| Objective:This study aimed to probe the effects of ginseng total saponins(GTS)on the corticosterone(CORT)-induced plasticity impairment of hippocampal glycogen metabolism and astrocyte(AS),and explore the antidepressant mechanism of GTS.Methods:Experiment 1.Stereology-based study on the structural plasticity of hippocampal astrocytes in a mouse model of depression.20 C57BL/6N mice were divided randomly and evenly into 2 groups:the control group(Control)and the CORT group,with 10 in each group.Continuously subcutaneous injection of CORT(20 mg·kg-1·d-1)was given at random time to the mice in the CORT group for five weeks to induce the depression model.While those in the control group were given the same dosage of normal saline.Behavioral test and content test of serum CORT were done after the model making was successful.Then the brains were taken out after cardiac perfusion for measuring the hippocampal volumes,the number,volume and neurite length of GFAP-positive cells with the application of glial fibrillary acidic protein(GFAP)immunohistochemistry and stereological analysis.Experiment 2.Assay for hippocampal glycogen levels in the mouse model of depression30 C57BL/6N mice were divided randomly and evenly into 2 groups;the control group(Control)and the CORT group,with 15 in each group.After the success of model making,each group was further randomly divided into three sub groups,with 5 rats in each sub-group.Brain glycogen(GC)content,glycogen phosphorylase(PYGM)and glycogen synthase(GS)activity were tested in the first sub group.The mice in the second sub-group were sacrificed in the same way as those in the first sub-group,with the hippocampus being taken out and put into the EP tube,and the expression of brain-tybe glycogenphosphorylase(PYGM)and glycogen synthase(GS)protein being tested by using Western-blot analysis.The mice in the third sub-group were still killed in the same way as those in the first sub-group,with hippocampus being taken out and put into the EP tube,and the gene expression of brain-tybe glycogen phosphorylase(PYGM)and glycogen synthase(GS)being tested by using Q-PCR.Experimental 3.Study on the protective mechanism of GTS to astrocyte plasticity in the mouse model of depression90 C57BL/6N mice were divided randomly and evenly into 6 groups:the control group(Control),the CORT group,the control group(Control)and the CORT group,the low-dose group of GTS(12.5 mg · kg-1 · d1-)plus CORT(GTSL),the moderate-dose group of GTS(25 mg · kg-1 · d-1)plus CORT(GTSM),the high-dose group of GTS(50 mg · kg-1 · d-1)plus CORT(GTSH),and the fluoxetine(10 mg · kg-1 · d-1)plus CORT(FLU)group.Each group had 15 rats.Intragastric administration was given to each dose group for 21 days.After the success of the model making,these mice went through forced swimming test(Forced Swimming,Test,FST)and suspension test(Suspenseful Test,TST)on the twentieth and the twenty-first day.In addition,on the twenty-second day the orbital blood was extracted to measure the serum CORT level.All animal were sacrificed on the twenty-third day.In accordance with the requirements,the mice in the each group were randomly divided into three sub-groups,with 5 rats in each group.The hippocampuses of the mice in the first sub-group were taken out after cardiac perfusion and the hippocampal volumes,the number,volume and neurite length of GFAP-positive cells were measured with the application of glial fibrillary acidic protein(GFAP)immunohistochemistry and stereological analysis.The mice in the second sub-group were killed by immersing them in the liquid nitrogen and the hippocampuses were grinded into powder in the liquid nitrogen after being separated from the brains swiftly.Then the hippocampuses were put into the EP tube for testing the brain glycogen(GC)content,glycogen phosphorylase(PYGM)and glycogen synthase(GS)activity.The mice in the third sub-group were killed in the same way as those in the second sub-group,with hippocampal tissue being put into the EP tube to detect the expression of PYGM and GS protein and gene level.Experiment 4.Study on the protective mechanism of GTS to astrocyte plasticity in normal mice20 C57BL/6N mice were divided randomly and evenly into 2 groups:the control group(Control)and the high-dose GTS(50 mg · kg-1· d-1)group.Mice in the high dose group of GTSH were given the intragastric administration for 21 days,with the dosage being 50 mg-kg-1·d-1.After the success of mode making,all the animals were put to death according to the requirements of the experiment on the twenty-second day.The animals in each group are randomly divided into two sub-groups,with 5 rats in each group.Mice in the first sub-group were required to take out the brain after cardiac perfusion and the hippocampus volume and the number and volume and dendritic length of positive cells of GFAP were measured with the application of glial fibrillary acidic protein(GFAP)immunohistochemistry and stereological analysis.Mice in the second sub-group were required to be killed by being exposed to liquid nitrogen.And then the brains of mice would be detached quickly after these mice were taken out.After grinding the hippocampus into powder,the hippocampus was put into the EP tube to detect the content of animal dextrin.ResultsExperiment 1.Depressive like behavior in mice induced by long-term injection of corticosterone,injury of astrocytes in the hippocampus of structural plasticityCompared with Control group,the static time of mice in CORT group during forced swimming and forced tail suspension was significantly prolonged(P<0.01)and the serum levels of CORT increased significantly(P<0.01).Compared to Control group,the hippocampus vilumes of mice in the CORT group were found to be significantly reduced(P<0.01),together with a significant decrease in the number of GFAP positive AS(P<0.01)and in the neurite length of GFAP positive AS(P<0.01).Immunohistochemical staining(GFAP)test showed that the CORT group's AS positive hippocampal volume was significantly reduced when compared to that of the control group(P<0.01).Experiment 2.the levels of glycogen in the hippocampus with long term injection of corticosteroneCompared with Control group,mice hippocampus GC content of CORT group decreased significantly(P<0.01),while both PYGM activity and GS activity in hippocampus increased significantly(P<0.01).Compared with the Control group,the expression of CORT GS protein in the hippocampus of mice was significantly increased(P<0.01),the expression of PYGM protein in the hippocampus was significantly increased(P<0.01),the expression of GS gene in the hippocampus was significantly increased(P<0.01),and the expression of PYGM gene in the hippocampus was significantly increased(P<0.01).Experiment 3.GTS can increase glycogen in depression model mice in hippocampus,antagonistic astrocyte plasticity damageCORT group's serum corticosterone level was higher than that of the Control group(P<0.01),and no evident influences were noticed during the model making when different dosages of GTS were administrated.Both the FLU group and the three GTS groups of different dosages could significantly antagonize mouse model of FST and TST(P<0.01),shorten the immobility time and notably inhibit the PYMG activity(P<0.05,P<0.01,P<0.05,P<0.05).Compared with the CORT group,both the GC content and GS activity in hippocampus of the mice in the FLU,GTSM and GTSH groups were significantly increased(P<0.01),the expression of PYGM protein were notably resisted in hippocampus of model mice(P<0.01),and significant down-regulation of PYGM gene expression were detected in hippocampus of mice(P<0.01).Compared with the CORT group,FLU,GTSM and GTSH groups experienced significantly up-regulated expression of GS protein and GS gene in the hippocampus of mice(P<0.05,P<0.05,P<0.01).In addition,the hippocampus volumes of mice in the CORT+FLU,CORT+GTSM and CORT+GTSH groups were increased significantly(P<0.01),with an evident increase in the number of GFAP positive cells(P<0.01).Compared with CORT group,the GFAP positive AS volume of mice in the CORT+FLU and CORT+GTSH group increased notably(P<0.05),with a notable increase in the neurite length of hippocampal GFAP positive AS(all P<0.01).Experiment 4.GTS does not affect the structural plasticity of hippocampal astrocytes in normal miceThe hippocampus stereology detection results about each mouse of different groups were compared with t test.Compared with Control group,GTSH had no obvious change in mice hippocampal volume(P>0.05),the number of hippocampal GFAP positive AS showed no significant changes(P>0.05),and hippocampal GFAP positive AS volume(P>0.05)had no significant changes.So it was with the neurite length of hippocampal GFAP positive AS(P>0.05)and hippocampal GC levels(P>0.05).ConclusionThis experiment has observed the anti-depression effects of GTS and explored the mechanism by adopting repeated CORT injection to simulate chronic stress and induce the depression animal model based on hippocampal glycogen levels and astrocytes structural plasticity.It is shown that long-term CORT can induce depression-like behavior and GTS has anti-depressant effects.GTS has the following characteristics:?possess the anti-depressant like behavioral effects,such as the behavior of FST and TST experiments,and high dose of GTS can reduce the depressive behavior of normal mice and corticosterone in depressive model mice.?The injury of hippocampus can be resisted by increasing glycogen content in AS.? The increased expression of GS protein and gene as well as the decreased expression of PYGM protein and gene displayed that high dosage of GTS can increase the glycogen protein and gene level.?It is effective in improving the hippocampal volume,the number of GFAP positive cells as well as their cell volumes and neurite lengths to improve the plasticity of hippocampal AS.This study suggests that the role of GTS in anti-depression may be associated with the improvement of glycogen content and structural plasticity of AS. |
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