Figure 6.

Nrf2 plays a key role in the anti-inflammatory effects of fisetin. (A) Representative Western blots for Nrf2, ATF4 and actin in nuclear extracts from BV-2 cells treated with 25 ng/mL LPS alone or in the presence of 5 µM fisetin ± the indicated concentrations of FeCl₂ or CuCl₂ for 4 h. (B) Quantitation of results from four independent experiments identical to (A). ** indicates p < 0.01 and *** indicates p < 0.001 relative to fisetin alone. (C) Representative Western blot of BV-2 cells treated for 48 h with control siRNA (ct si) or Nrf2 siRNA (Nrf2 si). (D–F) BV2 cells transfected with control siRNA (ct si) or Nrf2 siRNA (Nrf2 si) were treated overnight with 25 ng/mL LPS and 5 µM fisetin and/or the indicated concentrations of FeCl₂ or CuCl₂. Cell culture supernatants were cleared and assayed for NO by the Griess assay or pro-inflammatory cytokines using specific ELISAs. Results are presented as the percent (%) of the value obtained with LPS alone which was set at 100%. NO (D); IL6 (E) or TNFα (F). Results in (D–F) are the average of three independent experiments. * indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.001 relative to fisetin + LPS alone. # indicates p < 0.05 relative to Nrf2 siRNA with fisetin and LPS only.