YC-1 attenuates LPS-induced proinflammatory responses and activation of nuclear factor-kappaB in microglia.
ABSTRACT: BACKGROUND AND PURPOSE: An inflammatory response in the central nervous system mediated by the activation of microglia is a key event in the early stages of the development of neurodegenerative diseases. LPS has been reported to cause marked microglia activation. It is very important to develop drugs that can inhibit microglia activation and neuroinflammation. Here, we investigated the inhibitory effect of YC-1, a known activator of soluble guanylyl cyclase, against LPS-induced inflammatory responses in microglia. EXPERIMENTAL APPROACH: To understand the inhibitory effects of YC-1 on LPS-induced neuroinflammation, primary cultures of rat microglia and the microglia cell line BV-2 were used. To examine the mechanism of action of YC-1, LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production, iNOS, COX-2 and cytokine expression were analyzed by Griess reaction, ELISA, Western blotting and RT-PCR, respectively. The effect of YC-1 on LPS-induced activation of nuclear factor kappa B (NF-kappaB) was studied by NF-kappaB reporter assay and immunofluorocytochemistry. KEY RESULTS: YC-1 inhibited LPS-induced production of NO and PGE2 in a concentration-dependent manner. The protein and mRNA expression of iNOS and COX-2 in response to LPS application were also decreased by YC-1. In addition, YC-1 effectively reduced LPS-induced expression of the mRNA for the proinflammatory cytokines, TNF-alpha and IL-1beta. Furthermore, YC-1 inhibited LPS-induced NF-kappaB activation in microglia. CONCLUSIONS AND IMPLICATIONS: YC-1 was able to inhibit LPS-induced iNOS and COX-2 expression and NF-kappaB activation, indicating that YC-1 may be developed as an anti-inflammatory neuroprotective agent.
Project description:Female sex is associated with improved outcome in experimental brain injury models, such as traumatic brain injury, ischemic stroke, and intracerebral hemorrhage. This implies female gonadal steroids may be neuroprotective. A mechanism for this may involve modulation of post-injury neuroinflammation. As the resident immunomodulatory cells in central nervous system, microglia are activated during acute brain injury and produce inflammatory mediators which contribute to secondary injury including proinflammatory cytokines, and nitric oxide (NO) and prostaglandin E2 (PGE2), mediated by inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively. We hypothesized that female gonadal steroids reduce microglia mediated neuroinflammation. In this study, the progesterone's effects on tumor necrosis factor alpha (TNF-?), iNOS, and COX-2 expression were investigated in lipopolysaccharide (LPS)-stimulated BV-2 microglia. Further, investigation included nuclear factor kappa B (NF-?B) and mitogen activated protein kinase (MAPK) pathways. LPS (30 ng/ml) upregulated TNF-?, iNOS, and COX-2 protein expression in BV-2 cells. Progesterone pretreatment attenuated LPS-stimulated TNF-?, iNOS, and COX-2 expression in a dose-dependent fashion. Progesterone suppressed LPS-induced NF-?B activation by decreasing inhibitory ?B? and NF-?B p65 phosphorylation and p65 nuclear translocation. Progesterone decreased LPS-mediated phosphorylation of p38, c-Jun N-terminal kinase and extracellular regulated kinase MAPKs. These progesterone effects were inhibited by its antagonist mifepristone. In conclusion, progesterone exhibits pleiotropic anti-inflammatory effects in LPS-stimulated BV-2 microglia by down-regulating proinflammatory mediators corresponding to suppression of NF-?B and MAPK activation. This suggests progesterone may be used as a potential neurotherapeutic to treat inflammatory components of acute brain injury.
Project description:Cyclooxygenase-2 (COX-2) is induced under inflammatory conditions, and prostaglandin E2 (PGE2) is one of the products of COX activity. PGE2 has pleiotropic actions depending on the activation of specific E-type prostanoid EP1-4 receptors. We investigated the involvement of PGE2 and EP receptors in glial activation in response to an inflammatory challenge induced by LPS.Cultures of mouse microglia or astroglia cells were treated with LPS in the presence or absence of COX-2 inhibitors, and the production of PGE2 was measured by ELISA. Cells were treated with PGE2, and the effect on LPS-induced expression of TNF-α messenger RNA (mRNA) and protein was studied in the presence or absence of drug antagonists of the four EP receptors. EP receptor expression and the effects of EP2 and EP4 agonists and antagonists were studied at different time points after LPS.PGE2 production after LPS was COX-2-dependent. PGE2 reduced the glial production of TNF-α after LPS. Microglia expressed higher levels of EP4 and EP2 mRNA than astroglia. Activation of EP4 or EP2 receptors with selective drug agonists attenuated LPS-induced TNF-α in microglia. However, only antagonizing EP4 prevented the PGE2 effect demonstrating that EP4 was the main target of PGE2 in naïve microglia. Moreover, the relative expression of EP receptors changed during the course of classical microglial activation since EP4 expression was strongly depressed while EP2 increased 24 h after LPS and was detected in nuclear/peri-nuclear locations. EP2 regulated the expression of iNOS, NADPH oxidase-2, and vascular endothelial growth factor. NADPH oxidase-2 and iNOS activities require the oxidation of NADPH, and the pentose phosphate pathway is a main source of NADPH. LPS increased the mRNA expression of the rate-limiting enzyme of the pentose pathway glucose-6-phosphate dehydrogenase, and EP2 activity was involved in this effect.These results show that while selective activation of EP4 or EP2 exerts anti-inflammatory actions, EP4 is the main target of PGE2 in naïve microglia. The level of EP receptor expression changes from naïve to primed microglia where the COX-2/PGE2/EP2 axis modulates important adaptive metabolic changes.
Project description:Peripheral inflammation leads to immune responses in brain characterized by microglial activation, elaboration of proinflammatory cytokines and reactive oxygen species, and secondary neuronal injury. The inducible cyclooxygenase (COX), COX-2, mediates a significant component of this response in brain via downstream proinflammatory PG signaling. In this study, we investigated the function of the PGE2 E-prostanoid (EP) 4 receptor in the CNS innate immune response to the bacterial endotoxin LPS. We report that PGE2 EP4 signaling mediates an anti-inflammatory effect in brain by blocking LPS-induced proinflammatory gene expression in mice. This was associated in cultured murine microglial cells with decreased Akt and I-kappaB kinase phosphorylation and decreased nuclear translocation of p65 and p50 NF-kappaB subunits. In vivo, conditional deletion of EP4 in macrophages and microglia increased lipid peroxidation and proinflammatory gene expression in brain and in isolated adult microglia following peripheral LPS administration. Conversely, EP4 selective agonist decreased LPS-induced proinflammatory gene expression in hippocampus and in isolated adult microglia. In plasma, EP4 agonist significantly reduced levels of proinflammatory cytokines and chemokines, indicating that peripheral EP4 activation protects the brain from systemic inflammation. The innate immune response is an important component of disease progression in a number of neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In addition, recent studies demonstrated adverse vascular effects with chronic administration of COX-2 inhibitors, indicating that specific PG signaling pathways may be protective in vascular function. This study supports an analogous and beneficial effect of PGE2 EP4 receptor signaling in suppressing brain inflammation.
Project description:We investigated the mechanism of suppression of inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) by ergolide, sesquiterpene lactone from Inula britannica. iNOS activity in cell-free extract of LPS/IFN-gamma-stimulated RAW 264.7 macrophages was markedly attenuated by the treatment with ergolide. Its inhibitory effect on iNOS was paralleled by decrease in nitrite accumulation in culture medium of LPS/IFN-gamma-stimulated RAW 264.7 macrophages in a concentration-dependent manner. However, its inhibitory effect does not result from direct inhibition of the catalytic activity of NOS. Ergolide markedly decreased the production of prostaglandin E(2) (PGE(2)) in cell-free extract of LPS/IFN-gamma-stimulated RAW 264.7 macrophages in a concentration-dependent manner, without alteration of the catalytic activity of COX-2 itself. Ergolide decreased the level of iNOS and COX-2 protein, and iNOS mRNA caused by stimulation of LPS/IFN-gamma in a concentration-dependent manner, as measured by Western blot and Northern blot analysis, respectively. Ergolide inhibited nuclear factor-kappaB (NF-kappaB) activation, a transcription factor necessary for iNOS and COX-2 expression in response to LPS/IFN-gamma. This effect was accompanied by the parallel reduction of nuclear translocation of subunit p65 of NF-kappaB as well as IkappaB-alpha degradation. In addition, these effects were completely blocked by treatment of cysteine, indicating that this inhibitory effect of ergolide could be mediated by alkylation of NF-kappaB itself or an upstream molecule of NF-kappaB. Ergolide also directly inhibited the DNA-binding activity of active NF-kappaB in LPS/IFN-gamma-pretreated RAW 264.7 macrophages. These results demonstrate that the suppression of NF-kappaB activation by ergolide might be attributed to the inhibition of nuclear translocation of NF-kappaB resulted from blockade of the degradation of IkappaB and the direct modification of active NF-kappaB, leading to the suppression of the expression of iNOS and COX-2, which play important roles in inflammatory signalling pathway.
Project description:To investigate the anti-neuroinflammatory activity of a novel synthetic compound, 7-methylchroman-2-carboxylic acid N-(2-trifluoromethyl) phenylamide (MCAP) against LPS-induced microglial activation in vitro.Primary mouse microglia and BV2 microglia cells were exposed to LPS (50 or 100 ng/mL). The expression of iNOS and COX-2, proinflammatory cytokines, NF-?B and p38 MAPK signaling molecules were analyzed by RT-PCR, Western blot and ELISA. The morphological changes of microglia and nuclear translocation of NF-?B were visualized using phase contrast and fluorescence microscopy, respectively.Pretreatment with MCAP (0.1, 1, 10 ?mol/L) dose-dependently inhibited LPS-induced expression of iNOS and COX-2 in BV2 microglia cells. Similar results were obtained in primary microglia pretreated with MCAP (0.1, 0.5 ?mol/L). MCAP dose-dependently abated LPS-induced release of TNF-?, IL-6 and IL-1?, and mitigated LPS-induced activation of NF-?B by reducing the phosphorylation of I?B? in BV2 microglia cells. Moreover, MCAP attenuated LPS-induced phosphorylation of p38 MAPK, whereas SB203580, a p38 MAPK inhibitor, significantly potentiated MCAP-caused inhibition on the expression of MEF-2 (a transcription factor downstream of p38 MAPK).MCAP exerts anti-inflammatory effects in murine microglia in vitro by inhibiting the p38 MAPK and NF-?B signaling pathways and proinflammatory responses. MCAP may be developed as a novel agent for treating diseases involving activated microglial cells.
Project description:In the course of a search for anti-inflammatory metabolites from marine-derived fungi, methylpenicinoline (1) was isolated from a marine isolate of Penicillin sp. Compound 1 inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production by suppressing the expression of inducible NO synthase (iNOS) in RAW264.7 macrophages and BV2 microglia. It also attenuated prostaglandin E2 (PGE2) production by suppressing cyclooxygenase-2 (COX-2) expression in a concentration-dependent manner (from 10 μM to 80 μM) without affecting cell viability. In addition, compound 1 reduced the production of the pro-inflammatory cytokine interleukin-1β (IL-1β). In a further study designed to elucidate the mechanism of its anti-inflammatory effects, compound 1 was shown to block nuclear factor-kappa B (NF-κB) activation in LPS-induced RAW264.7 macrophages and BV2 microglia by inhibiting the phosphorylation of inhibitor kappa B-α (IκB-α), thereby suppressing the nuclear translocation of NF-κB dimers, namely p50 and p65, that are known to be crucial molecules associated with iNOS and COX-2 expression. In addition, compound 1 inhibited the activation of mitogen-activated protein kinase (MAPK) pathways. Taken together, the results suggest that compound 1 might be a valuable therapeutic agent for the treatment of anti-inflammatory and anti-neuroinflammatory diseases.
Project description:We investigated the effect of cyclolinteinone, a sesterterpene from Caribbean sponge Cacospongia linteiformis, on inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2) protein expression in lipopolysaccharide (LPS)-stimulated J774 macrophages. Incubation of J774 cells with LPS (1 microgram/ml) caused an increase of both iNOS and COX-2 protein expression, which was prevented in a concentration-dependent fashion by cyclolinteinone (12.5, 25 and 50 microM). Electrophoretic mobility-shift assay indicated that cyclolinteinone blocked the activation of nuclear factor-kappaB (NF-kappaB), a transcription factor necessary for either iNOS or COX-2 induction. Cyclolinteinone also blocked disappearance of I(kappa)B-alpha from cytosolic fraction and nuclear translocation of NF-kappaB subunits p50 and p65. These results show that cyclolinteinone down-regulates iNOS and COX-2 protein expression by inhibiting NF-kappaB activation and suggest that it may represent a novel anti-inflammatory compound capable of controlling the excessive production of prostaglandins and nitric oxide occurring in several inflammatory diseases.
Project description:The farnesoid X receptor (FXR) is a nuclear receptor that plays key roles in hepatoprotection by maintaining the homeostasis of liver metabolism. FXR null mice display strong hepatic inflammation and develop spontaneous liver tumors. In this report, we demonstrate that FXR is a negative modulator of nuclear factor kappaB (NF-kappaB)-mediated hepatic inflammation. Activation of FXR by its agonist ligands inhibited the expression of inflammatory mediators in response to NF-kappaB activation in both HepG2 cells and primary hepatocytes cultured in vitro. In vivo, compared with wild-type controls, FXR(-/-) mice displayed elevated messenger RNA (mRNA) levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interferon-inducible protein 10, and interferon-gamma in response to lipopolysaccharide (LPS). Examination of FXR(-/-) livers showed massive necroses and inflammation after treatment with LPS at a dose that does not induce significant liver damage or inflammation in wild-type mice. Moreover, transfection of a constitutively active FXR expression construct repressed the iNOS, COX-2, interferon-inducible protein 10 and interferon-gamma mRNA levels induced by LPS administration. FXR activation had no negative effects on NF-kappaB-activated antiapoptotic genes, suggesting that FXR selectively inhibits the NF-kappaB-mediated hepatic inflammatory response but maintains or even enhances the cell survival response. On the other hand, NF-kappaB activation suppressed FXR-mediated gene expression both in vitro and in vivo, indicating a negative crosstalk between the FXR and NF-kappaB signaling pathways. Our findings reveal that FXR is a negative mediator of hepatic inflammation, which may contribute to the critical roles of FXR in hepatoprotection and suppression of hepatocarcinogenesis.
Project description:Gamisoyo-san (GMSYS) is a traditional herbal formula used to treat insomnia, dysmenorrhea, and infertility in Korea. The purpose of this study was to investigate the anti-inflammatory effect and action mechanisms of GMSYS in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.The anti-inflammatory effects of GMSYS were investigated using nitric oxide (NO) assay and ELISAs for prostaglandin E2 (PGE2), tumor necrosis factor-? (TNF-?), and interleukin-6 (IL-6). The anti-inflammatory action mechanisms of GMSYS were evaluated using Western blotting for inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and activation of nuclear transcription factor kappa B (NF-?B) and mitogen-activated protein kinases (MAPKs).GMSYS significantly inhibited the LPS-induced production of NO, PGE2, TNF-?, and IL-6 compared with the vehicle-treated cells. GMSYS consistently downregulated the expression of iNOS and COX-2 mRNA induced by LPS. In addition, pretreatment with GMSYS suppressed the LPS-induced activation of NF-?B and MAPKs such as p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK).Our results indicate that the anti-inflammatory effects of GMSYS in RAW 264.7 macrophages are associated with inhibition of the release of inflammatory mediators and cytokines through the suppression of MAPK and NF-?B activation. These findings suggest that GMSYS may be a useful therapeutic candidate for the prevention or treatment of inflammatory diseases.
Project description:We studied the effect of PPM-18, a chemically synthesized naphthoquinone derivative and also an anti-inflammatory agent, on the lipopolysaccharide (LPS)-activated inducible NO synthase (iNOS) expression in rat alveolar macrophages. Pretreatment of macrophages with PPM-18 (0.1-10 microM) significantly inhibited nitrite production, iNOS protein expression and iNOS mRNA accumulation. PPM-18 did not directly affect the enzymic activities of iNOS and other constitutive NOS forms. The LPS-induced increase in nuclear transcription factor kappaB (NF-kappaB) p65 and p50 in nucleus was suppressed by PPM-18 (10 microM). Moreover electrophoretic mobility-shift assays demonstrated that PPM-18 inhibited DNA binding to NF-kappaB induced by LPS in whole cells but not when added in the nuclear extract, suggesting that PPM-18 did not interfere directly with the binding of NF-kappaB to DNA and that some events had to be processed before NF-kappaB could bind DNA. Examination of NF-kappaB showed that PPM-18 stabilized the NF-kappaB inhibitor, IkappaBalpha, by preventing its degradation from NF-kappaB. Therefore the stabilization of IkappaBalpha might have contributed to the inhibition of NF-kappaB activation. These results also indicate strongly that NF-kappaB is involved in the production of NO on stimulation by LPS. PPM-18 significantly decreased the production of tumour necrosis factor alpha in response to LPS. PPM-18 protects mice against LPS-induced lethal toxicity. These results also indicate that PPM-18 is a potent inhibitor of iNOS expression by blocking the binding of NF-kappaB to promoter and exerts a beneficial effect in the mouse model of sepsis.