Fig. 5

Effects of 5-methoxyflavone on RAW264.7 cells M1 polarization and M2 repolarization. (A) RAW264.7 cells were treated with 5-methoxyflavone for 24 and 48 h. The viability of RAW264.7 cells was determined by the MTT assay. (B) Representative images of morphological changes of LPS-stimulated RAW264.7 cells with or without 5-methoxyflavone treatment were obtained under a microscope. (C) RAW264.7 cells were treated with LPS (100 ng/mL) in the presence or absence of 5-methoxyflavone for 24 and 48 h. Expression of Nrf2 and HO-1 was detected by western blotting. (D) Immunofluorescence analysis of 5-methoxyflavone-mediated Nrf2 nuclear translocation in LPS-stimulated RAW264.7 cells. (E-H) RAW264.7 cells were stimulated with a combination of LPS (100 ng/mL) plus IFN-γ (20 ng/mL) for M1 polarization and IL-4 (20 ng/mL) for M2 polarization. Flow cytometry analysis of M1 marker (iNOS) in LPS/IFN-γ-stimulated RAW264.7 cells with indicated concentration of 5-methoxyflavone treatment alone (E and F) or with combinations of 5-methoxyflavone plus brusatol treatment (G and H) for 24 h. ^###P < 0.001 versus the control group; ^***P < 0.001 versus the M1-polarized RAW264.7 cell group; ^δδδP < 0.001 versus the 5-methoxyflavone treatment alone. (I) After stimulation with LPS/IFN-γ for 0.5 - 8 h, RAW264.7 cells were lysed for analysis of the activation of P-STAT1. (J, K) Western blot analysis of P-STAT1 in LPS/IFN-γ-stimulated RAW264.7 cells with indicated concentration of 5-methoxyflavone treatment alone (J) or with combinations of 5-methoxyflavone plus brusatol treatment (K) for 30 min. (L) Immunostaining of iNOS and P-STAT1 in M1-polarized RAW264.7 cells treated with 5-methoxyflavone alone or in combination with brusatol treatment for 24 h. (M-P) M2-polarized RAW264.7 cells were generated by a 24 h treatment with IL-4 at 20 ng/mL, and then were repolarized into M1 phenotypes by incubation with LPS/IFN-γ for another 24 h. Flow cytometry analysis of M1 marker (iNOS) and M2 marker (CD206) in repolarization of M2-polarized RAW264.7 cells treated with 5-methoxyflavone alone (M and N) or in combination with brusatol treatment (O and P) for 24 h. ^###P < 0.001 versus the control group; ^ζζζP < 0.001 versus the M2-polarized RAW264.7 cell group; ^**P < 0.01, ^***P < 0.001 versus the M2-polarized RAW264.7 cells with LPS/IFN-γ stimulation group; ^δδP < 0.01 versus the 5-methoxyflavone treatment alone. (Q-R) Western blot analysis of P-STAT1 in repolarization of M2-polarized RAW264.7 cells with indicated concentration of 5-methoxyflavone treatment alone (Q) or with combinations of 5-methoxyflavone plus brusatol treatment (R) for 30 min. (S) Immunostaining of iNOS and P-STAT1 in repolarization of M2-polarized RAW264.7 cells treated with 5-methoxyflavone alone or in combination with brusatol treatment for 24 h