Network Pharmacology And Molecular Docking Reveal The Mechanism Of Scopoletin Against Non-small Cell Lung Cancer

Objective: To explore the tumor cell lines sensitive to Scopoletin and elucidate the anti-tumor mechanism of it,so as to provide a scientific basis for the further development and application of it.Methods: Systematic solvent extraction,thin layer chromatography(TLC),high performance liquid chromatography(HPLC)and liquid chromatography-mass spectrometry(LC-MS)were used to isolate and identify compound from Fennel.Human lung cancer cell line A549,colon cancer cell line HCT-116 and liver cancer cell line HepG2 were used as test objects.The tumor cell lines treated with compound were screened by MTT.The toxic effect of compound on human normal lung epithelial cell line BEAS-2B was further investigated.Through network pharmacology,“Compound-Target-Pathway”(C-T-P)network and “Protein-Protein Interaction”(PPI)network,the related targets of compound against non-small cell lung cancer(NSCLC)were analyzed.Then,the docking mode and action site between compound and the key targets were analyzed by molecular docking.And the core targets were further determined according to the binding energy.The effect of compound on the proliferation and apoptosis of A549 cells was investigated by plate cloning and Hoechst 33342 staining.Finally,western blot(WB)was used to verify the regulatory effect of compound on the pathways involved in the core targets.Results: TLC,HPLC and LC-MS results showed that Scopoletin was isolated and purified from Fennel,and its purity was 98%.MTT results showed that Scopoletin significantly inhibited the growth of A549,HCT-116 and HepG2 cells in a dose-dependent manner.When Scopoletin concentration reached 16 μg/ml,the survival rate of A549 cells was 50.38%.But,the survival rate of BEAS-2B cells treated with Scopoletin was 102%.It suggested that Scopoletin had a good inhibitory effect on NSCLC.The results of “C-T-P” network and “PPI” network constructed by network pharmacology showed that EGFR,B-Raf and AKT1 were the core targets of Scopoletin against NSCLC.Molecular docking results not only verified this point,but also described the binding energy and binding site of Scopoletin with the core targets.Compared with the control group,Scopoletin inhibited the proliferation of A549 cells in a dose-dependent manner.And Scopoletin significantly down-regulated p-EGFR,Ras,B-Raf,p-ERK1/2,p-AKT1 and Bcl-2(P<0.05 or P<0.01),and up-regulated Bim,Bax and Cleaved-caspase 3(P<0.05 or P<0.01)in a dose-dependent manner when compared with the control group.Conclusion: 1.Scopoletin inhibited the proliferation of A549 cells in a dose-dependent manner,and it had no toxicity to BEAS-2B cells.2.Scopoletin increased the apoptosis of A549 cells in a dose-dependent manner.3.EGFR,B-Raf and AKT1 were the core targets of Scopoletin in the treatment of NSCLC.4.The mechanism of Scopolamine against non-small cell lung cancer was as follows: Scopolamine regulated the expression of related proteins in Ras-Raf-MEK-ERK pathway and PI3K/AKT pathway by binding with EGFR,B-Raf and AKT1,and played an anti-NSCLC role.In this study,the efficacy and mechanism of Scopoletin in the treatment of NSCLC were clarified.