Bioinformatics Analysis And Experimental Research Of Depression Regulatory Network And Xiaoyaosan Antidepressant Module

1 BackgroundBioinformatics,as a discipline formed by the combination of life science and computer and information science,provides new ideas and methods for the storage,retrieval and analysis of biological data of human genome.Under the background of 'Big Data' era,its application is not limited to the research of human genome project,but involves genomics,proteomics,comparative genomics,macrogenomics,gene and protein expression and analysis,biochip expression spectrum analysis,protein interaction network,biological system simulation,system biology and network biology research and other aspects.Bioinformatics studies frontier and hot issues related to disease and health from the perspective of system,information and complexity at the level of molecular biology,and promotes the development of life science and even natural science.Depression is a disease name of modern medicine.Although there is no 'Depression'in traditional Chinese medicine,its clinical manifestations can be classified into'Depression disease','Hysteria' and 'Lily disease'.At present,the pathogenesis of depression is still lack of complete understanding,clinical also lack of relatively objective detection indicators.In the treatment of depression,nearly half of the patients had no significant effect.Therefore,using bioinformatics methods to develop the transcription factor-miRNA-lncRNA regulatory network,which is closely related to the diagnosis and treatment of depression,is of great significance to the mechanism research,diagnosis and treatment of depression.The Xiaoyao Powder contains eight kinds of Chinese materia medica,which has the functions of dispersing stagnated liver Qi for relieving Qi stagnation,nourishing blood and invigorating the spleen.The application of Xiaoyao Powder has been extended to clinical departments,including many kinds of physical and mental diseases.In the early stage,aiming at the possible pathogenesis of depression,the research has been focused on synaptic plasticity,HPA axis,neurotrophic factors,neuron microstructure,brain gut peptide,intestinal flora and metabonomics,etc.Part of the mechanism of antidepressant effect of Xiaoyao Powder has been revealed with the characteristics of 'multi-component,multi-target and multi-pathway'.However,due to the characteristics and complexity of'multi-component,multi-target and multi-pathway',the research of antidepressant mechanism of Xiaoyao Powder is facing difficulties and challenges.Specifically,these eight Chinese materia medica of Xiaoyao Powder contain more than hundreds of compounds,which can interact with each other.So,which components of these complex compounds in Xiaoyao Powder act on which targets,which specific regulatory pathways,and which systems and pathways are involved?Can the material basis and mechanism of the efficacy of many traditional Chinese medicine formulas,including Xiaoyao Powder,be explained in the language of modern medicine?This series of problems are difficult to use the traditional experimental means to study,but also become the bottleneck in the study of the material basis and mechanism of the modern Chinese medicine compound.In recent years,Hopkins,a British pharmacology Professor,put forward the concept of 'Network Pharmacology',using bioinformatics methods to analyze the synergistic relationship between drugs,diseases and targets of 'multi-component,multi-target and multi-pathway',which is consistent with the 'Holism' of traditional Chinese medicine and the characteristics of 'multi-component,multi-target and multi-pathway' of Chinese materia medica,which has opened up a new research idea for the study of the pharmacodynamic substance basis of traditional Chinese medicineTherefore,on the basis of our previous work,with the aid of bioinformatics analysis methods,we carried out the construction and analysis of Xiaoyao Powder related networks,analyzed the active ingredients and potential targets,predicted the biological processes and signal pathways that may be involved,and explored the potential pharmacological mechanism.Finally,combined with the results of bioinformatics,we carried out targeted animal experiments to verify.The material basis and mechanism of Xiaoyao Powder were revealed with higher efficiency and more scientific strategy.2 MethodsThis study is mainly composed of bioinformatics analysis and experimental research.Bioinformatics analysis:(1)Retrieval of gene chip expression data set of depression patients in GEO database;data preprocessing and differential expression analysis;screening of potential depression related genes;construction of weighted gene coexpression network and identification of significant feature function modules;functional enrichment analysis of significant feature function modules and construction of protein interaction network;construction of transcription factor-miRNA-lncRNA regulatory network;cluster analysis and molecular typing;(2)Jointly retrieve the information of Xiaoyao Powder in TCMID,ETCM and TCMSP databases;to construct the network of'component target pathway/disease';to retrieve the target information related to depression in CTD database;to construct the network of 'component-target' and 'compound target-depression disease target' of Xiaoyao Powder;to construct the clustering function module of 'compound target-depression disease target';bioinformatics analysis of clustering function significant module;ADME based active component screening and virtual molecular dockingExperimental research:(1)The depression model rats were induced by CUMS for 6 weeks.The model was evaluated by macroscopical characterization,weight,open field experiment,sugar water preference experiment and forced swimming experiment,and the intervention effect of Xiaoyao Powder was observed;(2)The expression of CNR1,CNR2 and BDNF protein in prefrontal cortex of rats in each group was detected by immunohistochemistry;the expression of Cnrl mRNA,Cnr2 mRNA and Bdnf mRNA in prefrontal cortex of rats in each group was detected by real-time fluorescence quantitative PCR;the content of camp,PKA and p-CREB in prefrontal cortex of rats in each group was detected by ELISA3 ResultsBioinformatics analysis:(1)Twenty eight depression related genes were screened out,a weighted gene coexpression network of depression was constructed,and a significant function module negatively related to depression was identified;there were significant enrichment in 5 KEGG pathways and 11 gene ontological;five core genes(FOS,GNGT1,JUN,EGR1 and CCl4)were screened from protein-protein interaction network;the transcription factor-miRNA-lncRNA multi-factor regulatory network of depression was constructed;according to the 'degree' value of the multi-factor regulatory network,the top ten genes were analyzed,including three core genes,six lncRNAs and one miRNA;according to the differential expression of five genes(TCTEX1D4,AREG,C6orf222,PPP1R15A and TNFSF9)in depression samples,it is found that depression can be further divided into two types;(2)The 'component-target-pathway/disease' network,the'component-target' and 'compound target-depression disease target' network of Xiaoyao Powder were constructed and bioinformatics analysis was carried out;the 'compound target-depression disease target' had 23 modules by network module cluster analysis,among which 8 modules contained the common target of Xiaoyao Powder and depression;module 1 has the highest score,including common target and most significant enrichment pathway CNR2 and cAMP signaling pathway,respectively;according to the results of ADME module screening and virtual molecular docking experiments,ten active compounds have high affinity with four common targets of Xiaoyao Powder and depression.Experimental research:(1)The depression model rats were successfully reproduced by CUMS 6 weeks.After drug administration,the depression like behavior of rats in Xiaoyao Powder group was relieved.Compared with the model group,the rats in Xiaoyao Powder group were generally in a better state,with significant weight growth.In the sugar water experiment,the sugar water preference rate was increased.In the open field experiment,the total distance of exercise,the time of staying in the central area and the times of entering the central area were increased,and the immobility time in the forced swimming experiment was shortened;(2)The expression of CNR1,CNR2,BDNF,cAMP,PKA and p-CREB,the Cnrl mRNA,Cnr2 mRNA and Bdnf mRNA in prefrontal cortex in model group were significantly lower than those in normal group;compared with the model group,the expression of CNR1,CNR2,BDNF,the Cnrl mRNA,Cnr2 mRNA and Bdnf mRNA in prefrontal cortex of rats in Xiaoyao Powder group were significantly increased;the content of cAMP,PKA and p-CREB in prefrontal cortex of rats in Xiaoyao Powder group increased significantly.4 ConclusionBioinformatics analysis:Through the integration of bioinformatics analysis results,FOS,GNGT1,JUN,EGR1 and CCL4 were selected as the core genes related to depression.FOS,PTGS2,JUN,XIST,NEAT1,SNHG16,NORAD,MALAT1,ARHGAP27P1-BPTFP1-KPNA2P3 and hsa-miR-106a-5p were the key genes in the multi factor regulatory network of transcription factor-miRNA-lncRNA,TCTEXID4,AREG,C6orf222,PPP1R15A and TNFSF9 are identified as the potential genes to distinguish depression molecular typing;(2)Eight modules including the common target of Xiaoyao Powder and depression were obtained by network cluster analysis.The specific targets involved were CNR2,HTR2A,HTR7,DRD5,SLC6A4,IL2,PTGS2,CAT,ADORA2A,MTHFR,XDH,NR3Cl and ACS14.The most significantly enriched KEGG pathways were cAMP signaling pathway,neuroactive ligand receptor interaction signaling pathway,glutathione signaling pathway metabolic pathway,cocaine addiction,nonalcoholic fatty liver,purine metabolism,longevity regulation,peroxisome proliferator activated receptor signaling pathway,etc.It shows that the antidepressant effect of Xiaoyao Powder has the characteristics of 'multi-component,multi-target and multi-pathway';(3)Through ADME module screening and virtual molecular docking experiments,it was found that the ten active compounds and four active compounds contained in Xiaoyao Powder have high affinity with the common target of depression,which are potential active compounds with antidepressant effect.Among them,the compounds with high affinity to target CNR2 are Phaseolinisoflavan,3'-Methoxyglabridin and Liquidigenin;the compounds with high affinity to target NR3Cl are Isoramanone,Gamma sitosterol,Beta sitosterol and Pyrethrone ?.The compounds with high affinity to target SLC6A4 are Tetrahydropalmatine,and the compounds with high affinity to target CAT are Eburicol and Stigmasterol.Experimental research:On the basis of bioinformatics analysis,Xiaoyao Powder can affect the expression of Cnrl mRNA and Cnr2 mRNA in prefrontal cortex of CUMS depression model rats,and may play an antidepressant role by increasing the level of BDNF through cAMP-PKA-CREB pathway.