| Part Ⅰ Effects of Metformin on the degeneration of auditory cortex in D-gal induced mimetic aging rat modelObjective:To explore the effects of metformin on the degeneration of the auditory cortex on the basis of D-gal induced mimetic aging rat model.Methods:Two hundred Male Sprague Dawley(SD)rats(one-month-old),were divided into three groups randomly: a control group,a D-gal group and a D-gal + metformin group(Met group).The rats in the D-gal group and Met group were treated with D-galactose(dissolved in normal saline,500 mg/kg/day)via subcutaneous injection for 8 weeks,while the rats in the control group were administrated with an equal volume of the normal saline on the same schedule.The rats in the Met group were subjected to the daily administration of D-gal(subcutaneously)and Metformin(intragastrically: Metformin was dissolved in drinking water,150 mg/kg/d)for the same 8 weeks.After the 8-week establishment of modeling(3 months of age),the rats in the three groups were further randomly divided into 3-month-old(just after the injection),9-month-old(6 months post-injection)and15-month-old(12 months post-injection)subgroups(n=21-23 for each group),Metformin were intragastrically administrated to the rats in the Met group for 8 weeks before every sacrifice(9-month-old Met group;15-month-old Met group),the Control group and the D-gal group received the same dose of water without Metformin on the same schedule.The auditory threshold was tested by evoking auditory brainstem response(ABR)4 times during the whole experiment(1 months,3 months,9 months and 15 months old);the colorimetry was used to determine the level of Malondialdehyde(MDA),the activity of superoxide dismutase(SOD),Glutathione(GSH),catalase(CAT);Real-time PCR(RT-PCR)assay was used to quantify the mitochondrial DNA(mt DNA)4834bp common deletion;a Transmission Electron Microscope(TEM)was used to determine the ultrastructural morphology of the neurons in the auditory cortex;Nissle staining was used to test the density of neurons in the auditory cortex;Utilizing western blot to detect the levels of apoptosis related protein-p53 and Cleaved-Caspase-3 in auditory cortex.Results: There was no significant difference in ABR thresthold among all groups before treatment.The ABR threshold was significantly elevated in the 15-month-old rats compared with the 3-month-old rats.There was a significant increase in the average ABR threshold in the D-gal group compared with the control group,while metformin significantly suppressed this elevation,particularly in the 15-month-old rats.In9-month-old rats,the average thresholds were increased at 8k Hz,16 k Hz,32 k Hz after D-gal injection compared with control group,and in 15-month-old rats,significant increases in the thresholds at 16 k Hz,24 k Hz,32 k Hz were observed after injection with D-gal,while the hearing thresholds decreased significantly in the Met group;when comparing with the same age groups respectively,it can be seen that the CD levels of 3-month-old and9-month-old D-gal groups were drastically elevated when compared with control groups,the level of CD accumulated with age,also CD levels in the Met group were lower than that in the age-matched control groups during the whole process;by the TEM assay,irregular nucleus,accumulated lipofuscin,swollen and vacuolated mitochondria were observed in D-gal group much earlier than age-matched control group,D-gal-induced ultrastructural neurodegeneration were more serious than that in the age-matched Met groups;Nissl staining features has been highlighted,and it showed that the numbers of neurons in the auditory cortex regions were significantly reduced following the D-gal injection compared with control group,but these alterations were significantly attenuated by the Metformin treatment;compared with the age-matched control group,the levels of MDA were significantly increased in D-gal group,the level of MDA were decreased in Metformin group when compared to the age-corresponding D-gal group;the activity of GSH,SOD and CAT were decreased in the blood serum of 3,9 and 15-month-old D-gal group respectively when compared to the age-corresponding control groups,while the levels of GSH,SOD and CAT increased in the Met group compared to that of the age-matched D-gal groups;the results of western blotting showed that the expressions of p53 and cleaved caspase-3 were increased significantly in the D-gal group as compared with the control group,and the levels of p53 and cleaved caspase-3 were declined by Metformin-treatment.Conclusion: D-gal-injection induces mt DNA CD accumulation,ultrastructure of neurons degeneration,and D-gal declines antioxidant enzyme activities and the density of neurons,increases the level of cell apoptosis,and accelerates the degeneration of the neurons in the auditory cortex to accelerate the occurrence of age-related hearing loss.Exogenous intervention with metformin reduces associated oxidative stress and apoptosis levels as well as the regulation of related signaling pathways to delay the senescence of auditory cortex.Thus,treatment with metformin may offer a new avenue for protecting the auditory cortex against aging.Part Ⅱ The role of Metformin in preventing the process of the degeneration of auditory cortex and its correlation with UPR and AMPK/ERK1/2 signaling pathwaysObjective: Age-related hearing loss is a devastating disorder that severely affects the quality of life of elderly individuals,but its underlying molecular mechanisms of its occurrence and development has not been fully elucidated.To explore the changes of UPR and AMPK/ERK1/2 in the auditory cortex with aging and the effects of Metformin in preventing the process of the degeneration of auditory cortex and its relationship with UPR and AMPK/ERK1/2 signaling pathways.The purpose of the study was to explore the potential anti-aging mechanism of Metformin in the aging process of auditory cortex.Methods: One-month-old SD rats were divided randomly into three groups: a control group,a D-gal group and a D-gal+Metformin group(Met group)(n=55-56).The rats in the D-gal group and Met group received daily single subcutaneous injection of D-gal(dissolved in normal saline,500 mg/kg/day,8 weeks),while the control group were injected subcutaneously with an equal volume of the vehicle normal saline(0.9%,8 weeks).Met group were subjected to Metformin(intragastrically: Metformin was dissolved in drinking water,150mg/kg/d,Dongrui Chemical Co.,Suzhou,China)for the same 8 weeks(3-month-old mimetic aging+ Metformin group).After the schedule was finished(3 months of age),the three groups were divided into three age subgroups: 3-month-old(just after the injection),9-month-old(6 months after the injection)and 15-month-old(12 months after the last injection).Metformin was intragastrically administered to the rats in the Met groups(the 9-month-old Met group and the 15-month-old Met group)for 8 weeks before each sacrifice time point.The control group and the D-gal group received the same dose of water without metformin on the same schedule.Then we utilized Western blot to detect the levels of mitochondrial-UPR related proteins,HSP90 and HSP60,and the levels of Endoplasmic Reticulum-UPR related proteins,GRP78 and CHOP;also detected the phosphorylation level of AMPK/ERK1/2 in auditory cortex.Results: We found that the UPR-related proteins-HSP90,HSP60,GRP78 and CHOP were upregulated in the 3-month-old D-gal groups,and their expression levels decreased in Met groups.MT-UPR chaperone proteins-HSP90 and HSP60 were still upregulated in the15-month-old D-gal groups,and these expression levels decreased significantly in Met groups;meanwhile,the expression levels of the ER-UPR chaperone proteins GRP78 and CHOP decreased in the 15-month-old D-gal group compared with the control group but increased in the 15-month-old Met group compared with the D-gal group.In 3-month-old D-gal rats,the level of phosphorylation of AMPK increased significantly,and they were down-regulated in 15-month-old group when compared to control groups,and were reversed by Metformin treatment;there was a significant decreased level of ERK1/2phosphorylation in the 3-month-old D-gal group compared with the control group,while,the level was increased in Met group when compared with the D-gal group,in the15-month-old group,the phosphorylation of ERK1/2 level increased in the D-gal group and decreased in the Metformin group.Conclusion: Unfolded protein response and AMPK/ERK1/2 signaling pathways changed with age in the auditory cortex.Treatment with Metformin is able to regulate UPR and AMPK/ERK1/2 signaling pathways,and that can enhance the antioxidant capacity of the auditory cortex cells to reduce the damage of oxidative stress,consolidate protein homeostasis,and delay the aging process of the auditory cortex. |
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