Inhibitor of Hyaluronic Acid Synthesis 4-Methylumbelliferone as an Anti-Inflammatory Modulator of LPS-Mediated Astrocyte Responses

Abstract

Astrocytes are glial cells that play an important role in neuroinflammation. Astrocytes respond to many pro-inflammatory stimuli, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4). Regulatory specificities of inflammatory signaling pathways are still largely unknown due to the ectodermal origin of astrocytes. Recently, we have shown that hyaluronic acid (HA) may form part of astrocyte inflammatory responses. Therefore, we tested 4-methylumbelliferone (4-MU), a specific inhibitor of HA synthesis, as a possible regulator of LPS-mediated responses. Rat primary astrocytes were treated with LPS with and without 4-MU and gene expression levels of inflammatory (interleukins 1β, (IL-1β), 6, (IL-6), tumor necrosis factor alpha TNFα,) and resolution interleukin 10 (IL-10) markers were evaluated via real-time PCR and western blot. The release of cytokines and HA was determined by ELISA. Oxylipin profiles were measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. Our data show that 4-MU (i) has anti-inflammatory effects in the course of TLR4 activation, decreasing the cytokines level TNFα, IL-6 and IL-1β and increasing IL-10, (ii) downregulates prostaglandin synthesis but not via cyclooxygenases COX-1 and COX-2 pathways, (iii) modulates HA synthesis and decreases LPS-induced HA synthase mRNA expression (HAS-1, HAS-2) but does not have an influence on HAS-3, HYAL1 and HYAL2 mRNAs; (iv) the effects of 4-MU are predominantly revealed via JNK but not p38, ERK mitogen-activated protein kinases (MAPKs) or nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathways. For the first time, it is shown that 4-MU possesses the useful potential to regulate an inflammatory astrocyte response.

Keywords: 4-methylumbelliferone (4-MU); astrocytes; cyclooxygenase (COX); hyaluronic acid; interleukin 10 (IL-10); interleukin 6 (IL-6); neuroinflammation; oxylipins; toll-like receptors (TLRs).

Figure 1

Effect of 4-MU on the inflammatory response. The primary rat astrocyte cultures were pretreated with 4-methylumbelliferone (4-MU, 400 μM) for 30 min and then stimulated with lipopolysaccharide (LPS) (100 ng/mL) for 4 h. (A): the mRNA levels of inflammatory markers (tumor necrosis factor alpha (TNFα), interleukin 10 (IL-10), complement component 3 (C3), interleukin 1 beta (IL-1β), interleukin 6 (IL-6) and inducible nitric oxide synthase (iNOS)) were determined by quantitative real-time PCR (qPCR). The values were normalized to β-actin mRNA levels. The results are expressed as fold-changes, relative to untreated cells. (B): the TNFα, IL-10 and IL-6 protein release were measured by ELISA in supernatant samples. The results are expressed as pg/mg. The values represent a mean ± SEM from three independent experiments. * p < 0.05, compared with the LPS-stimulated cells.

Figure 2

Effect of 4-methylumbelliferone (4-MU) on the oxylipins release and cyclooxygenases expression in the LPS-stimulated astrocytes. Primary rat astrocytes were pretreated for 30 min with 4-MU (400 μM) and then stimulated with lipopolysaccharide (LPS, 100 ng/mL) for 4 h or 24 h. Concentrations of oxylipins in supernatants were measured using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). (A) The heat map shows relative amounts of each lipid mediator compared to the control. The vertical axis indicates the stimuli, while the horizontal axis indicates the relative amount (log2) of each lipid mediator. Metabolites were divided into: Lipoxygenase (LOX), cyclooxygenase (COX) and cytochrome (CYP) pathways involved in their synthesis. (B) The bars show relative amounts of COX-derived lipid mediators. (C) COX-1 and COX-2 protein levels were evaluated by western blotting and normalized to the loading control β-actin. The example is representative for three independent experiments. Values represent the mean ± SEM from three independent experiments. * p < 0.05, compared with the unstimulated cells, # p < 0.05, compared with the LPS-stimulated cells.

Figure 3

Modulation of LPS-induced hyaluronic acid (HA) release and mRNA expression levels of HA metabolizing enzymes, HA synthases (HAS1, HAS2, HAS3), hyaluronidases (HYAL1 and HYAL2) by 4-methylumbelliferone (4-MU). (A): the primary rat astrocyte cultures were pretreated with 4-MU, 10 μM, 100 μM or 400 μM) for 30 min and then stimulated with lipopolysaccharide (LPS, 100 ng/mL) for 4 h. The HA release is measured by ELISA in supernatant samples. The results are expressed as fold-changes, relative to untreated cells. (B–D): astrocytes were pretreated with 4-MU, 400 μM for 30 min and then stimulated with LPS (100 ng/mL) for 4 h (B–D) or 24 h (B). (B): the HA release was measured by ELISA in supernatant samples. The results are expressed as ng/mL. (C,D): the mRNA levels of HAS1, HAS2, HAS3, HYAL1 and HYAL2 were determined by quantitative real-time PCR (qPCR). The values are normalized to β-actin mRNA levels. The results are expressed as fold-changes, relative to untreated cells. * p < 0.05, compared with the naive cells, # p < 0.05, compared with the LPS-stimulated cells.

Figure 4

Comparison of NF-kB, p38, JNK and ERK1/2 MAPK activity in the LPS-stimulated astrocytes, treated with 4-MU. Astrocytes were pretreated for 30 min with 4-methylumbelliferone (4-MU, 400 μM) and subsequently kept for 4 h without any additional stimulation or with lipopolysaccharide (LPS, 100 ng/mL). p38, p-p38, pJNK, JNK, pERK1/2, ERK1/2 mitogen-activated protein kinases (MAPKs), nuclear factor kappa-light-chain-enhancer of activated B cells (p-NF-KB and NF-kB) protein levels were evaluated by western blotting at indicated time (given in h) and normalized to the loading control β-actin. (A) Representative Western blots demonstrating phospho-p38 (p-p38), phospho-JNK (p-JNK), phospho-ERK1/2, phospho-NF-kB and total p38 (p38), total JNK (JNK), total ERK1/2 and total NF-kB protein levels. The example is representative for three independent experiments. (B–E) Results are expressed as fold-changes, relative to untreated control astrocytes. Values represent mean ± SEM from three independent experiments. * p < 0.05, compared with the unstimulated cells, # p < 0.05 compared with the LPS-stimulated cells.