| Purpose: Appropriate exercise can improve cognitive function in the elderly people,such as,declarative memory,long term memory,etc..It also can improve cerebral energy metabolism,prevent atrophy of the brain with aging,which may reduce morbidity of Alzheimer’s disease by maintaining the brain-volume.This phenomenon is particularly evident in the hippocampus dentate gyrus.The hippocampus dentate gyrus of adult neurogenesis is subject to the process fine-tuned by a variety of neurotransmitters and neurohormones and the glycogen synthase kinase 3β(GSK3β)is a key node in this process.The main task of this project is to explore the role of GSK3β plays in voluntary exercise promoting the learning and memory function,and to further molecular mechanisms in this process.Method: 1.The effect of voluntary exercise on GSK3β(1)Animals were divided in two groups: control group and voluntary exercise group.The voluntary exercise model mouse was established in the cage with a running wheel and resting places.(2)Mirros water maze tests was used to detect the learning and memory function of two groups of mice.(3)Western blot was used to detect the expression of GSK3β and phosphorylation in the brain of two groups.(4)Immunohistochemistry was used to detect the distribution and expression of GSK3β in hippocampus.2.The role of GSK3β in the voluntary exercise promoting learning and memory function.(1)Artificial cerebrospinal fluid(ACSF)and SB216763 were injected into the hippocampus by ntracerebroventricular injection.Grouped the mouse into control group,voluntary exercise + ACSF(RW+ACSF),voluntary exercise + SB216763(RW+SB),and established model by using of running wheel cage and normal mouse cage,respectively.(2)Compare the learning and memory function among the three groups by Mirros water maze tasts.(3)The expression and distribution of the DCX and Neuro D positived newborn neurons in hippocampus were detected by immunohistochemistry.The expression and distribution of Brdu in hippocampus of three groups of mice were detected by immunofluorescence.(4)The expression of CREB and its phosphorylation in the hippocampus of three groups were detected by western blot.(5)Immunofluorescence was used to detect the co-expression of GSK3β and Ser9 with DCX respectively in hippocampus of three groups.3.The GSK3β related signal pathways activated by the voluntary exercise.(1)The expression and activity of IRS-1 / PI3 K / Akt signaling pathway were detected by western blot.Immunohistochemical method was used to detect the distribution of IRS-1 in the hippocampus of mice.And the co-expression level of IRS-1 and DCX was detected by immunofluorescence.(2)The changes of AMP / ATP in the brain of three groups were detected by ELISA assay.Western blot was used to detect the changes of AMPK expression and phosphorylation.DARP D1 / c AMP / PKA signaling pathway and were detected by western blot.(3)Using ELISA assay detects changes of c AMP content in the hippocampus of mice in each group;western blot assay D1 dopamine receptor(dopamine receptor D1,DARP D1)signaling pathway,DARP D1 / c AMP / PKA signaling pathway,and the DARP D1 / Rho A.The expression of dopamine D1 receptor in hippocampus was detected by immunohistochemistry.The expression of dopamine D1 receptor and DCX was detected by immunofluorescence.Result: 1.The effect of voluntary exercise on GSK3β.1.1 Moirrs water maze results showed that in 7-day training experiment the latency in the exercise group was better than the control group from the third day.In the last day of the memory test experiment,the track in voluntary exercise group was better than control and percentage of target quadrant residence time was significantly higher than control group in voluntary exercise group(p <0.05).1.2 Western blot and immunohistochemistry showed that the expression level of GSK3β in the exercise group was significantly higher than the control group in the hippocampus(p <0.01),and the expression level of Ser9 was significantly decreased(p < 0.01).The expression level of Tyr216,which is the phosphorylation activating site of GSK3β,was significantly increased(p <0.01).2.The role of GSK3β in the voluntary exercise promoting learning and memory function.2.1 Moirrs water maze results showed that in the RW + ACSF group,the latency of the 7-day training experiment was better than the control group from the third day.In the last day of the memory test experiment,the track in RW + ACSF group was better than control and its percentage of target quadrant residence time was significantly higher than control group in RW + ACSF group(p>0.05).The RW + SB group reverse this effect(p<0.01).2.2 The results of wester blot showed that there was no significant difference in CREB expression between the three groups in the hippocampus,and the expression of p-CREB was significantly higher in the RW + ACSF group than in the control group(p <0.01).The RW + SB group has been a marked decline when compared with RW + ACSF group(P<0.01).2.3 Immunohistochemical results showed that the expression levels of DCX,Neuro D and Brdu in the hippocampus of RW + ACSF group were significantly higher than those in the control group(p<0.01).Compared with RW + ACSF group,the levels of DCX and Brdu were significantly decreased(P<0.01),and the Neuro D has decreased to some extent(p>0.05)in RW + SB group.2.4 Immunofluorescence results showed that DCX and GSK3β had a highly consistent change in the hippocampus of each group,and GSK3β could be fully expressed with DCX.Ser9 and DCX had a complete trend of change,and Ser9 and DCX could not be completely Co-expression.3.The GSK3β related signal pathways activated by the voluntary exercise.3.1 The results of western blot showed that the activity levels of IRS-1,PI3 K and Akt signaling molecules in the insulin signal pathway were significantly improved(P <0.01).In the RW + SB group,the expression of GSK3β was significantly decrease than RW + ACSF group(p<0.05),but the activity and activity of GSK3β were significantly decreased(p<0.05).Immunohistochemical results showed that the distribution of p-IRS-1 in the hippocampus of RW + ACSF group was significantly increase than that in the control group(p<0.01).The distribution of p-IRS-1 in RW + SB group was significantly decrease compared with RW + ACSF group(p <0.05).Besides,although immunofluorescence showed that IRS-1 and DCX had a high degree of consistent expression in the hippocampus of each group,but DCX and IRS-1 were not co-expressed.3.2 The results of the ELISA showed that the ratio of AMP / ATP was significantly increased in RW + ACSF group(p<0.01).In the RW + SB,the ratio havs a certain degree of decline compared with RW + ACSF group(p>0.05).The expression level of AMPK in RW + ACSF group was significantly higher than that in RW + ACSF group(P <0.05)(P <0.01),and RW + SB group had a certain degree of decline compared with RW + ACSF group.3.3 ELISA test results showed that,in RW + ACSF group,the c AMP content has a higher level than control group(p <0.05);RW + SB decreased to some extent(p compared> 0.05).Western blot results showed that the activity of DARP D1,Rho A,PKA signaling molecules in the DARP D1 signaling pathway was significantly higher in the RW + ACSF group(P<0.05).In the RW + SB group,the activity and expression and activity of GSK3β were significantly decreased(P <0.01).The expression and distribution of DARP D1 were significantly increased in RW + ACSF group(p <0.05).The RW + SB group had a certain degree of decrease compared with RW + ACSF group(P> 0.05).Immunofluorescence showed that DARP D1 and DCX had a highly consistent change in the hippocampus among the three groups.But only some of the DARP D1 could be fully co-expressed with DCX and the other were not.Conclusion: 1.Voluntary exercise can enhance the cognitive function and adult neurogenesis in the hippocampus of adult mice.2.Voluntary exercise to promote adult neurogenesis to improve cognitive ability in the process,GSK3β expression and activity levels will be significantly improved.3.Voluntary exercise can promote the activation of insulin signaling pathway,but not the main mechanism,which vouluntary exercise promote the adult neurogenesis by regulating the expression and activity of GSK3β.4.Dopamine D1 receptor signaling pathway can be activated after the voluntary exercise,and may be main mechanism of change that voluntary exercise promote adult neurogenesis and promote the learning and memory function by the regulation of GSK3β expression and activity. |
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