Multi-Omics Analysis of Phytolacca acinosa Fruit Fermentation Broth Against Hepatocellular Carcinoma and Evaluation of Phytolaccagenin as a Candidate Bioactive Constituent

Phytolacca acinosa Roxb. is a traditional Chinese medicine. In traditional practice, its root has long been used for “promoting diuresis to reduce edema” and “detoxifying and resolving masses”, and certain constituents of the root have been reported to exhibit in vitro anti-tumor activities. However, the fruit of Phytolacca acinosa represents a newly explored medicinal resource, and the anti-hepatocellular carcinoma (HCC) activity of its fermentation broth (PAFFB) remains largely unexplored. This study evaluated the anti-HCC efficacy of PAFFB and its underlying mechanisms by integrating pharmacodynamics, multi-omics (transcriptomics, proteomics, and metabolomics), and UPLC-Q-TOF-MS/MS profiling. In a diethylnitrosamine (DEN)-induced HCC rat model, PAFFB (0.75, 1.5, and 3 g/kg/d for 4 weeks) dose-dependently improved body weight and survival, alleviated liver injury, reduced tumor burden, and restored liver function, while exhibiting less apparent toxicity than cyclophosphamide in the parameters assessed (body weight, survival, and hepatic function and histopathology). Joint pathway enrichment analysis revealed that PAFFB modulated DEN-induced pathological alterations through the coordinated regulation of cholesterol–bile acid metabolism, inflammatory signaling, and energy metabolism. Mechanistically, PAFFB restored TLR-4/MYD88 expression, suppressed p65 and p38 phosphorylation, and upregulated CYP7A1, PPARα, and FXR. Among the 120 identified constituents, 21 compounds were absorbed into the bloodstream; phytolaccagenin suppressed Hepa1-6 proliferation, colony formation, and migration, induced apoptosis, ROS accumulation, and lipid peroxidation in vitro, and inhibited Hepa1-6 syngeneic tumor growth in mice without obvious toxicity. In conclusion, PAFFB exerts anti-HCC effects by regulating metabolic reprogramming and inflammatory signaling, and phytolaccagenin represents a candidate bioactive constituent of PAFFB.