| Objective:Yueju pill is a classic Traditional Chinese Medicine formula which could treat Yu syndrome in TCM.Many results of clinical studies have supported that Yueju pill has an antidepressant effect.Our previous clinical and animal studies demonstrated that Yueju can produce a rapid antidepressant effect.We found Yueju can rapidly increase the expression of PACAP in the hippocampus,the depression-related brain region,and activate the downstream signal CREB on which the antidepressant effect of Yueju is dependent.Therefore,we hypothesize that the up-regulation of PACAP and downstream signaling pathways may induce the rapid antidepressant effect of Yueju.Our project will utilize multiple methods including:western blot,intracranial micro injection PACAP antagonist etc.,to specifically investigate whether the rapid antidepressant efficiency of Yueju,include:1.Is dependent on the up-regulated expression of PACAP in the hippocampus;2.Is associated with activation of downstream signaling of PACAP,such as PKA,CREB,etc;3.Is associated with synaptic plasticity changes induced by PACAP.By studying the role of PACAP in the rapid antidepressant effect of Yueju,we may identify PACAP as a novel target for the rapid antidepressant effect,and provide a scientific basis for the clinical use of Yueju.Methods:1.In electrophysiology experiment,there were three groups:Sali+DDW,Sali+YJ,H89+YJ.Analyzed and compared fEPSP slope of three groups after continuous electrical stimulation in the hippocampal CA1 region of mice.To identify the time-course change of PACAP,Kunming mice were tested TST at 30min,2hr and 24hr.The samples of these mice were used half in WB test,half in RT-PCR test.We also tested the cAMP concentration in hippocampus of YJ treatment KM mice at 30min,2hr and 24hr.2.There were four groups in PACAP intracerebroventricular microinjection experiment:a.Surgery+ saline group,b.Surgery+ PACAP(18.75 pmol),c.Surgery+PACAP(75 pmol),d.Surgery+PACAP(300 pmol).30 minutes after administration,animal behavioral tests were test,including TST and FST.These tests lasted for 10 days.There were 4 groups in PACAP antagonist experiment:saline(injection)+saline(oral)group,saline(injection)+ YJ(oral)group,PACAP6-38(injection)+saline(oral)group,PACAP6-38(injection)+ YJ(oral)group.30 minutes after administration,animal behavioral tests were test,including TST and FST.These behavioral tests sustained to 48 hours after administration.There were four groups in PACAP RNAi experiment:a.scramble(injection)+ saline(oral)group,b.scramble(injection)+ saline(YJ)group,c.PACAP RNAi(injection)+ saline(oral)group,d.PACAP RNAi(injection)+ saline(YJ)group.30 minutes after administration,animal behavioral tests were test,including TST and FST.These behavioral tests sustained to 48 hours after administration.Western blot was used to detect the protein expression of PACAP,PKA,CREB and PSD95 in hippocampus of mice after 30 minutes of PACAP RNAi administration.3.Mice were singly housed and received 3 weeks of mild stress.A single administration of Yueju pill and ketamine were performed 2 hours,24 hours and 48 hours post the last CMS procedure,mice received a battery of behaviors in the order of SPT,OFT,TST,FST and NSF.Western Blot was performed to examine the expression of PACAP and BDNF in hippocampus of KM mice.4.Tail suspension test(TST)was used to test the rapid antidepressant effect of YJ-PE in KM mice at 30min and 24h post a single administration.Western Blot was performed to examine the expression of BDNF and TrkB in hippocampus of KM mice.Four fractions of GJ were extracted using standardized procedure.The four fractions were screened for rapid antidepressant potential,using the behavioral paradigm of forced swimming test(FST)and tail suspension test(TST)assessed at 24 hours post a single administration.A single dose of the putatively effective fractions was further tested in mice exposed to chronic mild stress(CMS),followed with a comprehensive behavioral testing including TST,FST,sucrose preference test(SPT),and novelty suppressed-feeding(NSF).To test the association of BDNF signaling with rapid antidepressant effects of effective factions,the expressions of BDNF and its receptor Tropomyosin receptor kinase B(TrkB)in the hippocampus were assessed at different times post a single administration of effective fractions.Tail suspension test(TST),forced swimming test(FST)and Novelty suppressed-feeding(NSF)were used to screen the rapid antidepressant potential of effective fractions of GJ-PE in KM mice at 24h post a single administration.Tail suspension test(TST)was also used at 30min and forced swimming test(FST)was used at 2h to test the initial onset time of effective fractions of GJ-PE in KM mice.Western Blot was performed to examine the expression of BDNF and p-eEF2 in hippocampus of KM mice at 2h and 24h.In Tail suspension test(TST),forced swim test(FST)and novelty suppressed-feeding test(NSFT)were performed in mice at 24 hours after iridoids fraction administration.High(200mg/kg),medium(100mg/kg),low(50mg/kg)doses were used to preliminarily screen the iridoids fraction of Gardenia jasminoides Ellis which has fast onset antidepressant potential.To examine the onset time of the antidepressant effect of the petroleum ether extract of Gardenia jasminoides Ellis,mice were accessed in the TST both 30 minutes and 2 hours after administration.NSFT,TST and FST were respectively performed at 3-day,5-day,and 7-day after drug administration.NSFT were performed after 10-day and 14-day of administration.Chronic model:mice in model group were under chronic mild stress.At 24 hours after medium dose of iridoids fraction of Gardenia jasminoides Ellis,mice were accessed in OFT(open field test),SPT(sucrose preference test),NSFT,TST and FST.NSFT,TST and FST were respectively performed at 3-day,5-day,and 7-day after drug administration.NSFT were performed after 10-day and 14-day of administration.Western Blot was performed to examine the expression of BDNF at 30min,2h,24h in hippocampus of KM mice and PKA,p-CREB and CREB at 2h and 24h in hippocampus of KM mice.Mice in the test group were administrated with iridoids fraction of Gardenia jasminoides Ellis after intraperitoneal injection of PKA blocker H89.TST were carried out at 30minutes and 2 hours after drug administration.Results:1.In electrophysiology experiment,the slope of fEPSP was significantly enhanced in the saline+YJ group,compared with saline or H89,however,there were no significant differences between the saline+ DDW and H89+ YJ groups.Both the gene and protein expression of PACAP was up-regulated at 30 min,but retruned to normal at 2 hr and remained at the baseline level at 24 hr.Yueju pill didn’t influence the concentration of cAMP level in hippocampus of Kunming mice at 30min,2hr or 24hr.2.Intracerebroventricular PACAP administration showed persistent antidepressant effects in TST,FST and NSF.Bilateral hippocampal PACAP antagonist or PACAP RNAi microinjection blocked rapid antidepressant-like effects induced by Yueju.Depressant responses which are induced by hippocampal knockdown of PACAP expression was related to the down-regulation of PACAP,PKA,CREB,PSD95.A single administration of YJ treatment could reverse these molecular changes.3.After 3 weeks CMS administration,mice showed depression-like response as shown previously(included a reduction in body weight,sucrose preference and unit food comsuption,and an increasing in latency to feed,immobility time in TST and FST.A single dose of Yueju rapidly and persistently remedied these behavioral deficits,as shown at 2hrs,24hr,and 48 hr.There was a significant reduction of PACAP expression as well BDNF expression in the hippocampus of mice subjected to CMS.Interestingly,a single dose of Yueju significantly normalized PACAP expression from 2 to 48 hrs in the CMS mice.Similarly,the expression of BDNF also recovered by Yueju administration in a lasting manner.4.An acute administration of YJ-PE decreased the immobility time of KM mice in TST and increased the expression of BDNF and TrkB as soon as 30min post a single administration and continued at least for 24 hours.Both petroleum ether(GJ-PE)and n-butyl alcohol fraction(GJ-BO)fractions of GJ displayed rapid antidepressant potential in the FST.In the TST,the antidepressant effects of GJ-PE lasted for a longer time than GJ-BO.Acute administration of either GJ-PE or GJ-BO significantly reversed the behavioral deficits in the tests of TST,FST,SPT and NSF in chronically stressed mice,confirming both fractions conferred rapid antidepressant efficacy.Interestingly,GJ-PE,but not GJ-BO,increased the expression of BDNF and TrkB in the hippocampus post a single administration.An acute administration of GJ-PE 1 decreased the immobility time of KM mice in FST at 2h and 24h and decreased the latency time in NSF at 24h.GJ-PE3 decreased the latency time in NSF at 24h.GJ-PE4 increased the unit food consumption in NSF at 24h.At 2h post a single GJ-PE1 treatment,the expression of BDNF was significantly up-regulated while the expression of p-eEF2 was significantly down-regulated.At 24h post a single GJ-PE1 treatment,the expression of BDNF significantly down-regulated while p-eEF2 expression significantly up-regulated.Middle dosage of iridoids fraction(equivalent dose of rapid antidepressant effect dose of Gardenia jasminoides Ellis)treatment significantly reduced the immobility time of TST at 30min.The antidepressant effects of iridoids fraction could last for 7 days in TST and FST.It could last to the 14th day in NSF.Similar to Yueju pill,iridoids fraction reversed the deficits in SPT,forced swim test,tail suspension test and novelty-suppressed feeding test.The antidepressant effects of a single dose of iridoids fraction could also last for at least 14 days.The results of molecule studies suggested a single dose of iridoids fraction of Gardenia jasminoides Ellis at 24 hours could activate the PKA-CREB pathway,but not BDNF.Blockade of PKA-CREB signaling pathway reversed the antidepressant effects of iridoids fraction at 24 hours.Conclusion:1.Hippocampal CA1 area is one of the specific regions which take effects in antidepressant effect after acute administration of Yueju pill at 24 hours.The immediately and non-persistent up-regulation of PACAP protein and mRNA in hippocampus of Kunming mice is related with the rapid antidepressant effects of Yueju.Yueju pill don’t influence the concentration of cAMP level in hippocampus of Kunming mice from 30min to 24hr.2.Three doses(high,middle,low dose)of PACAP all elicited instant antidepressant effect.The antidepresant effects of PACAP could at least last for 10 days.Bilateral hippocampal PACAP antagonist and PACAP RNAi treatment block the rapid antidepressant effect of Yueju pill.The significant down-regulation of PACAP,PKA,CREB and PSD95 was associated with depression-like behaviors.The rapid antidepressant effects of Yueju pill at 30min was related to the activating of PACAP,PKA,CREB and PSD95.3.Down-regulated hippocampal PACAP and BDNF expression was related with MDD.A single administration of Yueju treatment could reverse down-regulated expression of PACAP and BDNF in hippocampal region induced by Chronic mild stress(CMS).4.The up-regulation of BDNF and TrkB may be involved in rapid antidepressant effects of YJ-PE.Two standardized fractions GJ-PE and GJ-BO exhibited comparable rapid antidepressant-like effects in the CMS mice.However,only the effects of GJ-PE was associated with BDNF signaling.Iridoids fraction of Gardenia jasminoides Ellis could be the key fraction in rapid antidepressant of Yueju pill and Gardenia jasminoides Ellis.GJ-PE1 has the most significant rapid antidepressant potential among the four fractions of GJ-PE.The effective time of GJ-PE1 is 2h after drug treatment.The mechanism of the rapid antidepressant effect of GJ-PE1 at 2h is related to the up-regulation of BDNF and down-regulation of p-eEF2.GJ-PE3 and GJ-PE4 also have some features of rapid antidepressants.GJ-PE2 doesn’t have the rapid antidepressant potential.Similar to Yueju pill,rapid and enduring antidepressant effects of Iridoids fraction were dependent on activating of PKA-CREB signaling pathway.However,the antidepressant effect of Iridoids fraction of Gardenia jasminoides Ellis at early stage is not dependent on the rapid increasing of BDNF. |
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