The Study On The Mechanisms Underlying The Actions Of C-Myb And Paeoniflorin On Aminoglycoside-Induced Ototoxicity

Part ? The study on the mechanisms underlying the action of c-Myb on aminoglycoside-induced ototoxicityObjectiveHearing loss,one of the most common sensory disorders,not only affects the individual life quality,but also brings a heavy burden on families and the community.WHO has estimated that about 360 million people suffer from hearing loss globally.Hair cells(HCs),which can convert the mechanical sound waves into nerve impulse,play an essential role in hearing and the damage to or the degeneration of HCs is responsible for hearing loss in most cases.However,HCs are susceptible to multiple injuries,such as noise,ototoxic drugs and aging.Aminoglycosides are widely used in clinics to treat gram-negative bacterial infections,but ototoxicity,such as tinnitus,hearing loss and vestibular disorder,sets an obstacle to their clinical application.Neomycin,a member of the aminoglycosides,is primarily bactericidal and its antibacterial spectrum includes all strains of Micrococcus pyogenes,many gram-positive bacteria and gram-negative bacteria.Neomycin-induced ototoxicity mainly occurs in the cochlea with the vestibule receiving less influence,causing permanent bilaterally sensorineural hearing loss.ROS overproduction is considered to be the main mechanism underlying the neomycin-induced ototoxicity.Intracellular ROS overproduction can initiate intrinsic apoptotic pathway,subsequently resulting in HC death.c-Myb is a highly conserved transcription factor which belongs to the Myb family and acts an important part in cell proliferation,differentiation and apoptosis.In various cell types,Bcl-2 is considered to be the target gene of c-Myb and c-Myb can regulate the Bcl-2 expression thus interfering with the apoptotic pathway.Moreover,c-Myb overexpression can reduce the cellular ROS level and thereby alleviating oxidative stress.It has been well documented that c-Myb is expressed in chicken otic placode,controls the onset of Sox10 expression and acts as an important marker in otic placode development.However,there is still no report about the expression pattern and function of c-Myb in mammalian inner ear.Therefore,the present study was designed to investigate the expression pattern of c-Myb in inner ear and HEI-OC1 cells,to determine the relation between c-Myb and neomycin ototoxicity and to explore the underlying mechanisms.Methods1.C57BL/6 mice at postnatal day(P)1,7,11,14 and 30 were utilized in this experiment and immunostaining on stretched preparation of cochlea basilar membranes and frozen cochlear cryosections was applied to determine the expression patterns of c-Myb in inner ear.Myosin 7a and Sox2 were used as the HC marker and supporting cell marker respectively.2.Total mRNA and protein were extracted from the cochlear basilar membranes.qRT-PCR and western blot were used to measure the expression levels of c-Myb at P1,7,11,14 and 30.3.The expression of c-Myb in HEI-OC1 was identified by immunofluorescence,PCR and western blot.4.To establish animal models with hearing loss,C57BL/6 mice were randomly assigned into two groups.Mice in neomycin group were subcutaneously injected with 200 mg/kg neomycin from P 8 to 14 and mice in control group received sterile saline.At P 17,immunofluorescence,qRT-PCR and western blot were used to identify the expression level of c-Myb in neomycin and control group.5.We treated the HEI-OC1 cells with 2 mM neomycin and detected the c-Myb expression with qRT-PCR and western blot at 0 h,4 h,8 h,12 h and 24 h.6.shRNA transfection was applied to decrease c-Myb expression in HEI-OC1 and transfection efficiency was estimated through immunofluorescence,qRT-PCR and western blot.7.After transfection,HEI-OC1 cells were treated with 0 mM,1 mM,1.5 mM and 2 mM neomycin for 24 h or 48 h,and their cell viabilities were determined by MTT assay.8.At 48 h after transfection,the HEI-OC1 cells were treated with 2 mM neomycin for 24 h and cell apoptosis was detected by flow cytometry and TUNEL assay.9.At 48 h after transfection,the HEI-OC1 cells were treated with 2 mM neomycin for 24 h.qRT-PCR was used to measure the mRNA levels of Apaf-1,Bax,caspase-3 and caspase-9.Western blot detected the protein levels of cleaved caspase-3,cleaved caspase-9 and Bcl-2.10.At 48 h after transfection,the HEI-OC1 cells were treated with 2 mM neomycin for 24 h.MitoSOX red staining and flow cytometry were used to detect cellular ROS level.11.At 48 h after transfection,Bcl-2 protein level in HEI-OC1 cells was detected by western blot.Results1.Immunostaining on auditory epithelia and frozen cochlear cryosections showed that c-Myb existed in mammalian inner ear and specifically expressed in HCs.2.qRT-PCR and western blot results demonstrated that c-Myb was expressed in an age-dependent manner in mammalian inner ear and the expression level was highest in P 14.3.Immunofluorescence,PCR and western blot results showed that c-Myb was expressed in HEI-OC1 cell.4.Immunofluorescence and western blot results demonstrated that c-Myb expression in C57BL/6 mice was significantly decreased after neomycin treatment in contrast to the control group(**p<0.01).5.Immunofluorescence,qRT-PCR and western blot results showed that after neomycin exposure,c-Myb expression in HEI-OC1 cells was decreased in a time-dependent manner compared to that in the control group.6.HEI-OC1 cells were transfected with shRNA,and immunofluorescence,qRT-PCR and western blot results showed that c-Myb expression in shRNA-Myb group was decreased compared to that in the shRNA-Control group(***p<0.001).7.After transfection,HEI-OC1 cells were treated with neomycin and the cell viabilities were decreased in a concentration-and time-dependent manner.The cell viability in shRNA-Myb group was reduced than that in shRNA-Control group after neomycin treatment.8.Flow cytometry and TUNEL staining demonstrated that the apoptotic rate in shRNA-Myb group was significantly increased in comparison to the shRNA-Control group.9.qRT-PCR and western blot results showed that Bax,cleaved caspase-3,cleaved caspase-9 and Apaf-1 were increased in shRNA-Myb group than those in shRNA-Control group,while,the Bcl-2 expression was decreased.10.After HEI-OC1 cell transfection,the Bcl-2 expression in shRNA-Myb group was reduced without neomycin exposure.11.MitoSOX red staining demonstrated that the ROS levels in shRNA-Myb group were increased than that in the shRNA-Control group.ConclusionsTo our knowledge,this is the first report about the expression pattern and the function of c-Myb in mammalian inner ear.We have demonstrated that c-Myb exists in the mammalian inner ear and specifically expresses in the HCs and HEI-OC1 cell line in an age-dependent manner.c-Myb expression was decreased after neomycin injury,indicating that c-Myb is closely related to neomycin-induced ototoxicity and might exert a protective effect in normal HCs and HEI-OC1 cell.Without neomycin injury,c-Myb down-regulation in HEI-OC1 cells leads to the decreased expression of Bcl-2 and after neomycin treatment the ROS level in c-Myb down-regulated cells is obviously enhanced than that in the shRNA-Control group.Moreover,after neomycin exposure,the pro-apoptotic factors,such as caspase-3,caspase-9,Apaf-1 and Bax,were increased in c-Myb knockdown cells and the Bcl-2 expression was further decreased,which lead to the increased cell apoptosis and decreased cell viability.In conclusion,c-Myb knockdown can increase cell sensitivity to neomycin and our findings might provide a new target for the prevention of aminoglycoside-induced HC death.Part II The study on the mechanisms underlying the action of paeoniflorin on aminoglycoside-induced ototoxicityObjectivePaeoniflorin(PF),a monoterpene glycoside compound isolated from Paeoniae Radix,exerts various biological activities such as analgesic,anti-inflammatory and anti-depressant in many cell types.Recent studies have reported that PF also exhibits the anti-oxidative and anti-apoptotic effects.Aminoglycosides are widely used in clinics to treat gram-negative bacterial infections,but their ototoxicity,such as tinnitus,hearing loss and vestibular disorder,sets an obstacle to their clinical application.Neomycin,a member of the aminoglycosides,is primarily bactericidal and its antibacterial spectrum includes all strains of Micrococcus pyogenes,many gram-positive bacteria and gram-negative bacteria.Neomycin-induced ototoxicity mainly occurs in the cochlea with the vestibule receiving less influence,causing permanent bilaterally sensorineural hearing loss.ROS overproduction in hair cells(HCs)is considered to be the main mechanism underlying the neomycin-induced ototoxicity.Intracellular ROS overproduction can initiate intrinsic apoptotic pathway,subsequently resulting in HC death.The anti-oxidative and anti-apoptotic effects of PF have appeared on a series of publications,but there is still no report about the effect of PF on neomycin-induced ototoxicity.The present study was designed to investigate the effect of PF on neomycin ototoxicity and the underlying mechanisms.Methods1.In in vivo study,C57BL/6 mice in neomycin group received subcutaneous injection with 200 mg/kg neomycin from postnatal day(P)8 to 14.Mice in PF group were given 30 mg/kg PF intraperitoneal injection and mice in PF plus neomycin group received 30 mg/kg PF intraperitoneal injection at 2 h before the daily injection of neomycin.Mice in the control group were given sterile saline.At P 30,the cochleae were dissected out.2.Immunofluorescence was used to detect the morphology,arrangement and survival of HCs in in vivo study.3.HC apoptosis in control,neomycin and PF plus neomycin groups was detected by immunostaining with TUNEL assay and Myosin 7a.4.In in vitro study,we cultured the basilar membranes from C57BL/6 mice in P 3.The basilar membranes in neomycin group were treated with 2 mM neomycin for 24 h and were treated with 30 ?M PF in PF group.In PF plus neomycin group,the basilar membranes were pretreated with 30 ?M PF for 2 h and then neomycin for 24 h.5.Immunofluorescence was used to detect the morphology,arrangement and survival of HCs in in vitro study.6.Immunofluorescence with TUNEL assay and Myosin 7a was applied to measure the HC apoptosis.7.Western blot was used to measure the expressions of apoptotic factors in different groups.8.Immunostaining and western blot were applied to detect the expressions of p-ERK in HCs from control,neomycin and PF plus neomycin group.9.MitoSOX red staining was used to identify the ROS levels in HCs from control,neomycin and PF plus neomycin group.Results1.Immunofluorescence showed that HCs in control and PF group were in regular arrangement and had few cell loss.In neomycin group,HCs exhibited extensive degeneration and disordered arrangement.In PF plus neomycin group,HCs showed better arrangement and less cell loss.2.HCs counting demonstrated that there was no HC loss in control and PF group and the HC survival in neomycin group was decreased compared to the control group(#p<0.05).In PF plus neomycin group,the HC survival was increased in contrast to the neomycin group(*p<0.05).3.TUNEL staining demonstrated that HC apoptosis in neomycin group was significantly increased than the control group while in PF plus group the apoptosis was decreased compared to the neomycin group(*p<0.05).4.Western blot results showed that cleaved caspase-3 and cleaved caspase-9 were enhanced in neomycin group than those in control group,while these proteins were decreased in PF plus neomycin group in contrast to the neomycin group(*p<0.05).5.Immunofluorescence and western blot results demonstrated that p-ERK in neomycin group was increased than the control group(##p<0.01),whereas p-ERK was decreased in PF plus neomycin compared to the neomycin group(*p<0.05).6.MitoSOX Red staining showed that the ROS level in neomycin group was enhanced than the control group while reduced in PF plus neomycin group compared with the neomycin group.ConclusionsNeomycin-induced ototoxicity is closely linked with the ROS overproduction,ERK activation and mitochondrial apoptotic pathway.PF could reduce ROS generation,inhibit ERK signaling,suppress mitochondrial apoptosis and thus protect the HCs from neomycin ototoxicity.Our findings might provide a new agent for the prevention of aminoglycoside-induced ototoxicity.