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.
Highlights
A significant amount of research is currently being devoted to the establishment of the mechanisms of inflammation and the search for possibilities of process regulation
Our data demonstrated that treatment with the hyaluronic acid (HA) synthesis inhibitor 4-MU may be effective in relation to LPS-mediated inflammatory responses in astrocytes
Exposure of astrocytes to 4-MU led to a decreased synthesis of pro-inflammatory cytokines (TNFα, interleukin 6 (IL-6)) and prostaglandins
Summary
A significant amount of research is currently being devoted to the establishment of the mechanisms of inflammation and the search for possibilities of process regulation. Astrocytes are glial cells with homeostatic, metabolic and defensive functions and play an important role in the development of inflammatory responses in the brain [7]. Upon activation of TLR-mediated signaling pathways, astrocytes produce pro- and anti-inflammatory cytokines and polyunsaturated fatty acid derivatives, such as prostaglandins [7,8,9]. 4-MU has been reported to improve the course of diseases in mouse models of autoimmune diseases, such as the collagen-induced arthritis model [24] and a brain autoimmunity model [20] In view of these data, it is surprising that 4-MU has not been investigated in models of stimulating inflammation on cells of the nervous system. We have filled this gap and shown that 4-MU can effectively ameliorate TLR-mediated signaling by modulating cytokines and oxylipins’ release. The effectiveness of 4-MU as an anti-inflammatory agent stimulated the following assessment of possible participants in signaling cascades—phosphorylation of mitogen-activated protein kinases (MAPK) p38, JNK, ERK, transcription factor NF-kB, expression of the enzymes of HA metabolism
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