Hypericum japonicum Thunb. ex Murray inhibits hepatocellular carcinoma angiogenesis through the TF/FVIIa-mediated signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Hypericum japonicum Thunb. ex Murray (Hypericaceae), named 'Tianjihuang' is a traditional Chinese medicine with hepatoprotective, antibacterial, and antitumor effects. In Chinese ethnic medicines, H. japonicum is used as a raw material for treatment of acute and chronic hepatitis, and is often called "hepatitis herb". Given that chronic hepatitis is a well-established risk factor for hepatocellular carcinoma (HCC), this traditional use provides a strong ethnopharmacological rationale for investigating its anti-HCC mechanisms. However, the mechanism and active ingredients of H. japonicum extract inhibiting HCC and angiogenesis remain unclear.

AIM OF THE STUDY: This study aims to comprehensively evaluate the anti-HCC angiogenesis effects of H. japonicum extract through both in vivo and in vitro experiments, and to preliminarily investigate its underlying molecular mechanisms and target proteins.

MATERIALS AND METHODS: In vivo therapeutic efficacy of H. japonicum extract was validated in zebrafish. Network pharmacology, molecular docking and surface plasmon resonance (SPR) approaches were employed to predict potential targets and signaling pathways. In vitro, EA.hy926 cells were treated with conditioned medium (CM) derived from H. japonicum extract-exposed HepG2 cells to assess migration and tube formation capabilities. VEGF content in the CM was also detected. Effect of bioactive compound Quercetin-7-O-α-L-rhamnoside (Q7R) on coagulation factor VIIa (FVIIa) cellular localization was determined by immunofluorescence, while key signaling proteins were analyzed by Western blot.

RESULTS: In vivo zebrafish assay revealed that H. japonicum extract at 25 μg/mL suppressed embryonic intersegmental angiogenesis by 49.41%, demonstrating its potent anti-angiogenic activity. To elucidate the underlying mechanisms, we integrated UPLC-HRMS-based phytochemical profiling with network pharmacology, which identified tissue factor (TF) as a candidate target. Molecular docking further supported this finding by showing strong binding affinity between the bioactive constituents of H. japonicum and the TF/FVIIa complex. Functionally, conditioned medium from H. japonicum-treated HepG2 cells significantly impaired EA.hy926 endothelial cell migration and tube formation, concomitant with a marked reduction in VEGF levels. Notably, treatment with 10 μM Q7R, a flavonoid component of H. japonicum, attenuated FVIIa-stimulated VEGF hypersecretion in HepG2 cells by 52.2%. Mechanistic dissection revealed that Q7R acts through the TF/FVIIa/PAR2 axis to curb aberrant downstream PI3K-AKT and ERK signaling, thereby curtailing VEGF production and secretion, which in turn suppresses endothelial cell migration and tubulogenesis.

CONCLUSIONS: H. japonicum and its bioactive compound Q7R inhibit HCC angiogenesis by targeting the TF/FVIIa-mediated pathway and downstream PI3K/AKT and MAPK signaling, reducing VEGF expression. These findings reveal a novel mechanism of action for H. japonicum and support its potential as an anti-angiogenic agent for HCC therapy.