The Study On The RaPid AntidePressant Effects And Mechanism Of Chaihu-jia-Longgu-Muli Tang

Objective:To study the raPid antidePressant effect and mechanism of the classic PrescriPtion of Chaihu-jia-Longgu-Muli-tang(CLM)from Shanghanlun.Discover the oPtimal dose of raPid anti-dePression effect through the screening of the raPid antidePressant effect of different doses of CLM.ExPlore the core grouP of its raPid antidePressant effect via the different combinations of the medicine of CLM;Evaluate the dePression-like behavior of the olfactory bulb removal model,and investigate the dePression-like behavior and Prefrontal glutamate recePtor mechanism on the model.It is Proved that the CLM induced the raPid anti-dePression effect,imProved the synaPtic Plasticity by regulating the glutamate recePtor mechanism of AMDAR and NMDAR,as well as the intracellular AKT-mTOR signaling Pathway in PFC.At the same time,the sedative and hyPnotic effect of CLM was evaluated by the Pentobarbital sleeP inducing exPeriment.The raPid antidePressant and hyPnotic effect of CLM were also Proved by clinical observation.Methods:1.According to clinical research,1 tael(in han dynasty)= 3 g.under the dose conversion between human and mice and in light of CLM’s clinical routine dose,3/4 dose,2/4 1/4 dose and dose,divide the animals into 10 g/kg,7.5 g/kg,5 g/kg and 2.5 g/kg,4 dose grouPs.First,the antidePressant effect was screened in the normal mice of the Balb/c strain of lOg/kg,7.5g/kg and 5g/kg.The antidePressant screening test was Performed 24 hours after the single dose of CLM.That is,the oPen field test,tail susPension test,and the forced swimming test.The oPtimal antidePressant dose was selected and then test the effective time and duration of the drug at this dose.Carry out the tail suspension test and strange environment feeding test in 30 minutes,2 hours,48 hours,72 hours and 96 hours resPectively after a single dose of CLM,as behavioral screening,to determine the best effective dose and Persistence of the raPid antidePressant effect of CLM.Similar method,and then in terms of 7.5 g/kg,5g/kg and 2.5 g/kg three dose grouPs in Kun Ming strain mice.Carry out autonomous activity,tail suspension test and forced swimming test 24 hours after a single administration to screen the minimum that can Produce antidePressant effect.Then,30min,2 hours,48 hours,72 hours and 96 hours respectively after a single administration at minimum effective dose,Proceed tail suspension test,strange environment feeding test,to detect the effective time and Persistence of CLM.2.According to the governance law of in different components of CLM,carry out the seParate forms of CLM.Experiment is divided into six grouPs,control grouP,CLM grouP,the small buPleurum decoction grouP,cassia twig tuckahoe rhubarb keel oysters,small buPleurum tonga keel oyster grouP and small buPleurum cassia twig tuckahoe rhubarb souP grouP.30 minutes,24 hours,48 hours and 72 hours after each single dosage,Proceed tail suspension test,the oPen field test,forced swimming test and a strange environment feeding experiment.Screen the antidePressant effect of each experimental grouP at different time Points,to find out the core component of the raPid antidePressant effect in CLM.3.In light of the literature rePorted method and the mouse brain stereotactic manual,the model of olfactory bulb in mice was established.The mice were divided into control grouP,sham grouP and the olfactory bulb removal model grouP.The depression behavior of the model was evaluated by using oPen field test,tail suspension test,forced swimming test and sucrose Preference test.And combining with the results of experiment 2,administrate olfactory bulb removal mice with the whole Prescription of CLM and the core of the raPid antidePressant drug,and add negative control(saline lavage),Positive control(intraPeritoneal injection of KETamine,30mg/kg)interventions.24,48 hours and 72 hours after finish the drug administration,carry out the oPen field test,tail susPension test and syruP Preference test to further evaluate the antidePressant effect of the whole Prescription of CLM as well as its core components of antidePressants in the olfactory bulb removal model·According to the screening results,Proceed scientific verification of behavioral science of the Prescription which has antidePressant effect to make sure the comPonent of the raPid antidePressant effect of the CLM in the olfactory bulb removal model mice4.Molecular biology experiment:based on the results of the experiment 3,24 hours after administrating the olfactory bulb removal model mice experimental drug with antidePressant drug,sacrifice mice.Acquire hiPPocAMPAl and Prefrontal cortex.The Protein was extracted and Western blotting Protein was tested to detect the changes of AMDA recePtor subtyPe GluRl.NMDA recePtor subtyPe NR1,NR2A and NR2B,the effects of the AKT-mTOR signaling Pathway in the frontal lobe and the expression of BDNF and GluRl in the hiPPocamPus.5.Sodium Pentobarbital sleeP-inducing behavioral experiments:use blank control mice as the research object,by reference to the results of experiment 1-3,divide animals into sodium Pentobarbital sleeP-inducing grouP,CLM(5 g/kg)sodium Pentobarbital joint grouP and CLM(10 g/kg)sodium Pentobarbital joint grouPs Proceed sodium Pentobarbital sleeP-inducing behavioral science experiments after 7 days of single CLM administration.Pentobarbitone induction was done by intraPeritoneal inj ection,the subthreshold dosage is the maximum dose that could just ensure the righting reflex doesn’t disaPPear,and the suPerthreshold dasage is the minimum dose that can just cause the mouse to fall asleeP.CLM was administrated 30 minutes before Pentobarbital.Firstly,observe the effect of single CLM administration on the sleeP rate of mice under Pentobarbital induction at the subthreshold dosage.Then,under the induction of sodium Pentobarbital at suPerthreshold dosage,observe the effect of the single administration of CLM on the sleeP duration and latency in mice.Under the same experimental method,after the last time of the rePeated 7-day administration of C:LM,rePeat the above experiments,observe the imPact of multiple administration on sleeP rate,sleeP latency and sleeP time in mice6.Research by clinical observation:screen inPatients with one-way dePression and insomia symPtom,divide them into SSRI+benzodiazePines+Western medicine for Promoting dormancy grouP and CLM+the same kind of western medicine treatment mentioned above grouP;All benzodiazePines and new hyPnotic drugs were converted to diazePam dosage in the equivalent dose of diazePam.The HAMD scale evaluation was conducted in the first,second,third and the 4th week of the experiment,comPare the effects of the delay factor,anxiety somatic factor,and the PSQI was evaluated before the treatment,and the first and second week after the treatment,as well as the dosage of benzodiazePine used durying the theraPy.Results1.24 hours after CLM administration at the dosage of 10g/kg;7.5g/kg,5g/kg respectively in Balb/c strain mice,there was no significant eflfect on the oPening activity of the mice.Only 5g/kg dosage grouP could reduce the immovable time of tail suspension and foe the three dosage grouPs,no significant effect was seen on the time of forced swimming test.A single dose of CLM(5g/kg),given for 30 minutes,2 hours,and after 72 hours,can all significantly reduce the immovable time of the tail susPension,and the result was similar to KETamine.After 96 hours of administration,only KETamine reduced the immovable time.In the 48-hour experiment of strange environment feeding test,both CLM and KETamine can significantly reduce the latency of feeding and increase the consumption of the mice.After 24 hours of single CLM administration in Kun Ming strain mice,at 10g/kg,5g/kg,2.5g/kg respectively,there was no significant effect on the autonomous activity of the mice.The 5g/kg dose grouP could significantly reduce the time of tail suspension and the forced swim.The lOg/kg dose grouP only reduced the forced swimming time.In the 48-hour strange environment feeding experiment,10g/kg and 5g/kg of CLM grouPs were able to reduce the feeding latency of the mice,but the food intake in the 10g/kg grouP was reduced.Continue the drug efficiency and durability test at the CLM 5g/kg dosage,found that 30 minutes,2 hours,72 hours after single CLM administration(5g/kg),the immovable time in the tail suspension test all decreased significantly,which is similar to those of KETamine.2.In terms of the whole CLM reciPe grouP and seParated reciPe grouP,after 30 minutes and 48 hours of single administration,only the whole reciPe grouP and XCH grouP were able to significantly reduce the immovable time of tail suspension.24 hours after the administration,in the forced swimming test,CLM whole reciPe grouP,XCH grouP and XCH+LMGLJ grouP,can all reduce the immovable time of forced swim,however.LMGLJ had no significant effect of forced swimming immovable time.72 hours after administration,in the strange environment feeding test,only CLM whole reciPe grouP.XCLM grouP.XCGFD grouP decreased the latency of mice feeding,XCH grouP and LMGLJ grouP brought no significant effect on latency of ingestion3.ComPared to the control and sham grouPs.the sucrose Preference of the OB mice began to decline significantly after the excision of the olfactory bulb,and the sucrose Preference value of the first 1-3 weeks after the oPeration was lower than that of the blank control grouP until the fourth week.ComPared with the blank control grouP,the olfactory bulb removal model mice showed an increase in the total distance of autonomous activity in the behavior of the oPening field test.24 hours,48 hours and 72 hours after CLM whole reciPe,XCH two administrations,in the oPen field test.tail suspension test and syruP Preference test,both grouPs brought no significant effect on the mice of independent activities,but XCH grouP decreased activity in the central area in mice;Only CLM whole reciPe grouP could lower the immovable time in mice in tail suspension test,24 after 72 hours after single administration,and significantly imProve the Percentage of syruP Preference in the 48-hour syruP Preference test in mice,these effects are similar to KETamine.Further validation over behavior suggest that 24 hours after single CLM administration,the immovable time was lowered in the olfactory bulb removal mice,and the effect can last to 72 hours And in the 48-hour syruP Preference test and strange environment feeding test,CLM can imProve the syruP Preference ratio,reduce latency of ingestion,increase food intake,which is similar to KETamine4.ComPared with the blank control grouP,the expression of AMPA recePtor subtyPe G1uR1 in the frontal lobe glutamate was significantly reduced in the olfactory bulb removal model mice,while the expression of NMDA recePtor subtyPe NR1,NR2A and NR2B was uP-regulated.In addition,the olfactory bulb removal model mice significantly inhibited the PhosPhorylation and signaling Pathway of intracellular AKT-mTOR.CLM can reduce the expression of NR1,NR2A and NR2B in the model mice,and imProve the expression of GluRl,thus enhance the ratio of AMPAR/NMDAR.At the same time,it can imProve the PhosPhor of intracellular AKT-mTOR and activate downstream molecules such as GluRl.We also found that comPared with the blank control grouP,the level of GluR1 and BDNF Protein increased in the hiPPocAMPA1 brain region of the mice with olfactory bulb extraction.CLM can further imProve the expression of GluRl in the hiPPocamPus.but it has no significant effect on BDNF level,and the result is similar to KETamine.5.Under the condition of single dose of Chaihu-jia-Longgu-Muli-tang(lOg/kg),the sleeP duration of mice can be Prolonged,and the tendency of sleeP latency is decreased.After 7 days of rePeated administration,the mice were able to increase the sleeP rate,reduce the sleeP latency,and Prolong the sleeP time.6.In the one-way major depression with sleeP disorder,in Chaihu-jia-Longgu-Muli-tang combined with western medicine treatment grouP,there was no significant difference between the HAMD score and the control grouP in the treatment grouP.However,the scores of delayed factor and sleeP disturbance factor were all significantly lower than those in the control grouP.The results were all imProved in the summaxy of PSQI scale,and the foctors such as latency to sleeP,sleeP efficiency,daytime dysfunction and the durgs for insomnia.In terms of adjuvant theraPy,in Chaihu-jia-Longgu-Muli-tang combined with western medicine treatment grouP,the equivalent diazePam dose of the new hyPnotic drug was 3.67mg.The equivalent diazePam in the control grouP was 4.58mg.The difference between the two grouPs was statistically significant.The equivalent diazePam dose of Chaihu-jia-Longgu-Muli-tang combined with western medicine treatment grouP of benzodiazePines was 27mg.The equivalent diazePam in the control grouP was 29mg.There was no significant difference between the two grouPs.Conclusions:1.The 5g/kg dose of Chaihu-jia-Longgu-Muli-tang grouP showed stable and raPid antidePressant effect in Balb/c and Kun Ming mice after a single administration for 30 minutes,and the effect lasts for 3 days.That is similar to KETamine.2.It was found that both Chaihu-jia-Longgu-Muli-tang and XiaoChaihu showed raPid antidePressant effect,and XiaoChaihu is the core of the raPid anti-dePression effect of the whole Prescriptions.Part Guizhi Fuling Dahuang Longgu Muli does not show the antidePressant effect alone,but it may be of great significance to the Persistence of Chaihu-jia-Longgu-Muli-tang.3.Model of olfactory bulb removal of depression mice showed the characteristics of increased autonomic activity,and the ratio of sucrose Preference test decreased significantly,which has good model stability.Chaihu-jia-Longgu-Muli-tang showed a stable antidePressant effect on the olfactory bulb dePression model.However,Xiaochaihu did not show antidePressant effect in this model.and may have an anxious behavior reaction4.The molecular biology experiment found that the model of olfactory bulb removal of the Prefrontal glutamate recePtor AMPAR/NMDAR was abnormal.inhibition of AKT-mTOR PhosPhorylation and associated signaling Pathways.This may be the underlying mechanism that leads to dePressive symPtoms.Chaihu-jia-Longgu-Muli-tang could increase the ratio of frontal lobe AMPA/NMDA recePtor in the olfactory bulb removal model mice,increase the PhosPhorylation of mTOR molecules,activate the AKT-mTOR signaling Pathway.regulate the expression of glutamate membrane recePtor.enhance synaPtic Plasticity,and quickly resist dePression.The mechanism is similar to KETamine.As a result,Chaihu-jia-Longgu-Muli-tang may Play an anti-dePressant role mainly through the AMPA recePtor and mTOR signaling Pathway of glutamate energy system.5.Chaihu-jia-Longgu-Muli-tang(lOg/kg)has a certain sedative and hyPnotic effect6.Through the observation of clinical cases,we found that,in the unilateral severe dePression with insomnia,Chaihu-jia-Longgu-Muli-tang imProved insomnia and meanwhile synergize the raPid anti-dePressant effect of SSRI antidePressants,significantly imProved somatic symPtoms.And it can reduce the clinical dosage of sleeP inducer and Play the role of auxiliary sleeP inducing.