Total flavonoids of Prunella vulgaris exert anti-hepatocellular carcinoma effects through autophagy suppression mediated by activation of the PI3K/AKT/mTOR pathway.

[ETHNOPHARMACOLOGICAL RELEVANCE] Prunella vulgaris has been traditionally used in Chinese folk medicine for treating thyroid nodules, liver diseases, and other conditions. Our preliminary studies have identified its potential therapeutic effects against hepatocellular carcinoma (HCC). However, the active constituents and precise molecular mechanisms remain unclear. This study aims to elucidate its anti-HCC components and underlying mechanisms, thereby providing a scientific foundation for its novel application in HCC treatment.

[AIM OF THE STUDY] To determine the effect of the intervention and molecular mechanism of the total flavonoids of P. vulgaris in a rat model of primary hepatocellular carcinoma.

[MATERIALS AND METHODS] UPLC-MS/MS was used to identify the main chemical components of the total flavonoids of P. vulgaris. A rat model of primary hepatocellular carcinoma was established by administering N-diethylnitrosamine (DEN; 100 ppm) in drinking water continuously for 19 weeks, followed by 3 weeks of treatment with the total flavonoids of P. vulgaris (200 mg/kg, 100 mg/kg, 50 mg/kg) to evaluate their anti-liver cancer effect in vivo. Network pharmacology was used to determine the molecular mechanisms and targets of the total flavonoids of P. vulgaris against liver cancer. After treatment of HepG2 and SMMC7721 cells with 3 doses (200 μg/kg, 400 μg/kg, 800 μg/kg) of the test compounds, the anticancer effects were assessed in vitro using a real-time cell analyzer and cell cycle experiments. Subsequently, the PI3K/AKT/mTOR pathway was inhibited to validate the molecular mechanisms and targets of the total flavonoids of the anti-liver cancer effects of P. vulgaris.

[RESULTS] Luteolin, rosmarinic acid, kaempferol, and 11 additional compounds were determined to be the main components of the total flavonoids from P. vulgaris using UPLC-MS/MS. In vivo experiments revealed that the total flavonoids of P. vulgaris reduced the number of liver nodules in rats with DEN-induced primary liver cancer, decreased their liver weights, improved the pathological carcinogenesis of the liver, and inhibited autophagy in cancer tissue cells. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that the anti-liver cancer mechanism of the total flavonoids of P. vulgaris primarily involved cancer pathways and PI3K/Akt signaling pathways. Findings from in vitro studies demonstrated that the total flavonoids of P. vulgaris significantly inhibited the viability of SMMC7721 and HepG2 cells and blocked the cell cycle. After the addition of PI3K and mTOR inhibitors, the inhibitory effect of the total flavonoids of P. vulgaris on cell viability decreased, autophagy levels increased, and the number of autophagosomes increased significantly.

[CONCLUSION] The total flavonoids of P. vulgaris may inhibit the autophagy of hepatocellular carcinoma cells primarily via the PI3K/AKT/mTOR pathway, affecting the homeostasis of cancer cells and preventing cancer cell proliferation, thereby highlighting a potential novel strategy to treat liver cancer.