| ObjectiveIn this study,to study the maternal separation(MS),chronic unpredictable mild stress(CUMS)and maternal separation combined with chronic unpredictable mild stress(MS+CUMS)to prepare depression models,to observed the effects of weight and behavioral in adult Sprague-Dawley(SD)rats,and validate the validity of the model and whether early maternal separation increased depression-like behavior in adult rats by later-stress;To investigate the effect and mechanism with SNS on prevention and treatment of depression-like behaviors by MS+CUMS induced in rats,and provide reliable experimental basis evidence of Sinisan on patients with depression,especially adult depression with a history of adverse life stress for clinic.MethodsForty neonatal male SD rats were randomly divided into four groups of 10 rats each group:control(Control),maternal separation(MS),chronic unpredictable mild stress(CUMS),and maternal separation combined with chronic unpredictable mild stress(MS+CUMS).The MS and MS+CUMS groups received MS,and the normal and CUMS group was undisturbed during PND1-21.The total duration of separation was 360 min each day,which was regularly divided into two 180 min periods at 09:00-12:00 and 14:00-17:00.Except the control and the MS rats,the MS+CUMS and CUMS rats were exposed to one arbitrary mild stressor each day starting from PND28 to PND60.During PND53-PND59,to evaluate the effect of depression model through sucrose preference,open-field,and forced swimming test in rat.Sixty neonatal male SD rats were randomly divided into six groups of 10 rats each group:blank group(Control),MS+CUMS model group(Model),MS+CUMS+fluoxetine group(Positive:5mg/kg),MS+CUMS+Sinisan low dose group(SNS-Low:2.5g/kg),MS+CUMS+Sinisan medium dose(SNS-Medium:5g/kg)and MS+CUMS+Sinisan high dose SNS-High:l0g/kg).Except for the blank group,the rest of the rats were subjected to maternal separation stress twice a day for 3 hours during pnd1-21.Drug treatment rats were exposed to one randomly stressor and treatment each day starting from PND60 to PND100.Sucrose preference test,open-field test,and forced swimming test were evaluated the effects of SNS on the behavior changes in rats.Nissl staining was used to detect the number of neurons and immunohistochemical detection of CaSR and Syn expression in in hippocampus(HIP)and prefrontal cortex(PFC).Calcium sensitive receptor(CaSR),protein kinase C(PKC),ERK1/2,and synapse-associated proteins[PSD-95,GAP-43,and synaptophysin(Syn)]levels in the hippocampus and prefrontal cortex were assayed using Western blot.Results(一)Maternal separation increases depression-like behavior in CUMS rats.1.Weight:Compared with the control group,the weight gain in CUMS,MS and MS+CUMS groups were significantly reduced(P<0.05);the weight gain of rats significantly decreased in the MS+CUMS groups compared with that in the MS group(P<0.05).2.Sucrose preference:The sucrose preference of the MS,CUMS and MS+CUMS groups were significantly decreased compared with that in the control group(P<0.05).The MS+CUMS group only showed a lower trend without significant difference in sucrose preference compared with the MS and the CUMS groups(P<0.05).3.Forced swimming test:Compared with the normal group,the immobility time of the rats in the MS,CUSM,and MS+CUMS groups were significantly increased(P<0.05).The immobility time in the MS and CUMS groups also markedly decreased compared with that in the MS+CUMS group(P<0.05).4.Open field test:The total distance of the MS+CUMS groups significantly decreased compared with those in the normal,MS and CUMS rats(P<0.05).However,in the central region time,only the MS+CUMS groups significantly decreased compared with that in the control group(P<0.05),no significant difference was observed in the MS and CUMS groups(P>0.05).The central region time of the MS+CUMS rats only showed an decreased trend than the MS and CUMS groups(P<0.05).(二)Effects of Sinisan on CaSR regulates synaptic plasticity related pathways in MS+CUMS rats1.Weight:The model rats showed a significantly decreased weight gain compared with the control group(P<0.05).After 40 days of treatment,the weight gain of rats significantly increased in all the drug intervention groups compared with that in the model group(P<0.05).2.Behavior:The sucrose preference and the central region time of the model group significantly decreased(P<0.01,P<0.05),and significantly increased the immobility time(P<0.1)compared with that in the control group;But,compared with the normal group,the total distance of the model rats in the open field only showed a decreasing tendency but with no statistical significance(P>0.05).The sucrose preference,the central region time and total distance of the drug intervention groups markedly increased compared with the model rats(P<0.01,P<0.05 and P<0.05).Moreover,the immobility time of rats significantly reduced in the SNS treatment and Positive groups compared with that in the model group(P<0.01).3.Nissl staining:Compared with the control group,neurons in the model group were loosely arranged,low in number,and significantly lacked clear neurons boundaries(P<0.05).The number of neurons,which were neatly arranged and with normal morphology and clear boundary of the neurons,significantly increased after SNS and positive treatment compared with that in the model rats(P<0.05).4.Immunohistochemistry:The percentage of the CaSR and the Syn-positive areas in the HIP and PFC were markedly decreased in model rats compared with the normal group(P<0.05).In the HIP and PFC,the percentage of the CaSR and the Syn-positive areas increased evidently by SNS and positive treatment compared with that in the model group(P<0.05),except the percentage of the Syn-positive areas in the HIP showed a increasing tendency,but with no statistical significance in the SNS-Low group compared with that in the model rats(P>0.05).5.Western blot:the expression levels of CaSR,PKC,ERK1/2,PSD-95,GAP-43,and Syn proteins in the HIP and PFC of the model groups were markedly downregulated than those of the normal rats(P<0.05).SNS and fluoxetine treatment strongly upregulated the protein levels of CaSR,PKC,ERK1/2,PSD-95,GAP-43,and Syn compared with the stress-exposed rats in the model group(P<0.05).Conclusion1.Maternal separation combined with chronic unpredictable stress induced depression-like and anxiety-like behaviors in rats.2.Maternal separation increased the depressive-like behaviors in CUMS rats.3.SNS ameliorates depression-like behavior in rats by enhancing synaptic plasticity via the CaSR-PKC-ERK signaling pathway... |
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