Effects Of Quercetin From Ramie Root On Proliferation And Migration Of Caco-2 Cells And Its Mechanism Of Action

ObjectiveThe effects of quercetin,the main active component of ramie root,on the proliferation and migration of Caco-2 cells and the related mechanism of action were investigated by network pharmacology and experiments in vitro,which provided theoretical basis for subsequent animal experiments and clinical experiments.Methods1.Network pharmacology and molecular docking: the effective active ingredient targets of ramie root and related targets of colon cancer were obtained by TCMSP and Gene Cards,and the intersection of colon cancer targets and active ingredient targets was obtained based on Venn diagram.Cytoscape3.7.1 was used to construct a network diagram of "Drug-ingredient-target-disease" to screen key active ingredients.Using STRING database to construct PPI network and select core targets;The binding ability of key active ingredients and core targets was verified by molecular docking.The gene ontology(GO)enrichment analysis of the collected target proteins was carried out by using R language.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis using DAVID database.2.In vitro experiment verification: Caco-2 cells were treated with different concentrations of quercetin(0.01 μg/μL,0.1 μg/μL,0.25 μg/μL,0.5 μg/μL,1μg/μL)for 6 h,18 h and 24 h.The cell survival rate was detected by CCK8method;Caco-2 cells were treated with 0.5 μg/μL quercetin at 0 h,12 h,24 h,36 h and 48 h for scratch experiments to detect the effect of the drug on the migration ability of colon cancer cells;Caco-2 cells were treated with quercetin at concentrations of 0.1,0.5 and 1 μg/μL for 18 h.The relative expression levels of PI3K/Akt signaling pathway related genes and proteins were detected by real-time quantitative PCR and Western blot.Results1.Network pharmacology and molecular docking: Five potential chemical active ingredients and 186 potential targets closely related to active ingredients in ramie root were obtained through screening,104 of which were related to colon cancer;PPI network analysis showed that the key nodes of interaction between ramie root and colon cancer were AKT1,JUN,EGFR,CASP3,IL6,etc.Cytoscape 3.7.1 software constructs the visual network diagram of drug active ingredient-target and colon cancer disease-target,and it is concluded that quercetin plays the main role in ramie root treatment of colon cancer.Molecular docking results showed that the binding energies of quercetin with AKT1 protein,Jun protein,EGFR protein,CASP3 protein and IL6 protein were-9.5 kcal/mol,-5.7 kcal/mol,-8.9 kcal/mol,-7.7 kcal/mol and-7.3 kcal/mol,respectivelyol;Gene ontology(GO)enrichment analysis showed that the biological processes of GO in vivo mainly included the response to lipopolysaccharide and reactive oxygen species.The Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway and gene function enrichment analysis were conducted using DAVID database.A total of 143 potential signaling pathways of ramie root in the treatment of colon cancer were screened out(P<0.01).2.In vitro experiment:The results showed that 0.01-1 μg/μL quercetin had little effect on the survival rate of Caco-2 cells at 6 h,0.1,0.25,0.5and 1 μg/μL quercetin decreased the survival rate of Caco-2 cells at 18 h(P<0.01),and at 24 h,0.01,0.1,0.25,0.5 and 1 μg/μL quercetin significantly decreased the survival rate of Caco-2 cells(P<0.01).The scratch test results showed that the scratch healing rate of quercetin intervention group was 21.46% lower than that of the untreated group(P<0.01).Real-time quantitative PCR results showed that quercetin could decrease the expressions of PI3 K,AKT1 and CCND1 genes and increase the expression of CASP9 compared with blank control group(P<0.01).Westernblot showed that quercetin could decrease the phosphorylation levels of PI3K/Akt pathway related proteins p-PI3 K and p-Akt,decrease the expression of CCND1 and increase the expression of CASP9(P<0.05).conclusions1.Network pharmacology and molecular docking indicate that the core target of quercetin,the effective ingredient of ramie root,is AKT1 in the treatment of colon cancer,and may act on colon cancer through PI3K/Akt signaling pathway.2.In vitro cell experiments showed that quercetin,the active component of ramie root,inhibited Caco-2 by inhibiting PI3K/Akt signaling pathway,which verified the accuracy of the earlier network pharmacology...