The anti-inflammatory compound palmitoylethanolamide inhibits prostaglandin and hydroxyeicosatetraenoic acid production by a macrophage cell line

Abstract

The anti-inflammatory agent palmitoylethanolamide (PEA) reduces cyclooxygenase (COX) activity in vivo in a model of inflammatory pain. It is not known whether the compound reduces prostaglandin production in RAW264.7 cells, whether such an action is affected by compounds preventing the breakdown of endogenous PEA, whether other oxylipins are affected, or whether PEA produces direct effects upon the COX-2 enzyme. RAW264.7 cells were treated with lipopolysaccharide and interferon-γ to induce COX-2. At the level of mRNA, COX-2 was induced >1000-fold following 24 h of the treatment. Coincubation with PEA (10 μmol/L) did not affect the levels of COX-2, but reduced the levels of prostaglandins D₂ and E₂ as well as 11- and 15-hydroxyeicosatetraenoic acid, which can also be synthesised by a COX-2 pathway in macrophages. These effects were retained when hydrolysis of PEA to palmitic acid was blocked. Linoleic acid-derived oxylipin levels were not affected by PEA. No direct effects of PEA upon the oxygenation of either arachidonic acid or 2-arachidonoylglycerol by COX-2 were found. It is concluded that in lipopolysaccharide and interferon-γ-stimulated RAW264.7 cells, PEA reduces the production of COX-2-derived oxylipins in a manner that is retained when its metabolism to palmitic acid is inhibited.

Keywords: N‐acylethanolamine hydrolysing acid amidase; Palmitoylethanolamide; RAW264.7 cells; bootstrapped linear model; cyclooxygenase; fatty acid amide hydrolase; oxylipin; prostaglandin.

Figure 1

Effect of PEA and LPS + IFNγ treatment of RAW264.7 cells upon mRNA and the protein levels of COX‐2. RAW264‐7 cells (2.5 × 10⁵ per well) in six‐well plates were treated with LPS (0.1 μg/mL) + INFγ (100 U/mL) (or vehicle) and either 0 or 10 μmol/L PEA and cultured at 37°C for either 30 min (Panel A), 24 h (Panel B) or 20 h (Panel C); n = 6–16. In Panels A and B, the individual ∆C_t values (the difference in threshold cycle between the mRNA of interest and the housekeeper mRNA, in this case RPL19), are shown with the means represented by the bars. The right y‐axes show data where the mean value in the absence of either PEA or LPS + IFNγ are set to unity. The scale is antilogged since a change of ∆C_t of −1 unit corresponds to a doubling of mRNA. Under the figures are the P values (calculated using bootstrapped linear models for the data expressed as ∆C_t as described in Methods) for each time point, given the significant interaction LPS x Time in the main analysis (Table 2). ***P < 0.001, ^NS P > 0.05 for the comparisons shown in Panel A (i.e. where the LPS × PEA interaction was significant). In Panel C, the data are shown on a log scale, since the variances were unequal for the untransformed data. An one‐way ANOVA on the log‐transformed data gave F_2,15 = 163, P < 0.0001. The residual Q.Q plot was consistent with a normal distribution of the residuals. ***P < 0.001, ^NS P > 0.05 for the comparisons shown (Holm–Sidak's multiple comparison test).

Figure 2

Effect of PEA and LPS + IFNγ treatment of RAW264.7 cells upon mRNA levels of FAAH (Panels A, B) and NAAA (Panels C, D). For experimental details, see Legend to Figure 1. The incubation times were 30 min (Panels A, C) and 24 h (Panels B,D). *P < 0.05, ^NSP > 0.05.

Figure 3

Effect of PEA upon levels of (A) PGD₂; (B) PGE₂; (C) 11‐HETE; (D) 15‐HETE; (E) 9‐HODE and (F) 13‐HODE in LPS + IFNγ‐treated RAW264.7 cells. Cells (2.5 × 10⁵ per well) were added to six‐well plates with LPS (0.1 μg/mL well) and INFγ (100 U/mL) and cultured at 37°C for 24 h. PEA (3 μmol/L, P3; or 10 μmol/L, P10) or vehicle were added either at the beginning of this culturing period (“24 h”) or for 30 min after the LPS + INFγ incubation phase (“30 min”). The P values were from linear models for main effects alone (top three rows, ti = time component, with 30 min as the reference value) or for a model including interactions (bottom two rows), calculated using t‐distributions determined by bootstrap with replacement sampling (10,000 iterations) of the data under the null hypothesis. Possible and probable outliers, flagged in Boxplot (Tukey) plots, are shown as triangles and red squares, respectively. The possible outliers were included in the statistical analyses, whereas the probable outlier was excluded. The bars represent median values after exclusion of the probable outlier (n = 11–12). For 11‐HETE, the P values for the entire data set (i.e. including the probable outlier) were: ti, 0.87; P3, 0.86; P10, 0.0020; ti × P3, 0.83; ti x P10, 0.93.

Figure 4

Metabolism of PEA in LPS + IFNγ‐stimulated RAW264.7 cells. Untreated or LPS + INFγ‐treated (24 h) cells were preincubated for 10 min with either vehicle (“V”), 1 μmol/L URB597 (“U”), 30 μmol/L pentadecylamine (“P”) or a combination of the two compounds, prior to addition of 10 μmol/L [³H]PEA and incubation for 30 min. The recovered [³H]ethanolamine is expressed as % of the added tritium.

Figure 5

Effect of the combination of 1 μmol/L URB597 (“U”) and 30 μmol/L pentadecylamine (“P”) prior to addition of 0 or 10 μmol/L PEA upon the levels of (A) PGD₂; (B) PGE₂; (C) 11‐HETE and (D), 15‐HETE in LPS + IFNγ‐treated RAW264.7 cells. Cells (2.5 × 10⁵ per well) were added to six‐well plates with LPS (0.1 μg/mL well) and INFγ (100 U/mL) and cultured at 37°C for 24 h. URB597 and pentadecylamine (or vehicle) were added 10 min prior to addition of vehicle or 10 μmol/L PEA and incubation for 30 min. The raw data are shown as scatterplots (n = 17–18, bars indicate the medians). Possible and probable outliers are shown as triangles and squares, respectively. The possible outliers were included in the statistical analyses, whereas the probable outlier was excluded. The P values were calculated using t‐distributions determined by bootstrap with replacement sampling (10,000 iterations) of the data under the null hypothesis. For PGE₂, inclusion of the two probable outliers gave P values of: PEA, 0.57; U + P, 0.60; PEA × U + P, 0.24. For 11‐HETE, inclusion of the two probable outliers gave P values of: PEA, 0.023; U + P, 0.056; PEA × U + P, 0.19.

Figure 6

Effects of PEA upon the activity of human recombinant COX‐2. Substrates and concentrations were: (A) 10 μmol/L AA, N = 9; B, 2 μmol/L 2‐AG, N = 6; (C) 10 μmol/L 2‐AG, N = 6. Values are means ± S.E.M, (unless enclosed by the symbols) of the change in oxygen utilisation following addition of enzyme to the oxygen electrode chamber. The PEA concentrations are shown beside each curve.