The Study Of The Mechanism Of Acupoint Catgut Embedding In The Treatment Of Depression Based On Metabolomics Analysis

Objective1.A depression animal model was constructed through repeated subcutaneous injections of corticosterone(CORT),and the behavioral changes of CORT depression model mice were observed.2.The effect of acupoint catgut embedding(ACE)at Baihui(GV20)and Dazhui(GV14)acupoints on depression-like behavior,hippocampal pathology and neurobiochemical indicators,and hippocampal metabolic status in CORT depression model mice were observed.The antidepressant mechanism of ACE from the perspective of hippocampal metabolism was explored with the aim to give more available evidence for the ACE clinical application on depression.MethodsIn this study,after one-week adaptive feeding,a total of 80 SPF male Kunming mice were randomly divided into five groups with 16 mice in each group using a random number table.The detailed grouping was as follows:control group(CON),model group(MOD),ACE group(ACE),sham ACE group(SHAM),fluoxetine group(FLX).The CON group was injected with normal saline subcutaneously,and the other groups were used to construct the animal model of depression through repeated CORT subcutaneous injection for 21 continuous days.After the modeling was completed,on the 1st and 8th day of the intervention,the CON group and MOD group were not received any intervention except for grasping,fixation and disinfection.On the 1st and 8th days of the intervention,the ACE group was treated with ACE treatment at GV20 and GV14.In the SHAM group,the intervention was the same as that of the ACE group except that the absorbable medical surgical catgut was not placed in the embedding needle.Since the 1st day of intervention,the FLX group was given fluoxetine 15 mg/kg by intragastric administration once daily for 14 consecutive days.1.Tail suspension test(TST)and forced swimming test(FST)were utilized to evaluate the behavioral changes of CORT depression model mice.2.The effects of ACE treatment on the behavior of depression model mice were evaluated through measuring body weight and daily feed consumption,open field test(OFT),TST and FST.3.Nissl staining and the levels of corticotropin releasing hormone(CRH),adrenocorticotropic hormone(ACTH),5-hydroxytryptamine(5-HT),noradrenaline(NE),dopamine(DA)were evaluated by enzyme linked immunosorbent assay to explore the effects of ACE treatment on hippocampal pathology and the neurobiochemical levels of depression model mice.4.Non-targeted metabolomic analysis by liquid chromatography-tandem mass spectrometry(LC-MS/MS)was employed to evaluate the effect of ACE on the hippocampal metabolic status of depression model mice.5.The transcription level of ACE antidepressant differential metabolite enrichment pathway mTOR signaling pathway genes in the hippocampus of depression model mice was verified by real-time quantitative polymerase chain reaction(RT-qPCR)experiments.Results1.Behavioral changes of CORT repeated subcutaneous injection depression model miceCompared with the CON group,the body weight of MOD group were significantly decreased after CORT modeling(P<0.05).The immobility time of TST and FST in the MOD group were dramatically increased(P<0.01).2.The effect of ACE treatment on the behavior of depression model mice(1)In relative to the CON group,the body weight of mice in the MOD group was obviously decreased(P<0.05).In contrast to the MOD group,the weight of mice in the ACE group and FLX group were dramatically increased(P<0.05),while the SHAM group showed no change in body weight(P>0.05).In relative to the CON group,the daily feed consumption of mice in the MOD group was markedly decreased(P<0.05).In contrast to the MOD group,the daily feed consumption of ACE group and FLX group were increased significantly(P<0.05).There was no improvement in the SHAM group(P>0.05).(2)In the OFT,in contrast to the CON group,the total running distance,distance in center,ratio of time in center and the times in center of the MOD group were remarkably decreased(P<0.01 or P<0.05).In relative to the MOD group,the total running distance,distance in center and the ratio of time in center of ACE group were dramatically improved(P<0.05),and the times in center showed increasing trend(P>0.05).The above indicators of FLX group were obviously enhanced(P<0.01 or P<0.05).While,these of the SHAM group showed no marked change(P>0.05).(3)In the TST,in comparison with the CON group,the immobility time of MOD group was dramatically increased(P<0.01).In contrast to the MOD group,the immobility time of ACE group and FLX group were markedly decreased(P<0.05 or P<0.01).While,that of SHAM group existed no significant improvement(P>0.05).(4)In the FST,in relative to the CON group,the immobility time of MOD group was dramatically increased(P<0.05).In contrast to the MOD group,that of ACE and FLX groups were remarkably reduced(P<0.05).While,that of SHAM group showed no marked improvement(P>0.05).3.The effect of ACE treatment on the hippocampal pathology and neurobiochemical indexes of depression model mice(1)In contrast to the CON group,the neuron density in the CA1 and CA3 regions of the hippocampus of mice in the MOD group showed a decreasing trend,the cells arrangement was looser,the cytoplasm became lighter,the number of Nissl bodies decreased and a few cells showed nuclear shrinkage.In relative to the MOD group,the neuron density in the ACE and FLX groups were obviously increased,the shape and size of the cell bodies recovered better,the cytoplasm was darkly colored,the nucleus were round,and more Nissl bodies were existed.While,the neuron histopathologic damages of hippocampus in the SHAM group were not improved.(2)In contrast to the CON group,the CRH and ACTH levels in the MOD group were remarkably increased(P<0.01),and the levels of 5-HT,NE and DA were obviously decreased(P<0.05).In relative to the MOD group,ACE treatment remarkably reduced CRH and ACTH levels(P<0.05)and obviously enhanced the NE and DA levels(P<0.05),and showed an increasing trend on 5-HT level(P>0.05).FLX administration effectively reduced the ACTH and CRH levels(P<0.01)and increased the 5-HT,NE and DA content(P<0.05).While,these of SHAM group showed no obvious improvement(P>0.05).4.Non-targeted metabolomics to explore the effect of ACE on the hippocampal metabolism in depression model mice(1)In both positive and negative ion modes in the PLS-DA analysis model,screening conditions were set as the two principal components VIP≥1,univariate analysis FC≥1.2 or ≤0.83 and T test p-value<0.05.In the positive and negative ion modes,there were 181 and 16 kinds of differential metabolites respectively between the CON group and MOD group.And there were 162 and 52 kinds of differential metabolites respectively between the MOD group and ACE group.(2)In relative to the CON group,29 metabolites were up-regulated and 168 metabolites were down-regulated in the MOD group.In relative to the MOD group,117 metabolites were up-regulated and 97 metabolites were down-regulated in the ACE group.Among them,21 kinds of down-regulated metabolites in the MOD group such as leucine,valine and tyrosine were up-regulated in the ACE group.Glycerine and spermidine which were up-regulated in the MOD group were down-regulated in the ACE group.(3)Based on the enrichment analysis of the metabolic pathways of differential metabolites between the MOD group and ACE group based on the KEGG database,it was found that the differential metabolites of ACE for antidepressant were mainly enriched in mTOR signaling pathway,biosynthesis of branched-chain amino acids,endogenous cannabinoid pathway,purine metabolism pathway and other metabolic pathways.5.ACE antidepressant differential metabolite enrichment pathway mTOR signaling pathway gene transcription level verificationIn relative to the CON group,the expression of PI3K,Akt,mTOR,Raptor,BDNF,NGF,TrkB,SYN-1,SYT-1,PSD93 and PSD95 mRNA in the hippocampus of mice in the MOD group were markedly reduced(P<0.05 or P<0.01).In contrast to the MOD group,the expression of Akt,mTOR,Raptor,BDNF,NGF,TrkB,SYN-1,SYT-1,PSD93 and PSD95 mRNA in the hippocampus of mice in the ACE group were obviously enhanced(P<0.05 or P<0.01),and the expression of PI3K mRNA showed an increasing trend(P>0.05).The expression of PI3K,Akt,mTOR,Raptor,BDNF,NGF,TrkB,SYN-1,SYT-1,PSD93 and PSD95 mRNA in the hippocampus of mice in FLX group were remarkably increased(P<0.01).While,the expression of mTOR signaling genes mRNA showed no remarkable change in the SHAM group(P>0.05).Conclusions1.ACE treatment at GV20 and GV14 improved the behavioral disorder of depression model mice.2.ACE treatment at GV20 and GV14 improved the hippocampus pathological damage and abnormal neurobiochemical indexes of depression model mice.3.ACE treatment at GV20 and GV14 improved the metabolic state of hippocampus of depression model mice.4.ACE treatment at GV20 and GV14 promoted the mRNA transcription levels of mTOR signaling pathway genes,nerve growth factor-related genes and synaptic plasticityrelated genes in the hippocampal of depression model mice.