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. 2014 Feb 13;9(2):e88341.
doi: 10.1371/journal.pone.0088341. eCollection 2014.

Anti-inflammatory effect of unsaturated fatty acids and Ergosta-7,22-dien-3-ol from Marthasterias glacialis: prevention of CHOP-mediated ER-stress and NF-κB activation

David M Pereira  1 , Georgina Correia-da-Silva  2 , Patrícia Valentão  1 , Natércia Teixeira  2 , Paula B Andrade  1
Affiliations

Anti-inflammatory effect of unsaturated fatty acids and Ergosta-7,22-dien-3-ol from Marthasterias glacialis: prevention of CHOP-mediated ER-stress and NF-κB activation

David M Pereira et al. PLoS One. .

Abstract

There has been increasing awareness to the potential interest of drug discovery from marine natural products to treat several pathological conditions, including inflammation. In this work we describe the anti-inflammatory activity of several compounds present in the echinoderm Marthasterias glacialis (spiny sea-star), using the inflammatory model RAW 264.7 cells challenged with LPS. Lipidomic profiling of the organism revealed two major classes of compounds: fatty acids and sterols. Among these, the predominant compounds cis 11-eicosenoic and cis 11,14 eicosadienoic acids and the unsaturated sterol ergosta-7,22-dien-3-ol were evaluated. The mechanism of action of the compounds was distinct as they modulated different levels of the inflammation pathway. Classical inflammatory markers, such as COX-2, iNOS, IL-6 and NF-κB, were evaluated. We also studied the contribution of the CHOP pathway-mediated ER-stress to the inflammatory process. Overall, the sterol ergosta-7,22-dien-3-ol was the most active compound, however maximum activity was obtained when all compounds were tested in combination, thus suggesting a potentially synergistic activity of both classes of metabolites. This work establishes the echinoderm M. glacialis as an interesting source of anti-inflammatory molecules.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Evaluation of the effect of LPS and LPS+extract on cell morphology: A – phase contrast; B – Wright staining. Red arrows: cytoplasmic vesicles (autophagosomes); C - Acridine orange staining of control and LPS-treated macrophages.
White arrows: autophagosomes. Incubation with LPS causes the characteristic morphology of activated macrophages, which includes the advent of autophagosomes. Pre-incubation with the extract attenuates the number of autophagosomes. Original magnification: 400×.
Figure 2
Figure 2. IL-6 levels determined in culture media.
Challenge with LPS causes a marked increase in IL-6 levels, which are partly prevented by pre-incubation with the extract (156 µg/mL) for 2 hours. Ext: extract (156 µg/mL). The results correspond to the mean ± SD of three independent experiments performed in triplicate. ***P<0.001 (vs LPS).
Figure 3
Figure 3. Viability of macrophages after treatment with purified extract or individual compounds with or without LPS after 24 hours of incubation (A).
NO production in RAW 264.7 cells in the presence of a purified extract and of the major compounds in the extract, isolated and in combination after 24 hours of incubation (B). Compounds were used in the concentrations at which they occur in the extract; cis 11: 35 µM cis 11-eicosenoic acid; cis 11,14: 10 µM cis 11,14-eicosadienoic acid; E-7,22: 25 µM ergosta-7,22-dien-3-ol. Ext: extract (156 µg/mL); combination: Palmitc acid+cis 11-eicosenoic acid+cis 11,14-eicosadienoic+ergosta-7,22-dien-3-ol.The results correspond to the mean ± SD of three independent experiments performed in triplicate. *P<0.05; **P<0.01; ***P<0.001 (vs LPS).
Figure 4
Figure 4. Effect of purified extract (156 µg/mL) from M. glacialis (A) and major compounds in the extract (B) in the expression of iNOS, COX-2, CHOP and IKB-α in LPS-treated RAW 264.7 cells.
Densitometric analysis of the studied proteins after normalisation to β-tubulin levels. Results are expressed as mean ± SD of three experiments. *P<0.05; **P<0.01; ***P<0.001 (vs LPS). Ext: extract (156 µg/mL); PA: 20 µM palmitic acid; cis 11: 35 µM cis 11-eicosenoic acid; cis 11,14: 10 µM cis 11,14-eicosadienoic acid; E-7,22: 25 µM ergosta-7,22-dien-3-ol. combination: Palmitic acid+cis 11-eicosenoic acid+cis 11,14-eicosadienoic+ergosta-7,22-dien-3-ol.
Figure 5
Figure 5. Evaluation of intracellular ROS assessed by the fluorescent probe DCDHF-DA.
A – Fluorescence microscopy, qualitative evaluation of the effect of the extract in LPS-activated cells. B – Quantitative evaluation of the effect of the extract and its main compounds in LPS-challenged cells. The results correspond to the mean ± SD of three independent experiments performed in triplicate. ***P<0.001 (vs LPS). Ext: extract (156 µg/mL); cis 11: 35 µM cis 11-eicosenoic acid; cis 11,14: 10 µM cis 11,14-eicosadienoic acid; E-7,22: 25 µM ergosta-7,22-dien-3-ol. combination: Palmitic acid+cis 11-eicosenoic acid+cis 11,14-eicosadienoic+ergosta-7,22-dien-3-ol.
Figure 6
Figure 6. Proposed mechanism for the anti-inflammatory activity of different compounds present in M. glacialis.
See this image and copyright information in PMC

References

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