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, 15 (12), 2664-74

Palmitoylethanolamide Counteracts Reactive Astrogliosis Induced by β-Amyloid Peptide

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Palmitoylethanolamide Counteracts Reactive Astrogliosis Induced by β-Amyloid Peptide

Caterina Scuderi et al. J Cell Mol Med.

Abstract

Emerging evidence indicates that astrogliosis is involved in the pathogenesis of neurodegenerative disorders. Our previous findings suggested cannabinoids and Autacoid Local Injury Antagonism Amides (ALIAmides) attenuate glial response in models of neurodegeneration. The present study was aimed at exploring palmitoylethanolamide (PEA) ability to mitigate β-amyloid (Aβ)-induced astrogliosis. Experiments were carried out to investigate PEA's (10(-7) M) effects upon the expression and release of pro-inflammatory molecules in rat primary astrocytes activated by soluble Aβ(1-42) (1 μg/ml) as well as to identify mechanisms responsible for such actions. The effects of Aβ and exogenous PEA on the astrocyte levels of the endocannabinoidsand of endogenous ALIAmides were also studied. The peroxisome proliferator-activated receptor (PPAR)-α (MK886, 3 μM) or PPAR-γ (GW9662, 9 nM) antagonists were co-administered with PEA. Aβ elevated endogenous PEA and d5-2-arachidonoylglycerol (2-AG) levels. Exogenous PEA blunted the Aβ-induced expression of pro-inflammatory molecules. This effect was reduced by PPAR-α antagonist. Moreover, this ALIAmide, like Aβ, increased 2-AG levels. These results indicate that PEA exhibits anti-inflammatory properties able to counteract Aβ-induced astrogliosis, and suggest novel treatment for neuroinflammatory/ neurodegenerative processes.

Figures

Fig 1
Fig 1
PEA attenuates Aβ-induced astrocyte activation. Aβ-challenged (1 μg/ml) cells were treated with PEA (10−7 M) in the presence of the selective PPAR-γ antagonist (GW9662, 9 nM) or the selective PPAR-α antagonist (MK886, 3 μM). GFAP mRNA was evaluated 12 hrs following Aβ challenge; GFAP and S100B protein expression was evaluated after 24 hrs of treatments by Western blot and immunofluorescence analyses. S100B release in the cellular milieu was determined 24 hrs after Aβ challenge by ELISA assay. (A) Results of GFAP and S100B Western blot analysis and densitometric analysis of corresponding bands. β-actin was used as loading control. (B) Results of GFAP RT-PCR amplification and densitometric analysis of corresponding bands. GAPDH was used as standard control. (C) Measurement of S100B release by ELISA assay. (D) Analysis of GFAP and S100B protein expression by immunofluorescence (magnification 10 × ). Results are the mean ± S.E.M. of n = 4 separate experiments. ***P < 0.001 versus control; ###P < 0.01 versus Aβ-challenged cells; °°°P < 0.001 and °P < 0.05 versus Aβ+ PEA-challenged cells.
Fig 2
Fig 2
PEA blunts Aβ-induced neuroinflammation. Aβ-challenged (1 μg/ml) cells were treated with PEA (10−7 M) in the presence of the selective PPAR-γ antagonist (GW9662, 9 nM) or the selective PPAR-α antagonist (MK886, 3 μM). iNOS and COX-2 protein expression was evaluated 24 hrs following treatments by Western blot and immunofluorescence analyses. After 24 hrs of Aβ-challenge, it was evaluated the release in the cellular milieu of nitric oxide, measured as its stable metabolite NO2, and PGE2 by the Griess reaction and ELISA assay, respectively. (A) Results of iNOS and COX-2 Western blot analysis and densitometric analysis of corresponding bands. β-actin was used as loading control. (B) Determination of nitric oxide by the spectrophotometric assay based on the Griess reaction. (C) Measurement of PGE2 release by ELISA assay. (D) Analysis of iNOS and COX-2 protein expression by immunofluorescence (magnification 10 × ). Results from Western blot and immunofluorescence analyses are the mean ± S.E.M. of n = 3 separate experiments. Data from Griess reaction and ELISA assay are the mean ± S.E.M. of n = 5 independent experiments. ***P < 0.001 versus control; ###P < 0.001 versus Aβ-challenged cells; °P < 0.05 versus Aβ+ PEA-challenged cells.
Fig 3
Fig 3
PEA effects on Aβ-induced pro-inflammatory cytokine release. Aβ-challenged (1 μg/ml) cells were treated with PEA (10−7 M) in the presence of the selective PPAR-γ antagonist (GW9662, 9 nM) or the selective PPAR-α antagonist (MK886, 3 μM). TNF-α and IL-1β release was measured after 24 hrs of treatments by ELISA assay. Each bar shows the mean ± S.E.M. of n = 3 separate experiments. ***P < 0.001 versus control; ###P < 0.001 versus Aβ-challenged cells; °P < 0.05 versus Aβ+ PEA-challenged cells.
Fig 4
Fig 4
Anti-inflammatory actions of PEA depend upon MAPK inhibition. Aβ-challenged (1 μg/ml) cells were treated with PEA (10–7 M) in the presence of the selective PPAR-γ antagonist (GW9662, 9 nM) or the selective PPAR-α antagonist (MK886, 3 μM). p38 and JNK phosphorylated protein expression was analysed after 30 min. of treatments by Western blot. β-actin was used as loading control. Each bar shows the mean ± S.E.M. of n = 3 independent experiments. ***P < 0.001 versus control; ###P < 0.001 versus Aβ-challenged cells; °P < 0.05 versus Aβ+ PEA-challenged cells.
Fig 5
Fig 5
PEA inhibits the Aβ-induced activation of NF-κB and AP-1 nuclear transcription factors. Aβ-challenged (1 μg/ml) cells were treated with PEA (10−7 M) in the presence of the selective PPAR-γ antagonist (GW9662, 9 nM) or the selective PPAR-α antagonist (MK886, 3 μM). Nuclear transcription factors activation was evaluated after 30 min. of treatments by EMSA analysis. Figure shows the results of NF-κB and AP-1 complex shifts (upper panel) and densitometric analysis of corresponding bands (lower panel). Treatment of astrocytes with Aβ induced the activation of the nuclear factors NF-κB and AP-1 (lines 2). PEA significantly inhibited this effect (lines 3). This inhibition was attenuated by the PPAR-α antagonist, MK886, in a partial, although significant, manner (lines 5). Each bar shows the mean ± S.E.M. of n = 3 separate experiments. ***P < 0.001 versus control; ###P < 0.001 versus Aβ-challenged cells; °°P < 0.01 and °P < 0.05 versus Aβ+ PEA-challenged cells.
Fig 6
Fig 6
PEA effect on PPARs transcription and expression. PPAR-α and PPAR-γ transcription and expression were evaluated in primary astrocytes after 24 hrs of exposure to Aβ (1 μg/ml), in the presence or absence of PEA (10−7 M). (A) Results of PPAR-α and PPAR-γ Western blot analysis and densitometric analysis of corresponding bands. β-actin was used as loading control. (B) Results of PPAR-α and PPAR-γ RT-PCR amplification and densitometric analysis of corresponding bands. GAPDH was used as standard control. Each bar shows the mean ± S.E.M. of n = 4 separate experiments. ***P < 0.001 and *P < 0.05 versus control; ##P < 0.01 and #P < 0.05 versus Aβ-challenged cells.
Fig 7
Fig 7
PEA effect on cannabinoid receptors expression. The expression of CB1 and CB2 receptors was evaluated in primary astrocytes after 24 hrs of exposure to Aβ (1 μg/ml), in the presence or absence of PEA (10−7 M). Figure shows the results of Western blot and densitometric analysis of corresponding bands. β-actin was used as loading control. Each bar shows the mean ± S.E.M. of n = 4 independent experiments. **P < 0.01 and *P < 0.05 versus control.

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