Mechanistic Investigation of Aconitine Combined with Paeoniflorin Against Knee Osteoarthritis via the Ihh-Gli Signaling Pathway

• • Aconitine-paeoniflorin at 20+200 μg/kg reduced MMP13 serum levels and upregulated Col II with P<0.001 versus model, directly countering cartilage catabolism; this dual regulation addresses the inability of COX-2 inhibitors like celecoxib to modify disease progression. • • Ihh, Gli, Ptch1, and MMP13 gene and protein expressions were downregulated by 30–60% (P<0.05 to P<0.001) in joint tissues, confirming pathway-specific inhibition; this provides a druggable target for halting the Hedgehog-driven osteoarthritic cascade. • • Molecular docking revealed binding energies ≤−5 kcal/mol for aconitine and paeoniflorin with Ihh, Gli, ADAMTS5, and MMP13, indicating strong thermodynamic affinity; this supports structure-based optimization of the pair as a multi-target agent. • • No mortality or significant elevations in AST, creatinine, BUN, or urinary protein occurred across all dose groups, with histopathology showing normal heart, liver, and kidney morphology; this establishes a safety margin that mitigates the narrow therapeutic window of aconitine monotherapy.