FIGURE 7.
The anti-inflammatory effect of A4-NP or oxDHA is independent of PPARγ and can be abrogated by reduction with NaBH4 or adduction to glutathione. A, RAW cells were treated with vehicle or one of two PPARγ antagonists (GW9662, 1 μm, or T01007, 500 nm) for 30 min, then pretreated with A4-NP (10 μm) or oxDHA (40 μm), and stimulated with LPS as in previous experiments. p < 0.05 versus LPS alone. B and C, A4-NP (gray bars, 10 μm) or oxDHA (black bars, 40 μm in B and 10 μm in C) was subjected to reduction with NaBH4 (B) or incubated with GST and GSH (+GSH) for 2 h (C) and then purified by C18 SepPak and applied to RAW cells 30 min prior to LPS stimulation as in previous experiments. Control A4-NP and oxDHA was subjected to identical incubation and purification in the absence of NaBH4 or GSH. C18 SepPak purification removes residual GST, GSH, and NaBH4, as well as unoxidized DHA. Graphs in B and C are from a single experiment performed in triplicate and are representative of results from three independent experiments.