Effects And Mechanism Of Aging Associated PSMD11/Rpn6 In D-Galactose Induced Mimetic Aging Models

Part ? Foundation of D-Galactose induced mimetic aging models in vivo and in vitro Objective: To found the D-gal mimetic aging models in vivo and in vitro.Methods: a.Animal model: One hundred and fifty wistar rats of 4 weeks old were grouped and treated as follows:(1)The D-gal mimetic aging group(n=75): the wistar rats were was injected with D-gal subcutaneously for 8 weeks(500mg/kg/day),and then were adaptive fed for one week.Three age-related subgroups were divided according to the months after the mimetic aging rats were established,the 4-month-old groups(right after the injection),the 10-month-old groups(6 months after injection),and the 16-month-old groups(12 months after injection).(2)The normal saline group(n=75): the wistar rats were injected daily with the vehicle(0.9% saline)for 8 weeks,and divided into three subgroups accordingly.Taq-PCR was used to detect the relative expressions of mt DNA CD in different age groups.ABR was used to explore the hearing thresholds of all age subgroups.The age-related morphological changes of auditory cortex were observed with nissl staining and transmission electron microscopy,respectivey.Both western blot and oxyblot were used accordingly to detect the accumulated levels of carbonyl proteins in the auditory cortex of all age groups.Meanwhile the relative expressions of neuron toxic proteins,p-Tau and TDP43,were detected and analyzed by western blot.b.Cell model: The PC12 cells were treated with series concentrations of D-gluctose in the RPMI medium,and selected the optimal concentration via CCK8 to establish the mimetic aging group,while the control group was cultured with an equal RPMI medium.Aging related mt DNA CD changes were detected by Taq-PCR in the two groups.?-galactosidase staining was used to observe the positive neurons between the two groups as the treatment time increased.The accumulation of oxidative damaged proteins were detected with the oxyblot assay and western blot assay..Results: a.D-gal induced aging rats: mt DNA CD increased in the D-gal mimetic aging groups than corresponding control groups,and elevated with aging.The mt DNA CD accumulated by 1.19-fold(*P<0.05),1.24-fold(*P<0.05),1.9-fold(*P<0.05)in the auditory cortex of D-Gal with aging.Nissl?s staining reveales that the numbers of neurons reduced in the D-Gal mimetic aging groups compared with the age matched groups starting at 10 months and becoming most decremented at 16 months(*P<0.05).The ABR threshold was measured among different age groups,which increased with aging and was higher after D-gal injection after 10 months compared to the control.The NS groups showed an elevated threshold by 2.07-fold(**P<0.01)at 16 months compared to 4 months for the control group,and increased by 2.23-fold(**P<0.01)at 10 months and 2.69-fold(***P<0.001)at 16 months compared to 4 months for the D-Gal group.While in the 10 and 16 months old D-gal rats,the nuclear membranes became disrupted with the irregular nucleus and condensed chromatin,quite portion of the mitochondria appeared swollen and vacuolated,and the compact myelin were disrupted and swollen,meanwhile,all of which were also seen in 16 months old control rats.The oxidative carbonylated proteins increased in the D-gal induced groups than age-matched groups,and elevated with aging.The expressions in D-gal induced aging auditory cortex showed elevations of 1.17-fold(*P<0.05),1.19-fold(*P<0.05),and 1.52-fold(***P<0.001)compared to control groups respectively at 4 months,10 months and 16 months.The relative levels of phosphor-Tau(p-Tau)and TDP43 were also detected in the 16-month-old groups,and both were observed to accumulate in the D-Gal groups than in the control.b.D-gal induced PC12 cells: According to the results of CCK8 and ?-galactosidase staining,we selected the 15mg/ml as the optimal concentration.The ratio of mt DNA CD,expression of carbonylated proteins and ?-galactosidase staining reflected elevation in D-gal induced cells than control.Conclution: High dose of D-gal(500mg/kg/d or 15mg/m L)can induced mt DNA damage,oxidative stress,ultrastructural morphology changes and neuron apoptosis in auditory cortex and PC12 cells.Part ? The effect and mechanism of 19 S proteasome PSMD11/Rpn6 subunit in D-Galactose induced mimetic aging modelsObjective: To study the effect and the regulatory mechanism of 19 S proteasome PSMD11/Rpn6 subunit on the pathogenesis of central presbycusis in D-gal induced mimetic aging models.Methods: One hundred and fifty wistar rats of 4 weeks old were randomly grouped and treated as follows:(1)The D-gal mimetic aging group(n=75): the rats were was injected with D-gal subcutaneously for 8 weeks(500mg/kg/day),and then were adaptive fed for one week.Three subgroups were divided according to the ages after the mimetic aging rats were established,the 4-month-old groups(right after the injection),the 10-month-old groups(6 months after injection),and the 16-month-old groups(12 months after injection).(2)The normal saline group(n=75): rats were injected daily with the vehicle(0.9% saline)for 8 weeks,and divided into three subgroups accordingly.Western blot was used to detect the expressions of apoptosis-related P53,BAX and Bcl-2 proteins in each age-related subgroup.Changes in the level of ubiquitinated P53 in the two groups were detected by the immunoprecipitation(IP)assay.Western blot analysis of the whole ubiquitinated proteins related to proteasome function,19 S proteasome PSMD11 subunit,PSMD4 subunit and 20 S proteasome PSMA7 subunit were explored among different age subgroups.At the same time,the proteasome activities in each subgroup were maintained.The expression changes of AMPK?1 subunit,AMPK?2 subunit and P-AMPK?1/ 2 were explored and compared in different age-subgroups.And co-immunoprecipitation(Co-IP)technique was used to detect interaction between the PSMD11 subunit and AMPK 1/2 subunit.The PC12 cells were treated with 15 mg/ml D-gluctose in the RPMI medium for 72 h to establish the mimetic aging group,while the control group was cultured with an equal RPMI medium for 72 h.Then the cells were treated with AICAR/Compound C or Si-RNA/plasmid.Western blot analysis was used to detect the relative expressions of UPS related poly-ubiquitinated proteins,19 S proteasome PSMD11 subunit,PSMD4 subunit and 20 S proteasome PSMA7 subunit.At the same time,the changes of proteasome activity in living cells were detected and compared with specific fluorescent substrate.JC-1 analysis reflected the oxidative status in different treatment cell groups.AMPK?1 subunit and AMPK?2 subunit and p-AMPK were also detected and analyzed by western blot assay.Co-immunoprecipitation detection was used to confirm the relationship of PSMD11 and AMPK? in PC12 cells.Immunofluorescence and western blot were also used together to reflect the expression and location of the PSMD11-AMPK?2 complex in the treated PC12 cells.Results: a.D-gal induced aging rats: pro-apoptotic proteins,P53 and BAX,were activated and elevated at every age of the D-Gal induced groups than in the corresponding control groups.And the relative ratio of synchronous BAX/Bcl-2 increased in the D-Gal groups than in the control.The result of immunoprecipitation reflected the ubiquitinated P53 degradation level decreased with the reduction of MDM2 at D-gal induced aging groups than control.The poly-ubiquitinated proteins,19 S proteasome PSMD11 subunit,PSMD4 subunit and 20 S proteasome PSMA7 subunit increased at 4 and 10 months,but decreased at 16 months in the D-Gal groups compared to the control.The proteasome activities changed with the age increased,which increased in the D-gal groups by 1.34-fold(**P<0.01)at 4-months-old group and 1.33-fold(**P<0.01)at 10-months-old group than control,while decreased by 0.6-fold(***P<0.001).Meanwhile the AMPK?2 subunit and p-AMPK also increased at 4 and 10 months,but decreased at 16 months in the D-Gal groups compared to the control.However,the expressions of AMPK?1 protein increased in the D-gal groups than control at all ages.Co-immunoprecipitation reflected the direct combination of PSMD11 and AMPK?1/2.b.The poly-ubiquitinated proteins,19 S proteasome PSMD11 subunit,PSMD4 subunit and 20 S proteasome PSMA7 subunit,AMPK?2 and p-AMPK decreased in the D-gal induced PC12 cells than control,while AMPK?1 increased.JC-1analysis confirmed the PSMD11 can influence the oxidative stress in the cell.The ratio increased by 5.56-fold in the D-Gal group compared to the control.Further,the data manifest that the ratios decreased by 0.668-fold in the PSMD11 overexpression group,and increased by 1.99-fold in the down-expression group compared to the D-Gal induced group.Co-immunoprecipitation assay confirmed the direct combination of PSMD11 and AMPK?2 in the PC12 cells.In addition,the relative expressions of the PSMD11-AMPK?2 complex behaved accordance in the different treating subgroups.Conclusion: D-gal induced oxidative injuries,mt DNA damage,proteasome impair and apoptosis in auditory cortex and PC12 cells.PSMD11 can interact with AMPK?2 in resisting aging,which is a promising method.