Study On The Pharmacological Action And Mechanisms Of Zhusha-Anshen-Wan On PTSD And Comorbid Sleep Disturbance

Objective:Todeterminethe effectiveness and mechanism of Zhusha-An shen-wan(ZSASW)on the symptoms of PTSD and coexistingsleep disturbance through the experimental study.Methods:1 The influence of ZSASW on contextual fear extinction in PTSD rats 1.1The influence of ZSASW on contextual fear conditioning,extinction and extinction retention(Behavior paradigm 1)SD Rats(n=50)were dividedrandomly into sham,model,positive drug(Prazosin),high-and low-dose ZSASW group.10 rats in each group.Rats were exposed to SPS(Single prolonged stress)in order to copy the PTSD model.Drug intervention started at the eighth day after the experiment began(day 8).Rats in treating groups were administrated orallywith high-dose(1.08 g/kg),low-dose(0.54 g/kg)ZSASW and prazosin(0.5 mg/kg)respectively once a day,in continuous 6 days.Rat in sham and model groups were treated with corresponding volume of distilled water.Fear conditioning experiment monitoring system was used to record the freezing time which was used as the evaluation index to analyze the the influence of ZSASW on contextual fear conditioning,extinction and extinction retention.1.2 The influence of ZSASW on contextual fear extinction(Behavior paradigm 2)SD Rats(n=50)were dividedrandomly into sham,model,positive drug(Prazosin),high-and low-dose ZSASW group.10 rats in each group.Rats were exposed to SPS(Single prolonged stress)in order to copy the PTSD model.Drug intervention and extinction both started at the eighth day after the experiment began(day 8).Rats in treating groups were administrated orallywith high-dose(1.08 g/kg),low-dose(0.54 g/kg)ZSASW and prazosin(0.5 mg/kg)respectively once a day,in continuous 5 days.Rat in sham and model groups were treated with corresponding volume of distilled water.Fear conditioning experiment monitoring system was used to record the freezing time which was used as the evaluation index to analyze the the influence of ZSASW on contextual fear extinction.1.3 The influence of ZSASW onspontaneous motor activitySD Rats(n=60)were dividedrandomly into blank,high-dose ZSASW,low-dose ZSASW,model,model+ high-dose ZSASW,model+ low-dose ZSASW groups.10 rats in each group.Rats in treating groups were administrated orallywith high-dose(1.08 g/kg)and low-dose(0.54 g/kg)ZSASW respectively once a day,in continuous 6 days.Rats in blank and model group were treated with corresponding volume of distilled water.Movement time and distance in open field test were used as evaluation index to study the effects of ZSASW and SPS procedure on spontaneous motor activity.2 The influence of ZSASW on sleep in rats2.1 Method evaluation on the use of CLAMS Systems equipped with Sleep detection analysis module on analyzing sleep patternSD Rats(n=20)were dividedrandomly into EEG group and CLAMS group,10 rats in each group.EEG(Electroencephalograph)and CLAMS(Comprehensive Laboratory Animal Monitoring System)were used to record and analyze thesleep of normal rats.Sleep time(ST),Awakening number(AN)and brief awakening number(bAN)were used as evaluation index to compare the difference of these methods on analyzing sleep.2.2Establish the screening method of PTSD rats comorbid with sleep disordersSD Rats(n=60)were dividedrandomly into blank control group and SPS exposure group,20 rats in blank group and 40 rats in SPS exposure group.Rat's freezing time in novel environment test was used as evaluation index to differentiate high response(HR)rats and low response(LR)rats.Study the differences of sleep pattern between high and low response rats and the correlation between freezing time and sleep related indexes including ST,AN and bAN.2.3 The influence of ZSASW on sleep pattern in PTSD ratsCopy PTSD model ratsusing the SPS method and screen the HR rats in novel environment test.HR rats(n=40)were dividedrandomly into model,positive drug(Prazosin),high-and low-dose ZSASW group.10 rats in each group.Another 10 rats were selected as blank control group.Drug intervention started at the eighth day after the experiment began(day 8).Rats in treating groups were administrated orallywith high-dose(1.08 g/kg),low-dose(0.54 g/kg)ZSASW and prazosin(0.5 mg/kg)respectively once a day,in continuous 6 days.Evaluation indexes of ST,AN and bANwere recorded by CLAMS,and then these indexes were used to evaluate the influence of ZSASW on sleep pattern.3 The neurobiological mechanisms of ZSASW on alleviating the fear extinction disorder and sleep disorder based on LC-NA neurological function3.1 The neurobiological mechanisms of ZSASW on alleviating the fear extinction disorder based on LC-NA neurological functionSD Rats(n=65)were dividedrandomly into sham,model,positive drug(Prazosin),high-and low-dose ZSASW group.13 rats in each group.Rats were exposed to SPS(Single prolonged stress)in order to copy the PTSD model.Drug intervention started at the eighth day after the experiment began(day 8).Rats in treating groups were administrated orallywith high-dose(1.08 g/kg),low-dose(0.54 g/kg)ZS AS W and prazosin(0.5 mg/kg)respectively once a day,in continuous 6 days.Rat in sham and model groups were treated with corresponding volume of distilled water.Fear conditioning experiment monitoring system was used to record the freezing time,which was used as the evaluation index to analyze the the influence of ZSASW on contextual fear conditioning,extinction and extinction retention.Using fluorescence quantitative PCR(Polymerase chain reaction,PCR)and Immunofluorescence histochemistry(IFH)technology to detect the Tyrosine hydroxylase(TH),corticotrophin releasing factor receptor(CRFRi)and corticotrophin releasing factor(CRF)in rat's brain tissues,then analyze the influence of ZSASW on these observing indexes.3.2 The neurobiological mechanisms of ZSASW on alleviating the fear extinction disorder based on LC-NA neurological function 7 days after rats received SPS procedure,52 HR rats(SD male rats)were select out in novel environment test and divided randomly into model,positive drug(Prazosin),high-and low-dose ZSASW group.13 rats in each group.Another 13 rats(NoSPS exposure)were selected out as blank control group.Drug intervention started at the eighth day after the experiment began(day 8).Rats in treating groups were administrated orallywith high-dose(1.08 g/kg),low-dose(0.54 g/kg)ZSASW and prazosin(0.5 mg/kg)respectively once a day,in continuous 6 days.Evaluation indexes of ST,AN and bANwere recorded by CLAMS,and then these indexes were used to evluate the influence of ZSASW on sleep pattern.Use fluorescence quantitative PCR and IFH technology to detect the TH,CRFRi and CRF in rat's brain tissues and ELISA(Enzymelinkedimmunosorbentassay)method to detect the CORT in urine,then analyze the influence of ZSASW on these observing indexes.Results:1 The influence of ZSASW on contextual fear extinction in rats 1.1 The influence of ZSASW on contextual fear conditioning,extinction and extinction retention(Behavior paradigm 1)In contextual fear conditioning and extinctionsection,there was no obviously change in freezing time of all the testing groups.In fear condintion section,Compared with sham group,the freezing time of model group increased significantly in Min 1(first one minute)(P<0.01).Compared with model group,the freezing time in prazosin group and high-dose ZSASW group decreased significantly in Min 1(p<0.05 and p<0.01).1.2 The influence of ZSASW combined with multiple extinction training on contextual fear extinction(Behavior paradigm 2)Compared with sham group,the freezing time of model group increased significantly from day 10 to day 14(P<0.05 or P<0.01).Compared with model group,the freezing time in prazosin group decreased significantly in day 12,13 and 14(p<0.05)and the freezing time in high-dose ZSASW group decreased significantly in day13 and day14(p<0.05 and p<0.01).1.3 The influence of ZSASW onspontaneous motor activityThe result of two factor analysis of variance showed that neither SPS procedure and ZSASW have significant main effect onmovement distance and time,there is no significant interaction between SPS procedure and ZSASW treatment.2 The influence of ZSASW on sleep in PTSD rats2.1 The evaluation on the use of CLAMS Systems equipped with Sleep detection analysis module on analyzing sleep patternThere were no significant difference between EEG and CLAMS group with respect to ST,AN and bAN.2.2Establish the screening method of PTSD rats comorbid with sleep disorders9 days after SPS exposure,there was no significant difference between the blank control group and exposure group with respect to ST,but AN and bANincreased obviously in exposure group(P<0.05 and P<0.0 1).Compared with blank control group,ST,AN and bANin HRgroup decreased significantly(P<0.05 or P<0.01).14 days after SPS exposure,the bANin exposure group increased obviously compared with blank control group(P<0.05).Compared with blank control group,AN and bANin HRgroup decreased significantly(P<0.05 and P<0.01).The resultsof pearson's correlation analysisshowed that,for exposure and HR group the freezing time in novel environment test has obviouscorrelation with sleep-related indexes including ST,AN and bAN(P<0.05 or P<0.01).2.3 The influence of ZSASW on sleep pattern in PTSD rats9 day after SPS exposure,in light phase sleep related indexes in model group differ obviously with control group(P<0.05 or P<0.01).Sleep related indexes in prazosin,low and high-dose ZSASW groups show no significant difference compared with model group.In dark phase,compared with control group,the bAN in model group increased obviously(P<0.05).Compared with model group sleep related indexes in prazosin,low and high-dose ZSASW groups show no significant difference.14 day after SPS exposure,in light phase,AN and bAN in model group differ obviously with control group(P<0.01).Compared with model group,AN and bAN in prazosin and high-dose ZSASW groups decreased significantly(P<0.05 or P<0.01),while bAN in low-dose ZSASW group decreased significantly(P<0.05).In dark phase,compared with control group,the bAN in model group increased obviously(P<0.05).Compared with model group bAN in prazosin and high-dose ZSASW groups decreased obviously(P<0.05).3 The neurobiological mechanisms of ZSASW on alleviating the fear extinction disorder and sleep disorder based on LC-NA nervous system3.1 The neurobiological mechanisms of ZSASW on alleviating the fear extinction disorder based on LC-NA nervous systemCompare with sham group,the level of TH and CRFR1 in LC and CRF in CeA,as well as their mRNA increased significantly in model group(P<0.05 or P<0.01),moreover CRF mRNA in PVN increase obviously,too(P<0.05).Compared with model group,TH mRNA and protein,as well as CRF mRNA in CeA decreased significantly in prazosin group(P<0.05 or P<0.01).The TH mRNA and protein expression,as well as CRFRi in LC decreased significantly in high-dose ZSASW group(P<0.01 or P<0.05),moreover CRF mRNA in CeA decreased significantly(P<0.01).CRF mRNA in CeA decreased significantly in low-dose ZSASW group(P<0.01).3.2 The neurobiological mechanisms of ZSASW on alleviating the fear extinction disorder based on LC-NA nervous systemIn dark phase,the level of CORT of model rats in urine increased obviously compared with blank control group(P<0.05),but prazosin and ZSASW show no effect on it.In light phase,there was no significant difference between control and model groups.Compare with blank control group,the level of TH in LC and CRF in PVN,as well as their mRNA increased significantly in model group(P<0.05 or P<0.01).Compared with model group,TH mRNA and protein,as well as CRF in PVN decreased significantly in both prazosin and high-dose ZSASW groups(P<0.05 or P<0.01),moreover CRF in PVN decreased significantly in low-dose ZSASW groups(P<0.05).Conclusion:1 ZSASW can enhance the fear extinction retention and improve fear extinction,that resulting in increasing the efficiency of the fearextinction.2 ZSASW has therapeutic effect on the sleep disorders in PTSD rats3 The action of ZSASW on facilitating fear extinction is due to decreased CRFR1 in LC and.CRF in CeA,that result in decreased LC-NA function and less fear expression(freezing).In addition,the pharmacological action of ZSASW in improving sleep promote the fear extinction,that results in decreased fear expression.4 The action of ZSASW in improving sleep in PTSD rats may be related with reduced LC-NA neurological function,and the upstream regulatory mechanismcould be interpreted as decreased CRF in PVN.