Representative images of the immunolabeled OHSC with Iba-1 (red), GFAP (green), and DAPI (blue) in 200 μm. (A-E) Wild-type OHSC (OHSC WT) and (F-I) microglia-depleted OHSC (OHSC LIP-CL). (PDF 228 kb

Antônio Oliveira (4684915)

Ariel Marcotegui (4684900)

Bernd Fiebich (781952)

Eduardo Muñoz (42362)

Ellen Fortwängler (4684918)

Johannes Ditrich (4684912)

Juan Perazzo (4684906)

Soraya Saliba (3706465)

Journal of Neuroinflammation

Abstract Background N-arachidonoylphenolamine (AM404), a paracetamol metabolite, is a potent agonist of the transient receptor potential vanilloid type 1 (TRPV1) and low-affinity ligand of the cannabinoid receptor type 1 (CB1). There is evidence that AM404 exerts its pharmacological effects in immune cells. However, the effect of AM404 on the production of inflammatory mediators of the arachidonic acid pathway in activated microglia is still not fully elucidated. Method In the present study, we investigated the effects of AM404 on the eicosanoid production induced by lipopolysaccharide (LPS) in organotypic hippocampal slices culture (OHSC) and primary microglia cultures using Western blot, immunohistochemistry, and ELISA. Results Our results show that AM404 inhibited LPS-mediated prostaglandin E2 (PGE2) production in OHSC, and LPS-stimulated PGE2 release was totally abolished in OHSC if microglial cells were removed. In primary microglia cultures, AM404 led to a significant dose-dependent decrease in the release of PGE2, independent of TRPV1 or CB1 receptors. Moreover, AM404 also inhibited the production of PGD2 and the formation of reactive oxygen species (8-iso-PGF2 alpha) with a reversible reduction of COX-1- and COX-2 activity. Also, it slightly decreased the levels of LPS-induced COX-2 protein, although no effect was observed on LPS-induced mPGES-1 protein synthesis. Conclusions This study provides new significant insights about the potential anti-inflammatory role of AM404 and new mechanisms of action of paracetamol on the modulation of prostaglandin production by activated microglia

Soraya Wilke Saliba

Ariel R. Marcotegui

Ellen Fortwängler

Johannes Ditrich

Juan Carlos Perazzo

Eduardo Muñoz

Antônio Carlos Pinheiro de Oliveira

Bernd L. Fiebich

Directory of Open Access Journals

AM404, paracetamol metabolite, prevents prostaglandin synthesis in activated microglia by inhibiting COX activity

Effects of AM404 on cell viability in primary rat microglia cells. AM404 was added 30 min before stimulating the cells with LPS for 24 h, and cell death was measured by the intensity of the fluorescence emission in the culture using the CellTox™ Green Cytotoxicity assay kit. Each column and error bar represents the percentage of fluorescence (100% for just microglia cells). ***p < 0.001 with respect to just microglia cells (One-way ANOVA followed by the Newman-Keuls post-test). (PDF 16 kb

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Additional file 2: of AM404, paracetamol metabolite, prevents prostaglandin synthesis in activated microglia by inhibiting COX activity

N-arachidonoylphenolamine (AM404), a paracetamol metabolite, is a potent agonist of the transient receptor potential vanilloid type 1 (TRPV1) and low-affinity ligand of the cannabinoid receptor type 1 (CB1). There is evidence that AM404 exerts its pharmacological effects in immune cells. However, the effect of AM404 on the production of inflammatory mediators of the arachidonic acid pathway in activated microglia is still not fully elucidated. In the present study, we investigated the effects of AM404 on the eicosanoid production induced by lipopolysaccharide (LPS) in organotypic hippocampal slices culture (OHSC) and primary microglia cultures using Western blot, immunohistochemistry, and ELISA. Our results show that AM404 inhibited LPS-mediated prostaglandin E2 (PGE2) production in OHSC, and LPS-stimulated PGE2 release was totally abolished in OHSC if microglial cells were removed. In primary microglia cultures, AM404 led to a significant dose-dependent decrease in the release of PGE2, independent of TRPV1 or CB1 receptors. Moreover, AM404 also inhibited the production of PGD2 and the formation of reactive oxygen species (8-iso-PGF2 alpha) with a reversible reduction of COX-1- and COX-2 activity. Also, it slightly decreased the levels of LPS-induced COX-2 protein, although no effect was observed on LPS-induced mPGES-1 protein synthesis. This study provides new significant insights about the potential anti-inflammatory role of AM404 and new mechanisms of action of paracetamol on the modulation of prostaglandin production by activated microglia

Saliba, Soraya Wilke

Marcotegui, Ariel R

Fortwängler, Ellen

Ditrich, Johannes

Perazzo, Juan Carlos

Muñoz, Eduardo

de Oliveira, Antônio Carlos Pinheiro

Fiebich, Bernd L

Fondo Bibliográfico Digital Institucional

AM404, paracetamol metabolite, prevents prostaglandin synthesis in activated microglia by inhibiting COX activity.