| ObjectiveDepression is a common mental disease,clinical and experimental evidence indicates that a variety of gene function and expression differences associated with depression,a complex regulatory network formed by these genes involved in the pathogenesis of depression.As a safe and effective antidepressant therapy,acupuncture has been widely recognized in the world,but its mechanism is still not clear.Based on the preliminary research of our team,in this study,we established the rat model induced by chronic restraint stress(CRS),and the mechanism of depressive and acupuncture antidepressant was discussed at the level of behavioral and transcriptome.MethodsForty-eight male SD rats were randomly divided into normal control group,model group,fluoxetine group and acupuncture group.Except the normal control group,the rats in the other groups were received 28d restraint stress.Rats in Fluoxetine group were given fluoxetine administration(1.8mg/kg)1 h prior to restraint stress.Rats in Acupuncture group were treated with acupuncture at 1 h before the restraint stress(acupoints "Baihui" and "Yintang").The rats in each group were subjected to body weight measurement,open field test and sucrose preference test at day 0,day 7,day 14,day 21 and day 28 respectively.The effects of acupuncture intervention on the behavior of CRS model rats were studied by observing the changes in body weight,horizontal crossing number,number of vertical erection and sugar consumption.At the end of the animal experiment,the total RNA was removed from the hippocampus,frontal lobe and pituitary of the rats in each group.Then 12 RNA pool samples were collected according to different groups and different tissues.RNA-seq technology was used to complete the whole transcriptome sequencing of the 12 samples,and expression characteristics of the transcriptome and the expression of each gene were analyzed statistically.Based on the expression levels of the transcriptome of each sample,the different groups and the same tissues were compared and statistically.GO and KEGG were used to analyze the genes that were significantly different from each other,and the main biological functions and biological pathways were discussed.Results1.Before the animal experiment,there was no significant differences in the behavior of rats in each group(P>0.05).After modeling,compared with the normal control group,the body weight,the horizontal crossing number,the number of vertical erection and the sugar consumption were significantly lower in the model group(P<0.01,P<0.01,P<0.01,P<0.01).Compared with the model group,the body weight,the horizontal crossing number,the number of vertical erection and the sugar consumption were significantly increased in the fluoxetine group and the acupuncture group(P<0.01,P<0.01,P<0.01,P<0.01).2.The transcriptome of each sample was successfully constructed by RNA-seq technique.The gene cluster analysis showed that the expression patterns of the differentially expressed genes in the hippocampus,frontal cortex and pituitary gland of rats in different groups were significantly changed.3.Based on the expression levels of the transcriptome gene in each sample,the differences between the different groups and the same tissues were compared.The results of DEG analysis showed that each pair of samples were different in the presence of differentially down-regulated genes,some of these genes are consistent with the reported findings of depression,and most of the genes are associated with post-traumatic stress disorder,schizophrenia,Alzheimer's disease,Parkinson's disease and other psychiatric disorders.4.GO,KEGG analysis showed that there were significant differences between each pair of samples in the presence of GO term and KEGG pathway,which mainly includes immune response,inflammatory response,apoptosis,signal transduction,amino acid metabolism,lipid metabolism,transcription and so on.5.The significant difference between the expression of different genes and their significant enrichment of GO term,KEGG pathway overlap analysis found that gene,GO term,KEGG in different groups of the same organization and the same group of different organizations of different degrees of overlap between the samples.The overlap of the gene,GO,KEGG between acupuncture intervention and the CRS model was much higher than that of fluoxetine intervention and CRS modeling.Conclusions1.Chronic restraint stress can induce a series of depressive behavior in rats,including inhibition of body mass growth,decreased spontaneous activity,exploring behavioral levels,and increased the degree of anhedonia.Acupuncture and fluoxetine intervention can significantly improve the above situation.2.The expression profile of transcription was successfully constructed by RNA-seq technique,and the quality control and genome-wide analysis were performed.The data of this study were strictly controlled.3.DEG analysis showed that there were significant differences in the expression of various genes in the hippocampus,frontal cortex and pituitary gland of the rats in each group,suggesting that CRS modeling,fluoxetine intervention and acupuncture intervention had different effects on the gene expression patterns in rats.The analysis of the significant differences in the expression of gene overlaps between the samples showed that there were overlapping genes in each sample.It's indicate that:1)CRS induced depression like behavior in rats was significantly correlated with the expression of multiple genes in the hippocampus,frontal cortex and pituitary gland,2)Fluoxetine intervention and acupuncture intervention can affect the expression of these overlapping genes in hippocampus,frontal cortex and pituitary gland,3)Among the aforementioned overlapping genes,some of the genes have been reported or confirmed to be associated with depression and/or other psychiatric disorders(such as bipolar disorder,posttraumatic stress disorder,schizophrenia,autism,panic disorder,Alzheimer Disease,Parkinson's disease,multiple sclerosis,etc.),these genes may be related to the onset of depression and/or other psychiatric disorders,and may also be fluoxetine/acupuncture antidepressant effect on the hippocampus,frontal and pituitary effective target genes,4)In the antidepressant target gene,fluoxetine and acupuncture intervention have common target genes and their specific target genes.The number of effective target genes in acupuncture is much greater than that of fluoxetine.The results indicate that the antidepressant effect of acupuncture may be more extensive.4.The Gene ontology(GO)analysis showed that the enrichment of the differentially expressed down-regulated genes in hippocampus and frontal lobe contributes to the GO terms;the enrichment of the differentially expressed up-regulated and down-regulated genes in hypophysis contributes to the GO terms.The most enriched GO term of the differentially expressed genes were included in the GO terms.The results indicated that there was a close relationship between the function of the the differentially expressed genes and the most enriched GO terms.The overlap analysis of the most enriched GO terms of the differentially expressed genes showed that there were overlap GO terms betweeen each couple samples.The results of the present study confirmed that:1)There was a close relationship between the depression-related behaviors induced by CRS and the most enriched GO term of the differentially expressed genes in hippocampus,frontal lobe and hypophysis:including inflammatory response,stimulate response to glucocorticoids,immune response,RNA polymerase II promoter transcriptional regulation,DNA transcription regulation reaction,chemotaxis,neutrophils chemotaxis,gene expression regulation,negative regulation of apoptosis,etc,2)There was a close relationship between the antidepressant effects of acupuncture on depression-related behaviors induced by CRS and the most enriched GO term of the differentially expressed genes in hippocampus,frontal lobe and hypophysis:including inflammatory response,stimulate response to glucocorticoids,chemotaxis,immune response,negative regulation of apoptosis,chemokine activation,etc.5.The KEGG analysis showed that the KEGG pathway was enriched by the differentially expressed genes in hippocampus,frontal lobe and hypophysis.The result indicated that there was a close relationship between the biological pathway of the the differentially expressed genes and the most enriched KEGG pathway among each groups;The overlap analysis of the most enriched KEGG pathways of the differentially expressed genes showed that there were overlap KEGG pathways betweeen each couple of samples.The results of the present study confirmed that:there was a close overlap relationship between the biological pathway which were involved in the differentially expressed genes in hippocampus,frontal lobe and hypophysis.1)There was a close relationship between the depression-related behaviors induced by CRS and the biological pathway,including TLR signaling pathway,cytoplasm DNA sence signaling pathway,NLR signaling pathway,TNF signaling pathway,etc,2)There was a close relationship between the antidepressant effects of acupuncture on depression-related behaviors induced by CRS and the most enriched KEGG pathway of the differentially up-expressed genes in hippocampus,frontal lobe and hypophysis:including histidine metabolism,beta alanine metabolism,glycine,serine and threonine metabolism,porphyrin and chlorophyll metabolism,3)The signaling pathway of the down-expressed genes in hippocampus,frontal lobe and hypophysis:including TLR signaling pathway,Chemokine signaling pathway,TNF signaling pathway,cytoplasm DNA sence signaling pathway;The differentially down-expressed genes in hippocampus and frontal lobe in acupuncture and fluoxetine group includes TLR signaling pathway.Notably,the response genes of fluoxetine intervention were on the downstream of the TLR signaling pathway,while the response genes of acupuncture intervention were on the upstream and downstream of the TLR signaling pathway.The results indicated that the antidepressant effects of acupuncture might be involved in regulating the TLR signaling pathway. |
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