Anti-Inflammatory Effects of Spirulina platensis Extract via the Modulation of Histone Deacetylases
<p><span class="html-italic">Spirulina platensis</span> (SPE) reduce histone deacetylase (HDAC) mRNA expression and protein in a time and dose dependent manner in RAW 264.7 macrophages. (<b>A</b>) mRNA expression of HDACs in RAW 264.7 macrophages treated with 100 μg/mL of SPE for indicated amount of hours. <span class="html-italic">N</span> = 3, value = mean ± SEM * indicates significantly different from control (<span class="html-italic">p</span> < 0.05); (<b>B</b>) Western blot time course and (<b>C</b>) dose-response of HDACs in RAW 264.7 macrophages treated with 100 μg/mL of SPE for indicated amount of time or varying SPE concentrations. A represented blot of three independent experiments is shown.</p> "> Figure 2
<p>SPE reduced HDAC proteins in a dose and time-dependent manner in bone marrow-derived macrophages (BMDM). (<b>A</b>) Western blot analysis of HDAC2, 3, and 4 in BMDM treated with 100 μg/mL of SPE for indicated amount of time; (<b>B</b>) Western blot analysis of HDAC2, 3, and 4 in BMDM treated with indicated concentration of SPE for 3 h. A representative blot of 2–3 experiments is shown.</p> "> Figure 3
<p>Lysosomal and calpain-mediated degradation of HDAC4 by SPE. (<b>A</b>) Western blot analysis of HDAC4 in RAW 264.7 macrophages pretreated with 10 μg/mL of MG-132 (top), or 50 μM of chloroquine (bottom) for 1 h and then treated with 25 μg/mL of SPE in the presence of inhibitors; (<b>B</b>) Western blot analysis of HDAC4 in RAW 264.7 macrophages pretreated with calpeptin (10 μg/mL) for 1 h and then treated with 25 μg/mL of SPE in the presence of inhibitors; (<b>C</b>) Western blot analysis of HDAC4 in RAW 264.7 macrophages pretreated with CAMKII inhibitor KN-93 at the concentration of 5 μM for 1 h and then treated with 25 μg/mL of SPE in the presence of inhibitors. A represented blot of three independent experiments is shown.</p> "> Figure 4
<p>SPE increase acetylated histone H3 similarly to tricostatin A (TSA). (<b>A</b>) Western blot analysis of acetylated histone H3K9 in RAW 264.7 macrophages. Cells were pretreated with vehicle control (0.5% DMSO), 50 or 100 μg/mL of SPE for 12 h. The cells were then treated with vehicle control (0.5% DMSO), 25 nM TSA, 100 nM TSA, 50 or 100 μg/mL of SPE for 18 h; Blot image (top) and quantification (bottom) (<b>B</b>) mRNA expression of histone acetyltransferase p300 and GCN5 in RAW 264.7 macrophages pretreated with vehicle control (0.5% DMSO), or 100 μg/mL of SPE for 12 h and then stimulated with 100 ng/mL LPS for 18 h; <span class="html-italic">n</span> = 3 (<b>C</b>) mRNA expression of inflammatory genes in RAW 264.7 macrophages pretreated with vehicle control (0.5% DMSO), 25 nM TSA, 50, or 100 μg/mL of SPE for 12 h. Cells were then treated with vehicle control, 25 nM TSA, 50 or 100 μg/mL SPE alone or in combination with 100 ng/mL of LPS for 18 h. Different letters indicate significantly different (<span class="html-italic">p</span> < 0.05). Mean ± SEM, <span class="html-italic">n</span> = 3. Bars with different letters are significantly different (<span class="html-italic">p</span> < 0.05).</p> "> Figure 5
<p>Effects of HDAC3 and 4 knockdown on LPS-induced inflammatory gene expression. RAW 264.7 macrophages were transfected with scrambled control, siRNA against HDAC3 or HDAC4 for 24 h and then cells were stimulated with or without 100 ng/mL of LPS for 3 h for subsequent gene expression qRT-PCR. (<b>A</b>) Percent knockdown of HDAC3 (top left), expression of IL-1β (top right), expression of IL-6 (bottom left), and expression of TNFα (bottom right); (<b>B</b>) Percent knockdown of HDAC4 (top left), expression of IL-1β (top right), expression of IL-6 (bottom left), and expression of TNFα (bottom right). ). <sup>#</sup> indicates significantly different from scrambled control (<span class="html-italic">p</span> < 0.05); * indicates significantly different from scrambled control + LPS (<span class="html-italic">p</span> < 0.05).</p> "> Figure 6
<p>Chromatin Immunoprecipitation of p65 and H3K9/K14 at the promoter of inflammatory genes. ChIP of RAW 264.7 macrophages pretreated with 100 μg/mL of SPE for 12 h and then stimulated with 100 ng/mL of LPS for an additional 18 h in the presence of SPE. ChIP DNA was obtained by immunoprecipitation of p65 (left) and histone H3K9 (right) and quantified by qPCR with primers at the (<b>A</b>) IL-1β promoter and (<b>B</b>) TNFα promoter.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. SPE Preparation
2.2. Bone Marrow Isolation and Macrophage Differentiation
2.3. Cell Culture and Treatments
2.4. HDAC3 and 4 Knockdown by Small Interfering RNA (siRNA)
2.5. Quantitative Real-Time PCR (qRT-PCR)
2.6. Chromatin Immunoprecipitation (ChIP)
2.7. Western Blot Analysis
2.8. Statistical Analysis
3. Results
3.1. Reduction in HDAC2, 3 and 4 Proteins by SPE Preceded Decreases in Their mRNA Levels in Macrophages
3.2. The Lysosome, Calpain Proteases, and Ca2+/Calmodulin-Dependent Protein Kinase II Are Involved in the Rapid HDAC4 Degradation by SPE
3.3. SPE Increased Acetylated Histone H3 in RAW Macrophages
3.4. HDAC3 Deficiency Increased, While HDAC4 Deficiency Attenuated LPS-Induced Inflammatory Gene Expression in RAW Macrophages
3.5. SPE Reduced p65 Binding and H3 Acetylation in the Il-1β and Tnfα Promoter
4. Discussion
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
BMDM | bone marrow-derived macrophage |
CVD | cardiovascular disease |
GCN5 | general control non-derepressible 5 |
HAT | histone acetyltransferase |
HDAC | histone deacetylase |
IL-1β | interleukin-1β |
IL-6 | interleukin-6 |
LPS | lipopolysaccharide |
NF-κB | nuclear factor-κB |
SP | Spirulina platensis |
SPE | Spirulina platensis organic extract |
TLR4 | toll-like receptor 4 |
TNFα | tumor necrosis factor α |
TSA | trichostatin A |
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Pham, T.X.; Park, Y.-K.; Lee, J.-Y. Anti-Inflammatory Effects of Spirulina platensis Extract via the Modulation of Histone Deacetylases. Nutrients 2016, 8, 381. https://doi.org/10.3390/nu8060381
Pham TX, Park Y-K, Lee J-Y. Anti-Inflammatory Effects of Spirulina platensis Extract via the Modulation of Histone Deacetylases. Nutrients. 2016; 8(6):381. https://doi.org/10.3390/nu8060381
Chicago/Turabian StylePham, Tho X., Young-Ki Park, and Ji-Young Lee. 2016. "Anti-Inflammatory Effects of Spirulina platensis Extract via the Modulation of Histone Deacetylases" Nutrients 8, no. 6: 381. https://doi.org/10.3390/nu8060381