Pharmacological Targets And Clinical Significance Of Plumbagin To Treat Colorectal Cancer:A Systematic Pharmacology Study

OBJECTIVE: This article applies network pharmacology methods to obtain the "drug-target-protein-disease" network of plumbagin(PL)in the treatment of colorectal cancer(CRC),and explore the core pharmacological targets of PL in the treatment of CRC And the main biological process,KEGG pathway,and predict its mechanism.Based on the results of network pharmacology,follow-up clinical case observations and in vitro experimental studies are carried out to verify the targets and markers,and provide theoretical references for clinical research and treatment.Methods: 1.First,use the TCMSP database to find the drug ingredients,and find out the PL verified and predicted anti-cancer biological targets based on the ingredients in the SwissTargetPredict and SuperPred databases,and the Gene Cards database.The CRC-related targets were obtained through the DisGeNET database and the Gene Cards database.After obtaining the drug target and disease target respectively,use the Funrich software to map,take the intersection of the two targets,construct the PPI network on the STRING website,and get the protein interaction map,and then import the Cytoscape software to perform the PL treatment of the CRC target Visualize,screen the core targets,and finally use the DAVID database to enrich the core targets to obtain biological processes and signal pathways.2.Collect serological data and biopsy tissues of clinical patients for immunofluorescence staining,and locate cancer tissue specimens for the core targets predicted above.3.Human-derived HCT116 cells and SW620 cells were treated with high-concentration 2.5μM,medium-concentration 1.5μM,and low-concentration 0.5μM PL for 12 h to observe their cytotoxicity.DMSO was used as a solvent control group.4.Use CCK8 method to detect the effect of PL on cell proliferation inhibition.5.Use Hoechst33258 staining method to detect the effect of PL on cell apoptosis.6.Use immunofluorescence and Western blotting to detect the expression position and changes of core protein in cells after PL intervention.Results: 64 biological candidate targets of PL anti-CRC were screened out,and the network interaction diagrams of related targets were drawn.The 7 core target proteins with the highest degree of freedom in the topological parameters of protein interaction were obtained,namely: Tumor protein p53(TP53),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),Mitogen-activated protein kinase 1(MAPK1),E1A-associated protein p300(EP300),Poly ADP-ribose polymerase 1(PARP1),Nuclear factor kappa p65 protein(RELA),Bcl-2 like protein 1 Bcl-2(BCL2L1),conduct pathway enrichment analysis of the above core target proteins,and screen the top 20 biological processes and KEGG signaling pathways related to the core target of PL treatment of CRC.The main biological processes involved in the core target are the transcription of the RNA polymerase II promoter,the negative regulation of the transcription of the RNA polymerase II promoter,and the cell’s response to DNA damage stimuli.The main KEGG pathways involved are pancreatic cancer,chronic granulocytes,etc.Cell leukemia,prostate cancer,etc.The results of clinical patient serum statistical analysis showed that the tumor markers carcinoembryonic antigen and carbohydrate antigen CA199 of CRC patients increased significantly,and many indicators of blood routine fluctuated greatly.IF staining of CRC biopsy specimens showed that the expression of MAPK1 and PARP1 in CRC tumor samples was more obvious,and the expression of EP300 was less.The results of immunofluorescence staining of patient specimens showed that MAPK1 and PARP1 were more expressed in cancer sites,and EP300 was less expressed.The CCK8 experiment showed that under the intervention of PL,the proliferation of HCT116 cells and SW620 cells was significantly inhibited in a time and dose-dependent manner.The results of cellular immunofluorescence experiments and western blot experiments showed that PL intervention in HCT116 and SW620 cells both resulted in the down-regulation of MAPK1 and PARP1 expression(P<0.05),and the up-regulation of EP300 expression(P<0.05).Conclusion: The 64 targets of PL against CRC were obtained through network pharmacology,and the biological targets and molecular mechanisms were screened and verified.Part of the core targets MAPK1,EP300,PARP1 were verified in clinical specimens and in vitro cell experiments.These biological targets may become biomarkers for CRC detection and treatment.