Exploring the Anti-fibrotic Mechanism of Fugan Huaxian Decoction based on Ferroptosis

Objective: Liver fibrosis represents a pivotal stage in the progression of chronic liver diseases toward cirrhosis, primarily driven by the activation of hepatic stellate cells (HSCs). Ferroptosis, a novel form of iron-dependent programmed cell death, has been shown to alleviate liver fibrosis when induced in HSCs. Fugan Huaxian (FGHX) Decoction has demonstrated significant anti-fibrotic effects; however, whether its mechanism of action involves ferroptosis induction in HSCs remains unclear. Our research aims to investigate the anti-fibrotic effects of FGHX Decoction and its potential role in inducing ferroptosis in HSCs. Methods: A transforming growth factor-β1 (TGF-β1)-induced HSC-T6 cell activation model was established to mimic the liver fibrosis microenvironment. Ferroptosis-related markers, including Glutathione Peroxidase 4 (GPX4) and Solute Carrier Family 7 Member 11 (SLC7A11), were assessed via Western blotting. Intracellular levels of glutathione (GSH), malondialdehyde (MDA), and ferrous iron (Fe²⁺) were quantified using enzyme-linked immunosorbent assay (ELISA). Mitochondrial morphology was examined by transmission electron microscopy to evaluate ferroptosis-associated structural changes following FGHX Decoction treatment. Results: After 24 hours of treatment with FGHX Decoction–containing serum, GPX4 and SLC7A11 protein expression in HSC-T6 cells was significantly downregulated (P < 0.01). ELISA analysis revealed a marked decrease in intracellular GSH levels (P < 0.05), accompanied by a marked increase in MDA and Fe²⁺ levels (P < 0.05). Transmission electron microscopy revealed characteristic ferroptotic mitochondrial damage in FGHX Decoction–treated cells, including pronounced mitochondrial swelling, cristae disruption or disappearance, structural disorganization, and vacuolization. Conclusion: FGHX Decoction induces ferroptosis in HSC-T6 cells by downregulating GPX4 and SLC7A11, reducing GSH levels, and increasing MDA and Fe²⁺ levels, leading to mitochondrial damage. These findings suggest that the anti-fibrotic effects of FGHX Decoction may be mediated through the ferroptosis signaling pathway, providing a novel therapeutic strategy for liver fibrosis.