Study On The Mechanism Of Yinchenhao Decoction And Yinzhihuang Granule In Inhibiting Hepatitis-cancer Transformation Based On Bioinformatics

BackgroundChronic infection of viral hepatitis may lead to liver cirrhosis and liver cancer.In the process of diagnosis and treatment of clinical diseases,chronic hepatitis B(C)-cirrhosishepatocellular carcinoma is a common law of disease development and transformation.This law of disease development and transformation can be regarded as a typical process of"inflammatory cancer transformation".Yinchenhao Decoction(YCHD)is from Treatise on Febrile Diseases written by Zhang Zhongjing in Eastern Han Dynasty.It is composed of Herba Artemisiae Scopariae,Fructus Gardeniae and Radix et Rhizoma Rhei,and is the main prescription for treating damp-heat jaundice in TCM.Yinzhihuang Granule(YZHG)is a modern pharmaceutical dosage form based on YCHD.The formula is composed of extracts of four traditional Chinese medicines including Herba Artemisiae Scopariae,Fructus Gardeniae,Scutellariae Radix and Flos Lonicerae,and has the effects of clearing away heat and toxic materials,promoting diuresis and eliminating jaundice.The results of modern pharmacological studies show that YCHD has a significant protective effect on liver and gallbladder,and also has the functions of inhibiting liver fibrosis and resisting tumor.However,due to the complex composition of the formula,its specific mechanism of action is difficult to explain clearly.Analyzing the multi-component,multi-target,multi-channel synergy of YCHD from the perspective of network is helpful for further developing the basic experimental research of YCHD and expanding the application range of YZHG.Therefore,it is of great academic significance and clinical value to study the key genes and mechanism of liver cancer transformation caused by hepatitis B(C)by integrated bioinformatics,and the mechanism of YCHD and YZHG inhibiting liver cancer transformation caused by hepatitis B(C).ObjectiveIn this study,bioinformatics analysis was used to analyze the key genes and mechanism of liver inflammatory cancer transformation caused by hepatitis B(C).From the perspective of network,this paper comprehensively analyzes the synergistic effect of YCHD on inhibiting the transformation of liver inflammation and cancer caused by hepatitis B(C),hoping to provide reference for further basic experimental research of YCHD.In addition,based on the method of bioinformatics,this study hopes to predict the mechanism of YZHG inhibiting the transformation of liver inflammatory cancer caused by hepatitis B(C),and provide reference for further expanding the application scope of drugs.Methods1.Bioinformatics analysisGet the related gene chip data sets that meet the screening requirements in GEO database,and use limma package to analyze the differential expression of the screened gene chip data sets,and get the differentially expressed genes with statistical differences.Using STRING database to construct the protein interaction relationship of differentially expressed genes,the related protein information was obtained,and the protein interaction network was constructed in Cytoscape software.MCODE plug-in in Cytoscape software can be used to cluster and build functional modules in a huge gene network.GO and KEGG enrichment analysis were carried out on key differentially expressed genes,and functional annotations of the differentially expressed genes in multiple levels were obtained.In addition,KM plotter,GEPIA and other data websites are used to analyze the survival,expression level and relevance of the key differentially expressed genes.2.Network pharmacological and molecular docking analysisThe research literature about the related chemical components of YCHD and its variant YZHG was collected by searching the database,and the chemical components of YCHD and its variant were imported into PubChem database to obtain the simplified molecular linear input specification information(SMILES)of Chinese medicine chemical components,which was imported into SuperPred,SwissTargetPrediction and other databases to obtain the known or predicted targets of compounds.Merge the above data in Cytoscape,and obtain the potential targets of TCHD and YZHG to inhibit the transformation of liver inflammation and cancer caused by hepatitis B(C).Further analyze the potential targets obtained from the above analysis in the STRING database,obtain the protein interaction relationship of the potential targets,and construct the protein interaction network.MCODE,CytoHubba and other plug-ins can be used for modular analysis of protein interaction networks of potential targets.In addition,GO and KEGG enrichment analysis of key potential targets was carried out using DAVID database.The key potential targets and corresponding compounds were verified by molecular docking using autodock software.The chemical structure of small molecule drugs was downloaded from PubChem database,and converted into a file in mol2 format.After adding electric charge,it was saved as a pdbqt file.Screening protein conformation in pdb database,downloading PDB format files,deleting water molecules,separating original ligand small molecules,and performing hydrogenation and charging operations.Then determine the position of the active pocket.The PDB database includes the atomic three-dimensional structure of biological macromolecules(protein,DNA,RNA)determined by experiments.Autodock Vina is used to perform docking operations on ligands and receptors.According to the Grid Box coordinates and box size determined in the Autodock software operation,the components are screened and sorted according to the binding free energy.Finally,the receptor-ligand complex obtained by docking was visually analyzed in Pymol software.Results1.Bioinformatics analysis resultsIn the study of the mechanism of transformation of hepatitis B inflammatory cancer,22 overlapping differentially expressed genes were screened through comprehensive analysis of GEO data set and TCGA data of gene expression profile.In order to clarify their potential biological functions,22 overlapping differential genes were annotated.GO entries showed that overlapping differential genes were enriched in cell division,mitotic sister chromatid separation and nuclear entries.In order to further analyze the pathogenic mechanism of HBV,KEGG pathway of 22 overlapping differential genes was analyzed.The enrichment results of KEGG pathway showed that overlapping differential genes were mainly enriched in meiosis pathway and cell cycle pathway of oocytes.In this study,five key genes(CDK1,MAD2L1,CCNA2,PTTG1,NEK2)were identified.In addition,the prognostic gene markers composed of PTTG1,MAD2L1,RRM2,TPX2,CDK1,NEK2,DEPDC1 and ZWINT were constructed,which performed well in predicting the overall survival time.In the study of the transformation mechanism of hepatitis C inflammatory cancer,a microarray data set of oncogene expression profiles of hepatitis C-related cirrhosis and hepatitis C-related hepatocytes was selected from GEO data set of gene expression profiles.Integration analysis revealed six key genes closely related to the pathogenesis and prognosis of hepatocellular carcinoma:CCNA2,CCNB1,CCNB2,CDC20,CDK1,TOP2A.Six key genes were negatively correlated with the survival time of HCC patients,while the expression level of core genes was positively correlated.GO items of differential genes in key modules show that they are mainly enriched in chromosome separation in biological processes,cyclindependent kinase activity in molecular functions,and spindle in cellular components.The results of KEGG pathway showed that the differential genes were mainly concentrated in cell cycle,progesterone-mediated oocyte maturation,oocyte meiosis and cell aging.2.Results of network pharmacology and molecular docking analysisIn the study on the mechanism of YCHD inhibiting the transformation of liver inflammation and cancer caused by hepatitis B(C),44 effective components in YCHD were obtained by TCMSP search.Searching SMILES values corresponding to 44 effective components,and importing them into Superpred and SwissTargetPrediction databases for target prediction to obtain 510 targets.Use Cytoscape to build the network.The results show that there is a common target MMP2 in the process of inhibiting hepatitis B and hepatitis C by YCHD.YCHD may play an important role in the intervention of hepatitis B and its inflammatory cancer transformation.AURKA,CCNB2,CCNB1,CDK1,TOP2A may all play an important role in the process of YCHD inhibiting the transformation of hepatitis B and hepatitis C cirrhosis into hepatocellular carcinoma.KEGG pathway enrichment showed that key genes of modules 1,2 and 4 were mainly enriched in progesterone-mediated oocyte maturation pathway,oocyte meiosis pathway and p53 signal pathway.The key genes of module 3 are enriched in JAK-STAT signaling pathway,EGFR tyrosine kinase inhibitor resistance pathway,hepatitis C and so on.This may be related to the fact that the data source of module 3 is disease database,which shows that there are some limitations in the data source of our research.In the study of the mechanism of YZHG in treating hepatitis B,the compounds of YZHG were obtained from CNKI and PubMed databases,and the targets corresponding to the compounds were predicted by searching Superpred and SwissTargetPrediction databases.The target data of hepatitis B virus comes from TTD,PharmGKB and DisGeNET databases.Use Cytoscape 3.7.1 to visually analyze the above data.The biological network identified 13 potential targets.Molecular docking verification showed that CDK6,CDK2,TP53 and BRCA1 may be closely related to hepatitis B treatment.In addition,GO and KEGG analysis showed that YZHG may be related to the positive regulation of transcription,gene expression,hepatitis B pathway and viral carcinogenesis pathway.In the mechanism of YZHG inhibiting the transformation of liver inflammation and cancer caused by hepatitis B(C),firstly,the components of YZHG were searched in CNKI and PubMed,and then the compounds and their corresponding targets were predicted by searching Superpred and SwissTargetPrediction databases.The differential genes of liver inflammation and cancer transformation caused by hepatitis B(C)were obtained from TTD,PharmGKB and GEO databases.Use Cytoscape 3.7.1 to construct compounds-prediction target network and key module network.The results showed that YZHG had a common target MMP 2 in the treatment of hepatitis B and hepatitis C;YZHG may play an important role in the intervention of hepatitis B and its inflammatory cancer transformation and the transformation of hepatitis CDK1 cirrhosis into hepatocellular carcinoma.KEGG pathway enrichment showed that key genes of modules 1,2 and 4 were mainly enriched in progesterone-mediated oocyte maturation pathway and oocyte meiosis pathway.ConclusionIn this study,the methods of network pharmacology and bioinformatics were used to reveal the chemical composition,target and signal pathway of YCHD and YZHG in inhibiting the transformation of liver inflammatory cancer caused by hepatitis B(C).It is preliminarily explained that the mechanism of YCHD and YZHG inhibiting the transformation of liver inflammation and cancer caused by hepatitis B(C)may be related to synergistic regulation of several key targets and pathways.This study can provide clues and ideas for the study of the mechanism of action of traditional Chinese medicine in treating liver inflammatory cancer transformation,and provide reference for further developing the basic experimental study of YCHD and YZHG in inhibiting liver inflammatory cancer transformation and promoting the rational application of YCHD and YZHG in clinic.