| Background:According to the World Health Organization(WHO)World Cancer Progress Report 2020,colorectal cancer(CRC)is the third most prevalent and second most deadly disease in the world,and a serious threat to human health.The prevention and treatment of CRC is therefore particularly important.Research has shown that natural phytochemicals are effective in the prevention and adjuvant treatment of tumours,with low toxicity,few side effects and milder interventions.A number of studies have shown that Anthocyanins,which are derived from vegetables,fruits and other plants,are useful in the prevention and treatment of many types of tumours.Delphinidin(Dp)is an important active monomer in anthocyanins,with anti-inflammatory,antioxidant and anti-cancer properties,which are in high abundance in anthocyanins,but the effects of Dp on CRC cells and its mechanism are not fully understood and need to be further explored.Objective:The effect of Dp on colorectal cancer cells SW480 was investigated by in vitro cellular assays.We also analyzed the action targets of Dp through network pharmacology,matched the drug’s action targets with those of CRC disease by correlation,and searched for possible major action targets and molecular mechanisms of Dp’s action on CRC,with a view to providing new evidence for the preventive use of Dp against CRC and new support for network pharmacology to predict the action mechanisms of natural phytochemicals.Methods:1.The effect of Dp on SW480 cells was investigated by in vitro cellular assays using CCK-8 assay to detect cell proliferation;morphology and Hoechst 33258 staining assay to detect apoptosis;flow cytometry to detect cell cycle;scratch assay and Transwell to detect cell migration.2.To explore the possible molecular mechanisms of Dp against CRC based on network pharmacology;to identify the interaction between Dp and CRC in PubChem,Pharmmapper,SwissTargetPrediction,GenCard and OMIM databases,to map the interaction between Dp and CRC using Cytoscape software and STRING database.We used Cytoscape and STRING to map the "drug-disease-target" interaction network and to establish a protein interaction network to screen for core targets.GO and KEGG functional enrichment analyses were performed on key genes in the regulatory network to identify possible pathways of action.In addition,the core targets and pathways were initially validated by molecular docking and protein blotting assays.Results:1.Dp has a proliferative inhibitory effect on CRC cells SW480 in vitro(1)The effect of Dp on the proliferation of SW480 cells:After 48 h of administration,the cell viability of 13 μmol/L,15 μmol/L,18 μmol/L and 20 μmol/L Dp groups were 57.43%,52.60%,39.50%and 27.95%respectively,which were significantly decreased compared with the control group,indicating that Dp could inhibit the proliferation of SW480 cells and This indicated that Dp could inhibit the proliferation of SW480 cells in a concentration-dependent manner.(2)The effect of Dp on the morphology of SW480 cells:SW480 cells in the 15μmol/L and 20 μmol/L Dp groups were gradually loosened,the number of adherent walls decreased,the morphology changed from shuttle-shaped to round,and the number of floating cells increased after 48 h of administration.(3)The effect of Dp on apoptosis of SW480 cells:After staining with Hoechst 33258,it was found that SW480 cells in the 15 μmol/L and 20 μmol/L Dp groups showed the typical characteristics of apoptosis with dark and bright blue nuclei compared with the control group.(4)The effect of Dp on SW480 cell cycle:The number of SW480 cells in S-phase increased in the 15 μmol/L and 20 μmol/L Dp groups compared with the control group,indicating that Dp could block SW480 cells in S-phase.(5)The effect of Dp on the migratory ability of SW480 cells:The area of cell scratches in the 15 μmol/L and 20 μmol/L Dp groups was calculated after 0 h,24 h and 48 h of administration,respectively,and it was found that the area after the effect of Dp was significantly wider than that of the control group,indicating that Dp was able to inhibit the migratory ability of SW480 cells.(6)The effect of Dp on the migratory ability of SW480 cells:The cell numbers of Transwell chambers in the 15 μmol/L and 20 μmol/L Dp groups were calculated after 48 h of administration,and it was found that the number of cells penetrating out of the chambers was reduced compared with that of the control group,indicating that Dp was able to inhibit the migratory ability of SW480 cells.2.Network pharmacology screening of Dp anti-SW480 core targets and pathways(1)231 Dp targets were screened through PubChem,pharmmapper and SwissTargetPrediction databases.(2)9395 CRC targets were screened by GenCards and OMIM databases.(3)Dp targets and CRC targets generated were entered into Venny2.1 software to obtain 95 common drug-disease targets.(4)95 common drug-disease intersecting targets were used in Cytoscape to build a "drug-disease-target" interaction network map,and AKT1 was identified as the most potentially active target.(5)A protein interaction network was constructed by PPI analysis of the intersecting targets using the String platform.The network has 95 nodes and 421 edges,and AKT1 is located at the centre of the network.(6)The topological analysis revealed that AKT1,SRC and EGFR may be the most central targets of Dp anti-CRC.(7)The biological process,cellular components and molecular functions of Dp anti-CRC were identified by GO entry analysis,and the main molecular mechanism of Dp anti-CRC was identified by KEGG enrichment pathway analysis.(8)The protein blotting assay revealed that Dp could reduce the expression of p-AKT protein in SW480 cells,which tentatively demonstrated that PI3K/AKT is one of the possible molecular mechanisms of Dp anti-CRC.Conclusion:The natural phytochemical Dp has a proliferation inhibitory effect on CRC cells SW480 in vitro,Dp can affect CRC through multiple targets and multiple pathways,AKT1 may be the most important target of Dp anti-CRC and PI3K/AKT may be the Ⅷsignaling pathway of Dp anti-CRC. |
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