| Objective: This study is to explore the mechanism of active compounds and targets of Si Ni San in the regulation of angiogenesis through network pharmacology,and to explore the mechanism of Si Ni San and one of its active components sinensetin on angiogenesis of liver cancer through experimental study,which aiming to provide a certain theoretical and experimental basis for the clinical application of Si Ni San in the treatment of liver cancer.Methods: 1.In this study,the active components of different components of Si Ni San and their intersection targets with angiogenesis were retrieved and screened through multiple databases.Compounds and related target proteins were screened through TCMSP database,and the network diagrams of active components,targets and disease targets of Si Ni San were constructed using Cytoscape.The PPI network of intersecting targets was constructed using the STRING database.GO enrichment and KEGG pathway enrichment were constructed to analyze the key targets and action mechanism of Si Ni San on angiogenesis.2.Hep G2-induced liver cancer model was established.The expressions of CD31 protein were detected by immunohistochemistry and Western Blot to identify the inhibitory effect of Si Ni San on angiogenesis of liver cancer.The expressions of VEGF,HIF-1α,and P-VEGFR2 were detected by Real-Time PCR,Western Blot,and immunofluorescence techniques,to explore the mechanism of Si Ni San against angiogenesis of liver cancer.3.Hep G2-induced liver cancer model was established.Immunohistochemistry and Western Blot were used to detect the expression of CD31 protein to identify the inhibitory effect of sinensetin on angiogenesis of liver cancer in vivo.CCK8,Annexin V/PI double staining method,scratch test,Tranwell and tube-formation were used to observe the effect of sinensetin on the proliferation,migration and tube-forming ability of HUVEC,and to identify the effect of sinensetin on angiogenesis in vitro.4.Real-Time PCR,Western Blot and other techniques were used to detect the expressions of VEGF and HIF-1α in hypoxia-treated Hep G2 with sinensetin,after the treatment with sinensetin in hypoxia condition,tumor conditioned medium of Hep G2 was collected and added to HUVEC.Tube formation assay was observed.and the expressions of P-VEGF2 and P-AKT proteins in HUVEC with sinensetin,molecular docking wad used to imitate the interaction between sinensetin and VEGFR2,to explore the mechanism of suppression of angiogenesis in liver cancer induced by sinensetin.Results: 1.108 active components and 92 effective targets such as TP-53,IL-6 and VEGF were screened out from Si Ni San.The top 14 significant gene ontology(GO)and top 21 significant genome encyclopedia(KEGG)pathways were concluded,which involved phosphatidylinositol 3-kinase and/or protein kinase B signaling pathway(PI3K-Akt signaling pathway),tumor necrosis factor signaling pathway(TNF signaling pathway),estrogen pathway(Estrogen signaling pathway),etc.2.The tumor weight of liver cancer and the expression of CD31 were reduced by Si Ni San or sinensetin in liver cancer tissues;VEGF protein level and m RNA level were reduced by Si Ni San or sinensetin in liver cancer.The expression of HIF-1α protein in liver cancer was also reduced by Si Ni San.3.The proliferation,the migration and tube-forming ability of HUVEC were inhibited and apoptosis was promoted by sinensetin in vitro.4.The expression of HIF-1α and VEGF was reduced by sinensetin in Hep G2 under hypoxia.Conditioned medium after sinensetin treatment inhibited the tube-forming ability of HUVEC.5.VEGF-induced phosphorylation of VEGFR2 and phosphorylation of AKT were reduced by sinensetin in HUVEC cells.Molecular docking results showed that sinensetin bind to VEGFR2 through a variety of chemical bonds,affecting its phosphorylation site.Conclusion: Si Ni San exhibits antiangiogenic effect in liver cancer by HIF-1α-VEGF signaling pathways.Sinensetin exhibits a significant antiangiogenic effect by inhibiting the vitality of endothelial cells and inducing apoptosis and inhibiting migratory aptitude and tube formation in HUVEC.Sinensetin potently inhibits angiogenesis in vitro by eliciting the blockade of VEGF expression in Hep G2-derived tumor and VEGFR2/AKT signaling pathways in endothelial cells. |
