Active Compounds and Potential Targets of Shuganning Injection in the Treatment of Hepatocellular Carcinoma by Network Pharmacology and in vitro Validation

Shuganning Injection (SGNI), a TCM (Traditional Chinese Medicine) injection with good hepato-protective effects, exerted therapeutic effects on HCC. However, the active compounds and effects of SGNI on hepatocellular carcinoma (HCC) remain unclear. To investigate the active compounds and potential targets of SGNI in the treatment of HCC, and explore the molecular mechanisms of main compounds. Network pharmacology was applied to predict the active compounds and targets of SGNI on cancer. The interaction between active compounds and target protein were validated by drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), and pull-down assay. The in vitro test of the effects and mechanism of vanillin and baicalein was elucidated by MTT, Western blot, immunofluorescence and apoptosis analysis. According to compound characteristics, targets, etc., two typical active ingredients (vanillin and baicalein) were selected as representatives to explore the effects on HCC. Vanillin (an important food additive), bound to NF-κB1 and baicalein (a bioactive flavonoid) bound to FLT3 (FMS-Like Tyrosine Kinase 3) were confirmed in this study. Vanillin and baicalein were both inhibited cell viability and promoted apoptosis of Hep3B and Huh7 cells. In addition, both vanillin and baicalein could enhance the activation of p38/MAPK (Mitogen-Activated Protein Kinase) signaling pathway, which may partially explain the anti-apoptosis effects of the two compounds. In conclusion, two active compounds of SGNI, baicalein and vanillin, promoted apoptosis of HCC cells via binding with NF-κB1 or FLT3, and regulating the p38/MAPK pathway. Baicalein and vanillin may be good candidates for HCC treatment on drug development.