Fig 1.
Effect of daidzein on pro-inflammatory gene expression in contact co-culture of 3T3-L1 adipocytes and RAW264 macrophages.
3T3-L1 preadipocytes were differentiated for seven days. RAW264 macrophages were co-cultured on adipocytes (Co-culture) in serum-free medium with (Daid) or without daidzein (Cont) for 24 h. (A) Ccl2, Il6, Tnf and adiponectin mRNA levels were quantified by real-time PCR and normalized to β-actin expression. Values are expressed as the fold change compared with a separate Cont culture arbitrarily set to 1. (B) The amounts of CCL-2, and IL-6 protein in the medium were determined by ELISA and normalized to the total cell protein content. **, p<0.01 versus Cont; §, p<0.05; §§, p<0.01 versus co-culture Cont.
Fig 2.
Effect of daidzein via PPAR-γ inhibition on pro-inflammatory gene expression in transwell co-cultured adipocytes.
3T3-L1 preadipocytes were differentiated for seven days. RAW264 macrophages were plated onto the transwell insert for 24 h. 3T3-L1 cells were differentiated in DMSO (Cont), 25 μM of daidzein (Daid), or Daid + 10 μM of GW9662 (Daid+GW). Ccl2, Il6 and adiponectin mRNA levels in 3T3-L1 adipocytes were quantified by real-time PCR and normalized to β-actin expression. Values are expressed as the fold change compared with a Cont culture without macrophages arbitrarily set to 1. **, p<0.01 versus Cont; Δ, p <0.1 versus co-culture Cont; #, p<0.05 versus co-culture Daid.
Fig 3.
Effect of daidzein on PPAR-α and PPAR-γ activity in HEK293T cells.
HEK293T cells were transfected with PPRE-containing reporter plasmid and (A) PPAR-α or (B) PPAR-γ expression plasmids, and then treated with the indicated concentrations (6.25–25 μM) of daidzein for 24 h. Luciferase activity was measured and expressed as a fold induction, that was corrected for transfection efficiency using renilla luciferase activity. Values are expressed as the fold change compared with the vehicle control that was arbitrarily set to 1. Δ, p<0.1; *, p<0.05; **, p<0.01 versus non-treated control.
Fig 4.
Effects of daidzein via PPAR-α/γ inhibition in palmitate- and CM- treated macrophages.
(A) RAW264 macrophages were cultured in the serum-free medium with the indicated concentrations (6.25–25 μM) of daidzein for 1 h, then stimulated with 400 μM palmitate for 4 h. (B) Cells were cultured in serum-free medium with DMSO (Cont), 25 μM of daidzein (Daid), Daid + 10 μM of GW6471 (Daid+α) or Daid + 10 μM of GW9662 (Daid+γ) for 1 h, then 400 μM palmitate was added and cells were incubated for another 4 h. (C) Cells were cultured in CM derived from hypertrophied adipocytes with Daid, Daid+α or Daid+γ for 24 h. Messenger RNA levels of each gene were quantified by real-time PCR and normalized to β-actin expression. Values are expressed as the fold change compared with the vehicle control that was arbitrarily set to 1. *, p<0.05; **, p<0.01 versus Cont. §, p<0.05; §§, p<0.01 versus palmitate Cont or CM Cont. Δ, p<0.1; ##, p<0.01 versus palmitate Daid or CM Daid.
Fig 5.
Effect of daidzein on palmitate-induced phosphorylation of JNK and p65 in RAW264 macrophages.
RAW264 macrophages were cultured in serum-free medium with (Daid) or without daidzein (Cont) for 1 h. Palmitate was added and cells were incubated for another 4 h. (A) Cells lysates were subjected to immunoblot analysis using specific antibodies. A representative immunoblot from two separate experiments with similar results is shown. (B) Densitometric analysis of protein bands in the western blots was done using Image J software. Optical density for each phosphorylated protein was calculated and normalized with the value of each total protein. **, p<0.01 versus Cont. §§, p<0.01 versus palmitate Cont.
Fig 6.
Putative mechanisms for the anti-inflammatory effect of daidzein on adipocyte-macrophage co-cultures.