| Object: To screen and predict the potential active ingredients,core targets and important signaling pathways of Arnebia euchroma(Royle)Johnst(AE)for the treatment of primary liver cancer through network pharmacology,and to explore the anti-hepatocellular carcinoma effects and mechanisms of potential active monomers of comfrey from the perspectives of regulating malignant biological behavior of hepatocellular carcinoma cells and modulating inflammatory microenvironment of hepatocellular carcinoma using in vitro and in vivo experiments,so as to provide a theoretical and experimental basis for the treatment of hepatocellular carcinoma with active ingredients of AE.Methods: Establishing a library of Xinjiang soft comfrey compounds by reviewing literature and searching databases such as TCMSP and TCMIP;screening active ingredients by FAFDrug4 platform;constructing ingredient target interactions network by Cytoscape software and databases such as OMIM and Genecard;enrichment analysis of key targets by DAVID database;molecular docking and The activity of the five naphthoquinones was verified by molecular docking and CCK-8 assays;the anti-hepatocellular carcinoma efficacy mechanism of β-hydroxyisovaleryl shikonin(β-HIVS),a naphthoquinone component,was initially verified in the H22 tumor-bearing mouse model.To further investigate the mechanism of the malignant behavioral action of β-HIVS against hepatocellular carcinoma in vitro,the cytotoxicity of β-HIVS on human hepatocellular carcinoma cells Hep G2 and normal hepatocytes WRL68 was investigated by MTT assay;colony generation assay was performed to investigate the effect of β-HIVS on the proliferation ability of Hep G2 cells;cell scratch assay and Transwell assay were performed to investigate the effect of β-HIVS on Hep G2 cell planar migration,spatial migration and invasion ability;DAPI staining and Annexin V-FITC/PI assay to examine the effect of β-HIVS on the apoptotic ability of Hep G2 cells;q RT-PCR assay to examine its effect on apoptosis and inflammation-related gene expression;Western blot assay to examine the effect ofβ-HIVS on the apoptotic ability of Hep G2 cells in The effect of β-HIVS on the expression of PI3K/Akt/NF-κB and MAPK signaling pathway-related proteins was investigated by Western blot,which clarified the mechanism of the in vitro anti-hepatocellular carcinoma effect of β-HIVS.Based on the inflammatory microenvironment of hepatocellular carcinoma,the in vivo anti-hepatocellular carcinoma effect of β-HIVS with better in vitro anti-hepatocellular carcinoma effect was investigated.A DEN/CCl4-induced primary hepatocellular carcinoma mouse model was established,and liver pathological changes were detected by detecting liver function,antioxidant,hepatocellular carcinoma marker serum index,HE staining and TUNEL staining,and Western blot method was used to detect hepatitis-hepatocellular carcinoma transformationrelated protein To determine the in vivo anti-hepatocellular carcinoma mechanism of β-HIVS.Results: The results of network pharmacological screening prediction and H22 tumor-bearing mouse model showed that the lipid-soluble naphthoquinone components in AE have good anti-hepatocarcinoma potential,in which both high and low dose groups of β-HIVS were able to inhibit tumor growth in tumor-bearing mice with good anti-hepatocarcinoma effect,and the key targets of AE exerting anti-hepatocarcinoma mechanism include PTGS2,TP53,Casp3,etc.,and the core pathways include PI3K/Akt,MAPK,VEGF,NF-κB,etc.In vitro results of anti-hepatocellular carcinoma malignant biology showed that the toxic effects of β-HIVS on hepatocellular carcinoma cells Hep G2 at the same administration concentration were higher than those of normal hepatocytes WRL68.β-HIVS could significantly inhibit the malignant proliferation,migration,invasion and apoptosis of Hep G2 cells(P < 0.01 or P < 0.05),significantly inhibit TNF-α,Bcl-2,upregulate Bad,Bax,Casp3 gene expression,in addition to exerting in vitro anti-hepatocellular carcinoma effects through regulating PI3K/Akt/NF-κB and MAPK signaling pathways.The in vivo anti-liver cancer results showed that β-HIVS could prolong the survival time of hepatocellular carcinoma mice,reduce serum transaminase and γ-GT levels,increase Alb levels to improve liver function,and increase serum SOD levels to alleviate lipid peroxidation process(P<0.01 or P<0.05).In addition,high dose of β-HIVS could significantly increase the TUNEL positive ratio of mouse liver cancer tissues(P< 0.01)and ameliorated chemically induced apoptosis in normal hepatocytes.β-HIVS was able to inhibit the phosphorylation levels of cancer cell invasion and metastasis-related proteins such as Akt and JNK,elevating the expression levels of apoptosis-initiating proteins Bad and Bax and decreasing the expression levels of apoptosis-inhibiting protein Bcl-2 alleviated the malignant aggressive lesions of liver cancer tissues.Conclusion: The β-HIVS in AE naphthoquinone component has good anti-hepatocellular carcinogenic potential and can inhibit the proliferation,migration,invasion and apoptosis process of hepatocellular carcinoma cells through regulating PI3K/Akt signaling pathway,and can inhibit the generation of inflammatory microenvironment of hepatocellular carcinoma through regulating MAPK/NF-κB signaling pathway and delay the development of hepatocellular carcinoma. |
