基于网络药理学和体外实验探讨凤尾草抑制膀胱癌细胞的作用机制OA

Objective To explore the potential mechanism of Pteris multifida in inhibiting bladder cancer cells based on network pharmacology and in vitro cellular experiments.Methods Active ingredients of Pteris multifida were screened from TCMSP and HERB databases,and drug targets were predicted by Swiss Target Prediction.Disease-related targets of bladder cancer were obtained by integrating three GEO bladder cancer microarray datasets as well as GeneCards,OMIM,TTD and NCBI databases.A protein-protein interaction(PPI)network was constructed to screen core targets.GEPIA database was used to analyze the expression and prognostic value of major genes in bladder cancer tissues.Gene Ontology(GO)functional enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were performed to predict the potential mechanism of Pteris multifida against bladder cancer.Molecular docking was adopted to evaluate the binding affinity between major active ingredients and core targets.CCK-8 assay and EdU assay were used to detect the proliferative capacity of T24 cells.Flow cytometry was applied to observe cell-cycle distribution and cell apoptosis.Immunofluorescence assay was used to detect the levels of cell-cycle and apoptosis-related proteins.RT-qPCR and Western blot were employed to determine the changes of core genes and corresponding proteins.Results After removing itraconazole(a synthetic drug included in the database),two core flavonoid components,quercetin and luteolin,were screened out.A total of 230 intersecting targets of Pteris multifida and bladder cancer were acquired.Cyclin-dependent kinase 1(CDK1),cyclin B1(CCNB1),cyclin-dependent kinase 6(CDK6),matrix metalloproteinase 2(MMP2),and poly(ADP-ribose)polymerase 1(PARP1)were identified as major targets.CDK1 and CCNB1 were highly expressed in bladder cancer tissues,and high CDK6 expression indicated poor survival prognosis of patients.KEGG enrichment analysis suggested that the cell-cycle pathway and p53 signaling pathway might serve as key pathways for Pteris multifida against bladder cancer.Molecular docking results showed that the binding free energies of quercetin and luteolin with CDK1 and MMP2 were all lower than-8 kcal·mol-¹,indicating stable binding conformations.Cellular experiments demonstrated that compared with the control group(ctrl),Pteris multifida significantly inhibited the proliferation of T24 cells in a time-and concentration-dependent manner(P<0.05).Pteris multifida induced G2-phase cell-cycle arrest(P<0.05)and cell apoptosis(P<0.05)in T24 cells.It down-regulated the mRNA and protein levels of CDK1,CCNB1,MMP2,CDK6,PARP1,and B-cell lymphoma-2(Bcl-2),whereas it up-regulated the mRNA and protein levels of tumor protein p53(p53),cyclin-dependent kinase inhibitor 1A(CDKN1A,p21),and Bcl-2-associated X protein(Bax)(P<0.05).Conclusion Pteris multifida can up-regulate the expression of mutant p53 and its downstream p21 in T24 cells,mediate G2-phase cell-cycle arrest and induce cell apoptosis.This anti-proliferative effect is partially dependent on the p53 signaling pathway,with p53-independent regulatory pathways also involved,thereby exerting an inhibitory effect on T24 bladder cancer cells.