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Fig. 5 Pinostrobin possibly binds to the TLR4/MD2 complex. (A) The ribbon model (pose 1, the strongest binding pose) represents that pinostrobin fits into the TLR4/MD2 complex (PDB; 3FXI, left). MD2 (red and chartreuse) and TLR4 (cyan and blue) are shown. Block dotted square shows the binding site and enlarged (right). (B) 2D interaction diagram from pose 1 was obtained using BIOVIA Discovery Studio Visualizer. Green line, carbon hydrogen bond; pink line, π-alkyl or alkyl interactions; light green circle, van der Waals interactions. (C) RAW 264.7 macrophages were pretreated with pinostrobin (0–20 μM) 2 h before incubation with 300 ng/mL LPS. Cellular protein was extracted 30 min after LPS treatment, and western blotting was performed. β-Actin was used for normalizing MyD88 and p-IRAK4 expression. The relative density was calculated using ImageJ software. ###, p < 0.001 vs. untreated cells; ***, p < 0.001 vs. LPS-treated cells.