Hu Gan Tang ameliorates metabolic-associated fatty liver disease by inhibiting ferroptosis through the Nrf2/GPX4 pathway

Ethnopharmacological relevance: Hu Gan Tang (HGT), a modified formulation of the traditional Chinese medicine "Si jun zi", is based on the principle of "strengthening the spleen, resolving dampness, and purging turbidity" for the treatment of metabolic-associated fatty liver disease (MAFLD). HGT has shown efficacy in alleviating liver damage in MAFLD patients. Nevertheless, its underlying molecular mechanisms remain unexplored.

Aim of the study: This study aimed to investigate the hepatoprotective effects of HGT against MAFLD by regulating hepatic Ferroptosis in both in vivo and in vitro models.

Materials and methods: The MAFLD rat model was established through exposure to a high-fat diet (HFD). HGT was administered intragastrically for eight consecutive weeks. Histological and biochemical tests were conducted to assess liver damage. In an in vitro study, HepG2 cells were incubated with free fatty acids (FFAs) in the presence or absence of HGT drug-containing serum. Ferroptosis-related parameters, including intracellular Fe²⁺ levels, MDA and SOD levels, ROS levels, and the expression levels of ferroptosis-related proteins and modulators, were quantified both in vivo and in vitro. Additionally, Nrf2 inhibitor (ML385) and GPX4 inhibitor (RSL3) were utilized to validate the underlying mechanisms of HGT's hepatoprotective effects.

Results: The results demonstrated that HGT alleviated liver injury and reduced lipid accumulation. Furthermore, HGT suppressed oxidative stress by decreasing MDA and ROS levels while increasing SOD levels in both in vivo and in vitro settings. Additionally, HGT inhibited Ferroptosis by reducing Fe²⁺ accumulation, upregulating xCT, GPX4, and FTH1 expression, and enhancing nuclear Nrf2 expression in liver tissue and steatotic HepG2 cells. Further research revealed that HGT reduced lipid accumulation, suppressed oxidative stress, and alleviated Ferroptosis in hepatocytes by activating the Nrf2/GPX4 signaling pathway. These findings provide a novel therapeutic mechanism for HGT in ameliorating MAFLD.

Conclusion: HGT significantly suppresses hepatic Ferroptosis, reduces oxidative stress, diminishes lipid accumulation, and ameliorates liver damage by activating the Nrf2/GPX4 pathway.

Ferroptosis; Hu Gan Tang; Metabolic-associated fatty liver disease; Nrf2/GPX4; Oxidative stress.