| Background Depression is a common mental disorder in clinical practice and is a mental disorder that seriously threatens the physical and mental health of human beings.Many studies have shown that the incidence of mental disorders such as depression in adulthood is related to adversity experience in early life such as the maternal separation and children in early life,child abuse and neglect,etc.With the rapid development of our society and the increasing pressure of social competition,more and more people are forced to live in the field and even abroad,which leads to the change of their family structure.Their offspring are left in the early or early stages of their birth or childhood by parents,especially their mothers.Epidemiological survey found that children separated from mother in early life have a higher risk of mental illness such as depression in adulthood.Neonatal maternal separation is a serious stress,affecting the normal development of the nervous system and others,leading to depression and other psychiatric disorders in adult.At present,antidepressants commonly used in clinical practice have the disadvantages of toxic and side effects.However,acupuncture and moxibustion treatment of mental disorders have achieved good results with less toxic and side effects.However,can electroacupuncture(EA)treat depression caused by neuropsychological disorders induced by maternal and infant separation and the mechanism of acupuncture in treating depression is unclear.1.Establishing maternal separation rat model to mimic maternal separation in early life stage in human,and to observe the effect of maternal separation in early life on the behavior and cognitive function of adult rats.2.In order to explore the therapeutic effect and mechanism of electroacupuncture treating of depressed rat suffered maternal separation,electroacupuncture(EA)was used to treat the maternal separation adult rat.3.A new generation of high-throughput transcriptome sequencing technology was used to perform high-throughput sequencing of prefrontal cortex brain regions in maternal separation model,and to obtain differentially expressed genes at the transcriptome level,to explore mechanisms of electroacupuncture on depression-like adult rats caused by maternal separation,and to reveal the signal pathways and regulatory aspects of electroacupuncture brain protection.In order to provide experimental evidence for the prevention and treatment of electroacupuncture treatment in depression caused by maternal separation in early life in clinical.ObjectiveMethodsMaternal Separation Rat Model Preparation:Newborn offspring rats undergo maternal separation every day for 4 hours during PND 1-20.Mathers of offspring rats were moved to another cage every day between 9:00-13:00.After 4 hours,the mother rats were returned to the atomic generation rat cage,lasted for 20 days.During PND1-10,the offspring rats were placed on a heated pad to maintain the body temperature of the offspring rats.During PND11-21,offspring rats were housed in squirrel cages.All rats were freely to eat and drink.In the blank control group,the offspring rats were normally reared,and no stimulation was provided except for the replacement of the rat cage on PND10.All rats were weaned at PND21.After weaning,the rats were reared on 4-5 calves per cage and free-diet until PND60.At PND60,female rats will be removed and male rats will be used for experiments.Group: The offspring rat suffered maternal separation were randomly divided into MS group,MS+EA group,MS+Sham-EA group,and MS+Flu group.Control rats were normal offspring offspring rats(without maternal separation).To reduce the difference caused by different littermates,each group of rats wasderived from a different generation of mother rats.Treatment: MS+EA group: The electroacupuncture was appied at Baihui and Yindang with a Korean type electroacupuncture needle,whith the current was 2 m A,the frequency was 2 HZ,during of the electroacupuncture time was 20 min,and every other day,a total of 21 days were treated;MS+Sham-EA group: Acupuncture needles were taped to the surface of Baihui and Yintang on acupuncture points.The needle did not penetrate the skin and was connected to a Korean electric acupuncture instrument,but no electricity was applied.The time of intervention in the pseudo-electroacupuncture group was 20 minutes,and every other time,lasted 21 days;MS+Flu group: Intraperitoneal injection of fluoxetine(10mg/kg)was applied once every 21 days for a total of 21 days;The Contro group was injected with an equal volume of 0.9% Na Cl every other day for a total of 21 days.Each group of rats underwent isoflurane anesthesia during the intervention and the anesthesia time was 20 min.Measurement:(1)Weekly monitoring of changes in body weight in rats(from post-weaning to the end of electroacupuncture treatment),using behavioral studies(sugar-water preference test,forced swim test,cross-overhead test,and prepulse inhibition test)to evaluate animals In the model,radioimmunoassay was used to measure the levels of CORT and ACTH in rat plasma to observe the neuroendocrine changes in rats.(2)The Morris water maze and conditional fear test were used to observe the effects of electroacupuncture on cognitive function of adult rats separated from mothers in early life,and the changes of synaptic LTP in hippocampal SC-CA1 region were recorded by in vivo electrophysiological technique.(3)Preparation of depression model induced by maternal separation in the early life stage and intervented it by EA.High-throughput transcriptome sequencing technology was used study of the prefrontal cortex brain regions in the Contro group,MS group,and MS+EA group rat.Differential expressed genes were analysised with Series Cluster,GO,KEGG to explore the possiblemechanism of EA in improving differential gene expression of depression-like symptoms of adult rats caused by maternal separation in early life,to reveal the signal pathways and regulatory mechainsm of EA in protecting brain,and to provide experimental evidence for the prevention and treatment of EA treating depression in clinical.ResultsThe main results are as follows: 1.The first experiment results:(1)The body weight of rats in each group was measured once a week starting from the day of weaning(PND21).No significant difference was observed in body weight between 3-11 weeks after birth(P>0.05),suggesting that repeated neonatal maternal separation and EA at Baihui and Yintang had no significant effect on the vegetative growth of rats.(2)Maternal separation in early life stage leaded to anhedonia and desperate behavior in adult rats.The adult rats sufferred from maternal separation showed a decreased preference for sugar water(P<0.01),and prolonged the immobility time in the forced swimming test(P<0.01);After 3 weeks of treatment of EA at Baihui and Yintang,compared with MS group,the consumption of sugar water in MS + EA group was increased(P<0.01),and the immobility time in forced swimming test was decreased(P<0.01,P<0.05);No significant difference of the sugar consumption and the immobility time were observed in the MS+Sham-EA group rats(P>0.05,P>0.05).(3)In the elevated test,there was no significant difference between the groups(P>0.05).Although the time of stay in the open arm decreased after adulthood,There was no significant difference between the two groups(P> 0.05),which suggested that PND1-20 prenatal rats did not induce significant anxiety and schizophrenia in early life.After electro-acupuncture intervention for 21 days,the rats in each group had an increasing trend in the open arm time,but the difference was not statistically significant.Electro-acupuncture did not significantly affect the behavior of the pre-pulse suppression test in preterm infants(P> 0.05).(3)In the elevated test,although the time of staying in the open arm of MS group was reduced,but no significant difference was observed among groups(P>0.05).In the pre-pulse inhibition experiment,there was no significant difference among the five groups(P>0.05).After EA intervention for 21 days in adults,the time of open arms and PPI in MS+EA group had an increasing trend,but the difference was not statistically significant(P>0.05,P>0.05).It suggested that maternal separation rats during PND1-20 did not induce significant anxiety and schizophrenic symptoms in adult rats.(4)The contents of CORT and ACTH in the plasma of rats were detected by radioimmunoassay.It was found that the content of CORT and ACTH in plasm in adult rats were significantly increased after maternal separation in early life(P<0.05,P<0.05).It was suggested that maternal separation stress in early life led to HPA axis hyperactivity.Compared with MS group,the levels of CORT and ACTH in plasma of rats in MS + EA group decreased significantly(P<0.05,P<0.01),while MS + Sham-EA group levels of CORT and ACTH in plasm did not significantly improved(P>0.05,P> 0.05).So,EA at Baihui and Yintang can inhibit HPA axis hyperfunction caused by early life maternal separation.In summary,the results of this study showed that maternal separation during PND1-20 for 4 hours a day can cause depression-like behavior in adult rats and EA at Baihui and Yin Tang can improve depression-like symptoms in adult rat sufferred early maternal separation.2.The second experiment results:(1)EA improved spatial learning and memory disorder in adult rat sufferred maternal separation in early life Morris water maze test result:Lantency in all group rats was gradual reducted in the Morris water maze test.Compared with the Control group,latency of MS group and MS+Sham-EA group was increased(P<0.01,P<0.05,resp).It suggested spatial learning and memory dysfunction in maternal separation rats.Compared with MS group,the latency of MS+EA group rats was significantly reduced(P<0.01).The percentage of time in the target quadrant was significantly lower in the MS group and the MS+Sham-EA group than in the Control group(P<0.05,P<0.05);compared with the MS group,the percentage of time in the target quadrant of MS+EA group and MS+Flu group was significantlyincreased(P<0.05,P<0.05).There was no significant difference of the percentage of time in the target quadrant between the MS+EA group,MS+Flu group and the Control group(P>0.05,P>0.05).(2)EA improved hippocampal-dependent cognitive dysfunction in adult rat sufferred maternal separation Fear conditioning test: In order to further verify the role of EA in the improvement of hippocampal-dependent cognitive function in adult rat sufferred maternal separation,the experimental rats were also subjected to fear conditioning test.Context dependent memory: Compared with the Control group,the freezing time in the MS group and the MS+Sham-EA group was significantly lower(P<0.01,P<0.05),while freezing time of the MS+EA group and MS+Flu group was no significant difference(P>0.01,P>0.05).Compared with the MS group,the freezing time of the MS+EA group was significantly increased(P<0.05).There was no significant difference in freezing time between Sham-EA groups and MS group(P>0.05).The tone dependent memory test: There was no significant difference in the freezing time among all groups(P>0.05).It suggested that EA improved hippocampal-dependent memory impairment in adult rat suffered maternal separation in early life.Because of the formation of context memory mainly depends on the hippocampus,while the formation of tone-dependent fear memory mainly depends on the amygdala.Combined the results of the Morris water maze test,it indicated that maternal separation may impair the hippocampal-dependent cognitive function in adult rats.EA improved hippocampal-dependent cognitive impairment in adult rats suffered separation separation in early life.(3)Electrophysiological recording in vivo results: In order to directly evaluate the effect of EA on cognitive dysfunction which associated with hippocampal neural plasticity in rats suffered maternal separation in early life,electrophysiological recording in vivo was used to record the LTP changes in the SC-CA1 region of the hippocampus in all rats.Compared with the Control group,the average slope of f EPSPs in the hippocampus of MS group and MS+Sham-EA group rats was significantly lower(P<0.05,P<0.05),while the average slope of f EPSPs in MS+EA group rats was not significantly difference(P>0.05).Compared with MS group,the average slope of f EPSPs in MS+EA group wassignificantly increased(P<0.01),while the average slope of f EPSPs in MS+Sham-EA group and MS+Flu group was not statistically different(P<0.05).3.The third experiment results:(1)Differential gene analysis showed that there were 253 differentially expressed genes in Control group,MS group and MS+EA group and there were 24 differentially expressed genes of three groups.There were 4 differentially expressed genes in Control group vs MS group and MS group vs MS+EA group,such as complexin 3,ras-related protein Rab-1B-like,synaptotagmin VI and uncoupling protein 3.(2)GO analysis: The biological processes(BP)involved in the differential express genes between MS and Control groups mainly include prostate gland epithelium morphogenesis,fatty acid metabolic process,positive regulation of activated T cell proliferation,protein heterooligomerization,female pregnancy,transport;the molecular functions(MF)mainly include syntaxin binding,butyrate-Co A ligase activity,SNARE binding;Cellular Component(CC)involved in the two groups mainly include mitochondrial membrane,mitochondrial matrix,mitochondrion,signal peptidase complex,aminoacyl-t RNA synthetase multienzyme complex,mitochondrial proton-transporting ATP synthase complex.BP involved in the differential express genes between MS and MS+EA groups mainly include peripheral nervous system development,developmental growth,response to morphine,phosphatidylinositol phosphorylation;MF involved mainly include alpha1-adrenergic receptor activity,calcium-dependent phospholipid binding,type 2 angiotensin receptor binding,alkyl sulfatase activity,oxytocin receptor binding,sequence-specific DNA binding transcription factor recruiting transcription factor activity,syntaxin binding,acetyltransferase activator activity;CC involved mainly include pre-sno RNP complex,intercalated disc,T-tubule,terminal bouton.(3)Series Cluster:Compared with the Control group,the differential genes in the MS group were down-regulated,and up-regulated after EA intervention(in MS+EA group).BP involved by these differential expressed gene were limb jointmorphogenesis,esophagus smooth muscle contraction,acrosomal vesicle exocytosis,heparan sulfate proteoglycan metabolic process,negative regulation of prostatic bud formation,glial cell-derived neurotrophic factor receptor signaling pathway,drug export,regulation of fibroblast growth factor receptor signaling pathway,embryonic process involved in female pregnancy,glomerular basement membrane development,negative regulation of fibroblast growth factor receptor signaling pathway,urate metabolic process,drug transport,glomerular filtration,innervation.MF involved were syntaxin binding,alkyl sulfatase activity,neurotransmitter transporter activity,N-acetylglucosamine-6-sulfatase activity,arylsulfatase activity,xenobiotic-transporting ATPase activity,sulfuric ester hydrolase activity,drug transmembrane transporter activity,clathrin binding,SNARE binding,ATPase activity,coupled to transmembrane movement of substances,calcium-dependent phospholipid binding,calcium ion binding,calcium-dependent protein binding,protein homodimerization activity.CC involved were perinuclear endoplasmic reticulum,synapse,Golgi stack,cell junction,extrinsic component of membrane,synaptic vesicle membrane,plasma membrane,synaptic vesicle,mitochondrial membrane.Compared with the Control group,the differential genes in the MS group were up-regulated,and down-regulated after EA intervention(in MS+EA group).BP involved by these differential expressed gene were regulation of renal sodium excretion,lactation,cellular sodium ion homeostasis,positive regulation of renal sodium excretion,drinking behavior,vasoconstriction,regulation of blood vessel size,ERK1 and ERK2 cascade,positive regulation of blood pressure,fatty acid metabolic process,positive regulation of multicellular organism growth,response to cold,cellular response to hormone stimulus,positive regulation of mast cell differentiation.MF involved were type 2 angiotensin receptor binding,oxytocin receptor binding,acetyltransferase activator activity,5-aminolevulinate synthase activity,complement component C1 q binding,V1 B vasopressin receptor binding,fatty acid ligase activity,neurohypophyseal hormone activity,V1 A vasopressin receptor binding,oxidative phosphorylation uncoupler activity,alpha1-adrenergic receptor activity,1-phosphatidylinositol-5-phosphate 4-kinase activity,angiotensin receptor binding,hormone activity,butyrate-Co A ligase activity.CC involved were membrane attack complex,mitochondrial matrix,haptoglobin-hemoglobin complex,hemoglobin complex,mitochondrial inner membrane.4.KEGG analysis: there were 5 pathways enriched in KEGG in Controlgroup and MSgroup,such as Butanoate metabolism,biotin metabolism,p53 signaling pathway,bile secretion,fox O signaling pathway.3 pathways wer enriched in KEGG in MS group and MS+EA group,they were alcoholism,viral carcinogenesis,renin-angiotensin system.Compared with GO enrichment analysis,the KEGG signal pathways enriched were fewer and more isolated,so KEGG pathway enrichment analysis results may be less value.the result may not be objective for fully explain the pathways that mediate the effect of EA treating depressed rat suffered maternal separation.Conclusion 1.Repeated maternal separation in early life lead to depression-like behavior in adult rats and EA at Baihui and Yintang points improved the depression-like behavior of adult rats induced by maternal separation,inhibited HPA axis hyperfunction in depression-like rat.The result suggested that effect of EA on depression-like adult rats induced by maternal separation in early life may be related to the regulation of HPA axis function by EA.2.EA ameliorates cognitive deficit and improves hippocampal synaptic plasticity in adult rat with neonatal maternal separation.3.The results of High-throughput sequencing showed that effect of EA improving the depression-like symptoms sufferred maternal separation in early life may be related to differentially expressed genes in PFC,such as complexin 3,ras-related protein Rab-1B-like,synaptotagmin VI and uncoupling protein 3.The multiple signaling pathways involved in differentially expressed genes may be important pathways for EA improving depression-like symptoms in adult rat sufferred early maternal separation.4.Combined three experimental results above,the results suggest that the effects of EA at Baihui and Yintang points in treating the depression-like symptoms of adult rat sufferred maternal separation may be through multiple targets and multiple pathways.The next phase of research should be designed to verify mechanism of EA effect based on this h High-throughput sequencing analysis results.The result suggested that EA at Baihui and Yintang points may be an effective method for the treatment of adult depression sufferred early life stress and provided experimental evidence for treatment of adult depression patients sufferred maternal separation in clinical. |
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