Figure 2

Fisetin probably binds to TLR4 and inhibits the TLR4-mediated intracellular signaling pathway. (A) Molecular docking of fisetin (pose 1) with the TLR4/MD2 complex (PDB ID: 3FXI). (B) BV2 microglial cells were seeded on 3% gelatin-coated coverslips and treated with the indicated concentrations of fisetin (0–5 µM) for 2 h prior to stimulation with 1 µg/mL LPS for 2 h and subsequent stimulation with 1 mM ATP (LPS/ATP) for an additional 2 h. Cells were fixed with 4% paraformaldehyde and immunostained for TLR4 using Alexa Fluor 647-conjugated secondary antibody. The images were captured using a CELENA S Digital Imaging System. (C) In a parallel experiment, the total proteins were extracted and western blotting was performed for detecting the expression of MyD88 and IRAK4 proteins. β-Actin was used as a loading control. The results indicate the mean ± standard error median (SEM), and is representative of the results obtained from three independent experiments. ^### p < 0.001 vs. untreated cells; *** p < 0.001 and ** p < 0.01 vs. LPS/ATP-treated cells.