Exploring the Anti-Hepatoma Effect of Fermentation Liquor of Fresh Fruit of Phytolacca Acinosa Roxb. in Vitro Based on PI3K/Akt/NF-κB Signaling Pathway

OBJECTIVE  To explore the effect of fermentation liquor of fresh fruit of Phytolacca acinosa Roxb. on Bel-7402 hepatoma cells based on phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/nuclear factor(NF)-κB signaling pathway.

METHODS  Fermentation liquor of fresh fruit of Phytolacca acinosa Roxb. was isolated by the combined use of ultrasonic extraction, ethanol precipitation, cation exchange resin and macroporous adsorption resin. Human hepatoma cell line Bel-7402 was used as the research object. The effects of fermentation liquor of fresh fruit of Phytolacca acinosa Roxb. and its components on its proliferation were investigated by cell counting kit-8(CCK8), in order to obtain the main active substances and appropriate doses for follow-up experiments. Using plate clone formation test, cell scratch test, flow cytometry and Western blotting to detect the effects of alkaloid components on clone formation ability, migration ability, apoptosis and cell cycle of Bel-7402 hepatoma cells and its mechanism.

RESULTS  Under the same concentration gradient, the alkaloid component of fermentation liquor of fresh fruit of Phytolacca acinosa Roxb. had the best inhibitory effect on the proliferation of Bel-7402 hepatoma cells, and the concentration of IC₅₀ was about 0.75 mg·mL⁻¹, and the alkaloids of 0.375, 0.75, 1.5 mg·mL⁻¹ were selected as the low, middle and high dose groups in the follow-up experiment. In follow-up experiments, it was found that alkaloid components could reduce the clone formation and migration ability of Bel-7402 hepatoma cells, promote apoptosis, and block cells in S phase and G₂/M phase. Further studies showed that alkaloids could up-regulate the expression levels of p53 protein and pro-apoptotic protein Bax in Bel-7402 hepatoma cells, and down-regulate the expression levels of PI3K, Akt, NF-κB P65, cyclooxygenase 2(COX-2), vascular endothelial growth factor(VEGF) protein and anti-apoptotic protein Bcl-2, resulting in an increase in Bax/Bcl-2 ratio.

CONCLUSION  The main active substances of fermentation liquor of fresh fruit of Phytolacca acinosa Roxb. in the treatment of hepatocellular carcinoma in vitro is alkaloid components, which may exert its anti-tumor effect by inhibiting the expression of proteins related to PI3K/Akt/NF-κB signaling pathway, and then increase the ratio of Bax/Bcl-2 to promote apoptosis and decrease VEGF to inhibit cell proliferation.