| Part I The protective effects of salidroside against manganese-induced toxicity of cochlear cells in vitro Objective: To investigate the protective effect of salidroside against toxicity in manganese-exposed cochlear cells in vitro.Methods: Cochlear explants were collected from 3-day-old rats and cultured with 1,5,10,50,100,500 and 1000 μM salidroside for 48 h,and their morphology was observed by immunofluorescence staining.The cochlear explants were cultured with 1 m M manganese with or without 1,5,10 or 100 μM salidroside for 48 h,stained by immunofluorescence and observed by morphological analysis,found the best protective concentration of salidroside.The following groups were used: the control group,the 5 μM salidroside control group,the 1 m M manganese-treated group,and the 1 m M manganese + 5 μM salidroside group.The afferent synapses of inner hair cells were stained to observe the protective effect of salidroside against manganese-induced synaptic toxicity in inner hair cells.TUNEL staining and cleaved-caspase3 staining were used to observe the protective effect of salidroside against apoptosis of cochlear cells exposed to manganese for 48 h.Reactive oxygen species(ROS)changes in each group were observed for 24 h.Results: At all concentrations of salidroside tested,the morphology of hair cells,nerve fibers and spiral ganglion neurons in vitro was undamaged.After treatment with 1 m M manganese with or without 1,5,10 or 100 μM salidroside for 48 h,the numbers of hair cells,nerve fibers and spiral ganglion neurons had decreased significantly in the 1 m M manganese-treated group compared with those in the control group.The numbers of hair cells,nerve fibers and spiral ganglion neurons in the manganese +(1,5 or 10 μM)salidroside groups were greater than those in the manganese treated group.Synaptic staining of inner hair cells showed that the number of synapses in the manganese-treated group was significantly reduced compared with that in the control group,while the number of synapses in the manganese + salidroside group was higher than that in the manganese-treated group.TUNEL-positive cells appeared among the cochlear hair cells after manganese exposure,while the number of TUNEL-positive cells and cleaved-caspase3 positive cells among the hair cells and spiral ganglion neurons was significantly decreased in the manganese + salidroside group.The ROS level and the fluorescence intensity were significantly increased in the manganese-treated group,while the ROS level and the fluorescence intensity were significantly decreased in the manganese + salidroside group.Conclusions:Salidroside at concentrations of 1-1000 μM does not damage cochlear cells.Salidroside at concentrations of 1,5 and 10 μM has a protective effect against manganese-induced toxicity of cochlear cells in vitro.Salidroside has a protective effect against manganese-induced toxicity of synapses.Salidroside has an inhibitory effect on apoptosis induced by manganese.Salidroside reduces ROS production caused by manganese.Part II The protective effects of salidroside against manganese-induced hearing loss in adult C57 mice Objective: To investigate the protective effect of salidroside against hearing loss and changes in cochlear cell structure induced by manganese exposure in C57 mice.Methods: Twenty-four female C57 mice were randomly divided into the following four groups: the control group(n=6),the 150 mg/kg salidroside control group(n=6),the 16.5mg/L manganese-treated group(n=6)and the 16.5 mg/L manganese + 150 mg/kg salidroside group(n=6).The manganese-treated group and the manganese + salidroside group were given manganese by drinking water.Once every other day,the salidroside control group and the manganese + salidroside group were given 0.2 m L salidroside by intraperitoneal injection,while the control group and the manganese-treated group were intraperitoneally injected with 0.2 m L saline.Before and every two weeks after the treatments,C57 mice were given auditory brainstem response(ABR)tests to measure the binaural hearing threshold,and to observe the protective effect of salidroside against hearing loss induced by manganese exposure.After the model was established,brain,liver,kidney and bone samples from the mice were dried and digested,and the manganese concentration(μ g/g)in the tissues was measured by ICP-MS.At the same time,the cochlear tissue was fixed and decalcified,and the hair cells were labelled with Myosin 7a to observe the effect of salidroside on the morphology of hair cells exposed to manganese.After fixation,decalcification and dehydration,spiral ganglion neurons were labelled withβ-Tubulin to observe morphological changes in spiral ganglion neurons in the C57 mice.Results: Click and tone burst ABR results showed that the hearing threshold of mice exposed to manganese was significantly higher than that of the mice in the control group,and the influence of manganese on the hearing threshold of mice was sustained and stable.After salidroside intervention,the hearing threshold of the mice in the manganese +salidroside group decreased significantly.The content of manganese in the brain,liver,and kidney was not different among the groups,while the content of manganese in bone was significantly higher in the manganese-treated group and manganese + salidroside group than that in the control group.The number of missing hair cells was significantly increased in the manganese-treated group compared with the control group and the manganese + salidroside group.The number of spiral ganglion neurons was not significantly different among the four groups.Conclusions: Manganese exposure can cause hearing loss in mice,while salidroside has a protective effect against hearing loss caused by manganese exposure.Manganese accumulates in the bone of mice after manganese exposure.Salidroside has a protective effect against the loss of outer hair cells after manganese exposure.The morphology of spiral ganglion neurons of mice does not change after manganese exposure.Part III: The protective mechanism of salidroside against manganese-induced ototoxicity in vitro Objective: To explore the protective mechanism of salidroside against manganese-induced ototoxicity by culturing HEI-OC1 cells and cochlear explants in vitro.Methods: The concentrations of manganese were 0,0.025,0.05,0.075,0.10,0.25,0.5,0.75,1,2 and 3 m M.The CCK-8 values of HEI-OC1 cells were determined at 24 h and 48 h post-treatment and the concentration of manganese resulting in a 50% cell survival rate for use as the experimental concentration of manganese.The concentrations of salidroside were 0,25,50,75,100 and 200 μM.The CCK-8 values of HEI-OC1 cells were determined at 24 h and 48 h post-treatment,and the concentration of salidroside resulting in a cell survival rate greater than or equal to 100% was selected for use as the experimental concentration of salidroside.The CCK-8 assay was used to detect the effect of salidroside on the survival rate of manganese-treated HEI-OC1 cells.Immunofluorescence staining was used to detect the protective effect of salidroside against manganese-induced hair cell injury to examine the mechanism of salidroside against manganese-induced cochlear hair cell injury.Forty-eight 3-day-old SD rats were divided into the following groups: the control group,the 5 μM salidroside group,the 1m M manganese-treated group and the 1 m M manganese +5 μM salidroside group and the cochlear explants were cultured for 24 h and 48 h.The cochlear proteins were extracted and probed by western blotting for the protein expression of γ-H2 AX,NFκB-p65,Bax,Bok and Bcl-2 of the Bcl-2 family,as well as Nrf2 and Keap1.Results: The cell survival rate was not different between the 0.1 m M manganese + 25μM salidroside group and the 0.1 m M manganese-treated group.The western blot results showed that the protein expression of γ-H2 AX and NFκB-p65 in the manganese-treated group was significantly higher than that in the control group after 48 h of culture,but not in the manganese + salidroside group.The expression of the Bcl-2 protein was not detected in either the manganese-treated group or the manganese + salidroside group after48 h of culture,while Bax protein expression was significantly increased in the manganese-treated group and the manganese + salidroside group.The expression of the Bok protein in the manganese-treated group was significantly higher than that in the manganese + salidroside group after 24 h of culture,while the expression of this protein in both groups was the same after 48 h of culture.After 48 h,Nrf2 protein expression was significantly higher in the manganese-treated group than in the control group,while Keap1 protein expression was lower in the manganese-treated group than in the control group,and salidroside inhibited the changes of Nrf2 or Keap1 expression induced by manganese.Conclusions: Salidroside has no protective effect against HEI-OC1 cell injury induced by manganese in this model.Salidroside attenuates oxidative stress injury and inhibits the apoptosis of the cells after manganese exposure after 48 h of culture,and the Nrf2-ARE signaling pathway may be involved in salidroside-mediated protection. |
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