An Integrated Study On The Antidepressant Mechanism Of Xiaoyaosan Based On Multi-omics And Network Pharmacology

Topics basis:Depression is a complex and difficult disease,which is clinically manifested as depression and lack of pleasure,which substantially affects the mental health and physical of humans.At present,there are many hypotheses about the pathogenesis of depression,which overlap each other,revealing the complexity of the etiology and pathogenesis of depression from different angles.Traditional Chinese Medicine(TCM)has obvious advantages in the treatment of depression with its overall concept and characteristics of syndrome differentiation and treatment,and it has accumulated a lot of valuable experience.Xiaoyaosan originated from the Taiping Huimin Hejiju Fang in the Song Dynasty.It is a representative prescription for soothing the liver,relieving depression,and reconciling the liver and spleen.It has been widely concerned in the treatment of depression.Modern clinical and experimental studies have proved that the prescription has definite antidepressant effects and has become one of the classic prescriptions commonly used in the treatment of depression.However,the current reports of Xiaoyaosan mostly study from the perspective of single target research and interpretation of its mechanism of action.There is insufficient attention to the multi-target characteristics of depression,and it is difficult to sort out and reveal its antidepressant mechanism.Similar to other Traditional Chinese Medicine prescriptions,Xiaoyaosan is also a complex system with multiple components,multiple targets,and multiple mechanisms.The study of its mechanism are not well understood so farThe current research on Xiaoyaosan antidepressants mainly has the following problems:1.The research on the antidepressant mechanism of Xiaoyaosan is unitary.Metabolomics or molecular biology techniques are mostly used,and there is a lack of data integration analysis methods at the same level.2.Multi-omics data is incomplete,and there is a lack of research data from multi-level "transcriptome-proteome-metabolome" integrated analysis.3.Xiaoyaosan uses a "multi-component" effect on depression and a "multi-target" model to play a treatment effect.There is no "multi-component-multi-target" research model to clarify the antidepressant mechanism of Xiaoyaosan.Therefore,this study starts from the perspective of the holistic view of Traditional Chinese Medicine and uses the overall research ideas of multi-omics and network pharmacology.Starting from multiple levels and perspectives,it systematically explains the antidepressant mechanism of Xiaoyaosan.For such a complex system,the participation of systems biology and modern pharmacological experimental technology is needed.Firstly,characterize the chemical components of Xiaoyaosan and construct a chronic unpredictable mild stress(CUMS)rat depression model.To verify the antidepressant effect of Xiaoyaosan and sample collection for omics analysis.Secondly,construct the research method of Xiaoyaosan antidepressant metabolic phenotype and the method of optimizing and sorting metabolic phenotype pathway.To clarify the antidepressant mechanism of Xiaoyaosan from the level of metabolic phenotype.Third,transcriptomics and proteomics techniques are used.Screen the differential genes and differential proteins in the hippocampus of CUMS rats after Xiaoyaosan was administered.Multi-omics level integrated analysis of Xiaoyaosan's antidepressant mechanism and carried out molecular biology verification.Finally,the ADME prediction and the contribution index model of network pharmacology were used to screen the active ingredients of Xiaoyaosan.From the perspective of "active molecules-key pathways",the antidepressant mechanism of Xiaoyaosan is clear.The research results provide new ideas and new methods for the use of integrated analysis of omics and network pharmacology to study the mechanism of action of Chinese medicine.It provides a basis for in-depth research on the pharmacological mechanism of Xiaoyaosan's antidepressant and provides a theoretical basis for the clinical application of Xiaoyaosan.It has important scientific significance and value for the modernization of Chinese medicine prescriptions and the research and development of new drugs.Objective:(1)Metabolic phenotypes are analyzed at the same level.Construct a method for the study of metabolic phenotype and the optimization and sorting of metabolic phenotype pathways.To clarify the antidepressant mechanism of Xiaoyaosan from the metabolic phenotype level.(2)Association analysis of "transcriptomics-proteomics-metabolic phenotype".Find the association with the metabolic phenotype at the longitudinal level.Elucidate the antidepressant mechanism of Xiaoyaosan from the multi-omics and multi-level perspective of "gene-protein-metabolism".(3)From the perspective of network pharmacology based on experimental data.Identify the disease network of depression and look for the active molecules in Xiaoyaosan.Explore the antidepressant active molecules and mechanism of Xiaoyaosan from the perspective of "multi-component-multi-target".Method:(1)The UPLC-MS/MS method was used to qualitatively identify the chemical components in Xiaoyaosan,and clarify the chemical substance basis of Xiaoyaosan.Construct a CUMS rat depression model to verify the antidepressant effect of Xiaoyaosan.Collect hippocampal tissue and serum samples of rats to provide sample support for subsequent omics experimental research and molecular experimental verification.(2)Using metabonomics technology,analyze the metabolites and metabolic pathways of Xiaoyaosan regulating the hippocampus of CUMS depression rats.Using metabolic phenotype network and module function analysis,focus on the antidepressant metabolic characteristics of Xiaoyaosan.Construct the antidepressant phenotype network of Xiaoyaosan to clarify the antidepressant metabolic phenotype mechanism of Xiaoyaosan.To construct an optimal ranking method for the antidepressant metabolic phenotype pathways of Xiaoyaosan,and to clarify the important metabolic phenotype pathways of Xiaoyaosan antidepressants.(3)Using transcriptomics technology,analyze the differential genes and important pathways in the hippocampus of CUMS depression rats regulated by Xiaoyao Powder,and clarify the antidepressant mechanism of Xiaoyao Powder at the gene level.Using proteomics technology,analyze the differential proteins and important pathways in the hippocampus tissue of CUMS rats regulated by Xiaoyao Powder,and clarify the antidepressant mechanism of Xiaoyaosan at the protein level.The Multi-omics data integration analysis method is used to explore the key pathways of Xiaoyaosan's antidepressant.Xiaoyaosan is in the "gene-protein-metabolism" multi-level antidepressant pathway.Using molecular biology methods to verify that Xiaoyaosan may play an antidepressant effect by regulating the oxidative phosphorylation and glutamatergic synaptic pathways in the hippocampus of CUMS depression rats.(4)Adopt the ADME analysis method to screen the active molecules of Xiaoyaosan.Using network pharmacology target prediction,disease network analysis,and contribution index model.Clarify the important components,important targets,and key pathways of Xiaoyaosan's antidepressant.Molecular docking and molecular biology methods(RT-PCR and Western blot)were used to verify that the important components of Xiaoyaosan play an antidepressant effect by regulating the glutamatergic synaptic pathway in the hippocampus of CUMS depression rats.Results:(1)In this study,UPLC-MS/MS was used to identify the components of Xiaoyaosan.62 components were identified through the method of combining reference materials and mass spectrometry data in the literature with experimental data.Among them,there are 14 ingredients in Radix Bupleuri,8 ingredients in Radix Paeoniae Alba,11 ingredients in Radix Angelicae Sinensis,4 ingredients in Rhizoma Atractylodis Macrocephalae,3 ingredients in Poria,14 ingredients in Radix Glycyrrhizae,4 ingredients in Herba Menthae,and 3ingredients in Rhizoma Zingberis Recens.This method is directly characterized and identified and has obvious advantages compared with chemical separation methods.The Xiaoyaosan group(21.2 g /kg)can significantly reverse the weight,sucrose preference test,the crossing number,the erecrt times,the central grid residence time,and the number of times to enter the central area of rats caused by CUMS,and the staying time of the central grid of rats is significantly reduced.(2)The results of the Xiaoyaosan antidepressant hippocampal metabolomics study showed that compared with the control group,there were 25 differential metabolites in the hippocampus of CUMS depressed rats that showed significant changes.After the intervention of Xiaoyaosan,16 different metabolites were significantly recalled.Metabolic phenotype studies have found that there are 97 metabolisms of Xiaoyaosan,of which 34 are the same and different metabolites regulated in different biological samples,and 11 of them have the same changing trend.The key metabolic pathways involved are energy metabolism and neurotransmitter pathways.The CNM algorithm was used to extract nine metabolic function modules from the Xiaoyaosan callback metabolic network to participate in the research on the anti-depressive mechanism of Xiaoyaosan.For the first time,the docking score-weighted pharmacological index(DSWMP)was used to construct a pathway optimization ranking method for the antidepressant metabolic phenotype of Xiaoyaosan.(3)Transcriptomics results showed that compared with the model group,there were737 differentially expressed genes in the hippocampus of the rats in the Xiaoyaosan administration group.Among them,263 genes were up regulated and 474 genes were down regulated.Compared with the differential genes identified in the model group/normal control group,it was found that there were 106 differential genes in Xiaoyaosan's callback,and 16 significant callback pathways.These pathways involve ?-aminobutyric acid synapses,serotonergic synapses,glutamatergic synapses,and calcium signaling pathways.Xiaoyaosan may exert antidepressant effects by regulating Adcy8,Gad1,Gad2,Htr2 a,Chrna7,Erbb3,Map4k4,Nr4a1,Zfpm2,LOC100911372,Twist1,Ntng1,Robo3,and Slit2 on these pathways.The proteomics results showed that compared with the model group,there were 28 differentially expressed proteins in the hippocampus of the rats in the Xiaoyaosan administration group.Among them,13 are up-regulated proteins and 15 are down-regulated proteins.Compared with the differential proteins identified in the model group/normal control group,it is found that there are 11 differential proteins in Xiaoyaosan callback,including RGD1311899,Krt10,Ndufab1,Cplx1,Fkbp8,Pafah1b3,Ndufs6,Sh3bgrl3,Pmm2,Fth1,and Tbca.KEGG pathway analysis of differentially expressed proteins mainly involves pathways such as oxidative phosphorylation,synaptic vesicle circulation,lipid metabolism,fructose and mannose metabolism,amino sugar and nucleotide sugar metabolism,etc.Multi-omics integration analysis found that:transcriptomics and proteomics data results show that Xiaoyaosan exerts antidepressant effects by regulating the oxidative phosphorylation pathway of CUMS depressed rats.The specific mechanism is that Xiaoyaosan can significantly reverse the decrease of Mrcc ?activity in the hippocampus of CUMS depression rats,the level of Uqcrc2 mRNA is significantly decreased,and the level of Ndufs6 protein is increased.The results of transcriptomics and metabolic phenotype data indicate that Xiaoyaosan exerts antidepressant effects by regulating the glutamatergic synaptic pathway in CUMS depressed rats.The specific mechanism is that Xiaoyaosan can significantly reverse the increase of glutamate content in the hippocampus of CUMS depression rats.Xiaoyaosan can significantly reverse the decrease of glutamate decarboxylase 1(Gad1),glutamate receptor1(Glur1)activity,and the increase of glutamate synthetase(Gls)and glutamate NMDA receptor epsilon-1 subunit(Grin2a)activity.Xiaoyaosan can significantly reverse the significant decrease in Slc1a3 mRNA level,the decrease in Eaat2,Erk1/2,and CREB protein levels,and the increase in Nmdar1 and Mglur1 protein levels.(4)The ADME method was used to screen the active ingredients in Xiaoyao Powder for the first time.There are 16 active chemical ingredients in Xiaoyaosan,which have good pharmacokinetic characteristics.The contribution index of network pharmacology is further used to screen the contribution index of the seven components of Xiaoyaosan that are greater than the average contribution index.These components are 6-shogaol,Paeoniflorin,Ferulic acid,Ligustilide,Saikosaponin a,Saikosaponin c,and Liquiritigenin.Molecular docking verified that 7 components have a good affinity with the pathways GRM5 and HTR2 A.Molecular experiments further found that these active components of Xiaoyaosan may regulate Htr2 a,Nmdar1,Pkc,Camk ?,and Caspase-3 in the glutamatergic synaptic pathway.Conclusion:(1)The antidepressant metabolic phenotype of Xiaoyaosan contains 97 metabolites and48 pathways.Metabolic phenotype integration analysis found that Xiaoyaosan antidepressant has the consistency of regulating different metabolites,the network of metabolic phenotype pathways,and the modularity of metabolic phenotypic functions.The docking score-weighted pharmacological index is used to optimize the ranking of the pathways of the antidepressant metabolic phenotype of Xiaoyaosan.(2)Xiaoyaosan can regulate 106 genes at the transcriptional level and 11 proteins at the protein level to exert antidepressant effects.The integrated analysis of transcriptomics and proteomics found that Xiaoyaosan exerts an antidepressant effect by regulating the oxidative phosphorylation pathway of CUMS depressed rats.The integrated analysis of transcriptomics and metabolic phenotype data found that Xiaoyaosan exerts antidepressant effects by regulating the glutamatergic synaptic pathway in CUMS depressed rats.(3)7 active components(6-shogaol,Paeoniflorin,Ferulic acid,Ligustilide,Saikosaponin a,Saikosaponin c and Liquiritigenin)in Xiaoyaosan may exert antidepressant effects by regulating glutamatergic synaptic pathways.