The Role Of Longevity Protein P66Shc In The D-galactose-induced Inner Ear Aging Of Mice

Part I Establishment of mice model with inner ear aging induced by D-galactoseObjective:To explore the establishment of the D-gal-induced inner ear aging mice.Method:18 kunming strain mice were randomly assigned to three groups. Then Mice were treated with daily subcutaneous injection of D-gal solutions (Low dose (LD) group, 800mg/kg of D-gal; High dose (HD) group, 1000mg/kg of D-gal) or normal saline (NS) (for control group) of equal volume at neck for 10 weeks. At the beginning and in the ending of the treatment, the hearing functions of mice were tested with ABR. The SOD activity and MDA levels of plasma were quantified. Real-time PCR assay was used to quantify the mitochondrial DNA 3873 bp deletion. The products of Real-time PCR were gel-purified, cloned and analyzed.Result:There was no significant difference in ABR threshold among the three experiment groups (p>0.05). The SOD activity in the plasma of low and high dose of D-gal-treated mice were significantly lower than that in the control groups (p<0.01). The MDA levels in the plasma of low and high dose of D-gal-treated mice significantly higher than that in the control groups (p<0.01). The mtDNA 3873 bp deletion in the low and high dose of D-gal-treated mice were were significantly higher than that in the control groups (p<0.01).Conclusion:The aging effect in inner ear of mice can be induced by D-galactose. Partâ…¡The expression of p66shc and age-related changes in levels of SHC and serine 36-phosphorylated p66shc in the inner earObjective:To explore the expression location and level of p66shc and age-related changes in levels of SHC and serine 36-phosphorylated p66shc in the cochlea.Method:4 adult Kunming strain mice were sacrificed to detect the location of the expression of SHC in cochlea by immunofluoresence.Then Real-time PCR was performed to detect mRNA levels of p66shc in cochlea and liver of 5 adult Kunming strain mice Western blotting was performed to analyze the total protein levels of p66shc and serine 36-phosphorylated p66shc (Ser36-P-p66shc) in the cochlea of 3 day,1 month,3 month and 12 month old mice.Result:SHC expressed in the stria vascularis. The expression levels of p66shc in cochlea were 40-fold to the levels in liver. The protein levels of SHC in the cochlea of 3 day old mice were higher than the other age mice, and the Ser36-P-p66shc levels were higher in 1, 3 and 12 month old mice.Conclusion:p66shc expressed at a high level in the cochlea of mice and the expression of SHC and Ser36-P-p66shc was age-related. Partâ…¢The role of p66Shc in D-galactose-induced accumulation of mitochondrial DNA 3873 bp deletion in the inner ear of miceObjective:To investigate the potential role of p66shc in presbycusis by using D-galactose (D-gal)-induced aging mice.Method:84 male Kunming strain mice were randomly assigned to three groups. Then Mice were treated with daily subcutaneous injection of D-gal solutions (Low dose (LD) group,800mg/kg of D-gal; High dose (HD) group, 1000mg/kg of D-gal) or normal saline (NS) (for control group) of equal volume at neck for 10 weeks. Then Real-time PCR was performed to detect mRNA levels of p66shc and FOXO3a. Western blotting was performed to analyze the total and mitochondrial protein levels of p66shc and total protein levels of serine 36-phosphorylated p66shc (Ser36-P-p66shc), FOXO3a and p-FOXO3a. Immunofluoresence was performed to detect the location of the expression of Ser36-P-p66shc in the cochlear lateral wall.Result:The mRNA levels of p66shc and FOXO3a were significantly increased in the D-gal-treated groups compared to the control group. The relative mRNA levels of p66shc in the low and high dose D-gal groups were increased about 1.53-fold and 2.12-fold, and the relative mRNA levels of FOXO3a were increased about 1.44-fold and 2.01-fold, respectively. Compared to control group, total protein levels of p66shc in low and high dose group was increased about 1.38-fold and 1.78-fold, and expression levels of Ser-P-p66shc were increased about 1.169-fold and 1.419-fold, respectively. Ser36-P-p66shc mainly expressed in the cytoplasm of the cells of stria vascularis. Compared to control group, total protein levels of FOXO3a in low and high dose group was increased about 1.22-fold and 1.39-fold, and expression levels of p-FOXO3a were increased about 2.29-fold and 2.84-fold, respectively.Conclusion:oxidative stress related increased expression of p66shc and Ser36-P-p66shc and the translocation of p66shc to mitochondria may associate with the accumulation of mtDNA 3873 bp deletion in the inner ear. FOXO3a may associate with the role of p66shc in D-gal-induced inner ear aging mice.