Conjugated Linoleic Acids Have Anti-Inflammatory Effects in Cultured Endothelial Cells
<p>(<b>A</b>) Viability of EA.hy926 cells after preincubation for 48 h with medium a containing 0.1% of ethanol (Control) or different concentrations (1, 10 or 50 µM) of linoleic acid (LA), <span class="html-italic">cis</span>-9, <span class="html-italic">trans</span>-11 linoleic acid (CLA9,11) or <span class="html-italic">trans</span>-10, <span class="html-italic">cis</span>-12 linoleic acid (CLA10,12) followed by incubation with a medium without fatty acids for 24 h. Bars are mean ± SD of 9 samples from 3 experiments. Data were analysed using one-way ANOVA with Tukey’s post hoc test. **** <span class="html-italic">p</span> < 0.0001 vs. Control. (<b>B</b>,<b>C</b>) Microscope images of EA.hy926 cells incubated with medium containing 0.1% ethanol (<b>B</b>) or containing <span class="html-italic">trans</span>-10, <span class="html-italic">cis</span>-12 linoleic acid at 50 µM (<b>C</b>).</p> "> Figure 2
<p>Concentrations (% of control) of MCP-1 (<b>A</b>), ICAM-1 (<b>B</b>), IL-6 (<b>C</b>), IL-8 (<b>D</b>) and RANTES (<b>E</b>) in the medium of EA.hy926 cells incubated for 48 h with medium containing 0.1% of ethanol (Control) or fatty acid at 1 or 10 µM, followed by incubation with a medium without fatty acids for 24 h. Bars are mean ± SD of 9 samples from 3 experiments. Data were analysed using the one-way ANOVA with Tukey’s post hoc test. * <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01; *** <span class="html-italic">p</span> < 0.001; **** <span class="html-italic">p</span> < 0.0001; where asterisks are shown immediately above a bar they refer to the difference from the control and where asterisks are shown above a horizontal line they refer to the differences between the two groups indicated by that line. LA, linoleic acid; CLA9,11, <span class="html-italic">cis</span>-9, <span class="html-italic">trans</span>-11 linoleic acid; CLA10,12, <span class="html-italic">trans</span>-10, <span class="html-italic">cis</span>-12 linoleic acid.</p> "> Figure 3
<p>Expression of <span class="html-italic">NFκB1</span> (<b>A</b>), <span class="html-italic">NFκBIA</span> (for IκBα, (<b>B</b>)), IκBKB (for IκK-β, (<b>C</b>)), <span class="html-italic">PPPAR-α</span> (<b>D</b>), <span class="html-italic">PTGS2</span> (for COX-2, (<b>E</b>)) and <span class="html-italic">IL-6</span> (<b>F</b>) genes in EA.hy926 cells preincubated for 48 h with 1 or 10 µM of fatty acid in a medium containing 0.1% of ethanol (Control) followed by incubation with a medium without fatty acids for 6 h. Cq values were normalized by the geometric mean of reference targets (YWHAZ and RPL13A genes). Bars are mean ± SD of 9 samples from 3 experiments. Data were analysed using the one-way ANOVA with Tukey’s post hoc test. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, **** <span class="html-italic">p</span> < 0.0001; where asterisks are shown immediately above a bar they refer to the difference from the control and where asterisks are shown above a horizontal line they refer to the differences between the two groups indicated by that line. LA, linoleic acid; CLA9,11, <span class="html-italic">cis</span>-9, <span class="html-italic">trans</span>-11 linoleic acid; CLA10,12, <span class="html-italic">trans</span>-10, <span class="html-italic">cis</span>-12 linoleic acid.</p> "> Figure 4
<p>Adhesion of THP-1 cells (% of control) to EA.hy926 cells incubated for 48 h with a medium containing 0.1% of ethanol (Control) or different concentrations (1 µM (<b>A</b>), 10 µM (<b>B</b>)) of fatty acid, followed by incubation with a medium without fatty acids for 6 h and then 1 h co-incubation with THP-1 cells. Bars are mean ± SD of 9 samples from 3 experiments. Data were analysed using the one-way ANOVA with Tukey post hoc test. ** <span class="html-italic">p</span> < 0.01; *** <span class="html-italic">p</span> < 0.001; **** <span class="html-italic">p</span> < 0.0001; where asterisks are shown immediately above a bar they refer to the difference from the control and where asterisks are shown above a horizontal line they refer to the differences between the two groups indicated by that line. LA, linoleic acid; CLA9,11, <span class="html-italic">cis</span>-9, <span class="html-italic">trans</span>-11 linoleic acid; CLA10,12, <span class="html-italic">trans</span>-10, <span class="html-italic">cis</span>-12 linoleic acid.</p> "> Figure 5
<p>Images of THP-1 cell adhesion to EA.hy926 cells. Adhesion of THP-1 cells to EA.hy926 cells without pre-incubation with fatty acid (control (<b>A</b>)) or with 48 h prior exposure to 10 µM linoleic acid (<b>B</b>), <span class="html-italic">cis</span>-9, <span class="html-italic">trans</span>-11 linoleic acid (<b>C</b>), <span class="html-italic">trans</span>-10, <span class="html-italic">cis</span>-12 linoleic acid (<b>D</b>), followed by incubation with a medium without fatty acids for 6 h and then 1 h co-incubation with calcein-labelled THP-1 cells. Attached THP-1 cells were visualised by fluorescence microscopy (Nikon Elipse Ti) at a magnification of 100× under transmitted light.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Viability of EA.hy926 Cells Incubated with FAs
2.2. FA Incorporation into EA.hy926 Cells
2.3. Effects of FAs on the Concentrations of Inflammatory Mediators in the Medium of Cultured ECs
2.4. Effects of FAs on the Expression of Inflammation-Related Genes
2.5. Effects of FAs on THP-1 Adhesion to EA.hy926 Cells
3. Discussion
4. Materials and Methods
4.1. Endothelial Cells
4.2. Fatty Acid Treatment
4.3. MTT Assay for Cell Viability
4.4. Fatty Acid Composition Measurement by Gas Chromatography
4.5. Measurement of Inflammatory Mediators in Cell Culture Supernatants Using Multiplex Magnetic ELISA
4.6. RNA Isolation, cDNA Synthesis, and Real-Time PCR
4.7. Adhesion of THP-1 Monocytes to ECs
4.8. Data Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Valenzuela, C.A.; Baker, E.J.; Miles, E.A.; Calder, P.C. Conjugated Linoleic Acids Have Anti-Inflammatory Effects in Cultured Endothelial Cells. Int. J. Mol. Sci. 2023, 24, 874. https://doi.org/10.3390/ijms24010874
Valenzuela CA, Baker EJ, Miles EA, Calder PC. Conjugated Linoleic Acids Have Anti-Inflammatory Effects in Cultured Endothelial Cells. International Journal of Molecular Sciences. 2023; 24(1):874. https://doi.org/10.3390/ijms24010874
Chicago/Turabian StyleValenzuela, Carina A., Ella J. Baker, Elizabeth A. Miles, and Philip C. Calder. 2023. "Conjugated Linoleic Acids Have Anti-Inflammatory Effects in Cultured Endothelial Cells" International Journal of Molecular Sciences 24, no. 1: 874. https://doi.org/10.3390/ijms24010874