Ailanthone ameliorates CCl4-induced liver fibrosis by targeting PKM2-mediated macrophage M1 polarization and glycolytic reprogramming

Background: Liver fibrosis is a chronic wound-healing response characterized by excessive extracellular-matrix deposition. Pro-inflammatory M1 macrophages drive fibrogenesis, whereas M2 macrophages support repair. Ailanthone (AIL), a plant-derived natural compound, has been suggested to regulate macrophage polarization, but its role and mechanism in liver fibrosis remain unclear.

Methods: Mice with CCl₄-induced fibrosis received AIL for 3 weeks; liver injury, fibrosis, and serum biochemistry were assessed. In vitro, LPS-stimulated RAW264.7 macrophages and TGF-β1-activated LX-2 cells were used to assess AIL's effects on PKM2/Akt signaling and glycolysis through Western blot, qPCR, enzyme assays, siRNA, and the PKM2 activator TEPP-46.

Results: AIL reduced Collagen deposition and lowered α-SMA, Collagen III, and fibronectin expression, while normalizing ALT and AST levels in fibrotic mice. AIL also suppressed TGF-β1-induced LX-2 activation and reduced LPS-driven M1 polarization in macrophages (TNF-α, IL-1β, IL-6, iNOS). AIL lowered PKM2 expression and activity and inhibited aerobic glycolysis (lactate, PK activity, GLUT-1, HIF-1α, LDHA). PKM2 knockdown or tetramer stabilization with TEPP-46 produced similar effects, confirming PKM2 as the critical target.

Conclusion: By targeting PKM2, AIL regulates macrophage metabolism and polarization while concurrently reducing hepatic stellate-cell activation and fibrosis. These findings position AIL as a promising PKM2-directed candidate for anti-fibrotic therapy.