| Objective:Based on the network pharmacology research,this project summarized the information of bioinformatics and common databases,combined with data mining and literature screening,to study the association between active ingredients,action targets and biological processes of Ganlong capsule,and to predict and screen its mechanism of action.We also used carbon tetrachloride（CCl4）to construct an animal model of liver fibrosis,and detected the differential proteins among rat liver tissue groups at the protein level by TMT quantitative proteomics technology,and used bioinformatics analysis methods to conduct functional analysis and protein interaction network analysis to investigate the key targets and pathways of the main active ingredients of Hepatosaurus capsule against liver fibrosis.Methods:1.The main compounds contained in Hepatron capsules were searched through a large amount of information,and the compounds were imported into Swiss Target Prediction database to speculate and screen the potential targets of the drugs.Dis Ge Net,Drug Bank,OMIM and Gene Cards databases were searched for potential targets related to liver fibrosis.The overlapping targets were the targets of hepatic fibrosis treatment with Ganlong capsule,and the overlapping targets were visualized using Cytoscape 3.9.0 software,which was combined with STRING 11.5 database to obtain PPI network relationships and plotted using Cytoscape software.The obtained targets were then imported into the DAVID database for relevant functional and pathway analysis,and the relevant pathways of Ganlong capsule against liver fibrosis were screened;2.Experimental animals were selected from SPF grade male SD rats,and CCl4was injected intraperitoneally into the rats to establish a liver fibrosis model;3.Four weeks of intervention with Ganlong capsule and colchicine as the test drug and positive control drug in rats with liver fibrosis;4.Observe and record the general status,body weight changes and the degree of liver pathological histological changes in each group of rats;5.Detection of four indicators of liver fibrosis and serum biochemical indicators of glutamic oxaloacetic transaminase（AST）and glutamic alanine transaminase（ALT）in rats by ELISA kit;6.TMT quantitative proteomics technique was applied to identify and quantify proteins in liver tissue specimens from rats in the normal group,liver fibrosis model group,colchicine group and hepatic dragon capsule group.The more prominent differential proteins among the total proteins were selected and functionally analyzed.Screening of proteins and pathways related to anti-liver fibrosis in Ganlong capsule based on GO functional analysis and KEGG pathway analysis.Results:1.Based on the literature search,52 active ingredients of Ganlong capsule were collected,and 44 possible active ingredient compounds of Ganlong capsule were screened by Swiss ADME database,and then the targets were predicted by Swiss Target Prediction database,and finally 265 drug targets were obtained,based on Drug Bank,OMIM,Gene Cards,Dis Ge Net database,1613 common targets were obtained,We obtained 1613 liver fibrosis-related targets based on Drug Bank,OMIM,Gene Cards,Dis Ge Net databases,and 97 common targets by combining the drug targets,which were imported into STRING 11.5 database to obtain PPI network diagram.Then,Cytoscape 3.9.0 was used to map the PPI network of target proteins again,and the targets in the PPI network were screened by the Degree value to predict the targets of liver fibrosis treatment with Hepalong capsules,and it was concluded that IL-6,HSP90AA1,CASP3,VEGFA,STAT3 and other targets might be the core targets for the drug treatment of liver fibrosis.Based on GO analysis,the biological process is mainly related to lipopolysaccharide response,protein phosphorylation,positive regulation of MAPK cascade,inflammatory response,ATP binding,enzyme binding,protein kinase activity,etc.KEGG enrichment analysis showed that the relevant targets mainly involve PI3K-Akt,MAPK,HIF-1,VEGF and other signaling pathways;2.After CCl4modeling,the rats in the model group showed the phenomenon of"fried hair",and the body hair was easily shed,and the liver fibers were severely adhered,dark red in color,and the liver index increased（P<0.05）.After treatment,the rats in the hepatosaurus capsule administration group showed improvement in physical signs and appetite,and the degree of liver adhesion was relieved,and the difference between the liver index and the normal group became smaller;compared with the normal group,the serum levels of transaminases and four items of liver fibrosis in the model group rats were significantly increased（P<0.05）,and after hepatosaurus capsule treatment,the serum levels of the above indexes were significantly reduced and tended to the normal group,and there was a dose dependence（P<0.05）,suggesting that Ganlong capsule can reduce the effect of liver fibrosis;3.The pathological staining results showed that Ganlong capsule could reduce liver fibrosis by reducing the abnormal proliferation of fibrous areas and inflammatory cell infiltration in the liver of rats with liver fibrosis,as well as improving collagen deposition and connective tissue proliferation in the liver of rats with liver fibrosis;4.The proteomic results showed that 6392 proteins were detected qualitatively and quantitatively using TMT tagging and LC-MS techniques.Compared with the model group,73 proteins were significantly differentially expressed in the experimental group of Ganlong capsule,covering 43 positively regulated proteins and 30 negatively regulated proteins.By bioinformatics gene ontology GO analysis,at the biological process（BP）level,Ganlong capsule were significantly correlated with cellular processes,bioregulation,metabolism and other processes;at the molecular function（MF）level,Ganlong capsule were significantly correlated with binding,transport and other functions At the cellular component（CC）level,Heparone capsule was significantly correlated with cellular and organelle components,suggesting that Heparone capsule exhibits anti-fibrotic effects through numerous targets and pathways.Based on the KEGG enrichment analysis of Ganlong capsule,significant changes were observed in important pathways such as chemo-carcinogenesis-reactive oxygen species,oxidative phosphorylation,HIF-1 and TGF-βsignaling pathways,and it was hypothesized that the mechanism of anti-fibrotic effect of Ganlong capsule involved amino acid synthesis and metabolism,regulation of extracellular matrix and apoptosis process.The protein interaction network analysis concluded that there may be a functional correlation between Heparagon capsules and Arg1,Cps1,Mat1a,Sds,Tdo2,Gls2.Conclusions:1.The network pharmacology initially predicted that IL-6,HSP90AA1,CASP3,VEGFA,STAT3 and other targets are the core targets of the drug for the treatment of liver fibrosis,and PI3K-Akt,MAPK,HIF-1,VEGF and other signaling pathways are the core pathways for the treatment of liver fibrosis,showing the diverse composition of Ganlong capsule with many targets and pathways,which provides a new way of thinking for It provides new thinking for the subsequent in-depth analysis of the pharmacological study of the anti-liver fibrosis effect of Ganlong capsule.2.Ganlong capsule are effective in reducing liver fibrosis in a dose-dependent manner.3.The proteomic study of Ganlong Capsule in anti-liver fibrosis found that the drug plays an anti-liver fibrosis role through oxidative phosphorylation,TGF-β,HIF-1and other signaling pathways,TGF-β,HIF-1 and other signaling pathways to ameliorate CCl4-induced liver fibrosis in rats.Further analysis showed that Arg1,Cps1,Mat1a,Sds,Tdo2 and Gls2 are the key proteins of Ganlong capsule in the treatment of liver fibrosis,and exert anti-fibrotic effects through the above proteins. |
