| Research purpose:Depression is a common psychiatric disorder.Early life stress is a risk factor for the development of depression and can cause permanent changes in neurodevelopmental trajectories,leading to impaired synaptic plasticity in the hippocampus and increased susceptibility to depression.Therefore,exploring the mechanisms of the effects of early life stress on the depressive disorders and seeking relevant pharmacological interventions will provide strategies for the prevention and treatment of depressive disorders.Previous studies by our group have found that Xiaoyaosan could improve depression and anxiety-like behavior in animals with liver depression and spleen deficiency by protecting nerve cells and increasing synaptic plasticity.In the present study,we modeled early life stress through maternal separation and re-stressed by chronic restraint stress to establish a model of depressive disorder with early life stress,and to investigate the effects and mechanisms of early life stress on susceptibility to depressive disordersMethods:Experiment 1:The effect of early stress on susceptibility to depressive disorders.SPF C57BL/6J pregnant mice(gestation day 17-18 days)were used as the dams for this experiment,and 36 male pups were subsequently selected as the study subjects and randomly divided into control group,CRS group,MS+CRS group.The procedure was as follows:dams were removed from their original cages and placed in new cages in another experimental room from 8:00 to 12:00 a.m.from postnatal dayl to 21(PND1-21),,separated from pups for 4 hours/day,and all pups were weaned at PND22,the sexes were identified,and male pups were selected for this study.PND22-38 were kept under adequate water and feed without any intervention or stimulation.PND39-60 MS+CRS and CRS groups were subjected to Chronic restraint stress for 3 hours/day.PND61-68 we detected depressive and anxiety-like behaviors,learning and memory abilities of mice through Open Field Test,Elevated Plus Maze,Novel Object Recognition Task,Morris Water Maze.The serum CORT levels of mice were measured by ELISA.Experiment 2:Exploring the effects and mechanisms of early stress on hippocampus synaptic plasticity in the of mice.On the basis of Experiment 1,we used RT-qPCR and Western Blot to detect the synaptic protein PSD95,synaptophysin mRNA and protein expression levels in the hippocampus of each group of mice;p-mTOR,t-mTOR,p-S6,t-S6 protein expression levels in the hippocampus of mice were detected using Western Blot.Experiment 3:Xiaoyaosan modulates BDNF-ERK-mTOR/S6 pathway to alleviate synaptic plasticity impairment in mice with early life stress depression disorder.SPF C57BL/6J pregnant mice were used as the dams for this experiment,and 48 male pups were subsequently selected as the study subjects and randomly divided into four groups.Except for the Control group,mice in MS+CRS group,Xiaoyaosan group and Fluoxetine group were used to establish the early life stress depression disorder model by MS+CRS.The corresponding drug were given in PND39-60 by gavage,Control group and MS+CRS group mice were given deionized water,Xiaoyaosan and Fluoxetine group miace were given Xiaoyaosan and fluoxetine hydrochloride suspension respectively.The mice were observed for macroscopic characterization and body weight changes were measured weekly.PND61-68 the behaviors were evaluated by behavioral tests such as Open Field Test,Elevated Plus Maze,Novel Object Recognition Task and Morris Water Maze to observe the effects of Xiaoyaosan on the behavior of model mice.We observed synaptic ultrastructural alterations in the CA1 region of the mouse hippocampus by TEM.The expression levels of synaptic proteins PSD95 and synaptophysin were detected by Western Blot.We detected the expression levels of BDNF,p-ERK,t-ERK,p-mTOR,t-mTOR,p-S6,t-S6 proteins by Western Blot.We used immunohistochemistry to localize and analyze their protein expression of the above molecules also.Results:(1)The early life stress increased the susceptibility to depressive disorder:①Open Field Test:The results showed that the CRS and MS+CRS groups were significantly different compared with the control group regarding the time spent in the central squares.In particular,both the CRS and MS+CRS mice spent less time in the center squares than the control mice.Moreover,the time was significantly lower in the MS+CRS group than in the CRS group.However,the distance that the CRS and MS+CRS groups moved was not significantly different from that in the control group.②Elevated Plus Maze:Both the CRS and MS+CRS groups had a decreased number of entries and percentage of time spent in the open arms compared with the control group.Moreover,the percentage of time spent in the open arms was lower in the MS+CRS group than in the CRS group,but no significant difference between the above two groups was observed in the number of entries into the open arms.③Novel Object Recognition Task:The RI value of mice in the CRS group was not significantly different from that in the control mice in the NORT,while the RI values of mice in the MS+CRS group were significantly different from those of the Control and CRS groups,respectively.④Morris Water Maze:Acquisition trial,the escape latency for mice in all experimental groups was shortened with the passage of training days.On the fourth day of training,the time to reach the platform was significantly higher in the MS+CRS group than in the control group.The latency in the CRS mice showed an increasing tendency,but the difference from the control group was not statistically significant.In the probe trial,we observed that the time spent in the target quadrant and the number of crossings were both significantly lower in the MS+CRS group than in the control group,but no difference was observed between the CRS and control groups.⑤Effect of early stress on serum CORT in mice:the result showed that the serum CORT levels were significantly higher in the CRS and MS+CRS groups,compared with the Control group,but there was no difference between the CRS and MS+CRS groups.(2)Early life stress reduced the expression levels of synaptic proteins PSD95 and synaptophysin in the hippocampal region of mice:the results showed that the hippocampal PSD95,synaptophysin mRNA and protein expression levels were reduced in mice in the CRS and MS+CRS groups compared with the control group.Compared with the CRS group,there was no difference in hippocampal PSD95 mRNA expression levels in the MS+CRS group,but lower PSD95 protein expression levels;synaptophysin mRNA and protein levels in the MS+CRS group were significantly lower than those in the CRS group.(3)Early life stress reduced the activity of mTOR/S6 pathway in the hippocampal region of mice:there were no differences in t-mTOR and t-S6 protein expression levels in the Control,CRS,and MS+CRS groups of mice.Compared with the Control group,p-mTOR(S2448)and p-S6(S240/244)were significantly reduced in the CRS and MS+CRS groups;compared with the CRS group,p-mTOR and p-S6 levels were more significantly reduced in the MS+CRS group.(4)Effects of Xiaoyaosan on macroscopic representation,body weight and behaviors of mice with early stress depression disorder.①Macroscopic manifestations:During the maternal separation of PND1-21,no significant abnormalities were observed in eating and activities of mice between the Control group,MS+RS group,Xiaoyaosan group and fluoxetine group.PND22-38,with the exception of the Control group,the mice in the other three groups showed intermittent irregular stools,increased tearing behavior in the cage,and increased irritability during weight measurement.PND39-60,the mice in the MS+CRS group,the Xiaoyaosan group and the fluoxetine group were given chronic restraint stress,and had significantly more hair loss,irritability,struggling and biting.With the prolongation of chronic restraint stress,at the 3rd week of restraint stress(i.e.PND53-60)mice in the MS+CRS group showed tying up and curling up,less struggling in the restraint tube,more urination and unformed defecation.In contrast,mice in the Xiaoyaosan and Fluoxetine groups still struggled strongly in the restraint tube.②Body weight:The body weight of mice in the four groups was at the same level at PND22.During the PND22-38,the body weight of mices increased significantly in each group of mice,but there was no significant difference in weight among the groups.At the PND46 and PND53,the body weight of mice in all four groups was increased,especially the Control group mice increased significantly.However,there was no difference in the comparison between the four groups of mice.At the PND60,the body weight of mice in the Control group,Xiaoyaosn group,and fluoxetine group still showed an increasing trend,while the body weight of mice in the MS+CRS group decreased,and there were differences compared with the Control group.The body weight of the mice in the Xiaoyaosan and Fluoxetine groups was slightly higher compared with the MS+CRS group,but none of the differences were statistically significant the two groups.③Open Field Test:Compared with the Control group,the time spent in the central squares was significantly shorter in the MS+CRS group.The time spent in the central squaresthe was increased after intervention with Xiaoyaosan and fluoxetine hydrochloride,and there was no significant difference between the Xiaoyaosan and Fluoxetine groups.④Elevated Plus Maze:The mice in the MS+CRS group had significantly less time in the open arm and number of entries they entered the open arm than the Control group,and Xiaoyaosan could increase the time spent in the open arm of MS+CRS mice,but has no effect on the number of times they entered the open arm,whlie the number of entries and the time spent in the open arm were significantly increased after intervention with fluoxetine.⑤Novel Object Recognition Task:The recognition index(RI value)of mice in the MS+CRS group was significantly lower than that in the Control group,and Xiaoyaosan could significantly increase the RI value of mice.⑥Morris Water Maze:The escape latency was prolonged in mice of the MS+CRS group compared to the Control group on days 3-4 of acquisition trial.Compared with the MS+CRS group,the escape latency was shortened in the Xiaoyaosan and fluoxetine groups,and there was no difference between the two groups compared.In the probe trial,the time spent in the target quadrant and the number of crossing escape platforms were reduced in the MS+CRS group of mice compared with the Control group.Further more,the time spent in the target quadrant and the number of crossing escape platforms were increased in the Xiaoyaosan and fluoxetine groups compared with the MS+CRS group,and there was no difference between the two groups.(5)Effects of Xiaoyaosan on synaptic ultrastructure in CA1 area of the mouse hippocampus:In the Control group,the synaptic structure was intact,the anterior and posterior membranes were clear,and the posterior membrane densities were abundant,while in the MS+CRS group,the anterior and posterior membrane boundaries were disrupted and unclear,and the posterior membrane densities were sparse,while the synaptic structure was still unclear and the posterior membrane densities increased after the intervention of Xiaoyaosan and Fluoxetine.(6)Effects of Xiaoyaosan on the expression of PSD95 and synaptophysin proteins in the hippocampus of mice:Compared with the Control group,the hippocampal synaptic protein PSD95 and synaptophysin protein expression levels were reduced in the MS+CRS group;the hippocampal PSD95 and synaptophysin protein levels were increased after the intervention of Xiaoyaosan and fluoxetine.(7)Effects of Xiaoyaosan on BDNF/ERK/mTOR-S6 signaling pathway in the hippocampus of mice:compared with the Control group,the expression levels of BDNF and p-ERK/t-ERK in the hippocampus of mice in the MS+CRS group were reduced.After 21 days of intervention with Xiaoyaosan and fluoxetine,BDNF and p-ERK/t-ERK protein expression levels were upregulated in the Xiaoyaosan and fluoxetine groups compared with the MS+CRS group.p-mTOR(S2448)and p-S6(S240/S244)expression levels were significantly lower in the MS+CRS group than in the Control group.The phosphorylation levels of mTOR and S6 proteins in the hippocampal tissues of mice were reduced,indicating that the mTOR-S6 pathway activity was inhibited in MS+CRS mice.Compared with the MS+CRS group,the relative expression levels of p-mTOR and p-S6 proteins were increased after drug intervention in the Xiaoyaosan and Fluoxetine groups.Immunohistochemical localization analysis of the above results showed that their positive neurons were distributed in the CA1 of hippocampus,in which BDNF,p-ERK,p-mTOR and p-S6 protein MOD values were significantly reduced in the CA1 of mice in the MS+CRS group,and Xiaoyaosan significantly increased the above indicators that had been reduced in the CA1 of hippocampus of mice in the MS+CRS group.Conclusion:(1)In this study,maternal separation was used to simulate early life stress and chronic restraint stress to simulate re-stress,confirming that early life stress increases susceptibility to re-stress and is a risk factor for the development of depressive disorders.(2)The mechanism by which early life stress increases susceptibility to re-stress may be related to inhibition of mTOR-S6 signaling pathway activity,reduced synthesis of PSD95 and synaptophysin,and reduced synaptic plasticity.(3)A mouse model of early stress depression disorder was established using Maternal separation combined with Chronic restraint stress(MS+CRS).Xiaoyaosan could improve depressive and anxiety-like behaviors and cognitive function impairment in mice with early life stress depression disorder,and this result indicats that liver-smoothing and spleen-invigorating method was effective in treating early stress depression disorder.(4)Xiaoyaosan can treat early life stress depression by promoting the activation of BDNF/ERK/mTOR-S6 pathway,increasing synapse-related protein and repairing synaptic structural damage. |
PDF Full Text Request