Fig 1.
DHA inhibits IL-6 expression stimulated by LPS or LPS plus PA.
A. RAW264.7 macrophages were treated with 100 μM of palmitic acid (PA), palmitoleic acid (POA), oleic acid (OA), linoleic acid (LA), or docosahexaenoic acid (DHA) in the absence or presence of 1 ng/ml of LPS for 24 h. After treatment, IL-6 in culture medium was quantified using ELISA. + vs. #, p<0.01; # vs. *, p<0.01. B. RAW264.7 macrophages were treated with both 1 ng/ml LPS and 100 μM of PA in the absence or presence of 100 μM of POA, OA, LA or DHA for 24 h. After treatment, IL-6 in culture medium was quantified using ELISA. # vs. *, p<0.01. C and D. RAW264.7 macrophages were treated with 1 ng/ml of LPS, 100 μM of PA or both 1 ng/ml LPS and 100 μM of PA in the absence or presence of different concentrations of DHA (C) or eicosapentaenoic acid (EPA) (D) for 24 h. After treatment, IL-6 in culture medium was quantified using ELISA. In panel C: ** vs. *, p<0.01; + vs. *, p<0.05; # vs. *, p<0.01; ++ vs. **, p<0.05; ## vs. **, p<0.01. In panel D: ** vs. *, p<0.01; + vs. *, p<0.05; # vs. **, p<0.01.
Table 1.
The effect of DHA on gene expression stimulated by LPS, PA or LPS plus PA.
Fig 2.
DHA inhibits the CER production increased by LPS and PA.
A. Diagram for CER de novo synthesis and sphingomyelin hydrolysis pathways. SPT, serine palmitoyltransferase; CerS, ceramide synthase; SMase, sphingomyelinase. B and C. LPS and PA synergistically stimulate total (B) and dhC16-CER production (C). RAW264.7 macrophages were treated with 100 μM of OA, DHA or PA in the absence or presence of 1 ng/ml of LPS for 12 h. After treatment, total and dhC16-CER were quantified using lipidomics. + vs. *, p<0.01; # vs. +, p<0.01. D-F. Either DHA or OA inhibited PA- or LPS plus PA-stimulated CER production. RAW264.7 macrophages were treated with 1 ng/ml LPS, 100 μM PA or both LPS and PA in the absence or presence of 100 μM DHA or OA for 12 h. After treatment, total CER (D), C16-CER (E) and dhC16-CER (F) were quantified using lipidomics. + vs. *, p<0.01; # vs. +, p<0.01; ++ vs. +, p<0.01; ## vs. #, p<0.01. G. DHA inhibits SPT1 mRNA expression stimulated by LPS and PA. RAW264.7 macrophages were treated with 1 ng/ml LPS, 100 μM PA or both LPS and PA in the absence or presence of 100 μM of DHA for 12 h. After treatment, SPT1 mRNA was quantified using real-time PCR. * vs. +, p<0.05; * vs. #, p<0.05; * vs. ^, p<0.05; ^^ vs. ^, p<0.01.
Table 2.
The effect of DHA on CER content regulated by LPS, PA or LPS plus PA.
Fig 3.
DHA has no effect on SM hydrolysis and ASMase activity stimulated by LPS and PA.
A. RAW264.7 macrophages were treated with 1 ng/ml of LPS, 100 μM of PA or both 1 ng/ml LPS and 100 μM of PA in the absence or presence of 100 μM of DHA for 12 h. After treatment, cellular sphingomyelin was quantified using lipidomics. * vs. #, p<0.01. B. RAW264.7 macrophages were treated with 1 ng/ml of LPS, 100 μM of PA or both 1 ng/ml LPS and 100 μM of PA in the absence or presence of 100 μM of DHA for 2 h. After treatment, cellular ASMase activity was determined as described in the Methods. * vs. +, p<0.05; * vs. #, p<0.05; * vs. ^, p<0.05.
Table 3.
The effect of DHA on sphingomyelin content regulated by LPS, PA or LPS plus PA.
Fig 4.
DHA inhibits IL-6 transcription and NFκB transcriptional activity.
A. RAW264.7 macrophages were transfected with plasmids containing an 1168-bp IL-6 promoter sequence and luciferase reporter gene for 24 h. After the transfection, the macrophages were treated with 1 ng/ml of LPS, 100 μM of PA or both LPS and PA for 24 h and cellular firefly and renilla luciferase activities were assayed. The ratios of firefly vs. renilla luciferase were calculated. * vs. cells with the same treatment in the absence of DHA, p<0.01. B. RAW264.7 macrophages were transfected with DNA construct containing the tandem repeats of NFκB transcriptional response element in the promoter and luciferase reporter gene for 24 h. After the transfection, the macrophages were treated with 1 ng/ml of LPS, 100 μM of PA or both LPS and PA for 24 h and cellular firefly and renilla luciferase activities were assayed. The ratios of firefly vs. renilla luciferase were calculated. The ratios of firefly vs. renilla luciferase activity were calculated. * vs. cells with the same treatment in the absence of DHA, p<0.01. C. RAW264.7 macrophages were transfected with DNA construct containing the tandem repeats of NFκB transcriptional response element in the promoter and luciferase reporter gene for 24 h. After the transfection, the macrophages were treated with 1 ng/ml of LPS or 50 μM of C2-CER for 24 h and cellular firefly and renilla luciferase activities were then assayed. The ratios of firefly vs. renilla luciferase activity were calculated. * vs. +, p<0.05; * vs. #, p<0.05.
Fig 5.
Myriocin inhibits IL-6 secretion stimulated by LPS or LPS plus PA.
A-C. RAW264.7 macrophages were treated with 1 ng/ml of LPS, 100 μM of PA or both LPS and PA in the absence or presence of 10 μM of myriocin for 12 h. After the incubation, total (A), C16-CER (B) and dhC16-CER (C) were quantified using lipidomics. D. RAW264.7 macrophages were treated with 1 ng/ml of LPS, 100 μM of PA or both LPS and PA in the absence or presence of 10 μM of myriocin for 24 h. After treatment, IL-6 in culture medium was quantified using ELISA. * vs. +, p<0.01; * vs. #, p<0.01; ** vs. *, p<0.01; ^^ vs. ^, p<0.01; ++ vs. +, p<0.01; ## vs. #, p<0.01.
Table 4.
The effect of myricin on CER synthesis stimulated by PA and LPS plus PA.
Fig 6.
Schematic diagram to show the proposed mechanism involved in the inhibition by DHA of proinflammatory gene expression stimulated by LPS or LPS plus PA in RAW264.7 macrophages.