| Part I Establishment of the D-galactose-diduced auditory cortex aging rat modelObjective:To establish of the D-galactose (D-Gal)-induced auditory cortex aging rat model.Methods:One hundred and sixty two male Sprague Dawley (SD) rats (8weeks old) with normal hearing were randomly divided into a control group (n=81) and a mimetic aging group (n=81). The mimetic aging group rats were given a subcutaneous injection of D-Gal (Sigma Chemical, St. Louis, MO)(500mg/kg/d) for8weeks; the control group rats were given an injection of normal saline (NS)(500mg/kg/d) on the same schedule. Then, the rats were sacrificed at the ages of4,10, and16months and divided into a4-month-old subgroup (just after the last treatment),10-month-old subgroup (6months after the last treatment), and16-month-old subgroup (12months after the last treatment). After the treatment, the auditory function was tested by auditory brain stem response (ABR); Real-time PCR (RT-PCR) assay was used to quantify the mitochondrial DNA (mtDNA)4834bp deletion; the colorimetry was used to determine the activity of total superoxide dismutase (T-SOD) and the ratio of Glutathione:Oxidant glutathione (GSH:GSSG); the level of Malondialdehyde (MDA) was examined by the colorimetry and high performance liquid chromatography (HPLC); The density of neurons was examined by toluidine blue staining, the cell apoptosis in the auditory cortex was examined using TUNEL; and the ultrastructural morphology of neurons was determined using a Transmission Electron Microscope (TEM).Results:There was no significant difference in ABR threshold between the control groups and the mimetic aging groups (p>0.05); Compared with the age-matched control groups, the activities of T-SOD and the ratios of GSHrGSSG were significantly decreased (P<0.05) in the mimetic aging group of10-and16-month-old rats; The levels of MDA were significantly increased in the mimetic aging group of4-,10-and16-month-old rats (P<0.05); The abnormal ultrastructure of neurons was more significant in the mimetic group when compared to the age-matched control group. accumulated lipofuscin, disrupted myelin, swollen mitochondria, condensed chromatin and irregular nuclear were found in mimetic aging group much earlier than age-matched control group; The density of auditory cortex neurons in16-month-old mimetic aging group was significantly lower when compared to the age-matched control group,(P<0.05). Besides, the quantity of TUNEL-positive cells in the mimetic aging group was much higher than in the control group at the ages of10and16month.(P<0.01).Conclution:High dose D-Gal leads to the activity of anti-oxidant enzyme decreases, oxidant stress becomes more severe, the mtDNA CD accumulates,the neurons in auditory cortex degenerate, the density of neurons reduces and the number of apoptosis cells increases. Chronic exposure to D-Gal accelerates the development of cell senescence in the auditory cortex. Partâ…¡ The role of mitochondrial anti-oxidant enzyme Trx2in the D-galactose-induced aging auditory cortexObjective:Presbycusis is the third most prevalent chronic disease in the world, just after arthritis and hypertension. It seriously affects the quality of life of the elderly. Previous studies indicated that oxidative stress plays an important role in the pathological process of presbycusis. However, the molecular mechanisms involved in central presbycusis still remain elusive.Methods:One hundred and fourteen male Sprague Dawley (SD) rats (8week old) with normal hearing were randomly divided into two groups:control group rats (n=57) and mimetic aging group (n=57). Mimetic aging group rats were injected subcutaneously with D-Gal (500mg/kg/d) for8weeks; control group rats were injected with an equal volume of normal saline (NS) on the same schedule. Then, both groups were divided into3age subgroups:4-month-old (just after the injection was finished),10-month-old (6months after the injection), and16-month-old (12months after the injection). RT-PCR was taken to examine the relative mRNA expression of Thiredoxin2(Trx2), thioredoxin-interacting protein (TXNIP) and apoptosis signal regulating kinase1(ASK1); Western blot was used to determine the relative protein expression of the Trx2, TXNIP, and ASK1. Also, we use this method to examine the changes of ASK1; Immunofluorescence was used to examine the location and expression of Trx2and ASK1in the auditory cortex; Immunohistochemistry was used to examine the location and expression of TXNIP; Immunoprecipitation was taken to determine the changes of the Trx2-TXNIP and Trx2-ASK1binding complexes.Results:The result of RT-PCR demonstrated:Compared to the age-matched control group, the mRNA levels of Trx2in the mimetic aging group at different ages were reduced by1.16-,1.40-(P<0.01), and1.45-fold(P<0.01), the mRNA levels of TXNIP were increased by1.39-(P<0.05),1.40-(P<0.01), and1.50-fold (P<0.05), and those of ASK1were increased by1.18-,1.25-(P<0.01), and1.26-fold (P<0.01), respectively. The mRNA levels of Trx2decreased with age (P<0.05). However, both ASK1and TXNIP mRNA levels increased with age. Compared to the4-month-old control group, the level of Trx2in the16-month-old control group was decreased by1.29-fold (P<0.05). However, those of ASK1and TXNIP increased by1.12-and1.59-fold (P<0.05) respectively. Compared to the4-month-old mimetic aging group, the level of Trx2in the16-month-old mimetic aging group decreased by1.29-fold (P<0.05). However, those of ASK1and TXNIP increased by1.42-and1.71-fold (P<0.01) respectively. The result of western blot showed:comparison of the4-,10-and16-month-old control groups demonstrated that Trx2protein expression in the age-matched mimetic aging group was reduced by1.50-,1.61-(P<0.05) and1.56-fold (P<0.05). However, the TXNIP protein expression was increased by1.34-(P<0.05),1.44-(P<0.05), and1.57-fold (P<0.05), and the ASK1protein expression was increased by1.15-,2.19-(P<0.01) and2.17-fold (P<0.01), respectively. Moreover, we found that the protein expression of Trx2, TXNIP and ASK1changed with age. Compared to the4-month-old control group, Trx2protein expression in the16-month-old control group was decreased by1.54-fold, but TXNIP and ASK1expression was increased by1.42-(P<0.05) and1.59-fold (P<0.05). Compared to the4-month-old mimetic aging group, Trx2protein expression in the16-month-old mimetic aging group was decreased by1.77-fold (P<0.01), but TXNIP and ASK1expression was increased by2.30-(P<0.01) and2.75-fold (P<0.05) respectively. The activity of ASK1increased in the mimetic aging rat (P<0.01). The immunoprecipitation result demonstrated:Compared to the age-matched control group, the levels of Trx2-TXNIP binding complex in the mimetic groups of4-,10-and16-month-old rats were increased by1.63-(P<0.05),1.21-(P<0.05) and1.26-folds (P<0.01), but the levels of Trx2-ASK1binding complex were decreased by1.09-(P<0.05),1.48-(P<0.01) and1.37-folds (P<0.05), respectively. Furthermore, the Trx2-TXNIP complex increased with age, while the Trx2-ASK1complex decreased with age. Compared to the4-month-old control group, the level of Trx2-TXNIP binding complex of the16-month-old control group was increased by2.38-fold (P<0.01), but the Trx2-ASK1binding complex decreased by2.61-fold (P<0.01). Compared to the4-month-old mimetic aging group, the level of Trx2-TXNIP binding complex of the16-month-old mimetic aging group increased by2.99-fold (P<0.01), but the Trx2-ASK1complex decreased by2.01-fold (P<0.01).Conclusion:decreased Trx2and the changes of the TXNIP-Trx2-ASK1signal pathway in the auditory cortex may have less ability to resist oxidative stress, resulting in an elevation in ROS generation. As a result, the ultrastructural morphology changed with aging, with oxidative stress accumulation, mitochondria dysfunction and abnormal auditory cortex cell apoptosis, which ultimately results in AHL. |
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