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The Omega-3 Fatty Acid Docosahexaenoic Acid Modulates Inflammatory Mediator Release in Human Alveolar Cells Exposed to Bronchoalveolar Lavage Fluid of ARDS Patients.

Cotogni P, Trombetta A, Muzio G, Maggiora M, Canuto RA - Biomed Res Int (2015)

Bottom Line: The proinflammatory cytokine release was reduced by the 1 : 2 ratio (P < 0.01 to <0.001) but was increased by the 1 : 7 ratio (P < 0.01).The 1 : 2 ratio reduced COX-2 and PGE2 (P < 0.001) as well as NF-κB translocation into the nucleus (P < 0.01), while it increased activation of PPARγ and IL-10 release (P < 0.001).Conclusion.

View Article: PubMed Central - PubMed

Affiliation: Anesthesiology and Intensive Care, Department of Medicine, S. Giovanni Battista Hospital, University of Turin, Via A.M. Dogliotti 14, 10126 Turin, Italy.

ABSTRACT

Background: This study investigated whether the 1 : 2 ω-3/ω-6 ratio may reduce proinflammatory response in human alveolar cells (A549) exposed to an ex vivo inflammatory stimulus (bronchoalveolar lavage fluid (BALF) of acute respiratory distress syndrome (ARDS) patients). Methods. We exposed A549 cells to the BALF collected from 12 ARDS patients. After 18 hours, fatty acids (FA) were added as docosahexaenoic acid (DHA, ω-3) and arachidonic acid (AA, ω-6) in two ratios (1 : 2 or 1 : 7). 24 hours later, in culture supernatants were evaluated cytokines (TNF-α, IL-6, IL-8, and IL-10) and prostaglandins (PGE2 and PGE3) release. The FA percentage content in A549 membrane phospholipids, content of COX-2, level of PPARγ, and NF-κB binding activity were determined.

Results: The 1 : 2 DHA/AA ratio reversed the baseline predominance of ω-6 over ω-3 in the cell membranes (P < 0.001). The proinflammatory cytokine release was reduced by the 1 : 2 ratio (P < 0.01 to <0.001) but was increased by the 1 : 7 ratio (P < 0.01). The 1 : 2 ratio reduced COX-2 and PGE2 (P < 0.001) as well as NF-κB translocation into the nucleus (P < 0.01), while it increased activation of PPARγ and IL-10 release (P < 0.001). Conclusion. This study demonstrated that shifting the FA supply from ω-6 to ω-3 decreased proinflammatory mediator release in human alveolar cells exposed to BALF of ARDS patients.

No MeSH data available.


Related in: MedlinePlus

Effects of ω-3/ω-6 PUFA ratios on NF-κB. (a) p65 NF-κB relative protein content in the cytoplasmic fraction of A549 cells, stimulated with BALF and treated with 50 μM 1 : 2 or 1 : 7 DHA/AA ratios. Data are expressed as “a.u.” (arbitrary units) of the densitometric values, normalized on the corresponding β-actin. The value of BALF was arbitrarily set as 100. Data are presented as mean ± standard deviation of 6 independent determinations (n = 6). The image is representative of all the WB experiments. (b) NF-κB relative protein content in the nuclear fraction of A549 cells, stimulated with BALF and treated with 50 μM 1 : 2 or 1 : 7 DHA/AA ratios. Data are expressed as “a.u.” (arbitrary units) of the densitometric values, normalized on the corresponding lamin A/C. The value of BALF was arbitrarily set as 100. Data are presented as mean ± standard deviation of 6 independent determinations (n = 6). (c) IκBα relative protein content in the cytoplasmic fraction of A549 cells, stimulated with BALF and treated with 50 μM 1 : 2 or 1 : 7 DHA/AA ratios. Data are expressed as “a.u.” (arbitrary units) of the densitometric values, normalized on the corresponding β-actin. The value of BALF was arbitrarily set as 100. Data are presented as mean ± standard deviation of 6 independent determinations (n = 6). The image is representative of all the WB experiments. PUFA, polyunsaturated fatty acid; NF-κB, nuclear factor-kappa B; BALF, bronchoalveolar lavage fluid; DHA, docosahexaenoic acid; AA, arachidonic acid; WB, western blot; IκB, inhibitor of NF-κB. *P < 0.05 1 : 2 DHA/AA versus all. **P < 0.01 1 : 2 DHA/AA versus all. ***P < 0.001 1 : 2 DHA/AA versus all.
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fig6: Effects of ω-3/ω-6 PUFA ratios on NF-κB. (a) p65 NF-κB relative protein content in the cytoplasmic fraction of A549 cells, stimulated with BALF and treated with 50 μM 1 : 2 or 1 : 7 DHA/AA ratios. Data are expressed as “a.u.” (arbitrary units) of the densitometric values, normalized on the corresponding β-actin. The value of BALF was arbitrarily set as 100. Data are presented as mean ± standard deviation of 6 independent determinations (n = 6). The image is representative of all the WB experiments. (b) NF-κB relative protein content in the nuclear fraction of A549 cells, stimulated with BALF and treated with 50 μM 1 : 2 or 1 : 7 DHA/AA ratios. Data are expressed as “a.u.” (arbitrary units) of the densitometric values, normalized on the corresponding lamin A/C. The value of BALF was arbitrarily set as 100. Data are presented as mean ± standard deviation of 6 independent determinations (n = 6). (c) IκBα relative protein content in the cytoplasmic fraction of A549 cells, stimulated with BALF and treated with 50 μM 1 : 2 or 1 : 7 DHA/AA ratios. Data are expressed as “a.u.” (arbitrary units) of the densitometric values, normalized on the corresponding β-actin. The value of BALF was arbitrarily set as 100. Data are presented as mean ± standard deviation of 6 independent determinations (n = 6). The image is representative of all the WB experiments. PUFA, polyunsaturated fatty acid; NF-κB, nuclear factor-kappa B; BALF, bronchoalveolar lavage fluid; DHA, docosahexaenoic acid; AA, arachidonic acid; WB, western blot; IκB, inhibitor of NF-κB. *P < 0.05 1 : 2 DHA/AA versus all. **P < 0.01 1 : 2 DHA/AA versus all. ***P < 0.001 1 : 2 DHA/AA versus all.

Mentions: The nuclear factor (NF-κB) is a key transcription factor involved in upregulation of inflammatory cytokines, adhesion molecules, and COX-2 genes [47]. Activation of NF-κB transcription factor has been implicated in a number of inflammation-related pathologies [14]. The p65 and p50 NF-κB heterodimers are maintained inactive in the cytosol by the binding with an inhibitory protein, namely, inhibitor of NF-κB (IκB) [48]. Proinflammatory stimuli induce phosphorylation, ubiquitination, and proteasome mediated degradation of IκB, allowing NF-κB translocation into the nucleus and NF-κB target gene transcription [48]. The inhibitory role of ω-3 PUFAs on NF-κB pathway has been demonstrated in several experimental models and pathological conditions [19]. Different authors demonstrated that ω-3 decreased TNF-α expression through the prevention of NF-κB activation by inhibiting IκB phosphorylation and consequently preventing NF-κB translocation into the nucleus [14]. The correlation between NF-κB activation and cytokine content in BALF of ARDS patients was previously demonstrated by Nys et al. [49]. In order to understand the role of 1 : 2 DHA/AA treatment on the NF-κB pathway, we analyzed the expression of p65 NF-κB both in the cytoplasmic and the nuclear fraction of A549 BALF-stimulated cells (Figures 6(a) and 6(b)). The results demonstrated that 1 : 2 DHA/AA treatment decreased NF-κB content, in the nuclear fraction in particular, indicating an inhibition of its activation. The results were confirmed by the finding that 1 : 2 DHA/AA treatment increased the content of IκBα, one of the isoforms of IκB (Figure 6(c)). These results suggested that the anti-inflammatory effects of 1 : 2 DHA/AA treatment were associated with the inhibition of the NF-κB inflammatory pathway. Since, genes of IL-1β, IL-6, and TNF-α, as well as COX-2, are regulated by NF-κB [50], we can speculate that the reduction of proinflammatory cytokine release and the inhibition of COX-2 expression were mediated by the inhibition of NF-κB transcriptional activity.


The Omega-3 Fatty Acid Docosahexaenoic Acid Modulates Inflammatory Mediator Release in Human Alveolar Cells Exposed to Bronchoalveolar Lavage Fluid of ARDS Patients.

Cotogni P, Trombetta A, Muzio G, Maggiora M, Canuto RA - Biomed Res Int (2015)

Effects of ω-3/ω-6 PUFA ratios on NF-κB. (a) p65 NF-κB relative protein content in the cytoplasmic fraction of A549 cells, stimulated with BALF and treated with 50 μM 1 : 2 or 1 : 7 DHA/AA ratios. Data are expressed as “a.u.” (arbitrary units) of the densitometric values, normalized on the corresponding β-actin. The value of BALF was arbitrarily set as 100. Data are presented as mean ± standard deviation of 6 independent determinations (n = 6). The image is representative of all the WB experiments. (b) NF-κB relative protein content in the nuclear fraction of A549 cells, stimulated with BALF and treated with 50 μM 1 : 2 or 1 : 7 DHA/AA ratios. Data are expressed as “a.u.” (arbitrary units) of the densitometric values, normalized on the corresponding lamin A/C. The value of BALF was arbitrarily set as 100. Data are presented as mean ± standard deviation of 6 independent determinations (n = 6). (c) IκBα relative protein content in the cytoplasmic fraction of A549 cells, stimulated with BALF and treated with 50 μM 1 : 2 or 1 : 7 DHA/AA ratios. Data are expressed as “a.u.” (arbitrary units) of the densitometric values, normalized on the corresponding β-actin. The value of BALF was arbitrarily set as 100. Data are presented as mean ± standard deviation of 6 independent determinations (n = 6). The image is representative of all the WB experiments. PUFA, polyunsaturated fatty acid; NF-κB, nuclear factor-kappa B; BALF, bronchoalveolar lavage fluid; DHA, docosahexaenoic acid; AA, arachidonic acid; WB, western blot; IκB, inhibitor of NF-κB. *P < 0.05 1 : 2 DHA/AA versus all. **P < 0.01 1 : 2 DHA/AA versus all. ***P < 0.001 1 : 2 DHA/AA versus all.
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Related In: Results  -  Collection

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fig6: Effects of ω-3/ω-6 PUFA ratios on NF-κB. (a) p65 NF-κB relative protein content in the cytoplasmic fraction of A549 cells, stimulated with BALF and treated with 50 μM 1 : 2 or 1 : 7 DHA/AA ratios. Data are expressed as “a.u.” (arbitrary units) of the densitometric values, normalized on the corresponding β-actin. The value of BALF was arbitrarily set as 100. Data are presented as mean ± standard deviation of 6 independent determinations (n = 6). The image is representative of all the WB experiments. (b) NF-κB relative protein content in the nuclear fraction of A549 cells, stimulated with BALF and treated with 50 μM 1 : 2 or 1 : 7 DHA/AA ratios. Data are expressed as “a.u.” (arbitrary units) of the densitometric values, normalized on the corresponding lamin A/C. The value of BALF was arbitrarily set as 100. Data are presented as mean ± standard deviation of 6 independent determinations (n = 6). (c) IκBα relative protein content in the cytoplasmic fraction of A549 cells, stimulated with BALF and treated with 50 μM 1 : 2 or 1 : 7 DHA/AA ratios. Data are expressed as “a.u.” (arbitrary units) of the densitometric values, normalized on the corresponding β-actin. The value of BALF was arbitrarily set as 100. Data are presented as mean ± standard deviation of 6 independent determinations (n = 6). The image is representative of all the WB experiments. PUFA, polyunsaturated fatty acid; NF-κB, nuclear factor-kappa B; BALF, bronchoalveolar lavage fluid; DHA, docosahexaenoic acid; AA, arachidonic acid; WB, western blot; IκB, inhibitor of NF-κB. *P < 0.05 1 : 2 DHA/AA versus all. **P < 0.01 1 : 2 DHA/AA versus all. ***P < 0.001 1 : 2 DHA/AA versus all.
Mentions: The nuclear factor (NF-κB) is a key transcription factor involved in upregulation of inflammatory cytokines, adhesion molecules, and COX-2 genes [47]. Activation of NF-κB transcription factor has been implicated in a number of inflammation-related pathologies [14]. The p65 and p50 NF-κB heterodimers are maintained inactive in the cytosol by the binding with an inhibitory protein, namely, inhibitor of NF-κB (IκB) [48]. Proinflammatory stimuli induce phosphorylation, ubiquitination, and proteasome mediated degradation of IκB, allowing NF-κB translocation into the nucleus and NF-κB target gene transcription [48]. The inhibitory role of ω-3 PUFAs on NF-κB pathway has been demonstrated in several experimental models and pathological conditions [19]. Different authors demonstrated that ω-3 decreased TNF-α expression through the prevention of NF-κB activation by inhibiting IκB phosphorylation and consequently preventing NF-κB translocation into the nucleus [14]. The correlation between NF-κB activation and cytokine content in BALF of ARDS patients was previously demonstrated by Nys et al. [49]. In order to understand the role of 1 : 2 DHA/AA treatment on the NF-κB pathway, we analyzed the expression of p65 NF-κB both in the cytoplasmic and the nuclear fraction of A549 BALF-stimulated cells (Figures 6(a) and 6(b)). The results demonstrated that 1 : 2 DHA/AA treatment decreased NF-κB content, in the nuclear fraction in particular, indicating an inhibition of its activation. The results were confirmed by the finding that 1 : 2 DHA/AA treatment increased the content of IκBα, one of the isoforms of IκB (Figure 6(c)). These results suggested that the anti-inflammatory effects of 1 : 2 DHA/AA treatment were associated with the inhibition of the NF-κB inflammatory pathway. Since, genes of IL-1β, IL-6, and TNF-α, as well as COX-2, are regulated by NF-κB [50], we can speculate that the reduction of proinflammatory cytokine release and the inhibition of COX-2 expression were mediated by the inhibition of NF-κB transcriptional activity.

Bottom Line: The proinflammatory cytokine release was reduced by the 1 : 2 ratio (P < 0.01 to <0.001) but was increased by the 1 : 7 ratio (P < 0.01).The 1 : 2 ratio reduced COX-2 and PGE2 (P < 0.001) as well as NF-κB translocation into the nucleus (P < 0.01), while it increased activation of PPARγ and IL-10 release (P < 0.001).Conclusion.

View Article: PubMed Central - PubMed

Affiliation: Anesthesiology and Intensive Care, Department of Medicine, S. Giovanni Battista Hospital, University of Turin, Via A.M. Dogliotti 14, 10126 Turin, Italy.

ABSTRACT

Background: This study investigated whether the 1 : 2 ω-3/ω-6 ratio may reduce proinflammatory response in human alveolar cells (A549) exposed to an ex vivo inflammatory stimulus (bronchoalveolar lavage fluid (BALF) of acute respiratory distress syndrome (ARDS) patients). Methods. We exposed A549 cells to the BALF collected from 12 ARDS patients. After 18 hours, fatty acids (FA) were added as docosahexaenoic acid (DHA, ω-3) and arachidonic acid (AA, ω-6) in two ratios (1 : 2 or 1 : 7). 24 hours later, in culture supernatants were evaluated cytokines (TNF-α, IL-6, IL-8, and IL-10) and prostaglandins (PGE2 and PGE3) release. The FA percentage content in A549 membrane phospholipids, content of COX-2, level of PPARγ, and NF-κB binding activity were determined.

Results: The 1 : 2 DHA/AA ratio reversed the baseline predominance of ω-6 over ω-3 in the cell membranes (P < 0.001). The proinflammatory cytokine release was reduced by the 1 : 2 ratio (P < 0.01 to <0.001) but was increased by the 1 : 7 ratio (P < 0.01). The 1 : 2 ratio reduced COX-2 and PGE2 (P < 0.001) as well as NF-κB translocation into the nucleus (P < 0.01), while it increased activation of PPARγ and IL-10 release (P < 0.001). Conclusion. This study demonstrated that shifting the FA supply from ω-6 to ω-3 decreased proinflammatory mediator release in human alveolar cells exposed to BALF of ARDS patients.

No MeSH data available.


Related in: MedlinePlus