Anti-Inflammatory Effects of Spirulina platensis Extract via the Modulation of Histone Deacetylases

Abstract

We previously demonstrated that the organic extract of Spirulina platensis (SPE), an edible blue-green alga, possesses potent anti-inflammatory effects. In this study, we investigated if the regulation of histone deacetylases (HDACs) play a role in the anti-inflammatory effect of SPE in macrophages. Treatment of macrophages with SPE rapidly and dose-dependently reduced HDAC2, 3, and 4 proteins which preceded decreases in their mRNA levels. Degradation of HDAC4 protein was attenuated in the presence of inhibitors of calpain proteases, lysosomal acidification, and Ca(2+)/calmodulin-dependent protein kinase II, respectively, but not a proteasome inhibitor. Acetylated histone H3 was increased in SPE-treated macrophages to a similar level as macrophages treated with a pan-HDAC inhibitor, with concomitant inhibition of inflammatory gene expression upon LPS stimulation. Knockdown of HDAC3 increased basal and LPS-induced pro-inflammatory gene expression, while HDAC4 knockdown increased basal expression of interleukin-1β (IL-1β), but attenuated LPS-induced inflammatory gene expression. Chromatin immunoprecipitation showed that SPE decreased p65 binding and H3K9/K14 acetylation at the Il-1β and tumor necrosis factor α (Tnfα) promoters. Our results suggest that SPE increased global histone H3 acetylation by facilitating HDAC protein degradation, but decreases histone H3K9/K14 acetylation and p65 binding at the promoters of Il-1β and Tnfα to exert its anti-inflammatory effect.

Keywords: Spirulina platensis; anti-inflammatory; epigenetics; histone deacetylases; inflammation.

Figure 1

Spirulina platensis (SPE) reduce histone deacetylase (HDAC) mRNA expression and protein in a time and dose dependent manner in RAW 264.7 macrophages. (A) mRNA expression of HDACs in RAW 264.7 macrophages treated with 100 μg/mL of SPE for indicated amount of hours. N = 3, value = mean ± SEM * indicates significantly different from control (p < 0.05); (B) Western blot time course and (C) 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.

Figure 2

SPE reduced HDAC proteins in a dose and time-dependent manner in bone marrow-derived macrophages (BMDM). (A) Western blot analysis of HDAC2, 3, and 4 in BMDM treated with 100 μg/mL of SPE for indicated amount of time; (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.

Figure 3

Lysosomal and calpain-mediated degradation of HDAC4 by SPE. (A) 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) 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; (C) 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.

Figure 4

SPE increase acetylated histone H3 similarly to tricostatin A (TSA). (A) 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) 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; n = 3 (C) 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 (p < 0.05). Mean ± SEM, n = 3. Bars with different letters are significantly different (p < 0.05).

Figure 5

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. (A) 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) Percent knockdown of HDAC4 (top left), expression of IL-1β (top right), expression of IL-6 (bottom left), and expression of TNFα (bottom right). ). ^# indicates significantly different from scrambled control (p < 0.05); * indicates significantly different from scrambled control + LPS (p < 0.05).

Figure 6

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 (A) IL-1β promoter and (B) TNFα promoter.