| Objective: Neurogenesis is the process in which the neurons newly derived from neural progenitor cells(NPCs)functionally integrate into the existing circuits.Adult neurogenesis existed in subventricular zone(SVZ)and subgranular zone(SGZ)of hippocampus.Adult hippocampal neurogenesis involves learning,memory,and emotional regulation.However,with the increase of age,the proliferation and differentiation ability of NPCs decreases,and the occurrence of neurogenic disorders.It is a key event in the occurrence of neurodegenerative diseases.Exercise can promote hippocampal neurogenesis and improve the cognitive function in mice,but the specific mechanism is not clear.Our early results showed that proteasome activity was correlated with functional maintenance of NPCs.Thus,it was speculated that exercise might promote neurogenesis by activating proteasome activity.To test this hypothesis,we used in vivo and in vitro experiments to analyze the molecular mechanism of exercise induced hippocampal neurogenesis in adults,so as to provide new strategies for clinical prevention and treatment of age-related diseases.Part 1 The effect of voluntary running on proteasome activity in the hippocampus of adult miceMethods: 1.Isolated hippocampal tissues of BALB/c mice at different ages,P0(< 24 h),P90(3 months)and P300(10 months)respectively.CT-L proteasome activity,C-L proteasome activity and T-L proteasome activity were detected respectively.The expression of proteasome subunits PSMB1,PSMB2,PSMB5 were detected by q PCR.2.Adult BALB/c mice(P90)were randomly divided into voluntary running group(Runner)and control group(Sedentary).Then the mice ran for 2 weeks,4 weeks and 8 weeks.Recorded the movement distance.The relative CT-L proteasome activity was detected using hippocampus tissues.After running,q PCR detected hippocampal proteasome subunit PSMB1,PSMB2,PSMB5 expression.Results: 1.With the increase of age,the hippocampal CT-L activity,C-L activity and T-L activity all decreased.Moreover,the expression of the hippocampal PSMB1,PSMB2,PSMB5 detected by q PCR also decreased with the increase of age.2.CT-L activity and C-L activity were increased in the exercise group after 2 weeks of voluntary wheel running,in which CT-L activity was increased by 1.4 times(P < 0.01)and C-L activity was increased by 1.3 times(P < 0.05).The activity of CT-L was the highest.At the same,the expressions of hippocampal PSMB1,PSMB2,PSMB5 were detected by q PCR after running in adult mice,in which PSMB5 was increased by abou1.3 times and PSMB2 by about 1.2 times,while the expressions of PSMB1 was not significantly changed.The m RNA level of PSMB5 also was the highest.CT-L activity in adult mice increased by about 1.2 times after running for 4 weeks and 8 weeks.These results suggest that voluntary wheel running can promote hippocampal CT-L andC-L proteasome activity,CT-L activity was the highest.3.The correlation between total exercise distance and relative CT-L activity was analyzedin 2 weeks and 8 weeks.The movement distance in the first 2 weeks was positively correlated with CT-L proteasome activity(r = 0.5,P = 0.08).However,there was no significant correlation between these two factors for prolonged exercise beyond 8 weeks(r = 0.19,P = 0.68),suggesting that prolonged running did not lead to a further increase in CT-L proteasome activation compared to the previous two weeks.Part 2 Effects of exercise up regulating IGF-1 activating chymotrypsin-like activity on neurogenesis,learning and memory in hippocampus of adult miceMethods: 1.Adult BALB/c mice(P90)were randomly divided into the Runner group and the Sedentary control group.After voluntary wheel running for 4 weeks,Elisa was used to detect the content of serum and hippocampal IGF-1 in the adult mice.2.Adult(P90)hippocampal NPCs were isolated and cultured in vitro,and the CT-L proteasome activity of NPCs was detected after different concentrations of IGF-1 treatment,and the expression of PSMB5 was detected by q PCR.Brd U and Tuj1 immunofluorescence staining were used to detect the proliferation and differentiation of NPCs.3.Adult mice(P90)were intraperitoneally injected with IGF-1 receptor(IGF-1R)inhibitor picropodophyllin(PPP)and its solvent control 10%DMSO,injected twice a day,20 mg/kg,continuously for 4 weeks.Meanwhile,voluntary wheel running was performed.Then the activity of CT-L proteasome activity was detected in adult hippocampal.Nrf2 nuclear translocation in hippocampal was detected by Western blot.Brd U(100 mg/kg/d)was intraperitoneally injected consecutive 7 days on the first day of running.After 4 weeks of voluntary wheel running and PPP-treatment,brain was perfusion and performed frozen section.Ki67,DCX,Brd U/DCX immunofluorescence staining was used to detect the proliferation,differentiation and survival of NPCs.The hippocampal learning and memory ability was tested by Y-maze spontaneous alternations and new object recognition.Results: 1.After voluntary wheel running for 4 weeks,Elisa showed that the contents of serum and hippocampus IGF-1 raised 1.3 times(P < 0.05)and 1.2 times(P < 0.01) respectively in running group.q PCR detection showed that the expression of IGF-1 m RNA increased 1.3 times(P < 0.05).These results suggest that exercise can upregulate the content of IGF-1.2.After the treatment of different concentrations of IGF-1 20,50 and 100 ng/m L,Brd U incorporation results showed that the proliferation capacity of NPCs was raised,in which 50 ng/m L and 100 ng/m L were increased by 1.4 times(P < 0.05)and 1.7 times(P < 0.01),respectively.Tuj1 staining showed that IGF-1 50 ng/m L and 100 ng/m L were increased by 1.4 times(P < 0.05)and 1.6 times(P < 0.001),respectively.CT-L activity also increased,as the same as the expression of PSMB5 m RNA.It was suggested that after the treatment of IGF-1,the CT-L activity was activated to promote the proliferation and differentiation of adult NPCs.3.During voluntary wheel-running for 4 weeks,adult mice were intraperitoneally injected with IGF-1R inhibitor PPP and solvent control 10% DMSO 20 mg/kg twice per day.The results of hippocampal CT-L activity showed that the hippocampal CT-L activity in the PPP-runner group was 30% lower than that of DMSO runner group(P < 0.05).4.Ki67 immunofluorescence staining showed that the average number of Ki67 positive cells in the DG of the hippocampus in the PPP-runner group was 219.6 ± 72.3,which was lower than that of the control group(319.2 ± 101.5)(P = 0.08).The number of DCX positive cells in DG of the hippocampus in the PPP group was 650.2 ± 195.9,significantly lower than that of the DMSO-runner group(P < 0.05).Metamorph analysis of the process length of DCX positive cells showed that the average length of DCX positive cells in DG of hippocampus in the PPP-runner group was 65.6 ?m ± 14.0 ?m,which was smaller than that of the DMSO-runner(103.0 ?m ± 22.2 ?m)(P < 0.01),showing a significant statistical difference.These results indicated that after PPP inhibited the activity of IGF-1,the proliferation and differentiation of NPCs in DG of hippocampus were inhibited.The Brd U/DCX double staining indicated the survival of NPCs in DG of the hippocampus in adult mice.The number of Brd U/DCX double positive cells,in the PPP-runner group was 128.2 ± 41.2,significantly decreased than that of DMSO control group 246.1 ± 82.5,suggesting the survivability of NPCs in hippocampus have been reduced when IGF-1 was inhibited.5.The hippocampal learning and memory ability were tested by Y-maze spontaneous alternations and novel object recognition.The results of Y-maze spontaneous alternations experiment showed that there was no difference in the number of arm entry between the PPP-runner group and the DMSO-runner control group,but the accuracy of Y-maze spontaneous alternations in the PPP-runner group was significantly lower than that in the DMSO-runner control group(P < 0.05).During the training period of novel object recognition detection,the PPP exercise group and DMSO exercise group spent similar time exploring objects,indicating that the activity of the two groups of mice was similar.During the detection period,the percentage of time spent exploring novel objects in the PPP exercise group was 61.2% ± 8.5%,significantly lower than that in the DMSO control exercise group(69.7% ± 7.9%)(P < 0.05).These results suggest that intraperitoneal injection of IGF-1R inhibitor PPP can inhibit hippocampal learning and spatial memory in adult mice.Part 3 The effect of IGF-1/Nrf2 on hippocampal neurogenesis,learning and memory in adult mice after voluntary runningMethods: 1.Nrf2 nuclear translocation in hippocampal tissue(P90 mice)was detected by Western blot after voluntary wheel running for 4 weeks.2.Adult(P90)hippocampal NPCs were isolated and cultured with IGF-1 in vitro,and Nrf2 nuclear translocation was detected by immunofluorescence staining.3.N2 a cells were cultured in vitro,and the CT-L proteasome activity was detected after adding different concentrations of IGF-1,and the nuclear translocation of Nrf2 was detected by Western blot.4.Nrf2-si RNA and its control si RNA transfected 8 pmol to N2 a cells for 48 hours by Lipofectamine? RNAi MAX Transfection Reagent in vitro.Then IGF-1 100 ng/m L was added for 24 hours to detect CT-L proteasome activity.5.The N2 a cells adhered into the wall about 70% to 80% fusion,N2 a cells were cotransfected the plasmids PSMB5-Luciferase,Renilla,pc DNA3-Myc3-Nrf2 and its control group PSMB5-Luciferase,Renilla,pc DNA3 for 48 hours using Turbofect transfection reagent.Or N2 a cells were co-transfecte the plasmids PSMB5-Luciferase and Renilla for 48 hours after the treatment of Nrf2-si RNA and its control group for 8 hours.Dual Luciferase/Renilla assay system kit detected the transcription activity of PSMB5 by Nrf2 after IGF-1 treated.6.Adult BALB/c mice(P90)were randomly divided into the AAV-sh RNA-Nrf2 autonomic wheel running group and the AAV-sh RNA-SC autonomic wheel running group.AAV-sh RNA-Nrf2 1 ?L/ side(2-3 x 1012 genotype /m L)was injected with bilateral hippocampal stereotaxic,and the control group AAV-sh RNA-SC was injected with the same amount.After 5 weeks,mice were going to voluntary wheel running for 4 weeks.Then the hippocampal CT-L proteasome activity was detected,and the expression levels of Ki67 and DCX in hippocampal dentate gyrus of the two groups were detected by immunofluorescence staining.The effects of inhibiting Nrf2 on the hippocampal proteasome activity and on the proliferation and differentiation of NPCs were observed.The hippocampal learning and memory ability was tested by Y-maze spontaneous alternations and novel object recognition.Results: 1.After voluntary wheel-running for 4 weeks in adult mice,the expression of Nrf2 in the hippocampus was detected by Western blot.The results showed that Nrf2 nuclear translocation in running group was increased by 1.3 times compared with the control group(P < 0.001),suggesting that voluntary wheel-running can promote Nrf2 nuclear translocation in the adult hippocampus.2.Adult hippocampal NPCs were isolated and cultured in vitro.After treatment with IGF-1(50,100 ng/m L),it was induced to adhere to the wall by passage.The results of Nrf2 immunofluorescence staining showed that the nuclear translocation of Nrf2 increased by 1.4 times compared with the control group after treatment with IGF-1(50,100 ng/m L)(P < 0.001).3.After the treatment of N2 a cells with different concentrations of IGF-1(10,20,50 and 100 ng/ml),the protein of N2 a cells was extracted.Western blot results showed that the nuclear translocation of Nrf2 increased by 1.2 times compared with that of the control group after the treatment of IGF-1 with 100ng/ml(P < 0.01).The results of CT-L activity test showed that after IGF-1 20,50,100 ng/ml treatment,CT-L activity increased by 1.2 times(P < 0.05)and 1.3 times(P < 0.001),indicating that Nrf2 nuclear translocation of N2 a cells increased and CT-L activity increased after IGF-1 treatment.4.During voluntary wheel running for 4 weeks,the PPP and control group 10% DMSO were intraperitoneal injected 20 mg/kg at 2 times per day,then extractd the hippocampus nuclear and cytoplasm protein.Western blot results showed Nrf2 nuclear translocation of the PPP-runner group decreased by 20%(P < 0.05)compared with the DMSO runner.These results suggest that Nrf2,as a downstream effector of IGF-1,was involved in exercise-induced hippocampal neurogenesis.5.After transfection of Nrf2-si RNA and control si RNA 8 pmol 48 hours by Lipofectamine? RNAi MAX Transfection Reagent,then IGF-1 100 ng/m L was added for 24 hours to detect CT-L activity in N2 a cells.The results showed that the activity of CT-L increased by 1.4 times after IGF-1 treatment(P < 0.001).Compared with the NC control group,there was no significant change in proteasome activity in Nrf2-si RNA group,but the proteasome activity in the Nrf2-si RNA group decreased when IGF-1 was used(P < 0.05).These results indicated that under the action of IGF-1,Nrf2 activated the CT-L proteasome activity.6.The plasmids PSMB5-Luciferase,Renilla,pc DNA3-Myc3-Nrf2 and the control group PSMB5-Luciferase,Renilla,pc DNA3 were co-transfected into N2 a cells by Turbofect transfection reagent.After overexpression of Nrf2,the transcriptional activity of PSMB5 increased by 3.5 times(P < 0.05),and the transcriptional activity of PSMB5 increased by 6.5 times(P < 0.05)after overexpressing Nrf2 and adding IGF-1 at the same time.The results showed that overexpressing Nrf2 further increased the transcriptional activity of PSMB5 after IGF-1 treatment.After transfection with Nrf2-si RNA and control si RNA 8 pmol for 8 hours,the plasmid PSMB5-Luciferase and Renilla were co-transfected for 48 hours by Turbofect Reagent.Then IGF-1 100 ng/m L was added for 24 hours.The transcription activity of PSMB5 was detected by Dual Luciferase/Renilla assay system kit.The results showed that the transcriptional activity of PSMB5 increased by 1.4 times after IGF-1 treatment(P < 0.05).Compared with NC group,the transcriptional activity of PSMB5 in Nrf2-si RNA group had no significant different.But when IGF-1 was treated,the transcriptional activity of PSMB5 in Nrf2-si RNA group was significantly reduced(P < 0.01).These results suggest that Nrf2 promotes the transcription of PSMB5 under the action of IGF-1.7.Adult BALB/c mice were injected with AAV-sh RNA-Nrf2 and control group AAVsh RNA-SC 1 ?L/ side(2-3 x 1012 genotype /m L)in hippocampus.After 5 weeks,mice were going to voluntary wheel running for 4 weeks.The CT-L proteasome activity test results showed that the CT-L proteasome activity of the AAV-sh RNA-Nrf2 group was 30% lower than that of the AAV-sh RNA-SC control group,suggesting that the knockdown of Nrf2 could inhibit the hippocampal CT-L proteasome activity.8.The results of Ki67 immunofluorescence staining showed that the average number of ki67-positive cells in DG of the hippocampus in the AAV-sh RNA-Nrf2 exercise group was 195.4 ± 85.0,which was significantly lower than that of the control group(303.8 ± 97.2)(P < 0.05).The number of DCX-positive cells in DG of the hippocampus in the AAV-sh RNA-Nrf2 group was 554.6 ± 238.8,significantly lower than that of the control group 887.2 ± 297.2(P < 0.05).At the same time,Metamorph analysis results showedthat the average length of DCX-positive cells in DG of the hippocampus of the AAVsh RNA-Nrf2 exercise group was 42.0 ?m ± 8.4 ?m,which was smaller than that of the control group of 64.2 ?m ± 15.4 ?m(P < 0.01).These results suggest that the knockdown of Nrf2 could inhibit the proliferation and differentiation of NPCs in DG of the hippocampus.9.The Y-maze spontaneous alternations and novel object recognition experiments mainly evaluated the spatial learning and memory ability of adult mice.The results of Y-maze spontaneous alternations showed that there was no difference in the frequency of arm entry between the AAV-sh RNA-Nrf2 exercise group and the control AAV-sh RNA-SC exercise group,but the accuracy of Y-maze spontaneous alternations in the AAVsh RNA-Nrf2 exercise group was significantly lower than that in the control AAVsh RNA-SC exercise group(P < 0.05).Moreover,during the training period of the novel object recognition experiment,the AAV-sh RNA-Nrf2 exercise group and the control AAV-sh RNA-SC exercise group spend similar time to explore objects,indicating that the activity of the two groups of mice was similar.During the detection period,the time spent exploring new objects in the AAV-sh RNA-Nrf2 exercise group decreased by 15% compared with the AAV-sh RNA-SC exercise group(P < 0.05).These results suggest that the knockdown of Nrf2 could inhibit learning and spatial memory in adult mice.Conclusion: 1.With the increase of age,the activities of CT-L,C-L and T-L all decreased.The activity of CT-L and C-L in hippocampus increased after voluntary wheel running for 2 weeks,among which CT-L activity the most increased.And the increase of CT-L activity tended to be stable after voluntary wheel running for 4 weeks.2.The content of IGF-1 was up-regulated by voluntary running wheel.In vitro culture of NPCs using IGF-1 could activate CT-L proteasome activity and promote the proliferation,differentiation and survival of NPCs in adult mice.3.After inhibiting IGF-1 with IGF-1R inhibitor,it can inhibit the increase of hippocampal CT-L proteasome activity and weaken the improvement of exercise-induced neurogenesis and cognitive function.4.In vitro culture of NPCs and N2 a cells using IGF-1 could induce Nrf2 nuclear translocation,up-regulate the transcriptional activity of PSMB5,and activate CT-L proteasome activity.5.Knockdown Nrf2 could inhibit the increase of hippocampal CT-L proteasome activity and weaken the neurogenesis and cognitive functions by IGF-1 induced. |
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