Role And Mechanism Of Nuclear Transcription Factor Nrf2 In D-galactose-Induced Premature Aging Of The Auditory Cortex

PartⅠ Establishment of D-galactose-induced auditory cortical aging modelObjective: To establish a model for aging of auditory cortexMethods: Male SD rats of 2 months old were randomly divided into two groups: control group(natural aging group)and D-galactose(D-gal)group(mimetic aging group).In the D-gal group,D-gal(500 mg/kg/d)was injected subcutaneously into the rat for 8 weeks;the control group was given the same volume of 0.9% saline.After the last injection,the control group and the D-galactose group were each divided into three age subgroups: the 4-month-old group,the 10-month-old group,and the16-month-old group.Transmission electron microscopy(TEM)assay was used to investigate the changes of the ultrastructure in the auditory cortex.Apoptosis was detected using a TUNEL assay in the auditory cortex.The senescence of rat auditory cortex cells was detected by β-galactosidase staining.Result: 1.In the control groups,neurons of the auditory cortex displayed no obvious ultrastructural changes in 4-and 10-month-old rats;in contrast,irregular nuclear structures,large amounts of lipofuscin,mitochondrial swelling,and demyelination of nerve fibers were observed in the 16-month-old rats.In the D-gal groups,the auditory cortex displayed no obvious ultrastructural changes in the 4-month-old rats.However,in the 10-month-old D-gal groups has similar ultrastructural changes compared with 16-month-old NS subgroup rats.Moreover,these abnormal ultrastructural changes were more significant in the 16-month-old D-gal group rats.2.In the natural aging group,TUNEL positive cells in the auditory cortex increased with age.Compared with the natural aging group,the number of TUNEL positive cells in the D-galactose group at 10 months and 16 months was significantly increased.3.In the natural aging group,a small amount of β-galactosidase-positive cells were observed at 4 months of age,positive cells increased at 10 months of age,and positive cells increased significantly at 16 months of age.Compared with the natural aging group of the same age,the β-galactosidase positive cells in the D-galactose group were significantly increased.Conclusions: With the aging process,the structure of the auditory cortex gradually degenerates,and the number of senescent and apoptotic cells increases gradually.D-galactose treatment accelerates neurodegeneration,senescence,and apoptosis in the auditory cortex.PartⅡ The role and mechanism of Nrf2 in premature aging of the auditory cortexObjective: Central presbycusis is the most common sensory disorder in the elderlypopulation,however,the underlying molecular mechanism remains unclear.NF-E2-related factor 2(Nrf2)is a key transcription factor in the cellular response to oxidative stress,however,the role of Nrf2 in central presbycusis remains to be elucidated.The aim of the present study was to investigate the pathogenesis of central presbycusis using a mimetic aging model induced by D-galactose(D-gal)in vivo.Methods: Malondialdehyde(MDA)was detected with a microplate reader.Taq Man quantitative real-time PCR assays was used to detect common deletions of mitochondrial DNA.DNA oxidative damage was detected with 8-hydroxydeoxyguanosine(8-OHd G).Immunofluorescence was used to detect the localization and expression of Keap1 and Nrf2.RT-PCR was used to detect the transcription levels of antioxidant genes(NQO1,HO-1,Mn SOD).Western blot was used to detect Cytochrome C and cleaved-caspase 3 levels.Result: 1.The MDA test showed that: compared with the 4-month-old groups,the MDA levels were increased in the 16 month-old control group;and the MDA levels in 16-month-old D-gal groups showed the more obvious increase.Moreover,compared with age-matched control groups,the MDA levels of D-gal group were significantly elevated in the 10-and 16-month-old groups.2.Real-time quantitative PCR revealed that CD accumulation increased with age;in addition,CD levels in the D-gal group increased significantly compared with the age-matched normal group.3.The 8-hydroxydeoxyguanosine(8-OHd G)assay showed that 8-OHd G levels in the D-gal group were significantly higher than those in the normal age-matched group;The 8-OHd G levels in the 16-month-old control group and the 10-month-old D-gal group were significantly higher than those in the 4-month-old group;the 8-OHd G levels in the 16-month old D-gal group were more significantly increased.4.Western blot analysis showed that,compared with the age matched natural aging group,cleaved caspase-3 and mitochondrial cytochrome c released into the cytoplasm increased in the auditory cortex of the D-galactose group.5.The Keap1 immunofluorescence showed that the Keap1 levels in the D-gal groups were higher than that in the control group;the Keap1 levels were significantly increased in the 16-month old groups compared with the 4-month old groups.6.The Nrf2 immunofluorescence test showed that: compared with the control groups,the nuclear Nrf2 levels in the 10-month-old and 16-month-old D-gal groups were decreased;in addition,compared with the 4-month-old group,the levels of nuclear Nrf2 were significantly reduced in the 16-month-old group.7.RT-PCR showed that: in the control group,the m RNA levels of NQO1,HO-1,and Mn SOD at 16 months old were lower than those at the 4-month old group.In D-galactose groups,the transcription levels of NQO1,HO-1,and Mn SOD at 16 months old were significantly lower than those at 4 months of age.Conclusions: Nrf2 signaling decreased in the auditory cortex of the aging rats.A decline in Nrf2-mediated antioxidant responses results in enhanced oxidative stress,increased mitochondrial DNA oxidative damage,mitochondrial dysfunction,and ultimately cell structure degradation and increased apoptosis.These changes may lead to the occurrence of central deafness.Part Ⅲ The role and mechanism of Nrf2 activator Oltipraz in delaying cell senescenceObjective: To explore the role of Nrf2 in senescenceMethods: The model of aging was induced with D-galactose.Cell viability was assessed with the Cell Counter Kit-8(CCK-8)assay.Real-time quantitative PCR was used to detect common deletions of mitochondrial DNA(CD).DNA oxidative damage was detected with 8-hydroxydeoxyguanosine(8-OHd G).Western blot was used to detect Cytochrome c and cleaved-caspase 3 levels.ROS was detected with DCFH-DA staining.JC-1 staining detected mitochondrial membrane potential.Apoptosis was examined with Annexin V-FITC/PI staining.β-Galactosidase staining was used to detect cell senescence.TEM assay was used to investigate the changes of the ultrastructure.Results:1.A mimetic aging model in vitro was induced by D-galactose(15 mg/ml)using PC12 cells.2.Oltipraz increased cellular antioxidant activity by activating the Nrf2 pathway.3.Oltipraz protected PC12 cells from D-gal induced mt DNA oxidative damage,common deletion,and mitochondrial dysfunction.4.Oltipraz attenuated D-gal-induced apoptosis and cellular senescence.Conclusions: Oltipraz reduced the oxidative stress of cells by activating the Nrf2 pathway,reduced mitochondrial DNA oxidative damage,maintained mitochondrial function,and ultimately delayed cell senescence.