| ObjectiveThis study adopted maternal separation(MS)paradigm to model experience of adverse stress in early life,and Sinisan intervention after modeling process.The study explored the change in key brain area under the effect of central nervous regulation related to liver regulating emotion,which revealed the effect and mechanism of JWSNS Sinisan on depression.Thereby the research could provide solid theoretical evidence of Sinisan on preventing and curing emotional diseases during young,adolescent and adult stage induced by early adverse life stress for clinic.Methods(1)The impact of maternal separation stress on behavior of SD rats80 SD rats(half male and half female)with the age of postnatal day(PND)1 were adopted to randomly divided into maternal separation(MS)group and control group,20 rats in per group with half male and half female.MS group was separated from their mothers for 6h/day separately from 9:00 to 12:00 and 15:00 to 18:00 during postnatal PND1 to PND21.The control group was fed normally without any intervention.All rats were weaned on PND21.From PND21 to PND60,animals were normal fed and tested body weight two times a week without any stimulation.Behavioral tests,including sucrose preference test(SPT),open field test(OFT),forced swimming test(FST),water maze test(WMT)and 0 maze test,were carried out separately at PND45、PND52、PND57、PND66、PND73.(2)The effect of SiNiSan on the depression-like behavior and BDNF-related pathway of rats experienced maternal separation108 Wistar rats with the age of postnatal day(PND)1 were adopted to randomly divided into six groups including control group,MS(model)group,MS+fluoxetine(positive)group,MS+SNS-Low dose(SNS-L)group,MS+SNS-medium dose(SNS-M)group,MS+SNS-high dose(SNS-H)group.Each group comprised 18 pups,which was randomly divided into three age stage:young,adolescent and adult with 6 rats per stage.Except control group without any intervention,other group rats were separated from their mothers 4h per day from 9:00 to 13:00 during PND1 to PND21.The six groups were separately administered with corresponding drugs intervention per day until PND 28(for young pups),PND42(for adolescent pups)and PND 56(for adult pups).The pups were weighed every day,and depression-like behavior was assessed by sucrose preference test,open field test,and forced swimming test separately during PND23-27(for young),PND37-41(for adolescent)and PND51-55(for adult).Serotonin 1A receptor(5-HT1AR),phosphorylated protein kinase A(p-PKA)substrate,cAMP response element-binding protein(CREB),phosphorylated-CREB(p-CREB)and brain-derived neurotrophic factor(BDNF)in the hippocampus were examined by Western blot,and in situ 5-HT1A receptor expression was measured by IHC.Results(1)The impact of maternal separation stress on weight gainDuring PND22 to PND52,the weight gain of male rats in MS group were significantly decreased(p<0.05);but the female MS group presented no obvious difference compared with control rats(p>0.05).(2)The impact of maternal separation stress on behavior of SD rats1)Sucrose preference Compared with control group rats,the sucrose preference of both male and female rats in MS group were significantly decreased(p<0.01).2)Forced swimming test Compared with control group rats,the immobility time in FST of male rats in MS group was significantly increased(p<0.05).Female rats in MS rats showed a more immobility time compared with female rats in control group,but the difference was not significantly significant(p>0.05).3)Open field test The male rats in MS group presented a significant decrease of activeness in OFT compared with the male rats in control group(p<0.05),but the central area time and distance of MS group rats were decreased without significant difference compared with control rats(p>0,05).In female rats,the activeness,central area time and distance of MS group were all decreased compared with control group,but the differences were not significantly significant(p>0.05).4)Water maze test The crossing platform times in WMT of MS male rats was significantly increased compared with the control male rats(p<0.05),the time spent in target quadrant only showed a increased trend(p>0.05).The crossing platform times and the time spent in target quadrant of MS female rats was significantly less than female rats in control group(p<0.05,0.01).5)0 maze test For the male rats,the percentage of open-arms entries of MS group was significantly decreased compared with control group(p<0.05);but the percentage of open-arms time of MS group presented a decreased trend without significant difference compared with control group(p>0.05).For female rats,the percentage of open-arms entries and open-arms time of MS group was both significantly decreased compared with control group(p<0.01).(2)The effect of SiNiSan on the depression-like behavior and BDNF-related pathway of rats experienced maternal separation1)Weight gain In young stage,compared with control group rats the weight gain of model group rats was significantly decreased from PND21 to PND28(p<0.05);the positive group rats had significantly increased more weight than model group rats(p<0.05)and the SNS-L,SNS-M and SNS-H group present an increased trend without significant difference compared with model rats(p>0.05).In adolescent stage,weight gain of model rats had significantly decreased compared with control rats(p<0.01);the weight of drug administration groups but SNS-L group showed an increased trend without significant difference compared with model group(p>0.05)and SNS-L group weight significantly increased(p<0.05).For adult rats,weight gain of model group significantly decreased compared with control group(p<0.05);the weight gain of SNS-L,SNS-M and SNS-H group significantly increased compared with model group(p<0.05,p<0.01,p<0.05),but the weight gain of positive group have no change compared with model group(p>0.05).2)Sucrose preference In young stage,the sucrose preference of model group was significantly decreased compared with control group(p<0.05);sucrose preference of drug administration groups but SNS-H group showed an increased trend without significant difference compared with model group(p>0.05)and sucrose preference in SNS-H group significantly increased(p<0.01).In adolescent stage,sucrose preference of model group was decreased compared with control group without significant difference(p>0.05);but sucrose preference of SNS-L and SNS-M group were significantly increased compared with model group(p<0.01,p<0.05).In adult stage,the sucrose preference of model rats significantly reduced than control rats(p<0.05);compared with model group,sucrose preference in SNS-L group presented significant increase(p<0.05).3)Forced swimming test Compared with control group,in young rats,the immobility time of model group was significantly increased(p<0.01);the immobility time in the drug intervention groups was lower than model group but no significant difference(p>0.05).For adolescent rats,immobility time of model group was significantly increased compared with control group and the drug groups were all significantly decreased compared with model group(p<0.01).In adult stage,compared with control group,immobility time in model rats was significantly increased(p<0.01);and immobility time in positive,SNS-L,SNS-M and SNS-H group were all significantly decreased compared with model group(p<0.01,p<0.05,p<0.01,p<0.01).4)Open field test In young and adolescent stage,the time and distance spent in central area of model rats showed a decreased trend compared with control rats and the index in drug administration groups were increased compared with model group,but these differences were not significantly significant(p>0.05);the trend of activity in young and adolescent rats was similar with central area time and distance but activity in adult rats presented no obvious change(p>0.05).Compared with control group,the time spent in central area of model rats in adult rats was significantly decreased(p<0.05)and central area time in SNS-H group was showed significant increase compared with model group(p<0.05);but the other drug intervention groups presented no significant difference than model group(p>0.05).5)The expression of 5-HT1AR、p-PKA/p-CREB、CREB、BDNF in cerebral hippocampus For young rats,the expression of 5-HT1AR and CREB in model group presented decreased trend compared with control group and drug administration groups showed increased trend compared with model group without significant differences(p>0.05).The expression of pCREB and BDNF in model group for adult rats was significantly downregulated compared with control group(p<0.05),but there were no significant differences between drug administration groups and model group(p>0.05),.The expression of p-PKA of young rats showed no marked differences among the groups(p>0.05).In adolescent rats,the expression of BDNF and pCREB significantly reduced compared with control group(p<0.05),the expression of BDNF in SNS-M group significantly increased compared with model group(p<0.05),but the expression of pCREB in drug group only showed increased trend compared with model group(p>0.05).The expression of CREB and p-PKA in model group presented decreased trend compared with control group and drug administration groups showed increased trend compared with model group without significant differences(p>0.05).No significant differences have been observed among all groups on the expression of 5-HTIAR(p>0.05).In adult stage,the expression of 5-HT1A receptor in model group was significantly decreased compared with control group(p<0.05)and in SNS treatment groups significantly increased compared with model group(p<0.05),but positive group had no significant differences compared with model group(p>0.05).The expression of BDNF in model group for adult was significantly decreased compared with control group(p<0.05)and the expression in SNS-L and SNS-H group significantly increased compared with model group(p<0.05),the other treatment group had no significant differences compared with model group(p>0.05).The expression of p-CREB in model group for adult was significantly decreased compared with control group(p<0.05)and the expression in SNS-L and SNS-M group significantly increased than model group(p<0.05),but positive and SNS-H group increased without statistic difference(p>0.05).The expression of CREB in model group for adult was decreased compared with control group without significant difference(p>0.05),and the expression in positive and SNS-L groups significantly increased than model group(p<0.05).No significant differences were found in the expression of p-PKA for adult rats just with the decreased trend of model group than control group and the increased trend of drug administration groups than model group(p>0.05).ConclusionThe maternal separation of the early life could induce depressive behaviors,including behavioral despair,anhedonia and so on,and accompany anxiety-like behaviors to some extent.Maternal separation successfully models the depressive animals induced by experiencing adverse event in early life.Sinisan could alleviate depression-like behaviors of young,adolescent and adult rats induced by early maternal separation through upregulating the 5-HT1AR/CREB/BDNF signaling pathway. |
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