| Section 1 Effect of puerarin on gentamicin-induced ototoxicity in C57BL/6 J miceResearch Target:(1)Establish an animal model of drug ototoxicity by treating C57BL/BJ mice with gentamicin.(2)Explore the effect of puerarin on the increase of hearing threshold and the loss of cochlear hair cells in C57BL/6J mice induced by gentamicin.Method:(1)Establishment of the animal model of gentamicin ototoxicity.Twenty-four 6-week-old C57BL/6J mice were randomly divided into 4 groups with 6 mice in each group:Control group,puerarin group(PU group),gentamicin group(GM group)and gentamicin+pueraringroup(GM+PUgroup).Controlgroup mice were intraperitoneally injected with 1mL normal saline once a day for 7 days.Mice in PU group were intraperitoneally injected PU 100mg/kg,once a day,for 10 days.Mice in GM group were intraperitoneally injected with GM 200 mg/kg once a day for 7 days.Mice in GM+PU group were first intraperitoneally injected PU 100mg/kg once a day for 3 days,and then intraperitoneally injected PU 100mg/kg and GM 200mg/kg once a day for 7 days.(2)Before and 8 weeks after treatment,ABR was used to detect the hearing threshold(3)At 8 weeks after the treatment,the cochlea was dissected and the basement membrane of the cochlea was taken out.The morphology,arrangement and survival number of hair cells in each group were identified by fluorescence staining.Result:(1)The ABR threshold in the GM group was 50-70 dB higher than that in the control group(***P<0.001).The ABR threshold in the GM+PU group was 15-30 dB lower than that in the GM group(**P<0.01).(2)Immunofluorescence staining showed that hair cells in the control group were neatly arranged without deletion.The hair cells in the GM group were disordered and partially missing,and the hair cells in the basal gyrus were more damaged than those in the parietal gyrus.The hair cell morphology of the GM+PU group was fair and the deletion was less.(3)The remaining hair cell counts showed that there was no significant loss of hair cells in the control and PU groups.The hair cells in the GM group were significantly less than those in the control group(*p<0.05).The remaining hair cells in the GM+PU group were higher than those in the GM group(*P<0.05).Conclusion:(1)Puerarin can protect the hearing loss induced by gentamicin in C57BL/6J mice.(2)Puerarin can reduce the damage of cochlea hair cells induced by gentamicin in C57BL/6J mice.Section 2 Effect and mechanism of puerarin on HEI-OC1 cells cytotoxicity induced by gentamicinResearch Target:Investigate the effect of puerarin on gentamicin-induced apoptosis of HEI-OC1 cells,Methods:(1)HEI-OC1 cells were treated with gentamicin for 24 h at 0 mM,0.2 mM,0.5 mM,1.0 mM and 2.0 mM respectively,then the survival rate of HEI-OC1 cells after treatment was determined by CCK-8 method.(2)HEI-OC1 cells were treated with puerarin at the concentration of 0μg/mL,5 μg/mL,10 μg/mL,20 μg/mL,50μg/mL,100 μg/mL,200 μg/mL and 300 μg/mL for 24 h.CK88 method was used to detect the survival rate of HEI-OC1 cells.(3)HEI-OC1 cells were pretreated with puerarin 0 μg/mL,50 μg/mL,100 μg/mL and 200μg/mL for 2 h,and then treated with gentamicin 1 mM for 24 h.The survival rate of HEI-OC1 cells after treatment was determined by the CCK-8 method.(4)HEI-OC1 cells were divided into the control group,hydrogen peroxide(H2O2)group,hydrogen peroxide+puerarin(H2O2+PU)group,gentamicin(GM)group,gentamicin+puerarin(GM+PU)group.GM,PU and H2O2 groups were treated with GM(1.0 mM),PU(100 μg/mL)and H2O2(0.1 mM)for 24 h,respectively.GM+PU group was pretreated with puerarin(100 μg/mL)for 2 h,followed by gentamicin(1.0 mM)for 24 h.H2O2+PU group was pretreated with puerarin(100 μg/mL)for 2 h,followed by hydrogen peroxide(0.1 mM)for 24 h.DCFH-DA staining and flow cytometry were used to measure ROS in each group.TUNEL staining and flow cytometry were used to analyze the early apoptosis of cells.Changes in mitochondrial membrane potential were detected by JC-1 staining.The mRNA and protein levels were detected by RT-PCR and Western blot.Results:(1)CCK-8 results showed that the survival rate of HEI-OC1 cells was about 50%when treated with gentamicin(1mM）for 24 h.Compared with the control group,the survival of HEI-OC1 cells treated with puerarin(100 μg/mL)was significantly enhanced(*P<0.05).(2)DCFH-DA staining and flow cytometry results demonstrated that the number of intracellular ROS in H2O2 and GM groups was significantly increased and time-dependent,compared with the control group(**P<0.01).Moreover,it was indicated that puerarin pretreatment could significantly reduce the ROS level in HEI-OC1 cells after the treatment of hydrogen peroxide and gentamicin(*P<0.05).(3)TUNEL staining and flow cytometry presented that apoptosis of HEI-OC1 cells was significantly increased after the treatment of hydrogen peroxide and gentamicin respectively(***P<0.001),and puerarin pretreatment could significantly reduce apoptosis(**P<0.01).(4)Treatment of HEI-OC1 cells with gentamicin concentration(1 mM)for 24 h could result in the decrease of the mitochondrial membrane potential of HEI-OC1 cells and green fluorescence enhancement and red fluorescence attenuation could be observed after JC-1 staining.In addition,pretreated with puerarin(100 μM)could attenuate the reduction of mitochondrial membrane potential in HEI-OC1 hair cell-like cells.(5)RT-PCR results demonstrated that the GM+PU group could increase the mRNA expression of Bcl-2 and decrease the mRNA expression of Bax and Caspase-3,,compared to the GM group.Western blot results showed that the expression of p53 in the GM group increased compared to the control group.Thus,puerarin could reduce the expression of p53 induced by gentamicin.Compared to the GM group,expression of Bax,Cleaved-caspase-3,p53 and Cyto C were lower in the GM+PU group,but the Bcl-2 expression was higher.Conclusion:In this study,it was found that after being treated with gentamicin,ROS levels in HEI-OC1 cells increased gradually over time,the apoptosis rate of HEI-OC1 cells increased and the survival rate decreasd.Puerarin could reduce gentamicin-induced apoptosis of HEI-OC1 cells.Moreover,puerarin reduced the damage of gentamicin to HEI-OC1 cells,and decreased the apoptosis rate of HEI-OC1 cells,increasing the survival rate.Therefore,as a protective agent,Puerarin could be used against deafness induced by gentamicin,providing a new treatment for aminoglycoside ototoxicity. |
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