Based On MicroRNA/STAT1 Expression To Explor The Antidepressant Mechanism Of Kaixin Jieyu Prescription

Depression is a common affective disorder characterized by marked and persistent depression.With the increasing pace of social life,people are facing increasingly heavy mental burden,and its incidence is increasing year by year.According to WHO statistics in 2012,there are about 350 million depressive patients around the world,nearly a million people commit suicide each year because of this disease.In 2000,depression had become the fourth largest disease burden in the world and was the leading cause of disability.However,the current treatment of depression is still very limited,and the existing antidepressant western medicine has obvious side effects such as sedation and blood pressure drop.Chinese medicine has played an important role in the treatment of depression,it has many advantages,such as multi target,multi link,holistic treatment of body and mind as well as small side effects,Through the study of the mechanism of antidepressant efficacy of Chinese herbal compound,and the development of new antidepressant drugs have become an important strategy for the prevention and treatment of depression.MicroRNA is a class of non coding single strand small RNA molecules widely found in eukaryotes in recent years,It participates in the pathophysiological process mainly by regulating the expression of its target genes.MiRNA is closely related to depression by involving in the regulation of neuronal differentiation,synaptic plasticity,brain-derived neurotrophic factor and the process of antidepression mechanism.Based on preliminary study of traditional Chinese medicine Kaixin Jieyu Prescription has significant clinical efficacy,the purpose of this study is to investigate the mechanism of miRNA regulation of target gene expression in Kaixin Jieyu Prescription treatment of depression.Objectives:To observe the expression of microRNA/signal transducer and activator of transcription 1 in hippocampus of chronic unpredictable mild stress depression model rats.Based on microRNA/STAT1 expression to explor the antidepressant mechanism of Kaixin Jieyu Prescription,exploring the new target of antidepressant drugs and providing a theoretical basis for traditional Chinese Medicine in prevention and treatment of depression.Methods:1 Animal models and groups:Adult healthy male SD rats were randomly divided into normal group,model group,chinese medicine group and western medicine group.The rats in the normal group were reared regularly;The rats in the model group were treated by chronic unpredictable mild stress for 42 days to establish a depression rat model;The rats in the chinese medicine group were treated with Kaixin Jieyu Prescription;The rats in the western medicine group was treated with fluoxetine hydrochloride.2 Medication:The treatment groups began the intragastric administration at the same time as the model was manufactured.The rats in the chinese medicine group were given Kaixin Jieyu Prescription 0.90g/kg/d;The rats in the western medicine group were treated with fluoxetine hydrochloride 2.0mg/kg/d.The model group gave the same volume of distilled water,and adjusted the amount of gastric perfusion every week according to the change of body weight.All rats were given intragastric administration for 42 days.3 Observation index:3.1 Behavioral observation:The behavioral changes of rats were evaluated by the sugar preference experiment and forced swimming test,to observe the effect of Kaixin Jieyu Prescription on the percentage of sugar consumption and the immobility time of forced swimming in model rats.3.2 The expression of miRNA in hippocampus of rat model was observed by using Agilent Rat miRNA microarray,The significant differential expression of miRNA is screened by p?0.05 and FC?2.Gene spring13.1 software is used to depict the volcano map and cluster analysis of miRNA,targetScan and microRNAorg database were used to predict the target gene of miRNA,and the DAVID bioinformatics database was used for target gene GO enrichment analysis and KEGG signaling pathway analysis..3.3 Through microarray results,we selected miRNA which is significantly different and may be related to Chinese medicine intervention.The expression of miR-223-3p,miR-664-3p,miR-874-3p,miR-665 and miR-125a-3p were verified by qRT-PCR,and the target genes of miR-223-3p,miR-664-3p and miR-874-3p were predicted,and the STRING database was used to analyze the protein function network of the protein.3.4 The expression of STAT1 and p-STAT1 in hippocampus of rats were detected by RT-PCR and Western imprinting,to study the therapeutic effect of Kaixin Jieyu Prescription.Results:1 There was no significant difference in body weight,percentage of consumption of water sugar and immobility time between each group before modelling.42 days later,compared with normal rats,the weight of depression model rats was significantly reduced,the difference was statistically significant(P<0.01),the percentage of sugar consumption was significantly reduced,the difference was statistically significant(P<0.05),and the immobility time of forced swimming test was significantly increased,the difference was statistically significant(P<0.01).Compared with the model group,the sucrose consumption percentage of the chinese medicine group increased significantly,the difference was statistically significant(P<0.05),the immobility time was significantly reduced,the difference was statistically significant(P<0.01),while no significant effect on the body weight of rats,the difference was not statistically significant(P>0.05);And the sucrose consumption percentage of the western medicine group increased significantly,the difference was statistically significant(P<0.01),the immobility time was significantly reduced,the difference was statistically significant(P<0.05),while no significant effect on the body weight of rats,the difference was not statistically significant(P>0.05)2 The results of miRNA microarray showed that there are 14 miRNA(miR-1249?miR-138-1-3p?miR-139-5p?miR-210-3p?miR-223-3p?miR-290?miR-296-5p?miR-2985?miR-341?miR-347?miR-652-5p?miR-664-3p?miR-667-3p?miR-874-3p)in the hippocampus of model rats was significantly up-regulated(FC?2,p?0.05),while the expression of 12 miRNA(miR-125a-3p?miR-136-5p?miR-138-2-3p?miR-150-3p?miR-181c-5p?miR-20b-5p?miR-23a-5p?miR-338-5p?miR-3560?miR-433-5p?miR-665?miR-872-5p)was significantly down regulated(FC?2,p?0.05).The expression level of 16 miRNA(miR-1249?miR-138-1-3p?miR-223-3p?miR-290?miR-296-5p?miR-2985?miR-347?miR-652-5p?miR-664-3p?miR-667-3p?miR-874-3p?miR-338-5p?miR-433-5p?miR-665?miR-125a-3p?miR-136-5p)of them was restored by Kaixin Jieyu Prescription in some degree(FC?2,p?0.05).3 The target genes of the significant difference miRNA were predicted by TargetScan and microRNAorg database,the model group compared with the normal group there are 2660 genes have been predicted,the Kaixin Jieyu Prescription group compared with the model group there are 2188 genes have been predicted.The GO functional analysis of their target genes showed that the model group compared with normal group,the biological process mainly involved in hypoxia response,the cell composition mainly involved in cytoplasmic stroma,the molecular function mainly involved in protein binding.The chinese medicine group compared with the model group,the biological process mainly involved in organic ring combination reaction,the cell composition mainly involved in cytoplasmic stroma,the molecular function mainly involved in protein binding.The KEGG pathway analysis of their target genes showed that the model group compared with normal group mainly involved in metabolic pathway,and the chinese medicine group compared with the model group mainly involved in antibiotic synthesis.4 The results of qRT-PCR showed that the expression of miR-223-3p,miR-664-3p and miR-874-3p were consistent with the microarray,The expression of miR-223-3p and miR-874-3p in hippocampus of model rats increased significantly,the difference was statistically significant(P<0.05),Kaixin Jieyu Prescription and fluoxetine hydrochloride can significantly inhibit its expression,the difference was statistically significant(P<0.05);compared with the normal group,miR-664-3p was significantly up-regulated in the model group,the difference was statistically significant(P<0.05),its expression in the Kaixin Jieyu Prescription group and the fluoxetine hydrochloride group were decreased compared with the model group,the difference was not statistically significant(P>0.05).and the 3 miRNA could regulate the hundreds of target genes.The results of the protein function network showed that STAT1 and other genes might be the key downstream functional gene.5 The results of qRT-PCR showed that the expression of miR-125a-3p and miR-665 were inconsistent with the microarray,the expression of miR-665 in hippocampus of model rats increased significantly,the difference was statistically significant(P<0.01),Kaixin Jieyu Prescription and fluoxetine hydrochloride can significantly inhibit its expression,the difference was statistically significant(P<0.01);and compared with normal group miR-125a-3p was significantly up-regulated in the model group,the difference was statistically significant(P<0.05),its expression in the Kaixin Jieyu Prescription group and the fluoxetine hydrochloride group were decreased compared with the model group,the difference was not statistically significant(P>0.05).6 The results of RT-PCR showed that compared with the normal group the expression of ST AT1 gene in hippocampus of model rats was significantly decreased,the difference was statistically significant(P<0.01);and the chinese medicine group compared with the model group,STAT1 gene expression was significantly increased,the difference was statistically significant(P<0.01).It can be seen that Kaixin Jieyu Prescription can significantly improve the expression of STAT1 gene in hippocampus of chronic stress depression rats.7 The results of Western blot showed that compared with the normal group the expression of p-STAT1 in hippocampus of model rats was significantly decreased,the difference was statistically significant(P<0.05);The chinese medicine group compared with the model group,p-STAT1 expression was significantly increased,the difference was statistically significant(P<0.05);The western medicine group compared with the model group,p-STAT1 expression was significantly increased,the difference was statistically significant(P<0.05).It is suggested that Kaixin Jieyu Prescription can promote the expression of p-STAT1 protein in hippocampus of model rats.Conclusions:1 The rat model of chronic unpredictable mild stress and solitary was a good model for depression,and the behavior of rats was obviously improved by treated with Kaixin Jieyu Prescription.2 The expression of miRNA in hippocampus of chronic stress depression rats was significantly changed,and the expression and phosphorylation of STAT1 were significantly decreased,which may be related to the pathogenesis of depression.3 Kaixin Jieyu Prescription can recover the expression of miRNA in hippocampus of model rats with different degrees,and promote the expression and phosphorylation of STAT1,which may be one of the mechanisms for the treatment.of depression.