The E₂/ERs-Related Mechanism Of Aerobic Exercise Improving Synaptic Plasticity In The Hippocampus And Cerebral Cortex Of OVX Mice

Objective: Menopausal women have lower circulating estradiol（E2）and are more susceptible to cognitive decline and emotional abnormalities.Epidemiological studies showed that the aging population was increasing in China,and cognitive decline damages the life quality.In recent years,the relationship between E2 and cognition has attracted extensive attention,while the mechanism remains to be explored.Literatures have found that aerobic exercise improved cognition in menopausal women and increased the circulating E2 level.Actin polymerization in the hippocampus and cerebral cortex improves synaptic plasticity and cognition.And will synaptic plasticity and actin polymerization be changed in the hippocampus and cerebral cortex after menopause? What are the effect and mechanism of aerobic exercise on E2/ estrogen receptors（ERs）,actin polymerization and synaptic plasticity in the hippocampus and cerebral cortex? Therefore,we explore the effect of 8-week treadmill exercise on E₂/ERs,synaptic plasticity,actin polymerization and related signaling pathway in the hippocampus and cerebral cortex of ovariectomized（OVX）mice,and to furtherly explore the relationship between E₂/ERs and synaptic plasticity through cell experiments.Methods: Forty-eight female C57BL/6 mice were randomly divided into SHAM operation（SHAM）group and OVX group,and then rested for two weeks after OVX.Twelve mice from SHAM and OVX groups were selected as 8-week treadmill exercise（EX）group,and the rest mice were divided as the control（CON）group.Y maze was used to test the working memory of mice in the end of 8-week exercise.24 h after last exercise,mice were sacrificed.blood samples,the hippocampus and cerebral cortex were collected.The morphology of dendrites in the hippocampal CA1 and DG area,prefrontal cortex was detected by Golgi staining.The levels of E2 in serum,hippocampus and cerebral cortex were determined by ELISA.ERα,ERβ and GPER,synaptic plastic-related proteins BDNF and Glu R1,actin polymerization marker F-actin and G-actin,actin polymerization-related signaling pathway proteins p-PAK/PAK and P-Cofilin/Cofilin in the hippocampus and cerebral cortex were tested using Western Blot.The hippocampus and prefrontal cells of newborn mice were isolated and cultured in vitro.The cells were intervened with E2,ERα antagonist（MPP）,ERβ antagonist（PHTPP）,GPER antagonist（G15）and aromatase inhibitor（Letrozole）.MTT assay was used to detect the effect of different concentrations of aromatase inhibitor（Letrozole）,ERs antagonists and aromatase inhibitor（100 n M,1 μM and 10 μM）on the activity of hippocampal and prefrontal cells.The expression of plasticity synaptic related-proteins BDNF and Glu R1,actin polymerization marker F-actin and G-actin,actin polymerization-related signaling pathway proteins P-PAK/PAK and p-Cofilin/Cofilin in the hippocampal and prefrontal cells were detected by Western Blot method.Results: 1.in vivo study:（1）Compared with the SHAM/CON group,E2 level in serum,ERα,ERβ and GPER protein expression in the hippocampus and E2 concentration and ERβ protein expression in cerebral cortex of OVX/CON group were decreased significantly.Compared with the SHAM/CON group,serum E2,E2 and GPER levels in the hippocampus,ERα and GPER proteins in cerebral cortex were significantly increased in SHAM/EX group;Compared with the OVX/CON group,E2 in serum,E2,ERα,ERβ and GPER in the hippocampus and cerebral cortex of OVX/EX group were significantly increased.（2）Compared with the SHAM/CON group,the percentage of spontaneous alternation（%）,numbers of entering new arm（%）and time of entering new arm（%）of mice in OVX/CON group did not change significantly,and the total number of entering arm decreased markedly.Compared with the SHAM/CON group,the percentage of spontaneous alternation（%）,number of entering the new arm（%）and time of entering the new arm（%）in SHAM/EX group did not change significantly.Compared with OVX/CON group,OVX/EX group had no significant changes in the percentage of spontaneous alternation（%）,the percentage of numbers of entering the new arm（%）and time of entering the new arm（%）,while the total number of entering the arm increased observably.（3）Compared with the SHAM/CON group,dendritic complexity and density of dendritic spines of pyramidal cells in the hippocampal DG area and prefrontal cortex were increased in the OVX/CON group,while density of dendritic spines in the CA1 area was decreased.Compared with the SHAM/CON group,the density of dendritic spines in the hippocampal CA1 and DG area and prefrontal cortex of the SHAM/EX group was increased.Compared with the OVX/CON group,the dendrite complexity and dendrite spinous density of the hippocampal DG area,prefrontal cortex were increased in the OVX/EX group.（4）Compared with the SHAM/CON group,the expression of BDNF protein in hippocampus and cerebral cortex of the OVX/CON group was decreased.Compared with the SHAM/CON group,Glu R1 protein expression in hippocampus and cerebral cortex of the SHAM/EX group was increased.Compared with the OVX/CON group,Glu R1 and BDNF protein expression in the hippocampus and cerebral cortex of the OVX/EX group were increased.（5）Compared with the SHAM/CON group,F-actin /G-actin,p-PAK/PAK and p-Cofilin /Cofilin in the hippocampus and cerebral cortex of the OVX/CON group was decreased.Compared with the SHAM/CON group,F-actin/G-actin,p-PAK/PAK and p-Cofilin /Cofilin in the hippocampus and cerebral cortex of the SHAM/EX group was increased.Compared with the OVX/CON group,F-actin /G-actin,p-PAK/PAK and p-Cofilin /Cofilin was increased in the hippocampus and cerebral cortex in the OVX/EX group.2.in vitro study:（1）MTT assay results showed that treating with G15,MPP and PHTPP for 2 h didn’t change the cell viability until the dose is 10 μM in the hippocampal and prefrontal cell.Intervention of Letrozole for 48 h didn’t change the cell viability until the dose is 10 μM in the hippocampal and prefrontal cell.（2）The expression of synaptic plasticity-related protein Glu R1 was decreased after Letrozole intervention for 48 h in the hippocampal and prefrontal cortex cells.The expressions of BDNF and Glu R1 proteins in the hippocampal and prefrontal cells were increased significantly with E2,G15,MPP and PHTPP intervention for 2 h,while it didn’t change the BDNF and Glu R1 protein expression significantly for 30 min or 1 h.（3）After intervention of Letrozole for 48 h in the hippocampal and prefrontal cortex cells,actin polymerization was decreased.E2 intervention for 30 min,1h and 2 h significantly increased the actin polymerization of the hippocampal and prefrontal cells,and MPPPHTPP and G15 intervention for 30 min,1 h or 2 h reversed the effect of E2.（4）E2 intervention for 30 min,1 h or 2 h increased the expression of p-Cofilin/Cofilin in the hippocampal and prefrontal cells,and increased the expression of p-PAK/PAK in the hippocampal cells,but the effect of E2 on the p-PAK/PAK in the prefrontal cells only appeared after 1 h and 2 h intervention.MPP-PHTPP or G15 reversed these changes.Conclusion:（1）The effect of aerobic exercise on autonomous activity ability,the density of dendritic spines,synaptic plasticity-related proteins,actin polymerization and PAK-Cofilin signaling pathway may be related to the increase of serum E2 concentration and E2 and ERs levels in the hippocampus and cerebral cortex of OVX mice.（2）ERα,ERβ and GPER mediated the effect of neuron-derived and exogenous E2 on actin polymerization,PAK-Cofilin signaling pathway and synaptic plasticity-related protein expression in the hippocampal and prefrontal cells.（3）ERα,ERβ and GPER mediate the short-term and long-term effect of E2 on actin polymerization and PAK-Cofilin signaling pathway in the hippocampal and prefrontal cells.