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. 2013 Jul;48(7):651-62.
doi: 10.1007/s11745-013-3799-x. Epub 2013 May 25.

Acetate Reduces PGE2 Release and Modulates Phospholipase and Cyclooxygenase Levels in Neuroglia Stimulated With Lipopolysaccharide

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Free PMC article

Acetate Reduces PGE2 Release and Modulates Phospholipase and Cyclooxygenase Levels in Neuroglia Stimulated With Lipopolysaccharide

Mahmoud L Soliman et al. Lipids. .
Free PMC article

Abstract

Acetate supplementation attenuates neuroglial activation, increases histone and non-histone protein acetylation, reduces pro-inflammatory cytokine expression, and increases IL-4 transcription in rat models of neuroinflammation and Lyme's neuroborreliosis. Because eicosanoid signaling is involved in neuroinflammation, we measured the effect acetate treatment had on phospholipase, cyclooxygenase, and prostaglandin E2 (PGE2) levels in BV-2 microglia and primary astrocytes stimulated with lipopolysaccharide (LPS). In BV-2 microglia, we found that LPS increased the phosphorylation-state of cytosolic phospholipase A2 (cPLA2), reduced the levels of phospholipase C (PLC) β1, and increased the levels of cyclooxygenase (Cox)-1 and -2. Acetate treatment returned PLCβ1 and Cox-1 levels to normal, attenuated the increase in Cox-2, but had no effect on cPLA2 phosphorylation. In primary astrocytes, LPS increased the phosphorylation of cPLA2 and increased the levels of Cox-1 and Cox-2. Acetate treatment in these cells reduced secretory PLA2 IIA and PLCβ1 levels as compared to LPS-treatment groups, reversed the increase in cPLA2 phosphorylation, and returned Cox-1 levels to normal. Acetate treatment reduced PGE2 release in astrocytes stimulated with LPS to control levels, but did not alter PGE2 levels in BV-2 microglia. The amount of acetylated H3K9 bound to the promoter regions of Cox-1, Cox-2, IL-1β and NF-κB p65 genes, but not IL-4 in were increased in BV-2 microglia treated with acetate. These data suggest that acetate treatment can disrupt eicosanoid signaling in neuroglia that may, in part, be the result of altering gene expression due chromatin remodeling as a result of increasing H3K9 acetylation.

Figures

Figure 1
Figure 1. Phospholipases levels in LPS-stimulated BV-2 cell cultures
Western blot analysis was performed to show changes in the levels of cPLA2 phosphorylation and total cPLA2, sPLA2 IIA, PLCβ1, PLCγ1 and PLCδ1 protein levels in BV-2 microglial cell cultures stimulated with LPS and/or treated with acetate. Panel A shows representative images of the Western blots. Panels B, D, E, F and G show the optical densities total cPLA2, sPLA2 IIA, PLCβ1, PLCγ1 and PLCδ1 normalized to the loading control α-tubulin. Panel C shows the optical density of phosphorylated cPLA2 normalized to total cPLA2. Bars represent means ± SD where statistical significance was set at p ≤ 0.05. Abbreviations are: a = compared to NaCl-treated group and b = compared to sodium acetate-treated group (n = 6 per group).
Figure 2
Figure 2. Phospholipase levels in LPS-stimulated primary astrocyte cell cultures
Western blot analysis was performed to show changes in the levels of cPLA2 phosphorylation and total cPLA2, sPLA2 IIA, PLCβ1, PLCγ1 and PLCδ1 protein levels in primary astrocyte cell cultures stimulated with LPS and/or acetate. Panel A shows representative images of the Western blots. Panels B, D, E, F and G show the optical densities total cPLA2, sPLA2 IIA, PLCβ1, PLCγ1 and PLCδ1 normalized to the loading control α-tubulin. Panel C shows the optical density of phosphorylated cPLA2 normalized to total cPLA2. Bars represent means ± SD where statistical significance was set at p ≤ 0.05. Abbreviations are: a = compared to NaCl-treated group, b = compared to sodium acetate-treated group and c = compared to LPS + sodium acetate-treated group (n = 6 per group).
Figure 3
Figure 3. Cox-1 and -2 levels in LPS-stimulated BV-2 and primary astrocyte cell cultures
Panel A shows representative images from a Western blot analysis to show the changes in the levels Cox-1 and Cox-2 in BV-2 microglia and primary astrocyte cell cultures stimulated with LPS and/or treated with acetate. Panels B and C show the optical densities of Cox-1 and Cox 2, respectively normalized to the loading control α-tubulin in BV-2 microglial cell cultures. Panels D and E show the optical densities of Cox-1 and Cox 2, respectively normalized to the loading control α-tubulin in primary astrocyte cell cultures. Bars represent means ± SD where statistical significance was set at p ≤ 0.05. Abbreviations are: a = compared to NaCl-treated group, b = compared to sodium acetate-treated group and c = compared to LPS + sodium acetate-treated group (n = 6 per group).
Figure 4
Figure 4. Prostaglandin E2 levels released from LPS-stimulated BV-2 microglia and primary astrocyte cultures
Media levels of secreted PGE2 from BV-2 microglia (panel A) and primary astrocyte cell cultures (panel B) stimulated with LPS and/or acetate as determined using an enzyme-linked immunoassay. Bars represent means ± SD where statistical significance was set at p ≤ 0.05. Abbreviations are: a = compared to NaCl-treated group, b = compared to sodium acetate-treated group and c = compared to LPS + sodium acetate-treated group (n = 6 per group).
Figure 5
Figure 5. Effect of H3K9 acetylation on binding to transcription start sites of inflammatory genes in BV-2 cultures
Binding levels of acetylated H3K9 to regions corresponding to ptgs1 (panel A), ptgs2 (panel B), p65 (panel C), il4 (panel D), and il1b (panel E) genes measured using Chromatin immunoprecipitation analysis followed by qrt-PCR. Bars represent means ± SD where statistical significance was set at p ≤ 0.05. Abbreviations are: a = compared to NaCl-treated group, b = compared to sodium acetate-treated group and c = compared to LPS + sodium acetate-treated group (n = 6 per group).

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