| Part one The effects of running exercise and fluoxetine on the behaviors of CUS-induced depression model miceObjective: Fluoxetine is one of the classic clinical drugs for the treatment of depression,but some problems exist in fluoxetine treatment such as delayed onset and aggravation of symptoms within the first two weeks.Even though running exercise has been shown to prevent or alleviate the onset and development of depression,its specific efficacy remains controversial.Therefore,this study explored the efficacy of running exercise and fluoxetine in the treatment of depression and compared the onset time of these two treatments.In order to provide a scientific basis for searching new antidepressants.Methods: 4-6 weeks old male C57BL/6 mice were selected to undergo sucrose water adaptation after 1 week of adaptive feeding,and the mice with taste disorders were screened out.The remaining mice were randomly divided into the control group(n = 14)and the CUS group(n = 119).After four weeks of CUS intervention,forty-two model mice were selected and randomly divided into the CUS/STD group(n = 14),CUS/RUN group(n = 14)and CUS/FLX group(n = 14).Mice in the CUS/RUN group received treadmill exercise for 4 weeks: 10 min per day and 5 days per week.Mice in CUS/FLX group received intraperitoneal fluoxetine injection: 1mg/ml,10 mg/kg/d for 28 days.The body weight and the sucrose preference test(SPT)were detected on every weekend.On the fourth weekend of exercise and the administration of fluoxetine,the tail suspension test(TST),forced swimming test(FST)and open field test(OFT)were performed.The depression-like and anxiety-like behaviors of the mice and the antidepressant effects of two treatments were determined using the behavior results.Results: 1.The weight gain of mice in the CUS group was significantly slower than that in the control group during the period of CUS.Beginning from the second week of CUS,the weight of mice in the CUS group was significantly lower than that in the control group(p < 0.01).During the period of running exercise and administration of fluoxetine,the weight of mice in the CUS/STD group,CUS/RUN group and CUS/FLX group was significantly lower than that in the normal control group(p < 0.01),and the weight of mice in the CUS/RUN group and CUS/FLX group was not significantly different from that in the CUS/STD group.2.The results of SPT showed that the sucrose preference percentage of mice in CUS group was lower than that of the control group at the fourth weekend of CUS(p < 0.01).At the third weekend of running exercise and fluoxetine treatment,the sucrose preference percentage of mice in CUS/RUN group was significantly higher than that in CUS/STD group(p < 0.01),while there was no significant difference between CUS /FLX group and CUS/STD group(p > 0.05).By the fourth weekend of running exercise and fluoxetine treatment,the sucrose preference percentages of mice in the CUS/RUN group and CUS/FLX group were significantly higher than that in the CUS/STD group(p < 0.05,p < 0.05).3.The results of FST and TST showed that the immobility time of mice in the CUS/STD group was significantly higher than that in the control group(p < 0.01),and the immobility times of FST and TST in the CUS/RUN group were significantly lower than that in the CUS/STD group(p < 0.05,p < 0.05).The immobility time of TST in the CUS/FLX group was also significantly lower than that in the CUS/STD group(p < 0.01),while there was no significant difference in the immobility time of FST between the CUS/FLX group and the CUS/STD group.4.The results of OFT showed that there was no significant difference in the total score of OFT among the control group,the CUS/STD group,the CUS/Running group and the CUS/FLX group.Conclusions: 1.Running exercise could significantly improve depression-like behavior in CUS-induced depression model mice,and running exercise could produce an antidepressant effect prior to fluoxetine.2.4-week running exercise could improve the depression-like behavior of CUS-induced depression model mice more comprehensively than fluoxetine.3.The weight gain of mice in the CUS/STD group was slower,while running exercise and fluoxetine could not reverse the phenomenon.4.The CUSinduced depression model mice in this study did not have anxiety-like behaviors.Part two The effect of running exercise and fluoxetine on the neurogenesis and synaptic plasticity in the hippocampus of CUS-induced depression model miceObjective: To explore the effects of running exercise and fluoxetine on the neurogenesis and the synaptic plasticity in the hippocampus of CUSinduced depression model mice in order to provide scientific bases for the antidepressant effect of running exercise.Methods: The mice of the control group,CUS/STD group,CUS/RUN and CUS/FLX group were given 11 days of continuous injection of Brd U(10 mg/m,50 mg/kg/d)from the beginning of running exercise and administration of fluoxetine.At the fourth weekend of running exercise and fluoxetine intervention,6 mice randomly selected in each group were perfused and then fixed by 4% paraformaldehyde after the SPT,FST and TST.One brain hemisphere of each mouse was randomly selected and dehydrated in the gradient concentration of sucrose water(10%,20%,30%).Then,the hemispheres were embedded with OCT and prepared into a continuous frozen section at 50 ?m.These frozen sections were randomly sampled at equal intervals according to the sampling fraction of 1/5.Five groups of isometric continuous sections containing hippocampal tissue were obtained.Two groups of the sections were randomly selected for the immunohistochemical staining of DCX(marker of immature neurons)and SP(marker of dendritic spines),and the quantitative study of DCX+ cells,SP+ dendritic spines of CA1,CA3 and DG in the hippocampus was conducted with the stereological method.Then,another two groups of the sections were randomly selected for the immunofluorescence staining of Brd U/Neu N/DCX(Brd U marks proliferation of cells,Neu N marks mature neurons)and MAP2(marker of dendrites).Fluorescent images were collected on a laser confocal scanning microscope.The numbers of cell(Brd U+,Brd U-/DCX+/Neu N+,Brd U+/DCX+/Neu N-,Brd U+/DCX-/Neu N+)per unit area in hippocampus of each mouse were quantified.Quantification of MAP2 immunofluorescence staining was performed with the Niselements AR Analys 4.2.Results: 1.The stereological quantitative results of DCX+ cells in the DG of hippocampus in four groups showed that the number of DCX+ cells in the DG of hippocampus in CUS/STD group was significantly increased when compared with the control group(p < 0.05),while running exercise and fluoxetine could significantly reduce the number of DCX+ cells(p < 0.05,p < 0.01).2.The quantitative results of the number of Brd U+ cells,Brd U-/DCX+/Neu N+ cells,Brd U+/DCX+/Neu N-cells and Brd U+/DCX-/Neu N+ cells per unit area in the DG of hippocampus in each group mice showed that there was no significant difference in Brd U+ cells in hippocampal DG among the control group,the CUS/STD group and the CUS/RUN group mice,while,CUS/FLX group mice had more Brd U+ cells than the CUS/STD group(p < 0.05).The number of Brd U+/DCX+/Neu N-cells per unit area and the percentage of the Brd U+/DCX+/Neu N-cells in the Brd U+ cells in the DG of the CUS/STD group were significantly increased when compared with the control group(p < 0.05,p < 0.01),and the number of Brd U+/DCX-/Neu N+ cells per unit area in the DG of the CUS/STD group was significantly decreased when compared with the control group(p < 0.05).Meanwhile,the number of Brd U+/DCX+/Neu N-cells per unit area and the percentage of the Brd U+/DCX+/Neu N-cells in the Brd U+ cells in the DG of the CUS/RUN group were significantly reduced when compared with the CUS/STD group(p < 0.01,p < 0.01),and the number of Brd U+/DCX-/Neu N+ cells per unit area and the percentage of the Brd U+/DCX-/Neu N+ cells in the Brd U+ cells in the DG of the CUS/RUN group were significantly increased when compared with the CUS/STD group(p < 0.05,p < 0.05).Same as the CUS/RUN group,the number of Brd U+/DCX+/Neu N-cells per unit area and the percentage of the Brd U+/DCX+/Neu N-cells in the Brd U+ cells in the DG of the CUS/FLX group were significantly reduced when compared with the CUS/STD group(p < 0.05,p < 0.01),while there were no significant difference in the number of Brd U+/DCX-/Neu N+ cells per unit area and the percentage of the Brd U+/DCX+/Neu N-cells in the Brd U+ cells in the DG between the CUS/FLX group and the CUS/STD group.In addition,there was no significant difference of Brd U-/DCX+/Neu N+ cells per unit area in the DG of hippocampus among the four groups.3.The fluorescence intensities of MAP2 in the three subregions of the hippocampus in CUS/STD group mice were significantly lower than those of the control group(p < 0.05,p < 0.05,p < 0.01),and the fluorescence intensities of MAP2 in the three subregions of the hippocampus of CUS/RUN group mice and CUS/FLX group mice were significantly higher than those of CUS/STD group mice(p < 0.05,p < 0.01,p < 0.05 and p < 0.05,p < 0.05,p < 0.05).4.The total numbers of SP+ dendritic spines in CA1,CA3 and DG of the hippocampus in CUS/STD group mice were significantly lower than those in the control group(p < 0.01,p < 0.05,p < 0.01),and the total numbers of SP+ dendritic spines in the three subregions of the hippocampus of CUS/RUN group mice were significantly higher than those of CUS/STD group mice(p < 0.05,p < 0.05,p < 0.01).However,the total number of SP+ dendritic spines in the whole hippocampus of the CUS/FLX group was significantly higher than that of CUS/STD group(p < 0.05),while the total numbers of SP+ dendritic spines in CA1 and CA3 were not significantly different from those of CUS/STD group mice.5.There were no significant differences in the protein expression levels of PSD95 and SYP in the hippocampus among four groups.Conclusion: 1.The DG of hippocampus in CUS-induced depression model mice had a maturation disorder of newborn neurons.2.Running exercise could promote the maturation of newborn neurons in DG of the hippocampus,and increase the plasticity of the dendritic spine synapses in the areas of CA1,CA3 and DG.The effect of fluoxetine on the maturation of newborn neurons was weaker than that of running exercise,and the effect of fluoxetine on the synaptic plasticity was mainly concentrated in DG rather than CA1 and CA3.3.The effects of running exercise on the maturation ability of newborn neurons and the synaptic plasticity were superior to that of fluoxetine,which might be one of the important cellular mechanisms for the faster and better antidepressant effect of running exercise.Part three Study on the mechanism for the fact that running exercise exerts antidepressant effect through promoting the maturation of the newborn neurons in hippocampusObjectives: To investigate the mechanism by which running exercise promotes the maturation of newborn neurons in order to provide scientific bases for searching the new therapeutic targets of depression.Methods: Firstly,the mouse,selected from the control group,the CUS/STD group and the CUS/RUN groups of part one,was put to death through breaking mouse neck,and the mouse hippocampus was rapidly isolated from brain tissue on the ice.RNA was extracted from a randomly selected one side of the hippocampus and reversely transcribed into c DNA,and the m RNA levels of PGC-1? in hippocampus were detected by fluorescence quantitative PCR.On the other side of the hippocampus,protein lysate was added in accordance with the mass to volume ratio of 1:10,and the supernatant was extracted after ultrasonic homogenizing.The protein expression of PGC-1? in the hippocampus was detected by Elisa kit.Secondly,PGC-1? in the hippocampus was knocked out.Male C57BL/6 mice aged 4-6 weeks were selected.The mice were randomly divided into the AAV-Control group(n = 32)and the AAV-PGC-1? group(n = 33).Then,the skull was drilled according to the mouse hippocampal coordinates(Bregma:-2.3 mm,L: ±1.8 mm,V:-2.0 mm),and the adenovirus vector(AAV2/9-GFP or AAV2/9-PGC-1?-GFP)with green fluorescent protein(GFP)was injected into the hippocampus of the mice(2 ?l per side of hippocampus,0.2 ?l/min).After the virus was stabilized,the mice in the AAV-Control group were randomly divided into the AAV-Control/SED group(n = 16)and the AAV-Control/RUN group(n = 16),and mice in the AAV-PGC-1? group were randomly divided into the AAV-PGC-1?/SED group(n = 16)and the AAV-PGC-1?/RUN group(n = 17).The mice in the AAV-Control/RUN group and the AAV-PGC-1?/RUN group were given a 4-week running exercise intervention(the running program was the same as the part one).The percentage of SPT of four group mice was measured at each weekend.After the stably expression of AAV,the expression of PGC-1? m RNA in the hippocampus of four groups was detected by fluorescence quantitative PCR.Besides,FST and TST were conducted after the stably expression of AAV and running exercise.Finally,the expression of gene and protein of FNDC5 and BDNF in the hippocampus of the mice in the control group,the CUS/STD group and the CUS/RUN groups of part one was detected by fluorescence quantitative PCR,Western blot and Elisa to explore the downstream mechanism of running exercise.Results: 1.The protein expression level of PGC-1? in hippocampus of the CUS/STD group was significantly lower than that of the control group(p < 0.01),and the protein expression level of PGC-1? in hippocampus of the CUS/RUN group were significantly higher than that of the CUS/STD group(p < 0.05).The m RNA level of PGC-1? in the hippocampus of the CUS/STD group was significantly lower than that of the control group(p < 0.05).However,the m RNA level of PGC-1? in the hippocampus of the CUS/RUN group was not significantly different from that of the CUS/STD group.2.(1)The m RNA level of PGC-1? in the hippocampus of the AAVPGC-1? group was significantly lower than that of the AAV-Control group after the stably expression of AAV(p < 0.05).(2)The results of SPT showed that the sucrose preference percentage of the AAV-PGC-1? group was lower than that of the AAV-Control group at the fourth weekend of AAV injection(p < 0.01).Four weeks later,the sucrose preference percentage of the AAVPGC-1?/SED group was significantly lower than that in the AAVControl/SED group(p < 0.01),and the sucrose preference percentage of the AAV-PGC-1?/RUN group was significantly lower than that in the AAVControl/RUN group(p < 0.01).There was no significant difference between the AAV-PGC-1?/RUN group and the AAV-PGC-1?/SED group.(4)The results of FST and TST showed that the immobility times in FST and TST of the AAV-PGC-1? group were significantly higher than those of the AAVControl group after stable expression of the virus(before running)(p < 0.05,p < 0.05).After running exercise,the immobility times of the AAV-PGC-1?/RUN group in FST and TST were significantly higher than those of AAV-Control/RUN group mice(p < 0.05,p < 0.05),and there was no significant difference of the immobility time in FST and TST between the AAV-PGC-1?/RUN group and the AAV-PGC-1?/SED group.(5)The quantitative results of the numbers of Brd U+ cell,DCX cell,Brd U-/DCX+/Neu N+ cell,Brd U+/DCX+/Neu N-cell and Brd U+/ DCX-/ Neu N+ cell per unit area in the DG of hippocampus in mice of each group showed that the Brd U+cells per unit area in the DG of the AAV-PGC-1?/SED group was significantly decreased when compared with the AAV-Control/SED group(p < 0.05),and the Brd U+ cells per unit area in the DG of the AAV-PGC-1?/RUN group was also significantly lower than that of the AAVControl/RUN group(p < 0.05).There was no significant difference in Brd U+ cells per unit area between the AAV-PGC-1?/RUN group and the AAVPGC-1?/SED group.There was no significant difference of DCX+ cell between the AAV-PGC-1?/SED group and the AAV-Control/SED group,but the DCX+ cell of the AAV-Control/RUN group was significantly lower than that of the AAV-PGC-1?/RUN group(p < 0.01).Although there was no significant difference of Brd U+/DCX+/Neu N-cells per unit area in DG among these four groups,the percentage of the Brd U+/DCX+/Neu N-cell in the Brd U+ cells in the DG of the AAV-PGC-1?/SED group was significantly higher than that of the AAV-Control/SED group(p < 0.05),and the percentage of the Brd U+/DCX+/Neu N-cell in the Brd U+ cell in the DG of the AAV-PGC-1?/RUN group was significantly higher than that of the AAV-Control/RUN group(p < 0.01).There was no significant difference in the percentage of the Brd U+/DCX+/Neu N-cell in the Brd U+ cells between the AAV-PGC-1?/RUN group and the AAV-PGC-1?/SED group.Additionally,Brd U+/DCX-/Neu N+ cell per unit area in the DG of the AAVPGC-1?/SED group was significantly decreased when compared with the AAV-Control/SED group(p < 0.05),and the number of Brd U+/DCX-/Neu N+ cell per unit area in the DG of the AAV-PGC-1?/RUN group was also significantly lower than that of the AAV-Control/RUN group(p < 0.05).Finally,there was no significant difference of the number of Brd U-/DCX+/Neu N+ cell per unit area in the DG of hippocampus among these four groups.3.There were no significant differences in the protein and m RNA expression levels of FNDC5 and BDNF in the hippocampus among the three groups.Conclusions: 1.PGC-1? was involved in the physiological process in which running exercise promotes the maturation of newborn neurons.2.After PGC-1? was knocked out in the hippocampus,the process of neurogenesis of newborn neurons in DG of mice was impaired and depression-like symptoms were induced.3.The effects of running exercise on the maturation of newborn neurons and the antidepressant effect disappeared after knocking out the PGC-1? in hippocampus,which suggested that running exercise required PGC-1? to promote the maturation of newborn neurons in the hippocampus of depressed mice,in order to exert the antidepressant effect.4.The downstream pathway FNDC5/BDNF of PGC-1? might not be the key mechanism for running exercise to promote the maturation of newborn neurons and exert antidepressant effects. |
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