Kaempferol reprograms pro-inflammatory macrophage polarization by targeting the PTGS2-PGE₂ axis: A multi-omics...

Metabolic dysfunction-associated steatohepatitis (MASH), a progressive liver disease driven by pro-inflammatory (M1) macrophage polarization, lacks effective pharmacotherapies. While the Chaihu-Baishao (CB) herb pair is a traditional remedy for liver disorders, its molecular mechanism is unknown. This study aimed to deconstruct CB's mechanism and validate its core bioactive component for MASH therapy. We employed an integrative strategy from multi-omics to in vivo validation. Transcriptomic analysis identified M1-polarized macrophages as central drivers of MASH. We then demonstrated that the CB extract ameliorates MASH pathology in a diet-induced mouse model. To elucidate the underlying mechanism, network pharmacology predicted kaempferol as the core bioactive compound and PTGS2 (COX-2) as its primary target. A direct kaempferol-PTGS2 interaction was rigorously confirmed by a suite of biophysical assays (CETSA, DARTS, MST), yielding a dissociation constant (Kd) of 5.16 µM. Functionally, kaempferol suppressed M1 macrophage polarization in vitro by inhibiting PTGS2-mediated prostaglandin E₂ (PGE₂) production, an effect reversed in a PGE₂ rescue experiment. Crucially, in vivo administration of kaempferol alone recapitulated the therapeutic benefits of the full CB extract, significantly improving liver pathology and suppressing hepatic PGE₂ levels. Our study provides a multi-scale validation, identifying kaempferol as the principal bioactive component of the CB herb pair. Kaempferol ameliorates MASH by directly targeting the PTGS2-PGE₂ axis to reprogram macrophage polarization, presenting a promising, mechanism-defined therapeutic lead with clear translational potential.