• Rutin significantly inhibits neutrophil extracellular trap (NET) formation in dHL60 cells by downregulating PAD4, MPO, and NE expression.
• NETs suppress osteogenic differentiation and promote apoptosis of MC3T3-E1 cells in vitro.
• Rutin treatment, especially combined with DNase I, rescues NET-induced inhibition of osteogenesis and reduces apoptosis in MC3T3-E1 cells.
• Molecular docking and dynamics simulations confirm stable binding of rutin to PAD4, MPO, and NE, suggesting targeted inhibition of NET formation.