The Role Of Mitochondrial DNA 4834 Bp Deletion In Presbyacusis And The Relating Mechanism

Part I Time sequence of relationship between mitochondrial DNA 4834 bp deletion in inner ear and hearing loss in the rat model induced by D-galactoseObjective:Mutations, especially deletions, in mitochondrial DNA (mtDNA) have been associated with diverse pathological states in humans, notably age-related hearing loss. Common deletions (CD) that are the result of recombination between direct repeats in human and rat (4977 bp and 4834 bp respectively) are known to increase in tissues of aged individuals. In previous studies, we have established a model of rat associated with mtDNA 4834 bp deletion in inner ear by D-galactose and inferred that common deletions might enhance the sensitivity of the inner ear to the aminoglycoside antibiotic.Methods:Auditory brainstem response (ABR) was used to detect the hearing threshold. Colorimetry was used to analyze activity of superoxide dismutase (SOD) and concentration of malondialdehyde (MDA). The percentage of mtDNA 4834 bp deletion in inner ear was identified by real-time PCR.Results:The data showed that the frequency of the 4834 bp deletion increase with age. Meanwhile, it is revealed that D-galactose has dose-depended effect on the frequency of the 4834 bp deletion increasing. Moreover, the elevation of ABR threshold was increased significantly in high dose D-galactose group after 6 month.Conclusion:These findings suggest that the ABR threshold may elevate when the percentage of the 4834 bp deletion exceeds a certain threshold. ROS is one of the causes of production of mtDNA 4834 bp deletion, but there might be other factors involve in the production of mtDNA 4834 bp deletion. PartⅡEffects of different percentage of dietary restriction on D-galactose induced presbycusisObjective:Chronic injection of D-galactose is a useful method for establishing an animal model of mimetic aging. However, there have been no studies about mitochondrial biogenesis in inner ear concerning the D-galactose aging model. The aims of the present study are to clarify this issue and whether DR is still effective in D-galactose induced aging rats with high frequency of CD in inner ear.Methods:Four week-old female wistar rats were injected with D-galactose (500mg/kg/d) for 8 weeks, and then were fed ad libitum or subjected to 70%,50% or 35% of the ad libitum diet for 12 months. ABR was detected 6 times during whole experiment (1 day,3 months,6 months,9 months and 12 months after 8 weeks injection of D-galactose). Cochlear antioxidant activity (SOD) and level of MDA were measured. The relative amount of cochlear mtDNA content and the frequency of mtDNA 4834 bp deletion were detected using real-time PCR. The mRNA expression levels of three transcription factors considered important in regulating mitochondrial biogenesis and respiratory function, peroxisome proliferator-activated receptor coactivator-1 (PGC-1), nuclear respiratory factor-1 (NRF-1), and mitochondrial transcription factor A (TFAM), were examined by real-time RT-PCR. The protein levels of NRF-1 and PGC-1 were measured by western blot.Results:Elevation of ABR threshold was significantly increased in rats at 6 months after injection of D-galactose. Oxidative damage was higher in D-galactose rats than in control, but was partially reversed by 70% and 50% of the ad libitum diet. The mtDNA content and the frequency of mtDNA 4834 bp deletion were increased in D-galactose rats, but were partially inhibited by 70% and 50% of the ad libitum diet. The 35% of ad libitum diet had the most production of ROS, the highest frequency of CD in inner ear and the poorest activity of SOD among all groups. The changes of PGC-1 (mRNA and protein levels) and TFAM (mRNA levels) were consistent with the changes of mtDNA content.Conclusion:The changes of mtDNA biogenesis in inner ear of D-galactose induced mimetic aging model are similar with the changes of that of the natural aging animals. Meanwhile,70% and 50% of the ad libitum diet can partially retard the progression of many age-related molecular and physiological processes in the D-galactose induced mimetic aging rats. However, the 35% of ad libitum diet has multiple malnutrition effect. PartⅢProtective roles ofα-lipoic acid in the rat model of mitochondrial DNA 4834 bp deletion in inner earObjective:To investigate the protective roles ofα-lipoic acid in the rat model of mitochondrial DNA (mtDNA) 4834 bp deletion in inner ear.Methods:Forty female Wistar rats at 4 weeks of age were randomly divided into four groups:group A (D-galactose group, n=10), which were treated with hypodermic 5% D-galactose (150 mg/kg/d) and then with intraperitoneal saline after half an hour for 8 weeks. Group B (D-galactose+α-lipoic acid group, n=10), which were treated with D-galactose and then with intraperitoneal 100mg/kg/d a-lipoic acid after half an hour for 8 weeks. Group C (a-lipoic acid group, n=10), which were treated with hypodermic saline and then with a-lipoic acid after half an hour for 8 weeks. Group D (control group, n=10), which were given saline only for 8 weeks. Auditory brainstem response (ABR) was used to detect the hearing threshold of rats. Colorimetry was used to analyze the activity of the superoxide dismutase (SOD) and concentration of malondialdehyde (MDA). The percentage of the mtDNA 4834 bp deletion in inner ear was identified by real-time PCR.Results:The difference in ABR threshold shift among all groups were not significant (P>0. 05). The frequency of the mtDNA 4834 bp deletion were 12.53%±1.70% in group A, 6.17%±1.04% in group B,4.89%±0.78% in group C and 5.24%±0.75% in group D. The difference between group A and other groups was significant (p<0.05), but among group B, group C and group D was not significant (p>0.05). The difference of the activity of SOD between group A and other groups was significant (p<0.05), but among group B, group C and group D was not significant (p>0.05). The difference of the concentration of MDA between group A and other groups was significant (p<0.05), but among group B, group C and group D was not significant (p>0.05).Conclusion:There is no significant hearing loss when the percentage of mtDNA 4834 bp deletion is lower than 12.5%.α-Lipoic acid can prevent the reactive oxygen species (ROS) induced mtDNA 4834 bp deletion in inner ear of rats.