Vitamin E (alpha-tocopherol) attenuates cyclo-oxygenase 2 transcription and synthesis in immortalized murine BV-2 microglia.
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ABSTRACT:
SUBMITTER: Egger T
PROVIDER: S-EPMC1223182 | biostudies-other | 2003 Mar
REPOSITORIES: biostudies-other
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Project description:In this study, we explored the upstream regulatory mechanisms underlying inflammation-induced mitochondrial dysfunction in microglial BV-2 cells. Our results demonstrate that Sirtuin 3 (Sirt3) expression was downregulated in response to LPS-induced neuroinflammation. In addition, overexpression of Sirt3 attenuated LPS-induced BV-2 cell death. Functional studies illustrated that Sirt3 overexpression promoted normal mitochondrial function and inhibited mitochondria-dependent apoptosis in LPS-treated BV-2 cells. At the molecular level, suppressor of ras val-2 (SRV2) promoted LPS-mediated mitochondrial damage by inducing mitochondrial fission. Sirt3 overexpression, which suppressed the transcription of SRV2 and thus suppressed mitochondrial fission, played an anti-apoptotic role in LPS-treated BV-2 cells. Furthermore, Sirt3 inhibited SRV2 expression via the Mst1-JNK pathway, and re-activation of this pathway abolished the protective effects of Sirt3 on mitochondrial damage and apoptosis. Taken together, our results indicate that Sirt3-induced, Mst1-JNK-SRV2 signaling pathway-dependent inhibition of mitochondrial fission protected against neuroinflammation-mediated cell damage in BV-2 microglia. Sirt3 might therefore be an effective treatment for neuroinflammation.
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Project description:Microglial cells are resident immune cells of the central nervous system (CNS), recognized as key elements in the regulation of neural homeostasis and the response to injury and repair. As excessive activation of microglia may lead to neurodegeneration, therapeutic strategies targeting its inhibition were shown to improve treatment of most neurodegenerative diseases. Benfotiamine is a synthetic vitamin B1 (thiamine) derivate exerting potentially anti-inflammatory effects. Despite the encouraging results regarding benfotiamine potential to alleviate diabetic microangiopathy, neuropathy and other oxidative stress-induced pathological conditions, its activities and cellular mechanisms during microglial activation have yet to be elucidated. In the present study, the anti-inflammatory effects of benfotiamine were investigated in lipopolysaccharide (LPS)-stimulated murine BV-2 microglia. We determined that benfotiamine remodels activated microglia to acquire the shape that is characteristic of non-stimulated BV-2 cells. In addition, benfotiamine significantly decreased production of pro-inflammatory mediators such as inducible form of nitric oxide synthase (iNOS) and NO; cyclooxygenase-2 (COX-2), heat-shock protein 70 (Hsp70), tumor necrosis factor alpha α (TNF-α), interleukin-6 (IL-6), whereas it increased anti-inflammatory interleukin-10 (IL-10) production in LPS stimulated BV-2 microglia. Moreover, benfotiamine suppressed the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and protein kinase B Akt/PKB. Treatment with specific inhibitors revealed that benfotiamine-mediated suppression of NO production was via JNK1/2 and Akt pathway, while the cytokine suppression includes ERK1/2, JNK1/2 and Akt pathways. Finally, the potentially protective effect is mediated by the suppression of translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus. Therefore, benfotiamine may have therapeutic potential for neurodegenerative diseases by inhibiting inflammatory mediators and enhancing anti-inflammatory factor production in activated microglia.
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Project description:BackgroundIt has been proved that cyclo-oxygenase-2 (COX-2) is rapidly induced by inflammatory mediators. However, it is not known whether overexpression of COX-2 in the liver is sufficient to promote activation or secretion of inflammatory factors leading to hepatitis.AimTo investigate the role forced expression of COX-2 in liver by using inducible COX-2 transgenic (TG) mice.MethodsTG mice that overexpress the human COX-2 gene in the liver using the liver-specific transthyretin promoter and non-TG littermates were derived and fed the normal diet for up to 12 months. Hepatic prostaglandin E(2) (PGE(2)) content was determined using enzyme immunoassay, nuclear factor kappaB (NF-kappaB) activation by electrophoretic mobility shift assays, apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labelling and proliferation by Ki-67 immunohistochemistry.ResultsCOX-2 TG mice exhibited strongly increased COX-2 and PGE(2), elevated serum alanine aminotransferase level and histological hepatitis. Hepatic COX-2 expression in the TG mice resulted in activation of NF-kappaB and inflammatory cytokine cascade, with a marked expression of the proinflammatory cytokines tumour necrosis factor (TNF)-alpha (9.4-fold), interleukin (IL)-6 (4.4-fold), IL-1beta (3.6-fold), and of the anti-inflammatory cytokine IL-10 (4.4-fold) and chemokine macrophage inflammatory protein-2 (3.2-fold). The inflammatory response of the COX-2 TG mice was associated with infiltration macrophages and lymphocytes, increased cell proliferation and high rates of cell apoptosis. Administration of the COX-2 inhibitor celecoxib in TG mice restored liver histology to normal.ConclusionEnhanced COX-2 expression in hepatocytes is sufficient to induce hepatitis by activating NF-kappaB, stimulating the secretion of proinflammatory cytokines, recruiting macrophage and altering cell kinetics. Inhibition of COX-2 represents a mechanism-based chemopreventive approach to hepatitis.
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Project description:Microglia experience dramatic molecular and functional changes when transferred from the central nervous system (CNS) to a cell culture environment. Investigators largely attribute these findings to the loss of CNS-specific microenvironmental cues that dictate the gene-regulatory networks specified by master regulator transcription factors such as V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MafB). MafB regulates macrophage differentiation and activation by activating or repressing target genes critical to these processes. Here, we show that basal MafB levels in the BV-2 microglial cell line depend on the availability of lipids in the cell culture environment. Depletion of lipids, either by serum deprivation or the use of lipid-depleted serum, reduced MafB protein levels in BV-2 cells. Using live imaging, we also observed the engulfment of apoptotic BV-2 cell debris by neighboring BV-2 cells, highlighting an additional potential source of lipids in the cell culture environment. This observation was supported by experiments showing reduced MafB protein levels in BV-2 cells cultured with various phagocytosis inhibitors (cytochalasin D, annexin V) and reduced BV-2 cell phagocytic activity with serum deprivation. In aggregate, our data suggest that serum exposure regulates the transcription factor MafB in BV-2 cells through direct and indirect mechanisms.
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Project description:UnlabelledBackgroundNeurons are known to employ the endogenous cannabinoid system to communicate with other cells of the CNS. Endocannabioid signaling recruits microglia toward neurons by engaging cannabinoid CB2 and abnormal cannabidiol (Abn-CBD) receptors. The Abn-CBD receptor is a prominent atypical cannabinoid receptor that had been discriminated by means of various pharmacological and genetic tools but remained to be identified at the molecular level. We recently introduced N-arachidonoyl glycine (NAGly) signaling via GPR18 receptors as an important novel signaling mechanism in microglial-neuronal communication. NAGly is an endogenous, enzymatically oxygenated metabolite of the endocannabinoid N-arachidonoyl ethanolamide (AEA). Our recent studies support strongly two hypotheses; first that NAGly initiates directed microglial migration in the CNS through activation of GPR18, and second that GPR18 is the Abn-CBD receptor. Here we present siRNA knockdown data in further support of these hypotheses.FindingsA GPR18-targetting siRNA pSUPER G418 GFP cDNA plasmid was created and transfected into BV-2 microglia. Successfully transfected GFP+ GPR18 siRNA BV-2 microglia displayed reduced GPR18 mRNA levels and immunocytochemical staining. Cell migration induced by 1 μM concentrations of NAGly, O-1602 and Abn-CBD were significantly attenuated in GFP+ cells.ConclusionsOur data provide definitive evidence that these compounds, characteristic of Abn-CBD receptor pharmacology, are acting via GPR18 in BV-2 microglia. A fuller understanding of the hitherto unidentified cannabinoid receptors such as GPR18; their molecular interactions with endogenous ligands; and how phytocannabinoids influence their signaling is vital if we are to comprehensively assess the function of the endogenous cannabinoid signaling system in human health and disease.
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Project description:Accumulating evidence suggests that the pathophysiology of depression might be associated with neuroinflammation, which could be attenuated by pharmacological treatment for depression. Omega-3 polyunsaturated fatty acids (PUFAs) are anti-inflammatory and exert antidepressant effects. The aim of this study was to identify the molecular mechanisms through which docosahexaenoic acid (DHA), the main omega-3 PUFA in the brain, modulates oxidative reactions and inflammatory cytokine production in microglial and neuronal cells. The results of this study showed that DHA reduced expressions of tumor necrosis factor-?, interleukin-6, nitric oxide synthase, and cyclo-oxygenase-2, induced by interferon-?, and induced upregulation of heme oxygenase-1 (HO-1) in BV-2 microglia. The inhibitory effect of DHA on nitric oxide production was abolished by HO-1 inhibitor zinc protoporphyrin IX. In addition, DHA caused AKT and ERK activation in a time-dependent manner, and the DHA-induced HO-1 upregulation could be attenuated by PI-3 kinase/AKT and MEK/ERK inhibitors. DHA also increased IKK?/? phosphorylation, I?B? phosphorylation, and I?B? degradation, whereas both nuclear factor-?B and I?B protease inhibitors could inhibit DHA-induced HO-1 expressions. The other major n-3 PUFA, eicosapentaenoic acid, showed similar effects of DHA on inflammation and HO-1 in repeated key experiments. In connecting with inflammation hypothesis of depression and clinical studies supporting the antidepressant effects of omega-3 PUFAs, this study provides a novel implication of the antidepressant mechanisms of DHA.
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Project description:Increasing evidence suggests that systemic inflammation triggers a neuroinflammatory response that involves sustained microglia activation. This response has deleterious consequences on memory and learning capability in experimental animal models and in patients. However, the mechanisms connecting systemic inflammation and microglia activation remain poorly understood. Here, we identify the autotaxin (ATX)/lysophosphatidic acid (LPA)/LPA-receptor axis as a potential pharmacological target to modulate the LPS-mediated neuroinflammatory response in vitro (the murine BV-2 microglia cell line) and in vivo (C57BL/6J mice receiving a single i.p. LPS injection). In LPS-stimulated (20 ng/mL) BV-2 cells, we observed increased phosphorylation of transcription factors (STAT1, p65, and c-Jun) that are known to induce a proinflammatory microglia phenotype. LPS upregulated ATX, TLR4, and COX2 expression, amplified NO production, increased neurotoxicity of microglia conditioned medium, and augmented cyto-/chemokine concentrations in the cellular supernatants. PF8380 (a type I ATX inhibitor, used at 10 and 1 µM) and AS2717638 (an LPA5 antagonist, used at 1 and 0.1 µM) attenuated these proinflammatory responses, at non-toxic concentrations, in BV-2 cells. In vivo, we demonstrate accumulation of PF8380 in the mouse brain and an accompanying decrease in LPA concentrations. In vivo, co-injection of LPS (5 mg/kg body weight) and PF8380 (30 mg/kg body weight), or LPS/AS2717638 (10 mg/kg body weight), significantly attenuated LPS-induced iNOS, TNFα, IL-1β, IL-6, and CXCL2 mRNA expression in the mouse brain. On the protein level, PF8380 and AS2717638 significantly reduced TLR4, Iba1, GFAP and COX2 expression, as compared to LPS-only injected animals. In terms of the communication between systemic inflammation and neuroinflammation, both inhibitors significantly attenuated LPS-mediated systemic TNFα and IL-6 synthesis, while IL-1β was only reduced by PF8380. Inhibition of ATX and LPA5 may thus provide an opportunity to protect the brain from the toxic effects that are provoked by systemic endotoxemia.
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Project description:Background: Naltrexone is an opioid receptor antagonist commonly used to treat opioid and alcohol dependence. The use of low dose naltrexone (LDN) was found to have anti-inflammatory properties for treatment of diseases such as fibromyalgia, Crohn's disease, multiple sclerosis and regional pain syndromes. Related to its anti-neuroinflammatory properties, the mechanism of action is possibly mediated via Toll-like receptor 4 antagonism, which is widely expressed on microglial cells. The aim of the present study was to assess the immunometabolic effects of naltrexone on microglia cells in in vitro conditions. All experiments were performed in the BV-2 microglial cell line. The cells were treated with naltrexone at 100 μM concentrations corresponding to low dose for 24 h. Cell viability was assessed for every drug dose. To induce additional activation, the cells were pretreated with LPS and IFN-γ. Immunofluorescence was used to analyse the classical microglial activation markers iNOS and CD206, while Seahorse was used for real-time cellular metabolic assessments. mTOR activity measured over the expression of a major direct downstream target S6K was assessed using western blot. LDN induced a shift from highly activated pro-inflammatory phenotype (iNOShighCD206low) to quiescent anti-inflammatory M2 phenotype (iNOSlowCD206high) in BV-2 microglia cells. Changes in the inflammatory profile were accompanied by cellular metabolic switching based on the transition from high glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). LDN-treated cells were able to maintain a metabolically suppressive phenotype by supporting OXPHOS with high oxygen consumption, and also maintain a lower energetic state due to lower lactate production. The metabolic shift induced by transition from glycolysis to mitochondrial oxidative metabolism was more prominent in cells pretreated with immunometabolic modulators such as LPS and IFN-γ. In a dose-dependent manner, naltrexone also modulated mTOR/S6K expression, which underlies the cell metabolic phenotype regulating microglia immune properties and adaptation. By modulating the phenotypic features by metabolic switching of activated microglia, naltrexone was found to be an effective and powerful tool for immunometabolic reprogramming and could be a promising novel treatment for various neuroinflammatory conditions.
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Project description:Chronic microglial activation and resulting sustained neuroinflammatory reaction are generally associated with neurodegeneration. Activated microglia acquires proinflammatory cellular profile that generates oxidative burst. Their persistent activation exacerbates inflammation, which damages healthy neurons via cytotoxic mediators, such as superoxide radical anion and nitric oxide. In our recent study, we have shown that benfotiamine (S-benzoylthiamine O-monophosphate) possesses anti-inflammatory effects. Here, the effects of benfotiamine on the pro-oxidative component of activity of LPS-stimulated BV-2 cells were investigated. The activation of microglia was accompanied by upregulation of intracellular antioxidative defense, which was further promoted in the presence of benfotiamine. Namely, activated microglia exposed to non-cytotoxic doses of benfotiamine showed increased levels and activities of hydrogen peroxide- and superoxide-removing enzymes-catalase and glutathione system, and superoxide dismutase. In addition, benfotiamine showed the capacity to directly scavenge superoxide radical anion. As a consequence, benfotiamine suppressed the activation of microglia and provoked a decrease in NO and (·)O(-) 2 production and lipid peroxidation. In conclusion, benfotiamine might silence pro-oxidative activity of microglia to alleviate/prevent oxidative damage of neighboring CNS cells.
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