ω-3 PUFAs antagonize NF-κB signaling in macrophages.
(A) ω-3 PUFAs suppress TLR4/NF-κB signaling in transfected 293T cells. 293T cells were transfected with TLR4/MD-2 expression vectors, and NF-κB luciferase reporter constructs. Transfected cells were pre-treated with ω-3 PUFA mixtures EPA/DHA (50 µM each) for 24 hours and then stimulated with LPS (100 ng/ml) in the presence or absence of ω-3 PUFA for additional 24 hours. NF-κB luciferase activity was measured using a Dual-Luciferase Reporter Assay. A.U.: Arbitrary Units. (B) ω-3 PUFAs suppress the NF-κB signaling in macrophages. Raw264.7 macrophages were transfected with NF-κB luciferase reporter constructs alone. The treatment is the same as described in (A). (C) ω-3 PUFAs act on the downstream signal(s) of TLR4 to inhibit NF-κB. Raw264.7 macrophages were transfected with CA-MyD88 expression vectors and NF-κB luciferase reporter constructs. (D) ω-3 PUFAs blocks NF-κB DNA binding. EMSA was conducted to examine the NF-κB DNA binding and was conducted as described in Materials and Methods. (E) ω-3 PUFAs blocks the NF-κB subunit p65 binding to the IL-6 promoter. ChIP assays were conducted to examine p65 DNA binding and were conducted as described in Materials and Methods. SYBR Green quantitative PCR was used to quantitate the immunoprecipitated DNA. For (A)–(C), all data are expressed as mean ± SE, n = 6. Groups labeled with the same superscripts are not statistically different from each other. Groups labeled with different superscripts are statistically different from each other.