Qiweizhigan granule ameliorates metabolic dysfunction-associated steatohepatitis by modulating the galectin 3/tumor necrosis factor receptor-associated factor 6-mediated ferroptosis

BACKGROUND Qiweizhigan granule (QWZG) is employed in clinical settings for the treatment of metabolic dysfunction-associated steatohepatitis (MASH). However, the precise biological mechanisms underlying its therapeutic effects are not yet fully elucidated. AIM To assess the efficacy and the mechanism of QWZG against MASH. METHODS Animal models were established, including normal group, a choline-deficient, L-amino acid-defined high-fat diet (CDAHFD) group, and low/medium/high-dose QWZG groups, as well as a rosiglitazone group. Through comprehensive biochemical, histopathological, RNA sequencing, and bioinformatics analyses, galectin 3 (LGALS3) was identified as a critical target of QWZG in the treatment of MASH. The level of LGALS3 was quantitatively assessed and validated using Western blotting, real-time quantitative PCR, and immunofluorescence. The role and function of LGALS3 in inflammation and MASH progression were further investigated through gene knockdown, overexpression, iron assay, and transmission electron microscopy. RESULTS QWZG significantly ameliorated liver pathology by reducing steatosis, inflammation, and fibrosis. RNA sequencing analysis identified 1507 co-expressed differentially expressed genes among the CDAHFD, normal, and QWZG groups. Among these, LGALS3 was identified as one of the most significantly altered differentially expressed genes. Both mRNA and protein levels of LGALS3 were elevated in the CDAHFD group compared to the normal group, whereas treatment with QWZG reduced their levels. Analysis of Human Protein Atlas database indicated that LGALS3 was predominantly expressed in Kupffer cells, and was validated by real-time quantitative PCR and immunofluorescence. Furthermore, the level of LGALS3 was significantly increased in lipopolysaccharide-induced RAW264.7 cells, where its overexpression and recombinant LGALS3 protein both significantly enhanced the expression of interleukin-6, interleukin-1β, and tumor necrosis factor-α. LGALS3 overexpression significantly inhibited glutathione peroxidase 4 (GPX4) expression, and exacerbated mitochondrial damage, whereas LGALS3 knockdown markedly increased GPX4 level, and significantly reduced the levels of both total iron and ferrous iron. QWZG treatment significantly reduced the levels of malondialdehyde and ferrous iron, increased the levels of superoxide dismutase and glutathione. In addition, QWZG treatment also significantly enhanced GPX4 expression. Mechanistically, LGALS3 knockdown was associated with reduced expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) and NOD-like receptor family pyrin domain containing 3, while its overexpression led to increased levels of these proteins. The TRAF6 inhibitor C25-140 effectively reversed the LGALS3-induced alterations in GPX4 expression and iron accumulation. Furthermore, QWZG treatment significantly decreased the levels of TRAF6 and NOD-like receptor family pyrin domain containing 3. CONCLUSION QWZG ameliorated the progression of MASH by modulating ferroptosis through the LGALS3/TRAF6/GPX4 axis.