Differential modulation of toll-like receptor agonists-induced iNOS expression by polyunsaturated and saturated fatty acids

Figures & data

Figure 1. The structure of EPA and ACA. (A) EPA structure. (B) ACA structure. (C) RAW264.7 cells were treated with EPA (10, 30, 50 μM) or ACA (10, 30, 50 μM) for 4 h. Twenty microliters of the CellTiter 96® AQ_ueous One Solution Reagent was added directly to culture wells. The plate was incubated at 37°C for 4 h in a humidified, 5% CO₂ atmosphere. The absorbance was recorded at 490 nm with a 96-well plate reader. Veh, vehicle; EPA, eicosapentanoic acid; ACA, arachidic acid.

Figure 2. EPA, but not ACA, suppresses the activation of NF-κB induced by TLR agonists. (A–C) RAW 264.7 cells were transfected with a NF-κB-luciferase reporter plasmid and pretreated with EPA (10, 30 μM) or ACA (10, 30 μM) for 1 h and then treated with LPS (10 ng/mL) (A), MALP-2 (10 ng/mL) (B), or Poly[I:C] (10 μg/mL) (C) for an additional 8 h. Cell lysates were prepared, and luciferase enzyme activities were determined. Values represent the mean ± SEM (n = 3). *, Significantly different from LPS alone, p < .01 (**) (A). #, Significantly different from MALP-2 alone, p < .05 (#) (B). +, Significantly different from Poly[I:C] alone, p < .05 ( + ) (C). Veh, vehicle; EPA, eicosapentanoic acid; ACA, arachidic acid.

Figure 3. EPA, but not ACA, suppresses iNOS expression induced by TLR agonists. (A–C) RAW 264.7 cells were transfected with iNOS-luciferase reporter plasmid and pretreated with EPA (10, 30 μM) or ACA (10, 30 μM) for 1 h and then treated with LPS (10 ng/mL) (A), MALP-2 (10 ng/mL) (B), or Poly[I:C] (10 μg/mL) (C) for an additional 8 h. Cell lysates were prepared, and luciferase enzyme activities were determined. Values represent the mean ± SEM (n = 3). *, Significantly different from LPS alone, p < .05 (*), p < .01 (**) (A). #, Significantly different from MALP-2 alone, p < .01 (##) (B). +, Significantly different from Poly[I:C] alone, p < .01 (++) (C). Veh, vehicle; EPA, eicosapentanoic acid; ACA, arachidic acid.

Figure 4. EPA inhibits iNOS protein induced by TLRs agonists, but ACA does not. (A–C) RAW264.7 cells were pretreated with EPA (10, 30 μM) or ACA (10, 30 μM) for 1 h and then further stimulated with LPS (10 ng/mL) (A), MALP-2 (10 ng/mL) (B), or Poly[I:C] (10 μg/mL) (C) for an additional 8 h. Cell lysates were analyzed for iNOS and β-actin protein by immunoblots. Veh, vehicle; EPA, eicosapentanoic acid; ACA, arachidic acid.

Figure 5. EPA, but not ACA, inhibits nitrite production induced by TLRs agonists. (A–C) RAW 264.7 cells were pretreated with EPA (10, 30 μM) or ACA (10, 30 μM) for 1 h and then treated with LPS (10 ng/mL) (A), MALP-2 (10 ng/mL) (B), or Poly[I:C] (10 μg/mL) (C) for an additional 20 h. The amounts of nitrite in the supernatant were measured using Griess reagent. Values represent the mean ± SEM (n = 3).*, Significantly different from LPS alone, p < .01 (**) (A). #, Significantly different from MALP-2 alone, p < .05 (#), p < .01 (##) (B). +, Significantly different from Poly[I:C] alone, p < .01 (++) (C). Veh, vehicle; EPA, eicosapentanoic acid; ACA, arachidic acid.