice were injected with 1×1010 GCs AAV-c-Myb or AAV-ie through the RWM combined with cisplatin treatment,the cisplatin+AAV-c-Myb group had significantly lower shifts in the ABR threshold at all frequencies from 4 kHz to 32 kHz compared to those in the cisplatin+AAV-ie group.The number of IHCs and OHCs lost in the cochlear basal turns was significantly reduced in the cisplatin+AAV-c-Myb group compared with that in the cisplatin+AAV-ie group.The average number of Ctbp2 puncta per IHC was significantly higher in the cisplatin+AAV-c-Myb group than that in the cisplatin+AAV-ie group.The number of TUNEL/myosin 7a doublepositive cells in the cochlear base was significantly lower in the cisplatin+AAV-cMyb group than that in the cisplatin+AAV-ie group.6.Immunofluorescence staining showed that the expression of c-Myb in the OHCs of c-Myb-HC conditional knockout mice(Prestin;c-Myb-cKO)at P30 was significantly downregulated compared to that in control mice,whereas the expression of c-Myb in the IHCs was not significantly different from that in the control group.After cisplatin treatment,the shifts in the ABR threshold in the cisplatin+prestin;c-Myb-cKO group were significantly higher than those in the cisplatin+WT group at all frequencies from 4 kHz to 32 kHz.The number of OHC lost in prestin;c-Myb-cKO mice was significantly higher than that in cisplatin-treated WT mice.The number of TUNEL/myosin7a double-positive OHCs in the cochlear base in the cisplatin+prestin and c-Myb-cKO groups was significantly higher than that in the cisplatin+WT group.7.After the cultured HCs were injured with cisplatin,the ratio of phosphorylated(P)Akt/Akt and the levels of P-PI3K(p85α)expression were significantly reduced in cochlear HCs treated with cisplatin+AAV-ie compared with those treated with cisplatin+AAV-c-Myb.Compared to the cisplatin+AAV-ie group,the expression levels of p85α and the ratio of p-Akt/Akt in the cochlear HCs were significantly increased after co-treatment with cisplatin and AAV-c-Myb,whereas treatment with LY294002 decreased the levels of p85α expression and the ratio of p-Akt/Akt in the cisplatin+AAV-c-Myb+LY294002 group.LY294002 treatment increased the number of HCs lost in the cisplatin+AAV-c-Myb+LY294002 group compared to that in the cisplatin+AAV-c-Myb group.ConclusionsThis study found that c-Myb expression is reduced in cisplatin-injured cochlear HCs.The overexpression of c-Myb in HCs by AAV significantly promoted HC survival,decreased HC apoptosis,and reduced intracellular reactive oxygen species(ROS)levels after cisplatin exposure in vitro and in vivo;it also improved hearing function in mice after cisplatin injury.The protective mechanism of c-Myb may be achieved by activating the PI3K/Akt signaling pathway in HCs exposed to cisplatin.The findings from this study suggest that c-Myb may serve as a novel target for the prevention of cisplatin-induced HC damage and hearing loss. |
