| Objective:Based on the method of network pharmacology and in vitro experimental verification,to explore the inhibitory effect of Siraitia grosvenorii kaempferol combined with Bacillus subtilis peptidoglycan on the proliferation of HepG2 cells and its possible mechanism.Methods:（1）The effective components and target prediction of Siraitia grosvenorii were screened by TCMSP database,the hepatocellular carcinoma related targets were searched by Genecards and other databases,and the two targets were obtained by Venny platform,and the"drug-component-disease-target"network map was constructed by Cytoscape software to screen the core components.According to the potential targets,the functional enrichment analysis of gene ontology（GO）and the enrichment analysis of genome encyclopedia（KEGG）pathway were carried out through DAVID database,and the core targets were obtained by biomolecule functional annotation system database and Cytoscape software.（2）The optimal concentration and ratio of kaempferol and peptidoglycan to inhibit the proliferation of HepG2 cells were screened by MTT assay,and the effects of peptidoglycan combined with kaempferol on the migration,apoptosis and expression of related molecules of HepG2 cells were studied by morphological observation,scratch test,flow cytometry,Western blot and qRT-PCR.Results:（1）Results of network pharmacology:11 active components and77 potential targets of Siraitia grosvenorii were screened by TCMSP database,7773 therapeutic targets of hepatocellular carcinoma were screened by Genecards database,62 potential targets of Siraitia grosvenorii in the treatment of hepatocellular carcinoma were obtained by Venny analysis,according to the"drug-component-disease-target"network map,the key active components of Siraitia grosvenorii were kaempferol,β-sitosterol,perlolyrine,mannitol and so on.PPI network analysis showed that the core targets of Siraitia grosvenorii in the treatment of hepatocellular carcinoma were AKT1,TNF,MAPK8,JUN,PTGS2,etc.GO functional enrichment analysis showed that BP,CC and MF were mainly related to organic cyclic compound reaction,positive regulation of apoptosis,membrane raft,enzyme binding and so on.KEGG pathway enrichment analysis showed that Siraitia grosvenorii’s treatment of hepatocellular carcinoma was mainly related to cancer signal pathway,hepatitis B signal pathway,TNF signal pathway,apoptosis signal pathway and so on.（2）The results of in vitro experiment:the results of MTT experiment showed that the best time and effective concentration of peptidoglycan combined with kaempferol to inhibit the proliferation of HepG2 cells were 48h,40μg·mL-1 and40μmol·L-1,respectively.Compared with kaempferol and peptidoglycan alone,the morphology of HepG2 cells became round,shrunken and refractive enhanced,the number of dead floating cells increased,the cell mobility decreased and the apoptosis rate increased.Western blot detection showed that the expression level of Bax was significantly up-regulated,while the expression levels of Bcl-2 and p-AKT1 were significantly down-regulated.QRT-PCR results showed that the expression level of Bax mRNA was significantly increased and the expression level of Bcl-2 mRNA was significantly decreased.Conclusions:（1）Network pharmacological analysis shows that the main components of Siraitia grosvenorii kaempferol,β-sitosterol,perlolyrine and mannitol have potential effects in the treatment of hepatocellular carcinoma through key targets such as AKT1,TNF,MAPK8,JUN and PTGS2,and its mechanism may be related to the regulation of cancer-related signal pathways by biological processes such as organic cyclic compound reaction,positive regulation of apoptosis process and enzyme binding.（2）The combination of peptidoglycan and kaempferol can significantly improve the inhibitory effect of peptidoglycan and kaempferol on the proliferation and apoptosis rate of HepG2cells,and reduce the ability of cell migration,and its mechanism may be related to the regulation of the expression of Bax,Bcl-2 and p-AKT1. |
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