Hua Zheng San Ji Fang promotes ferroptosis through Keap1/Nrf2 pathway in hepatocellular carcinoma cells.

[ETHNOPHARMACOLOGICAL RELEVANCE] Hua Zheng San Ji Fang (HZSJF) is a traditional Chinese medicinal formula comprising 11 authenticated herbs that have historically been used to treat liver ailments. Its multi-component and multi-targeted nature makes it a promising therapeutic agent for hepatocellular carcinoma (HCC), particularly via the modulation of ferroptosis, a regulated form of cell death.

[AIM OF THE STUDY] This study aimed to investigate the anticancer effects of HZSJF on hepatocellular carcinoma (HCC) cells and elucidate the underlying mechanisms, with a particular focus on ferroptosis and the Keap1/Nrf2 oxidative stress pathway.

[MATERIALS AND METHODS] HZSJF was extracted through a dual aqueous decoction and concentrated to 741 g/L. The effects of the extract were assessed on SK-HEP-1 and HepG2 cells using viability, proliferation, migration, and invasion assays. Ferroptosis biomarkers and signaling components were evaluated using biochemical assays, qRT-PCR, immunofluorescence, and western blotting. A subcutaneous xenograft model was used for in vivo validation.

[RESULTS] HZSJF significantly inhibited the proliferation, migration, and invasiveness of HCC cells. It induced ferroptosis by elevating lipid peroxidation and intracellular Fe, reducing GSH and SOD levels, and downregulating GPX4, FTH-1, and SLC7A11. Mechanistically, HZSJF upregulated Keap1 and suppressed Nrf2/HO-1 signaling, thereby enhancing oxidative stress. These effects were reversed by ferroptosis inhibitors and modulated by Keap1 overexpression. In vivo, HZSJF inhibited tumor growth and induced the molecular effects observed in vitro.

[CONCLUSIONS] HZSJF promotes ferroptosis and suppresses HCC progression by targeting the Keap1/Nrf2 axis. These findings support its potential as a natural multitarget therapeutic agent for liver cancer management.