| Part I Establishment of the mimetic aging SD rat model induced by D-galactoseObjective:To establish the aging rat model via injecting D-galactose.Methods:Ninety four-week-old Sprague Dawley rats were randomly assigned into two groups:a D-gal group and a control group. The rats in the D-gal group were injected with D-gal (500 mg/kg/d) subcutaneously for 8 weeks. Simultaneously, the control groups were injected with the vehicle (0.9% saline) in the same volume for 8 weeks. After the last injection, both the D-gal and NS control groups were divided into 3 age subgroups: 3-month-old (just after the last injection),9-month-old (6 months after the last injection), and 15-month-old (12 months after the last injection). After the injections, the auditory function was measured by auditory brain stem response (ABR). The activity of total superoxide dismutase (T-SOD) and Malondialdehyde (MDA) were quantified by the Microplate reader. The ratio of mitochondrial DNA (mtDNA) common deletion was analyzed by Tapman Real-time PCR assay. The ultrastructural morphology changes in the central auditory cortex were determined by using Transmission Electron Microscope. The density of neurons was examed by the toluidine blue staining.Results:In control groups, the ABR threshold of 15 month old groups were higher than that of other age groups in 8k,16k,24k and 32kHz frequencies, (p<0.05). In D-gal groups, the ABR threshold of 15 month old groups were higher than that of other age groups in all hearing frequencies, (p<0.05). No significant differences in ABR threshold were found between the control groups and the D-gal groups, (p>0.05). Compared with the age-matched control groups, the activities of T-SOD in the mimetic aging group of 9- and 15-month-old rats were decreased, (p<0.05). Compared to the age-matched control groups, the levels of MDA were increased in 3-,9- and 15-month-old mimetic aging groups, (P<0.05). Compared with the age matched control groups, the level of mtDNA CD in the 9-and 15-month-old mimetic aging group were increased, (p<0.05). Among control groups, the level of mtDNA CD in 15 month were higher than that in 3 month, (p<0.05). Compared with the age-matched control group, the ultrastructure changes of auditory cortex neurons in D-gal groups were more severe. Irregular nuclear, condensed chromatin, swollen mitochondria, disrupted myelin and accumulated lipofuscin were found in mimetic aging group much earlier than age-matched control group; The density of auditory cortex neurons in 15-month-old D-gal group was significantly lower than that in the age-matched control group, (P<0.05).Conclutions:The D-gal treated rats might more often suffered from oxidative stresses and the mutations of the mtDNA. The reduced density of neurons and the degeneration of neurons subcellular structure were more server than the control groups during aging process. A long time and high dose exposure to D-gal might accelerate the process of aging.Part ? The role of Sestrin2 protein and autophagy in the D-galactose-induced aging auditory cortexObjective:Age-related hearing loss (AHL) is the most common hearing deficit in elderly. It lead to adverse effects on the physical, cognitive, emotional, behavioral, and social function of older adults. The mechanisms involved in presbycusis were not clear. Our studied aimed to explore the activities of autophagy and its related proteins in central auditory cortex during aging.Methods:One hundred and twenty Sprague Dawley (SD) rats (4 weeks old) with normal hearing were divided into control group rats (n=60) and mimetic aging groups (n= 60) randomly. Mimetic aging group rats were injected with D-gal (500 mg/kg/d) for 8 weeks, the rats in control group were injected with 0.9% saline on the same schedule. After the last injections, the two groups were divided into 3 age subgroups:3- month-old (when the injection was completed),9-month-old (6 months after the last injection), and 15-month-old (12 months after the last injection). The activity of total superoxide dismutase (T-SOD) and Malondialdehyde (MDA) were quantified by the Microplate reader. The ratio of mitochondrial DNA (mtDNA) common deletion was analyzed by Tapman Real-time PCR assay. The RT-PCR was performed to evaluate the mRNA level of LC3B/Atg6, Beclinl/Atg8 and Sestrin2. The western blotting was done to exame the relative protein levels of the Sestrin2, Beclin1, LC3B, p-AMPK, AMPK, p-p70S6K and p70S6K. Immunofluorescence was used to determine the location of LC3B in the central auditory cortex.Results:Compared with the age-matched control groups, the activities of T-SOD in the mimetic aging group of 9- and 15-month-old rats were decreased, (p<0.05). Compared to the age-matched control groups, the levels of MDA were increased in 3-,9- and 15-month-old mimetic aging groups (P<0.05). Compared with the age-matched control groups, the level of mtDNA CD in the 9- and 15-month-old mimetic aging group were increased, (p<0.05). Among control groups, the level of mtDNA CD in 15 month were higher than that in 3 month, (p<0.05). The details of the genes expressions were as followed. Compared to the age-matched control group, the Atg6 mRNA levels in 9- month-old D-gal group were increased by 1.09 folds, the Atg8 mRNA levels in 3- and 9-month-old D-gal group were increased by 0.20 and 0.27 folds, Sestrin2 mRNA levels were increased by 0.33 and 0.52 folds, P<0.05. While at 15 months old, the D-gal Atg6 mRNA, Atg8 mRNA and the Sestrin2 mRNA were decreased by 0.76,0.33 and 0.83 folds, P<0.05. Compared to the 3-month-old control group, the mRNA levels of Atg8 in 9- and 15- month-old control groups were increased by 0.29 and 0.21 folds, the mRNA levels of Sestrin2 were increased by 0.62 and 0.83 folds, P<0.05. The activities the proteins:Compared to age matched control groups, the beclinl protein level in the 3-,9- D-gal group were increased by 0.34 and 0.52 folds, the LC3 ?/?-actin levels was increased by 0.71 and 1.21 folds, the Sestrin2 protein levels was increased by 1.03 and 0.27 folds, the p-AMPK/AMPK ratio were increased by 1.57 and 0.67 folds, while the p-p70S6K/p70S6K ratio were reduced by 1.86, 1.21 folds, P<0.05. However, the rats were 15 months old, we found that compared to controls, the 15 month D-gal Beclinl protein level was reduced by 1.80 fold, the LC3?/?-actin ratio was reduced by 1.80 folds, the Sestrin2 protein level were reduced by 1.37 folds, the p-AMPK/AMPK ratio were reduced by 2.11 folds, while the p-p70S6K/p70S6K ratio were increased by 1.89 folds, P<0.05. Comparisons of the 9-,15-month old control groups with the 3-month-old control group revealed that the LC3 ?/?-actin level were increased by 0.61 and 0.37 folds, the Sestrin2 protein level were increased by 0.58 and 0.58 folds, the p-AMPK/AMPK ratio were increased by 0.71 and 1.04 folds,while the p-p70S6K/p70S6K ratio were reduced by 1.63 and 1.62 folds, P<0.05. Conclusions:In the aging process of D-gal induced rats, the activity of autophagy changed with aging, possibly through the Sestrin2-AMPK-p70S6K pathway. In D-gal groups, the autophagy was induced as early as 3 months old, which might associated with the oxidation and the mtDNA CD accumulation. Along with accelerated senescence, autophagy activity decreased. While in the aging process of control rats, the autophagy-related changes appeared later than D-gal rats. |
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