Involvement of protein kinase Cδ and extracellular signal-regulated kinase-2 in the suppression of microglial inducible nitric oxide synthase expression by N-[3,4-dimethoxycinnamoyl]-anthranilic acid (tranilast) (original) (raw)
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Journal of Neuroimmunology, 1998
Ž . Nitric oxide NO has been implicated in a number of important brain functions, such as long-term potentiation LTP and long-term Ž . depression LTD , and in events associated with neurodegeneration and neuroprotection. In response to brain injury or disease NO Ž . production is increased by an inducible enzyme iNOS , which is only expressed under these conditions. Activated microglia are a major cellular source of iNOS in brain. Due to the important role of iNOS in brain injury and disease, a detailed understanding of intracellular events triggering the expression of iNOS in microglia would facilitate pharmacotherapeutic approaches. It is shown here, that iNOS Ž . mRNA, protein and NO product are induced in cultured microglia by lipopolysaccharide LPS . This induction is reduced by a number of substances elevating intracellular cyclic AMP levels. It is unabated, however, in the presence of substances inhibiting cyclooxygenase-1 Ž . andror cyclooxygenase-2 e.g., acetyl salicylic acid, SC 58125, L 745337 , but is decreased by approx. 50% with PDTC, a scavenger of Ž . Ž . reactive oxygen intermediates ROI that inhibits nuclear factor k B NF-kB activation. Furthermore, inhibitors of protein kinase C Ž . Ž PKC strongly inhibit iNOS mRNA and protein induction. PKC, therefore, constitutes a major second messenger component besides . NF-kB in the signaling pathway regulating iNOS expression in microglia. q
British Journal of Pharmacology, 2001
Microglial cells up-regulate inducible nitric oxide synthase (iNOS) expression in response to various pro-in¯ammatory stimuli including interferon-g (IFN-g), allowing for the release of nitric oxide (NO). Tranilast (N-[3,4-dimethoxycinnamoyl]-anthranilic acid) is an antiallergic compound with suppressive eects on the activation of monocytes. 2 Here, we show that N9 murine microglial cells express iNOS mRNA and protein and release nitric oxide into the culture medium in response to IFN-g (200 u ml 71) as measured by Northern and Western blot analyses and Griess assay. 3 Exposure to non-toxic doses of tranilast (30 ± 300 mM) leads to a concentration-dependent inhibition of IFN-g-induced (200 u ml 71) iNOS mRNA and protein expression. This is paralleled by a suppression of NO-release into the cell culture medium. 4 Inhibition of IFN-g-induced iNOS mRNA expression by tranilast is paralleled by an inhibition of nuclear factor-kB (NF-kB) activation and phosphorylation of inhibitory kB (IkB) as determined by Western blot analyses and NF-kB reporter gene assay. 5 These results suggest that tranilast-mediated suppression of microglial iNOS activity induced by IFN-g involves the inhibition of NF-kB-dependent iNOS mRNA expression.
British journal of …, 2001
Microglial cells up-regulate inducible nitric oxide synthase (iNOS) expression in response to various pro-in¯ammatory stimuli including interferon-g (IFN-g), allowing for the release of nitric oxide (NO). Tranilast (N-[3,4-dimethoxycinnamoyl]-anthranilic acid) is an antiallergic compound with suppressive eects on the activation of monocytes. 2 Here, we show that N9 murine microglial cells express iNOS mRNA and protein and release nitric oxide into the culture medium in response to IFN-g (200 u ml 71) as measured by Northern and Western blot analyses and Griess assay. 3 Exposure to non-toxic doses of tranilast (30 ± 300 mM) leads to a concentration-dependent inhibition of IFN-g-induced (200 u ml 71) iNOS mRNA and protein expression. This is paralleled by a suppression of NO-release into the cell culture medium. 4 Inhibition of IFN-g-induced iNOS mRNA expression by tranilast is paralleled by an inhibition of nuclear factor-kB (NF-kB) activation and phosphorylation of inhibitory kB (IkB) as determined by Western blot analyses and NF-kB reporter gene assay. 5 These results suggest that tranilast-mediated suppression of microglial iNOS activity induced by IFN-g involves the inhibition of NF-kB-dependent iNOS mRNA expression.
Neurochemistry International, 2014
D-glucopyranosyl-3-indolecarbonate (RG3I) is a chemical constituent isolated from the commonly used Asian traditional medicinal plant, Clematis mandshurica; however, no studies have been reported on its anti-inflammatory properties. In the present study, we found that RG3I attenuates the lipopolysaccharide (LPS)-induced DNA-binding activity of nuclear factor-jB (NF-jB) via the dephosphorylation of PI3K/Akt in BV2 microglial cells, leading to a suppression of nitric oxide (NO) and prostaglandin E 2 (PGE 2) production, along with that of their regulatory genes, inducible NO synthase (iNOS) and cyclooxygenase-2 (Cox-2). Further, the PI3K/Akt inhibitor, LY294002 diminished the expression of LPS-stimulated iNOS and COX-2 genes by suppressing NF-jB activity. Moreover, RG3I significantly inhibited LPS-induced reactive oxygen species (ROS) generation similar to the ROS inhibitors, N-acetylcysteine (NAC) and glutathione (GSH). Notably, NAC and GSH abolished the LPS-induced expression of iNOS and Cox-2 in BV2 microglial cells by inhibiting NF-jB activity. Taken together, our data indicate that RG3I suppresses the production of proinflammatory mediators such as NO and PGE 2 as well as their regulatory genes in LPS-stimulated BV2 microglial cells by inhibiting the PI3K/Akt-and ROS-dependent NF-jB signaling pathway, suggesting that RG3I may be a good candidate to regulate LPS-induced inflammatory response.
Molecular Brain Research, 2000
Glia in the brain respond to various toxins with an increased expression of inducible nitric oxide synthase (iNOS) and an increased production of nitric oxide (NO). Here, we report that lipopolysaccharide (LPS)-induced expression of iNOS was down-regulated post-transcriptionally through the destabilization of iNOS mRNA by the indolocarbazole compound, Go6976, in murine microglia. This Go6976 effect is specific for iNOS since tumor necrosis factor a was unaffected by the compound. Interestingly, the post-transcriptional effects ascribed to Go6976 were not observed with other inhibitors of protein kinase A, C (PKC), G, or protein tyrosine kinases. Instead, these kinases appear to affect the iNOS / NO system at the transcriptional level. In the past, Go6976 has been reported to be a rather specific inhibitor of PKC in vitro. Results from our experiments, through prolonged treatment with phorbol esters and with the various PKC inhibitors including phorbol ester-insensitive PKC isotype inhibitor, suggest that the Go6976-mediated post-transcriptional regulation of iNOS gene expression and NO production in microglia is not mediated through its reputed effects on PKC activity. Since the effects of various neurotoxins and certain neurodegenerative diseases may be manifested through alterations in the iNOS / NO system, post-transcriptional control of this system may represent a novel strategy for therapeutic intervention.
Regulation of microglia effector functions by tumor necrosis factor signaling
Glia, 2011
The exact biological role of the cytokine Tumor Necrosis Factor (TNF) in the central nervous system (CNS) is not well understood; but overproduction of TNF by activated microglia has been implicated in neuronal death, suggesting that TNF inhibition in the CNS may be a viable neuroprotective strategy. We investigated the role of TNF signaling in regulation of microglia effector functions using molecular, cellular, and functional analyses of postnatal and adult microglia populations in the CNS. No differences were found by flow cytometric analyses in the basal activation state between TNF-null and wild type mice. Although TNF-null microglia displayed an atypical morphology with cytoplasmic vacuoles in response to stimulation with lipopolysaccharide (LPS), the phagocytic response of TNF-null microglia to E.coli particles in vitro was normal and there were no signs of enhanced caspase 3 activation or apoptosis. Functionally, conditioned media from LPS-stimulated TNF-null microglia was found to have significantly reduced levels of IL-10, IL-6, IL-1β, IL-12 and CXCL1 relative to wild type microglia and exerted no cytotoxic effects on neurally differentiated dopaminergic MN9D cells. In contrast, incubation of wild type microglia with TNF inhibitors selectively depleted the levels of soluble TNF and its cytotoxicity on MN9D cells. To distinguish whether reduced cytotoxicity by LPS-activated TNF-null microglia could be attributed to deficient autocrine TNF signaling, we employed primary microglia deficient in one or both TNF receptors (TNFR1 and TNFR2) in coculture with MN9D cells and found that neither receptor is required to elicit LPS-evoked TNF production and cytotoxicity on dopaminergic cells.
Ellagic Acid Protects against Activation of Microglia by Inhibiting MAPKs and NF-κB Signalling
Indian Journal of Pharmaceutical Education and Research, 2020
Context: One of the hallmarks of neurodegenerative diseases is dysregulation of microglia resulting in neuroinflammation. Neuroinflammation is related to multiple neurodegenerative disorders, including stroke and Alzheimer's disease. Ellagic acid (EA) possesses antiapoptotic activity, anti-inflammatory, and antioxidant properties. It is most commonly found in fruits and nuts. EA has been linked to neuronal protection particularly through its anti-inflammatory and antioxidant actions. For example, EA inhibits enzyme activity which prevents the onset of Alzheimer's disease. Aim: In this study, the underlying protective mechanisms of EA in LPS-activated BV2 cells were investigated. Settings and Design: EA prepared in multiple concentrations was introduced for 24 hrs as pre-treatment in BV2 microglial cells. Lipopolysaccharide (LPS) at 1 µg mL-1 was used to stimulate BV2 microglial cells after the EA pre-treatment for an additional 24 hrs. After 24 hrs of stimulation, the cell lysate was harvested for bioassays. Materials and Methods: This study investigated the effects of EA on the viability of BV2 microglial cells using MTT assay. After LPS stimulation, the cell lysate was collected to measure nitric oxide (NO) levels using Griess assay kit and ELISA was used to measure TNFα levels. Western blotting was used to test protein levels of iNOS, MyD88, MAPKs and NF-κB. Statistical analysis used: Data are provided as means and standard errors of n = 3. A one-way analysis of variance was used to test the statistical significance of between group comparisons. The statistical significance of p<0.05 was set. Results: EA decreased the levels of TNFα and NO production in BV2 microglial cells induced by LPS. EA also reduced inducible nitric oxide synthase (iNOS) expression, and attenuated MyD88 and NF-κB expression. EA decreased the phosphorylation of p38, ERK and JNK and repressed the activation of CD11b and CD40 in a concentration-dependent manner. Conclusion: EA has a putative role in preventing neuroinflammation but its application in prevention of neurodegenerative diseases requires further investigations.
Up-regulation of Microglial CD11b Expression by Nitric Oxide
Journal of Biological Chemistry, 2006
Increased expression of CD11b, the -integrin marker of microglia, represents microglial activation during neurodegenerative inflammation. However, the molecular mechanism behind increased microglial CD11b expression is poorly understood. The present study was undertaken to explore the role of nitric oxide (NO) in the expression of CD11b in microglial cells. Bacterial lipopolysaccharide (LPS) induced the production of NO and increased the expression of CD11b in mouse BV-2 microglial cells and primary microglia. Either a scavenger of NO (PTIO) or an inhibitor of inducible nitric-oxide synthase (L-NIL) blocked this increase in microglial CD11b expression. Furthermore, co-microinjection of PTIO with LPS was also able to suppress LPSmediated expression of CD11b and loss of dopaminergic neuronal fibers and neurotransmitters in striatum in vivo. Similarly, other inducers of NO production such as interferon-␥, interleukin-1, human immunodeficiency virus type-1 gp120, and double-stranded RNA (poly(IC)) also increased the expression of CD11b in microglia through NO. The role of NO in the expression of CD11b was corroborated further by the expression of microglial CD11b by GSNO, an NO donor. Because NO transduces many intracellular signals via guanylate cyclase (GC), we investigated the role of GC, cyclic GMP (cGMP), and cGMP-activated protein kinase (PKG) in microglial expression of CD11b. Inhibition of LPS-and GSNO-mediated upregulation of CD11b either by NS2028 (a specific inhibitor of GC) or by KT5823 and Rp-8-bromo-cGMP (specific inhibitors of PKG), and increase in CD11b expression either by 8-bromo-cGMP or by MY-5445 (a specific inhibitor of cGMP phosphodiesterase) alone suggest that NO increases microglial expression of CD11b via GC-cGMP-PKG. In addition, GSNO induced the activation of cAMP response element-binding protein (CREB) via PKG that was involved in the up-regulation of CD11b. This study illustrates a novel biological role of NO in regulating the expression of CD11b in microglia through GC-cGMP-PKG-CREB pathway that may participate in the pathogenesis of devastating neurodegenerative disorders.
Amelioration of LPS-Induced Inflammation Response in Microglia by AMPK Activation
BioMed Research International, 2014
Adenosine 5 -monophosphate-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis via modulating metabolism of glucose, lipid, and protein. In addition to energy modulation, AMPK has been demonstrated to associate with several important cellular events including inflammation. The results showed that ENERGI-F704 identified from bamboo shoot extract was nontoxic in concentrations up to 80 M and dose-dependently induced phosphorylation of AMPK (Thr-172) in microglia BV2 cells. Our findings also showed that the treatment of BV2 with ENERGI-F704 ameliorated the LPS-induced elevation of IL-6 and TNF-production. In addition, ENERGI-F704 reduced increased production of nitric oxide (NO) and prostaglandin E2 (PGE2) via downregulating the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), respectively. Moreover, ENERGI-F704 decreased activated nuclear translocation and protein level of NF-B. Inhibition of AMPK with compound C restored decreased NF-B translocation by ENERGI-F704. In conclusion, ENERGI-F704 exerts inhibitory activity on LPS-induced inflammation through manipulating AMPK signaling and exhibits a potential therapeutic agent for neuroinflammatory disease.
Neurotoxicity Research, 2005
Pro-inflammatory molecules induce glial activation and the release of potentially detrimental factors capable of generating oxidative damage, such as nitric oxide (NO) and superoxide anion (O 2 •-). Activated glial cells (astrocytes and microglia) are associated to the inflammatory process in neurodegenerative diseases. A strong inflammatory response could escape endogenous control becoming toxic to neurons and contributing to the course of the disease. We evaluated in a hippocampal cells-microglia co-culture model, if the pro-inflammatory condition induced by lipopolysaccharide + interferon-γ (LPS+IFN-γ) promoted damage directly or if damage was secondary to glial activation. In addition, we explored the effect of the antiinflammatory cytokine transforming growth factor-β1 (TGF-β1), and pro-inflammatory cytokines, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) on the regulation of the inflammatory response of microglia. We found that LPS+IFNγ-induced damage on hippocampal cultures was dependent on the presence of microglial cells. In hippocampal cultures exposed to LPS+IFN-γ, TGF-β1 was induced whereas in microglial cell cultures LPS+IFN-γ induced the secretion of IL-1β. TGF-β1 and IL-1β but not TNF-α decreased the NO production by 70-90%. PD98059, an inhibitor of MAP kinase (MEK), reduced the IFN-γ-induced NO production by 40%. TGF-β1 and IL-1β reduced the IFN-γ-induced phosphorylation of ERK1,2 by 60% and 40%, respectively. However, the effect of IL-1β was observed at 30 min and that of TGF-β1 only after 24 h of exposure. We propose that acting with different timing, TGF-β1 and IL-1β can modulate the extracellular signal-regulated kinase ERK1,2, as a common element for different transduction pathways, regulating the amplitude and duration of glial activation in response to LPS+IFN-γ. Cross-talk among brain cells may be key for the understanding of inflammatory mechanisms involved in pathogenesis of neurodegenerative diseases.