The Expression Of Gal/GalR2 In The Hypothalamus Of CUMS Model Rats And The Regulation Mechanism Of Xiaoyaosan

BackgroundGalanin(Gal)is a kind of brain-gut peptide(BGP)with biological activity.It belongs to the class of neuropeptide.It consists of 29-30 amino acids.The N and C ends of its molecular structure are composed of glycine(Gly)and alanine(Alanine,Ala)residues,respectively.For the first time,researchers have extracted Gal from the small intestine of pigs.Gal is widely distributed in the Peripheral nervous system(PNS),the central nervous system(Central Nervous System,CNS)and the endocrine system(The),in the hippocampus,hippocampus and amygdala.Gal and its receptors were detected in the basal forebrain cortex and dorsal root ganglion(DRG).Gal is mediated by three subtypes of receptors,galanin receptor 1(GalR1),galanin receptor 2(GalR2),and galanin receptor 3(GalR3),which play a variety of physiological functions,including cognitive,memory and emotional regulation,feeding,seizures,and so on.Gal/GalR2 is most associated with depression.Gal is found to be involved in the occurrence and development of depression,and plays a very important role in this process.GalR2 is a G protein-coupled receptor,mainly distributed in hypothalamus,hippocampal amygdala,cortex and dorsal root ganglion.Researchers found that stimulating the expression of GalR2 in rats can play an antidepressant role.In addition,observation of GalR2 gene knockout mice showed that they were more likely to exhibit depressive behavior.In conclusion,Gal and GalR2 may be potential targets for the treatment of depression.Hypothalamus is an important center for maintaining the physiological activities of energy metabolism,and it participates in learning and memory,reward,emotion,diet regulation and other behaviors.Xiaoyaosan is a classical prescription for soothing the liver and relieving depression,and is also a commonly used traditional Chinese medicine preparation for the treatment of depression.Gal/GalR2 is widely distributed in the hypothalamus.Therefore,whether Xiaoyaosan can relieve and treat depression by regulating Gal/GalR2 remains to be further studied.ObjectiveIn this study,Chronic Mild Unpredictable Stress(CUMS)was used to establish rat depression model,and the animal model was evaluated in terms of macroscopical manifestations and experimental indicators.Gal and its receptor GalR2 in hypothalamus of CUMS depression model rats were detected by biological methods,and the changes of protein and gene levels of Gal and its receptor GalR2in hypothalamus of depression rats were observed.To study the effect and mechanism of Xiaoyaosan in the treatment of depression.MethodThe depression rat model was established by CUMS for 6 weeks.40 SPF male healthy SD rats were placed in the clean animal room of Beijing University of Traditional Chinese Medicine for 7 days.Five rats in each cage were fed with conventional diet and deionized water.The temperature of the laboratory was controlled at 22,the humidity was maintained at 30%-40%,and the light maintained normal circadian rhythm.According to body weight,all rats were randomly divided into four groups:normal group,model group,Xiaoyaosan group and fluoxetine group.The depression rat model was established by CUMS method.During the establishment of the model,the rats'external performance,changes in food intake,changes in body weight,preference for sugar and water,open-field experiments were observed and evaluated.After successful modeling,the rat hypothalamus was removed,and the expression of GalR2 receptor was detected by Western Blot(WB),the expression of GaL was detected by Enzyme linked immunosorbent assay(ELISA),and the expression of GaL was detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).The expression of Gal and its receptor GalR2 gene was detected.Result(1)After 6 weeks of CUMS modeling,the model rats developed symptoms such as dry hair,yellowing,restlessness,sparse stool,reduced food intake and slow weight gain.In open field experiment,the total number of punctures,modification times and standing times of model rats were significantly reduced compared with other groups,while the related performance of rats in Xiaoyaosan group and fluoxetine group was improved after the corresponding medication was administered.(2)RT-q PCR results showed that the expression of Gal in hypothalamus of model rats increased.Compared with model group,there was no significant difference in the expression of Gal between Xiaoyaosan group and fluoxetine group.The expression of GalR2 decreased in model group,and increased in Xiaoyaosan group and fluoxetine group compared with model rats,with significant difference.It indicated that Xiaoyaosan could up-regulate GalR2.(3)The results of ELISA showed that the content of Gal in hypothalamus of model rats was increased.There was no significant difference in the content of Gal between Xiaoyaosan group and fluoxetine group,and the expression of Gal protein and gene was slightly different.(4)Western Blot results showed that the expression of GalR2 protein in hypothalamus of rats in model group decreased,and the expression of GalR2 protein in Xiaoyaosan group and fluoxetine group increased in varying degrees compared with model rats,which also showed that Xiaoyaosan had an up-regulation effect on GalR2.The expression of protein was basically the same as that of gene.Conclusion(1)After 6 weeks of CUMS,the rat model of depression was successfully established.(2)There were different levels of Gal/GalR2 in hypothalamus of rats,suggesting that there were functional changes of Gal/GaIR2 in depression model.(3)Xiaoyaosan and fluoxetine can up-regulate the expression of GalR2 gene and protein to varying degrees and promote the binding of Gal and GalR2,which may be one of the biological bases of Xiaoyaosan in regulating depression,and GalR2 may be the main target of its role.