Screening Of Flavonoids From Astragalus Membranaceus Against HepG2 Cells Based On Network Pharmacology And Its Mechanism

Objective:This article uses network pharmacology methods and molecular docking technology to predict the six flavonoids of Astragalus membranaceus against liver cancer and their potential targets,and uses cell biology,molecular biology and gene chip technology to pass in vitro cell experiments,Verified the inhibitory effect of the above-mentioned components on HepG2 cells,among which Jaranol has a more significant effect;further using RT-qPCR,WB and other methods,the anti-liver cancer effect of Jaranol and its regulation of PI3K-Akt signal pathway and promotion Hepatocellular carcinoma cells are related to apoptosis.The research results of the thesis will help promote the development and utilization of astragalus flavonoids and their potential application value.Methods:The main components of Astragalus membranaceus were obtained by TCMSP database,the component targets were predicted by Swiss Target Prediction,the liver cancer targets were collected by Genecards database and OMIM database,the anti-liver cancer targets were mapped by Venny phase,and the protein interaction network(PPI)and"Astragalus membranaceus-component-pathway-liver cancer" interaction network of liver cancer targets were drawn by String database and Cytoscape 3.7.2 software.DAVID database was used to analyze the functional enrichment and pathway enrichment of core target genes.Surflex-Dock software was used to verify the molecular docking between the key components and core targets of Astragalus membranaceus.The effects of quercetin,Calycosin,kaempferol,formononetin,isorhamnetin and Jaranol on hepatoma cells(HepG2)were detected by MTT.The relative levels of gene expression of TP53,MAPK1,AKT1,IL6,MAPK8 and VEGFA were verified by RT-qPCR.MTT method was used to screen the low,middle and high concentration of Jaranol on hepatoma(HepG2)cells.Flow cytometry was used to detect the apoptosis of hepatoma(HepG2)cells induced by Jaranol.The contents of apoptotic proteins Bcl-2 and Bax were detected by,Western Blot.Transcriptome comparison of blank group and drug group,analysis of HepG2 cells treated with Jaranol on gene differential expression and signal pathway changes;RT-qPCR was used to verify the changes of relative mRNA content of related genes after metabolic pathway was regulated.Results:In this study,20 active components of Astragalus membranaceus were screened,involving 202 action targets and 100 KEGG signal pathways.GO analysis showed 487 biological functions.Network pharmacology analysis showed that Astragalus membranaceus may have anti-hepatoma effect through key targets such as TP53,MAPK1,AKT1,IL6,MAPK8 and VEGFA.Molecular docking shows that there is a certain combination between the target and the composition.MTT showed that the inhibitory effect of Jaranol on hepatoma cells(HepG2)was stronger than that of the other five components.RT-qPCR showed that the expression of 6 genes was up-regulated by different concentrations of Jaranol,which was consistent with the genes involved in KEGG pathway analysis.Jaranol significantly inhibited the proliferation of HepG2 cells in a time-and concentration-dependent manner.The results of flow cytometry showed that as the concentration of Jaranol increased,the rate of apoptosis increased,and the apoptosis rate of 75umoL concentration group reached 40%.Western Blot results showed that Jaranol could up-regulate Bax protein and down-regulate Bcl-2,in HepG2 cells.Apoptosis of hepatoma cells can be inhibited.The cells in the treated group were sequenced by transcriptome RNA and the results were analyzed.First of all,after the sample RNA purity is qualified,the overall quality of RNA-seq is evaluated,and then through gene volcano map and cluster map analysis,it is found that there are significant differences in gene expression after drug treatment,and further explore the changes of pathway after drug treatment.KEGG analysis showed that the PI3K-Akt signal pathway treated with Jaranol was significantly different from that of the blank group,and it ranked first in the pathway According to the literature,PI3K-Akt signal pathway is related to apoptosis.RT-qPCR confirmed the reliability of the sequencing results,and the six genes were all down-regulated,indicating that Jaranol inhibited HepG2 cell apoptosis,which may have affected the expression of some genes in the PI3K-Akt signaling pathwayConclusion:The mechanism of flavonoids from Astragalus membranaceus on hepatoma(HepG2)cells was screened based on network pharmacology.It was found that Jaranol had obvious anti-tumor effect,and the molecular mechanism of anti-hepatoma cells was found and proved,which not only provided a basis for anti-hepatoma,but also provided a reference for the future anti-hepatoma research of flavonoids.