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

Abstract

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 Fe²⁺, 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.