Figure 3

Docosahexaenoic acid (DHA) pretreatment inhibits the pro-inflammatory response to TNFα in rHypoE-7 cells. A (i). Western blots (α-phospho-TAK1 (pTAK1) and α-β-actin) of rHypoE-7 cells pretreated with 100 μM DHA or vehicle (DMSO) for 1 hr prior to the addition of 10 ng/mL TNFα for 10 min. (ii). DHA pretreatment (gray bars) prior to TNFα exposure significantly reduced pTAK1 levels relative to DMSO alone (white bars) (0.54 ± 0.06 and 1.51 ± 0.08 pTAK1/βactin, respectively). B. Relative to DMSO (white bars), DHA (gray bars) pretreatment (100 μM, 1 hr) significantly reduced the inflammatory transcriptional response to TNFα (10 ng/mL, 2 hr) as shown from mRNA levels of IκBα (i) (DMSO: 1.83 ± 0.22 and DHA: 1.07 ± 0.09 IκBα/histone mRNA, n = 4) and TNFα (ii) (DMSO: 2.33 ± 0.21 and DHA: 0.44 ± 0.04 TNFα/histone mRNA, n = 4). C (i). Western blots (α-TNFα, α-βactin) demonstrating TNFα protein levels in rHypoE-7 cells treated as described in Section B, with an additional recovery incubation (6 hr) in media containing FBS. As with mRNA levels, DHA pretreatment (gray bars) significantly reduced TNFα protein production relative to the DMSO vehicle control (white bars) (DMSO: 2.21 ± 0.26 and DHA: 0.62 ± 0.30 TNFα/βactin; n = 3 to 4) (ii). Data are shown as mean ± SEM; **P <0.01; ***P <0.01).