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Punicic acid a conjugated linolenic acid inhibits TNFalpha-induced neutrophil hyperactivation and protects from experimental colon inflammation in rats.

Boussetta T, Raad H, Lettéron P, Gougerot-Pocidalo MA, Marie JC, Driss F, El-Benna J - PLoS ONE (2009)

Bottom Line: Conventional anti-inflammatory therapies remain partially successful and may have side effects.This effect was mediated by the inhibition of Ser345-p47phox phosphorylation and upstream kinase p38MAPK.These data show that punicic acid exerts a potent anti-inflammatory effect through inhibition of TNFalpha-induced priming of NADPH oxidase by targeting the p38MAPKinase/Ser345-p47phox-axis and MPO release.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U773, Université Paris, Faculté de Médecine, Paris, France.

ABSTRACT

Background: Neutrophils play a major role in inflammation by releasing large amounts of ROS produced by NADPH-oxidase and myeloperoxidase (MPO). The proinflammatory cytokine TNFalpha primes ROS production through phosphorylation of the NADPH-oxidase subunit p47phox on Ser345. Conventional anti-inflammatory therapies remain partially successful and may have side effects. Therefore, regulation of neutrophil activation by natural dietary components represents an alternative therapeutic strategy in inflammatory diseases such as inflammatory bowel diseases. The aim of this study was to assess the effect of punicic acid, a conjugated linolenic fatty acid from pomegranate seed oil on TNFalpha-induced neutrophil hyperactivation in vitro and on colon inflammation in vivo.

Methodology and principal findings: We analyzed the effect of punicic acid on TNFalpha-induced neutrophil upregulation of ROS production in vitro and on TNBS-induced rat colon inflammation. Results show that punicic acid inhibited TNFalpha-induced priming of ROS production in vitro while preserving formyl-methionyl-leucyl-phenylalanine (fMLP)-induced response. This effect was mediated by the inhibition of Ser345-p47phox phosphorylation and upstream kinase p38MAPK. Punicic acid also inhibited fMLP- and TNFalpha+fMLP-induced MPO extracellular release from neutrophils. In vivo experiments showed that punicic acid and pomegranate seed oil intake decreased neutrophil-activation and ROS/MPO-mediated tissue damage as measured by F2-isoprostane release and protected rats from TNBS-induced colon inflammation.

Conclusions/significance: These data show that punicic acid exerts a potent anti-inflammatory effect through inhibition of TNFalpha-induced priming of NADPH oxidase by targeting the p38MAPKinase/Ser345-p47phox-axis and MPO release. This natural dietary compound may provide a novel alternative therapeutic strategy in inflammatory diseases such as inflammatory bowel diseases.

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Punicic acid inhibits the phosphorylation of p47phox on Ser345 in vivo.Rats received punicic acid dissolved in PBS (400 µg/0.5 ml) or PBS alone (0.5 ml) once a day for 10 days before TNBS treatment. Rats were anesthetized, then they received an intrarectal administration of TNBS (250 µl, 150 mg/Kg) dissolved in 50% ethanol in 0.9% NaCl . Control rats received only 50% ethanol vehicle. Animals were sacrificed 2 days after TNBS administration. Colons were fixed in formalin and paraffin-embedded tissue sections (5 µm) and were analyzed by confocal microscopy as indicated in the Methods section. The tissues were incubated overnight at 4°C with rabbit anti-phospho-Ser345p47phox polyclonal antibody (1∶1000), and mouse anti-gp91phox monoclonal antibody (1∶1000) diluted in 1% BSA/PBS. Following this incubation, the tissues were washed four times in PBS and incubated with Alexa Fluor 488-(green) conjugated goat anti-rabbit antibody (1∶200) diluted in 1% BSA/PBS and Alexa Fluor 568 (red) conjugated goat anti mouse (1∶200) for 1 h at room temperature in the dark. Stained cells were examined with a 63/1.4 numerical aperture objective under a Zeiss LSM510 confocal microscope and the images were imported into an LSM image browser for analysis. Merge corresponds to colocalisation of NOX2 and P-Ser345 as described in materials and methods.
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pone-0006458-g006: Punicic acid inhibits the phosphorylation of p47phox on Ser345 in vivo.Rats received punicic acid dissolved in PBS (400 µg/0.5 ml) or PBS alone (0.5 ml) once a day for 10 days before TNBS treatment. Rats were anesthetized, then they received an intrarectal administration of TNBS (250 µl, 150 mg/Kg) dissolved in 50% ethanol in 0.9% NaCl . Control rats received only 50% ethanol vehicle. Animals were sacrificed 2 days after TNBS administration. Colons were fixed in formalin and paraffin-embedded tissue sections (5 µm) and were analyzed by confocal microscopy as indicated in the Methods section. The tissues were incubated overnight at 4°C with rabbit anti-phospho-Ser345p47phox polyclonal antibody (1∶1000), and mouse anti-gp91phox monoclonal antibody (1∶1000) diluted in 1% BSA/PBS. Following this incubation, the tissues were washed four times in PBS and incubated with Alexa Fluor 488-(green) conjugated goat anti-rabbit antibody (1∶200) diluted in 1% BSA/PBS and Alexa Fluor 568 (red) conjugated goat anti mouse (1∶200) for 1 h at room temperature in the dark. Stained cells were examined with a 63/1.4 numerical aperture objective under a Zeiss LSM510 confocal microscope and the images were imported into an LSM image browser for analysis. Merge corresponds to colocalisation of NOX2 and P-Ser345 as described in materials and methods.

Mentions: To identify if the protective effect of punicic acid was due to its inhibitory action on neutrophil excessive activation, we analyzed in vivo neutrophil priming in colon sections by confocal microscopy using the anti-phosphoSer345-antibody. NOX2 antibody was used to detect total phagocytic NADPH oxidase. Confocal analysis clearly showed (Figure 6) that control untreated rat colon (Figure 6-PBS) expressed low NOX2 and phospho-Ser345 (p-Ser345) fluorescence intensity in accordance with few neutrophil infiltration while TNBS treatment induced bright NOX2 and p-Ser345 fluorescence intensity in accordance with massive neutrophil infiltration and priming or activation as assessed by the colocalization of anti-gp91phox/NOX2 and the anti-phosphoSer345-antibodies, respectively (Figure 6, p-Ser345, NOX2, Merge). Punicic acid significantly reduced neutrophil infiltration and priming/activation in the colon of TNBS-treated rats, as shown by decreased NOX2 and p-Ser345 fluorescence intensity compared to TNBS-treated rats that did not receive punicic acid.


Punicic acid a conjugated linolenic acid inhibits TNFalpha-induced neutrophil hyperactivation and protects from experimental colon inflammation in rats.

Boussetta T, Raad H, Lettéron P, Gougerot-Pocidalo MA, Marie JC, Driss F, El-Benna J - PLoS ONE (2009)

Punicic acid inhibits the phosphorylation of p47phox on Ser345 in vivo.Rats received punicic acid dissolved in PBS (400 µg/0.5 ml) or PBS alone (0.5 ml) once a day for 10 days before TNBS treatment. Rats were anesthetized, then they received an intrarectal administration of TNBS (250 µl, 150 mg/Kg) dissolved in 50% ethanol in 0.9% NaCl . Control rats received only 50% ethanol vehicle. Animals were sacrificed 2 days after TNBS administration. Colons were fixed in formalin and paraffin-embedded tissue sections (5 µm) and were analyzed by confocal microscopy as indicated in the Methods section. The tissues were incubated overnight at 4°C with rabbit anti-phospho-Ser345p47phox polyclonal antibody (1∶1000), and mouse anti-gp91phox monoclonal antibody (1∶1000) diluted in 1% BSA/PBS. Following this incubation, the tissues were washed four times in PBS and incubated with Alexa Fluor 488-(green) conjugated goat anti-rabbit antibody (1∶200) diluted in 1% BSA/PBS and Alexa Fluor 568 (red) conjugated goat anti mouse (1∶200) for 1 h at room temperature in the dark. Stained cells were examined with a 63/1.4 numerical aperture objective under a Zeiss LSM510 confocal microscope and the images were imported into an LSM image browser for analysis. Merge corresponds to colocalisation of NOX2 and P-Ser345 as described in materials and methods.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2714468&req=5

pone-0006458-g006: Punicic acid inhibits the phosphorylation of p47phox on Ser345 in vivo.Rats received punicic acid dissolved in PBS (400 µg/0.5 ml) or PBS alone (0.5 ml) once a day for 10 days before TNBS treatment. Rats were anesthetized, then they received an intrarectal administration of TNBS (250 µl, 150 mg/Kg) dissolved in 50% ethanol in 0.9% NaCl . Control rats received only 50% ethanol vehicle. Animals were sacrificed 2 days after TNBS administration. Colons were fixed in formalin and paraffin-embedded tissue sections (5 µm) and were analyzed by confocal microscopy as indicated in the Methods section. The tissues were incubated overnight at 4°C with rabbit anti-phospho-Ser345p47phox polyclonal antibody (1∶1000), and mouse anti-gp91phox monoclonal antibody (1∶1000) diluted in 1% BSA/PBS. Following this incubation, the tissues were washed four times in PBS and incubated with Alexa Fluor 488-(green) conjugated goat anti-rabbit antibody (1∶200) diluted in 1% BSA/PBS and Alexa Fluor 568 (red) conjugated goat anti mouse (1∶200) for 1 h at room temperature in the dark. Stained cells were examined with a 63/1.4 numerical aperture objective under a Zeiss LSM510 confocal microscope and the images were imported into an LSM image browser for analysis. Merge corresponds to colocalisation of NOX2 and P-Ser345 as described in materials and methods.
Mentions: To identify if the protective effect of punicic acid was due to its inhibitory action on neutrophil excessive activation, we analyzed in vivo neutrophil priming in colon sections by confocal microscopy using the anti-phosphoSer345-antibody. NOX2 antibody was used to detect total phagocytic NADPH oxidase. Confocal analysis clearly showed (Figure 6) that control untreated rat colon (Figure 6-PBS) expressed low NOX2 and phospho-Ser345 (p-Ser345) fluorescence intensity in accordance with few neutrophil infiltration while TNBS treatment induced bright NOX2 and p-Ser345 fluorescence intensity in accordance with massive neutrophil infiltration and priming or activation as assessed by the colocalization of anti-gp91phox/NOX2 and the anti-phosphoSer345-antibodies, respectively (Figure 6, p-Ser345, NOX2, Merge). Punicic acid significantly reduced neutrophil infiltration and priming/activation in the colon of TNBS-treated rats, as shown by decreased NOX2 and p-Ser345 fluorescence intensity compared to TNBS-treated rats that did not receive punicic acid.

Bottom Line: Conventional anti-inflammatory therapies remain partially successful and may have side effects.This effect was mediated by the inhibition of Ser345-p47phox phosphorylation and upstream kinase p38MAPK.These data show that punicic acid exerts a potent anti-inflammatory effect through inhibition of TNFalpha-induced priming of NADPH oxidase by targeting the p38MAPKinase/Ser345-p47phox-axis and MPO release.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U773, Université Paris, Faculté de Médecine, Paris, France.

ABSTRACT

Background: Neutrophils play a major role in inflammation by releasing large amounts of ROS produced by NADPH-oxidase and myeloperoxidase (MPO). The proinflammatory cytokine TNFalpha primes ROS production through phosphorylation of the NADPH-oxidase subunit p47phox on Ser345. Conventional anti-inflammatory therapies remain partially successful and may have side effects. Therefore, regulation of neutrophil activation by natural dietary components represents an alternative therapeutic strategy in inflammatory diseases such as inflammatory bowel diseases. The aim of this study was to assess the effect of punicic acid, a conjugated linolenic fatty acid from pomegranate seed oil on TNFalpha-induced neutrophil hyperactivation in vitro and on colon inflammation in vivo.

Methodology and principal findings: We analyzed the effect of punicic acid on TNFalpha-induced neutrophil upregulation of ROS production in vitro and on TNBS-induced rat colon inflammation. Results show that punicic acid inhibited TNFalpha-induced priming of ROS production in vitro while preserving formyl-methionyl-leucyl-phenylalanine (fMLP)-induced response. This effect was mediated by the inhibition of Ser345-p47phox phosphorylation and upstream kinase p38MAPK. Punicic acid also inhibited fMLP- and TNFalpha+fMLP-induced MPO extracellular release from neutrophils. In vivo experiments showed that punicic acid and pomegranate seed oil intake decreased neutrophil-activation and ROS/MPO-mediated tissue damage as measured by F2-isoprostane release and protected rats from TNBS-induced colon inflammation.

Conclusions/significance: These data show that punicic acid exerts a potent anti-inflammatory effect through inhibition of TNFalpha-induced priming of NADPH oxidase by targeting the p38MAPKinase/Ser345-p47phox-axis and MPO release. This natural dietary compound may provide a novel alternative therapeutic strategy in inflammatory diseases such as inflammatory bowel diseases.

Show MeSH
Related in: MedlinePlus