Qi Wei Zhi Gan formulation alleviates progressive fibrosis in metabolic dysfunction-associated steatohepatitis through suppressing Peroxidasin-collagen IV crosslinking.

BACKGROUND

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disorder involving steatosis, inflammation, and frequent fibrosis. Qi Wei Zhi Gan (QWZG) formulation, a traditional Chinese herbal formulation, has demonstrated therapeutic effects against MASH though its mechanisms remain not fully elucidated.

PURPOSE: This study investigates the anti-fibrotic effects of QWZG formulation and explores its mechanism related to extracellular matrix (ECM) remodeling.

METHODS

MASH was induced in mice by choline-deficient, l-amino acid-defined (CDAA) diet. QWZG efficacy was assessed via lipid profiles, liver function biomarkers, and histology. Proteomics identified candidate targets, validated by RT-qPCR, western blot, and immunofluorescence in primary hepatocytes. To explore potential mechanistic involvement, an in vivo experiment was performed using adenovirus-mediated Pxdn overexpression (Pxdn_OE) in CDAA-fed mice, with or without QWZG treatment. Human bulk, single-nucleus RNA sequencing (snRNA-seq) and spatial transcriptomics analyses support the key target and mechanism in MASH fibrosis.

RESULTS

QWZG treatment markedly reduced obesity, hepatomegaly, and lipid accumulation, and inflammation in MASH model. Proteomics revealed strong modulation of ECM and fibrosis pathway, with downregulation of Col1a1 and αSMA. Specifically, Pxdn emerged as the most upregulated protein in MASH mice and was suppressed following QWZG, an effect notably predominant in hepatocytes. Immunofluorescence further revealed colocalization of Pxdn and Col4a1 in primary hepatocytes. QWZG potently inhibited Pxdn-driven Col4a1 crosslinking, this suppression subsequently reduced the expression of fibrotic markers such as αSMA and Col1a1, indicating suppressed hepatic stellate cell activation. Adenovirus-mediated Pxdn_OE in CDAA-fed mice enhanced Pxdn-Col4a1 colocalization, indicating increased crosslinking activity, which was reversed by QWZG. SnRNA-seq and spatial transcriptomics further demonstrated a strong association between Pxdn expression and Col4a1, especially for fibrosis severity, supporting clinical relevance.

CONCLUSION

QWZG ameliorates liver fibrosis, which may be partly mediated by inhibition of Pxdn-mediated collagen IV crosslinking, reducing ECM deposition and improving basement membrane microenvironment. These results highlight the potential of QWZG as a novel therapeutic strategy for MASH.