Isofraxidin ameliorates CCl₄-induced liver fibrosis in mice by inhibiting the NF-κB pathway

To investigate the anti-fibrotic effects of Isofraxidin (IF) on liver fibrosis and its underlying mechanisms.

C57BL/6J mice (bred by The Jackson Laboratory) were induced with liver fibrosis using CCl4 and treated with different doses of IF (20 mg/kg and 40 mg/kg) via gavage. Colchicine served as the positive control. Liver fibrosis markers (α-smooth muscle actin,α-SMA; Fibronectin, FN; Collagen type I, Col-I) were assessed by histological staining, immunohistochemistry, and western blotting. Network pharmacology and molecular docking were adopted to conjecture underlying targets and signaling pathways of IF, followed by experimental validation.

IF at various doses improved liver function and exhibited dose-dependent anti-fibrotic effects. Network pharmacology identified Protein kinase Kinase B (PKB, Akt), Proto-oncogene tyrosine-protein kinase (Src), Epidermal growth factor receptor (EGFR), Heat Shock Protein 90 Alpha Family Class A Member 1 (HSP90AA1), Glycogen Synthase Kinase 3 beta (GSK3β), and Monoamine Oxidase B (MAOB) as the core targets. Molecular docking showed good binding affinity between IF and these targets. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway is a major pathway involved in IF's anti-fibrotic effects. Experimental validation confirmed that IF inhibited the expression of inflammatory factors (Tumor Necrosis Factor-alpha, TNF-α; Interleukin-1 beta, IL-1β; Interleukin-10, IL-10) and the phosphorylation of NF-κB and Inhibitor of Nuclear Factor Kappa B Alpha (IκBα).

IF effectively alleviated CCl4-induced liver fibrosis in mice by modulating key targets (AkT, Src, EGFR, HSP90AA1, GSK3B, and MAOB) and the NF-κB signaling pathway.