Artepillin C from Artemisia capillaris attenuates liver fibrosis by targeting NR3C2 and regulating the PPAR-γ/Smad...

Artemisia capillaris is a traditional Chinese medicinal herb and widely used for the treatment of jaundice, hepatitis, and other liver-related diseases. Contemporary pharmacological studies have revealed its hepatoprotective, anti-inflammatory, and anti-fibrotic activities. However, the bioactive constituents responsible for its anti-hepatic fibrotic effect remain poorly defined. Artepillin C (AC), one of the coumaric acid derivatives isolated from A. capillaris, has not yet been studied in liver fibrosis, and its mechanism of action is still unknown.

This study aimed to evaluate the anti-hepatic fibrotic effects of AC in vitro and in vivo, and to elucidate its molecular target and underlying mechanism.

The anti-fibrotic activity of AC was assessed in transforming growth factor-β1 (TGF-β1)-induced hepatic stellate cell (HSC) activation models using LX-2 and JS-1 cells, and validated in a carbon tetrachloride (CCl₄)-induced mouse model of liver fibrosis. The expression of extracellular matrix (ECM)-related markers were analyzed by Western blot, RT-qPCR, and immunofluorescence (IF). Transcriptomic analysis, target prediction and experimental screening were introduced to identify potential molecular targets of AC. The interaction between AC and target was validated by molecular docking and molecular dynamics (MD) simulation, which were supported by the cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS). Receptor agonist/antagonist treatment, and co-immunoprecipitation (Co-IP) were conducted to explore the anti-fibrotic mechanism of AC.

AC markedly attenuated TGF-β1-induced activation of HSCs without apparent cytotoxicity and reduced the expression of ECM proteins, including fibronectin (FN) and collagen I. In CCl₄-induced mice, AC alleviated liver injury and reduced ECM deposition. Transcriptomic analysis combined with target prediction suggested that nuclear receptor subfamily 3 group C member 2 (NR3C2) was a potential target of AC. Docking and MD simulation indicated that AC could interact with NR3C2, while the CETSA and DARTS further demonstrated that AC binds to NR3C2 and increases its resistance to thermal denaturation and protease digestion. Mechanistically, AC downregulated NR3C2 expression, weakened the interaction between NR3C2 and peroxisome proliferator-activated receptor-γ (PPAR-γ), and restored PPAR-γ levels at higher concentrations, thereby suppressing TGF-β1-induced Smad2/3 phosphorylation. Consistent with these results, activation of NR3C2 enhanced Smad2/3 phosphorylation, whereas inhibition of NR3C2 or PPAR-γ attenuated Smad2/3 phosphorylation.

This study suggests that AC, a natural product derived from A. capillaris, attenuates liver fibrosis by targeting NR3C2 and regulating the PPAR-γ/Smad axis. These findings provide mechanistic insight into the anti-fibrotic effects of AC and further expand the pharmacological understanding of A. capillaris-derived bioactive constituents.