Study On The The Effects Of Yiyi Fuzi Baijiang Powder On Apoptosis And Autophagy In Colon Cancer Cells Based On Network Pharmacology

Objective:The possible mechanisms of action of Yiyi Fuzi Baijiang Powder in the treatment of colon cancer were predicted in this research using network pharmacology,and the potential mechanisms were preliminary confirmed by in vitro experiments.Methods:The active components of each traditional Chinese medicine and their associated targets were screened out of Yiyi Fuzi Baijiang Powder sauce powder recipe using the TCMSP database.Colon disease targets are found using the datasets Gene Cards,Dis Ge NET,and Drug Bank,and differentially expressed genes from healthy and colon cancer samples are found using limmabao.Drug targets,disease targets,and differential genes were intersected to create intersection targets.To explore the pathways and gene ontology(GO)involved in the intersection target,enrichment analysis based on GO and the Kyoto Encyclopedia of Genes and Genomes was managed to perform using the R software cluster Profiler package.The intersection targets enriched to the top 10 KEGG and the top 10 GO were extracted separately,the intersection target-pathway network relationship was sorted out,and the network was beautified by Cytoscape software to obtain the intersection target-pathway network diagram.The intersection target-active ingredient-intersection target-disease network relationship was extracted separately,and the network was beautified using Cytoscape software to produce the traditional Chinese medicine-active ingredientintersection target-disease network diagram.We create a PPI(protein interaction relationship)network for the intersection targets using the STRING website to investigate whether there is an interaction between them.Protein crystal structure database,Auto Dock Tolls program,and organic small molecule biological activity data(Pub Chem)database were used to complete the molecular docking verification process.Vitro experiments were used to explore the effect of Yiyi Fuzi Baijiang Powder on colon cancer cells: human colon cancer cell lines(HCT116 and T-29)were cultured in vitro,and different doses(0 mg/ml,10 mg/ml,20 mg/ml,30 mg/ml,40mg/ml,50 mg/ml)were used to treat the cell for 24 h and 48 h,and the effect of Yiyi Fuzi Baijiang Powder on the proliferation of HCT116 and HT-29 cells was detected by CCK8 method.These apoptosis assays were detected using the TUNEL Apoptosis Assay.In vitro cultured human colon cancer cell lines(HCT116 and HT-29),two types of cells were treated with different doses(0 mg/ml,10 mg/ml,20 mg/ml,30mg/ml)of Yiyi Fuzi Baijiang Powder for 48 h,and Western Blot was used to detect the protein expression level of autophagy-related proteins like Beclin1,LC3,P62,and the protein expression level of MMP9,PI3 K,p-PI3 K,AKT,and p-AKT proteins.Results:A total of 192 drug targets and 1374 colon cancer disease targets were screened out from the database.Differential analysis using limma packages yielded 1717 differential genes.31 intersection targets were obtained by intersecting drug targets,disease targets and differential genes.KEGG and GO showed that Yiyi Fuzi Baijiang Powder can restrain colon cancer through a variety of pathways,including cell cycle,aging,autophagy,P53 signaling pathway,PI3K/AKT pathway,etc.PPI protein interaction analysis on intersection targets showed that genes such as CCND1,CDKN1 A,CXCL8,MMP9,and MYC were located at the center of protein interaction and had more connections than other proteins.The molecular docking of CCND1,CDKN1 A,CXCL8,MYC and MMP9 genes with the active molecules scattered by Yiyi Fuzi Baijiang Powder showed that the above genes could successfully dock with the active molecule and had high binding affinity.The results of in vitro experiments showed that: 1)Compared with the control group(0 mg/ml),10 mg/ml,20 mg/ml,30 mg/ml,40 mg/ml and 50 mg/ml concentrations of Yiyi Fuzi Baijiang Powder could significantly inhibit the growth of HCT116 and HT-29 cells,and it was dose-dependent.2)TUNEL cell apoptosis detection experiments showed that compared with the control group(0mg/ml),10mg/ml,20 mg/ml,30 mg/ml,40 mg/ml,50 mg/ml concentrations of Yiyi Fuzi Baijiang Powder were dose-dependent to promote the apoptosis of HCT116 and HT-29 cells.3)The results of Western blot experiments showed that the appropriate concentration of Yiyi Fuzi Baijiang Powder significantly increased the expression of proteins closely related to autophagy such as LC3 in HT-29 cells(P<0.05),significantly inhibited the expression of P62,MMP9 protein and significantly inhibited the expression of p-PI3 K and p-AKT proteins,which are in PI3K/AKT pathway(P<0.05).Conclusion:Network pharmacology confirmed that the mechanism of action of Yiyi Fuzi Baijiang Powder in the treatment of colon cancer through multiple targets and multiple pathways,the main mechanisms are related to cell cycle,aging,autophagy,P53 signaling pathway,PI3K/AKT pathway,and regulation of CCND1,CDKN1 A,MMP9,CXCL8,and MYC proteins.In vitro experiments have verified that the treatment of colon cancer with the PI3K/AKT pathway,MMP9 protein expression and autophagy are verified.Therefore,it is speculated that the dispersion of Yiyi Fuzi Baijiang Powder may be related to the PI3K/AKT pathway and MMP9 protein regulating autophagy,thereby promoting apoptosis in colon cancer cells.