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Eicosapentaenoic Acid Prevents LPS-Induced TNF- Expression by Preventing NF-B Activation

Graduate Center for Nutritional Sciences (Y.Z., L.H.C.), Internal Medicine (S.J.-B., S.B.), University of Kentucky, Lexington, KY

View larger version (32K): [in a new window]   Fig. 1. EPA prevents LPS-induced production of TNF-. THP-1 cells were stimulated with graded concentrations of LPS for 6 hours after pre-incubation with or without 60 µM of EPA for 24 hours. Cells were collected and TNF- levels were determined in cell culture supernatant by ELISA. Mean ± S.E., n = 3. *Significantly different from LPS group (p < 0.05). Data are representative of three experiments.

View larger version (17K): [in a new window]   Fig. 2. EPA prevents LPS-induced mRNA levels of TNF-. Cells were stimulated with 0.2 µg/ml of LPS after pre-incubation with or without 60 µM of EPA for 24 hours. TNF- and ß-actin mRNA levels were determined at various time points after LPS was added. Mean ± S.E., n = 2. *Significantly different from LPS group (p < 0.05). Data are representative of two experiments.

View larger version (14K): [in a new window]   Fig. 3. Effect of EPA on stability of TNF- mRNA. THP-1 cells were stimulated with 0.2 µg/mL of LPS after pre-incubation with or without 60 µM of EPA for 24 hours. Actinomycin D was added to the medium after cells were stimulated with LPS for 90 minutes. Cells were collected at various time points after addition of actinomycin D, and mRNA levels of TNF- and ß-actin were determined. Relative amount of mRNA (TNF-/ß-actin) at time 0 of actinomycin D addition was used as 100%. Mean ± S.E., n = 3. There was no significant difference between the means of two groups.

View larger version (48K): [in a new window]   Fig. 4. EPA prevents LPS-induced NF-B activation. (A) THP-1 cells pre-incubated with or without 60 µM of EPA for 24 hours were stimulated with 0.2 µg/mL of LPS for 45 minutes. Nuclear extracts were prepared and analyzed by EMSA. Data are representative of three experiments. (B) The competition assay was carried out by incubating nuclear extracts prepared from LPS-stimulated THP-1 cells with labeled probes (1), labeled probes plus excess unlabeled probes (2), or labeled probes plus excess mutated oligonucleotide (3). The mutated oligonucleotide has a "G""C" substitution in the NF-B/Rel DNA binding motif as described in "Material and Methods". (C) Supershift assay of NF-B. Antibodies of p65, p50 or control IgG was incubated with nuclear extracts of LPS-stimulated THP-1 cells and EMSA was performed.

View larger version (18K): [in a new window]   Fig. 5. EPA prevents LPS-induced translocation of p65 subunit of NF-B to nucleus. THP-1 cells pre-incubated with or without 60 µM of EPA were stimulated with 0.2 µg/mL of LPS for various time periods. Nuclear extracts were prepared and analyzed by Western blot. Data are representative of three experiments.

View larger version (23K): [in a new window]   Fig. 6. EPA prevents LPS-induced expression of B-directed luciferase. THP-1 cells transfected with pNF-B-Luc were pre-incubated with or without 60 µM of EPA for 24 hours before stimulation with 0.2 µg/ml of LPS for 6 hours. The efficacy of transfection was monitored by co-transfection of the cells with pSV-ß-galactosidase control vectors. Mean ± S.E., n = 6. Means with different letters are significantly different (p < 0.05). Data are representative of three experiments.

View larger version (27K): [in a new window]   Fig. 7. EPA prevents LPS-induced IB- phosphorylation and degradation. THP-1 cells pre-incubated with or without 60 µM of EPA for 24 hours were stimulated with 0.2 µg/mL of LPS for various time periods. Cytosolic extracts were prepared and analyzed by Western Blot using antibodies specifically recognize IB- (A) and phosphorylated IB- (B). Data are representative of three experiments.