Fuzheng Huayu formula attenuates ductular reaction and liver fibrosis potentially through suppressing Gli1 pathway

Fuzheng Huayu formula (FZHY) is clinically used for liver fibrosis treatment, but its pharmacological mechanisms remain incompletely understood.

This study explored the anti-fibrotic effect of FZHY, particularly its efficacy against cholestatic liver fibrosis, and the underlying molecular mechanisms.

The therapeutic potential of FZHY was evaluated using Mdr2^-/- mice and CCl₄/2-AAF-induced rats of liver fibrosis model rodents, and GANT61, a Gli1 inhibitor was used as a positive control drug. Histological analysis and double-immunofluorescence techniques were employed to detect the effects of FZHY on ductular reaction and liver fibrosis. In vitro, WB-F344 cells, a hepatic progenitor cell (HPC) line, were transduced with Gli1-overexpressing lentiviral vectors and induced with sodium butyrate (SB) to verify the mechanisms of FZHY.

In both Mdr2^-/- mice and CCl₄/2-AAF-induced rats, FZHY obviously ameliorated liver fibrosis, evidenced by reduced collagen deposition, decreased hepatic hydroxyproline content, and less inflammatory cell infiltration. FZHY reduced the expression of Epcam, CK19, and CK7. Double-immunofluorescence revealed an increased number of CK19⁺ and OV6⁺ cells in the CCl₄/2-AAF rats, which decreased after FZHY treatment. Mechanistically, FZHY downregulated Gli1 expression in the liver. Notably, FZHY exerted a similar effect on ductular reaction and liver fibrosis as the Gli1 inhibitor GANT61 in CCl₄/2-AAF-induced rats. In vitro, the inhibitory effect of FZHY on the differentiation of WB-F344 cells into a biliary phenotype was similar to the Gli1 inhibitor GANT61. Furthermore, Gli1 overexpression lentiviral transfection experiments confirmed that FZHY inhibited the differentiation of WB-F344 cells into a ductular phenotype in a Gli1-dependent manner.

FZHY exerts a remarkable antifibrotic effect, which may be mediated by inhibiting the differentiation of HPCs into a ductular phenotype, potentially through suppression of the Gli1 pathway. This finding provides further insight into the mechanism of FZHY and suggests a potential role of Gli1 signaling in its antifibrotic action.