LPS-induced Expression Of Pro-inflammatory Mediators Research Articles

NRF2 Plays a Crucial Role in the Tolerogenic Effect of Ethyl Pyruvate on Dendritic Cells.

Ethyl pyruvate (EP) is a redox-active compound that has been previously shown to be effective in restraining immune hyperactivity in animal models of various autoimmune and chronic inflammatory diseases. Importantly, EP has also been proven to have a potent tolerogenic effect on dendritic cells (DCs). Here, the influence of EP on the signaling pathways in DCs relevant for their tolerogenicity, including anti-inflammatory NRF2 and pro-inflammatory NF-κB, was explored. Specifically, the effects of EP on DCs obtained by GM-CSF-directed differentiation of murine bone marrow precursor cells and matured under the influence of lipopolysaccharide (LPS) were examined via immunocytochemistry and RT-PCR. EP counteracted LPS-imposed morphological changes and down-regulated the LPS-induced expression of pro-inflammatory mediators in DCs. While it reduced the activation of NF-κB, EP potentiated NRF2 and downstream antioxidative molecules, thus implying the regulation of NRF2 signaling pathways as the major reason for the tolerizing effects of EP on DCs.

3ʹ,4ʹ-Dihydroxyflavone mitigates inflammatory responses by inhibiting LPS and TLR4/MD2 interaction

BackgroundWe previously reported the potential inhibitory activity of 3ʹ,4ʹ-dihydroxyflavone (DHF) on nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-stimulated macrophages. PurposeWe investigated the underlying molecular mechanisms of DHF in LPS-activated macrophages and evaluated its effect on LPS-induced septic shock in mice. MethodsTo explore the anti-inflammatory effect of DHF, nitrite, PGE2, and cytokines were measured in vitro and in vivo experiments. In addition, to verify the molecular signaling pathway, quantitative real time-PCR, luciferase assay, nuclear extraction, electrophoretic mobility shift assay, immunocytochemistry, immunoprecipitation, molecular docking analysis, and myeloid differentiation 2 (MD2)-LPS binding assay were conducted. ResultsDHF suppressed the LPS-induced expression of proinflammatory mediators through nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and interferon regulatory factor 3 (IRF3) inactivation pathways in RAW 264.7 macrophages. Importantly, molecular docking analysis and in vitro binding assays showed that DHF interacts with the hydrophobic pocket of MD2 and then interferes with the interaction between LPS and toll-like receptor 4 (TLR4). DHF inhibited LPS-induced oxidative stress by upregulating nuclear factor erythroid 2-related factor 2 (Nrf2). Treatment of LPS-induced endotoxemia mice with DHF reduced the expression levels of pro-inflammatory mediators via the inactivation of NF-κB, AP-1, and signal transducer and activator of transcription 1 (STAT1) in the lung tissue, thus increasing the survival rate. ConclusionTaken together, our data first time revealed the underlying mechanism of the DHF-dependent anti-inflammatory effect by preventing LPS from binding to the TLR4/MD2 complex. Therefore, DHF may be a possible anti-inflammatory agent for the treatment of LPS-mediated inflammatory diseases.

Aucubin suppresses lipopolysaccharide-induced pro-inflammatory responses by blocking the NF-κB translocation signaling pathways in activated microglial cells

Aucubin is an iridoid glycoside with demonstrable hepatoprotective and anti-osteoporotic effects. Herein, using microglial cells and lipopolysaccharide (LPS) to induce inflammatory responses, we studied the signaling pathways involved in the anti-inflammatory action of aucubin and their influence on the expression of several genes known to be involved in inflammation. Aucubin inhibited LPS-stimulated pro-inflammatory responses by suppressing the production of nitric oxide and prostaglandin E 2 . Furthermore, aucubin inhibited inducible nitric oxide synthase and cyclooxygenase-2 at both the protein and mRNA levels. In addition, aucubin inhibited pro-inflammatory cytokine production in LPS-stimulated BV-2 microglial cells. Subsequent mechanistic studies revealed that aucubin inhibited the LPS-induced activation of nuclear factor-kappa B (NF-κB) translocation and phosphorylation of phosphatidylinositol 3-kinases (PI3K)/Akt as well as of mitogen-activated protein kinases (MAPKs), which are upstream molecules responsible for controlling inflammatory reactions. These results suggest that aucubin may exert anti-neuroinflammatory responses by suppressing the LPS-induced expression of pro-inflammatory mediators by blocking the activation of NF-κB, PI3K/Akt, and MAPK signaling pathways in microglial cells. • Aucubin inhibited LPS-induced pro-inflammatory mediators in microglial cells. • Aucubin suppressed LPS-induced phosphorylation of MAPKs and PI3K/Akt. • Aucubin blocked activation of NF-κB translocation and the DNA-binding activity of NF-κB. • Aucubin may exert anti-neuroinflammatory responses for neurodegenerative diseases.

Kaempferol 7-O-β-D-glucoside isolated from the leaves of Cudrania tricuspidata inhibits LPS-induced expression of pro-inflammatory mediators through inactivation of NF-κB, AP-1, and JAK-STAT in RAW 264.7 macrophages

Kaempferol 7-O-β-D-glucoside (KPG), a natural flavonol isolated from Cudrania tricuspidata, has been reported to exert anti-cancer effects; however, its anti-inflammatory effects have not yet been reported. In this study, we demonstrate the suppressive effect of KPG on the production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and mouse bone marrow-derived macrophages. KPG downregulated the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein level and iNOS, COX-2, TNF-α, IL-1β, and IL-6 at the mRNA level in LPS-treated RAW 264.7 macrophages. Moreover, we elucidated the underlying molecular mechanism, demonstrating that KPG attenuated LPS-induced nuclear factor-κB (NF-κB) activation by decreasing p65 nuclear translocation, inhibiting κBα (IκBα) phosphorylation/degradation and IκB kinaseα/β (IKKα/β) phosphorylation. KPG additionally reduced LPS-induced activator protein-1 (AP-1) activity by inhibiting c-Fos expression in the nucleus, though c-Jun was not affected. Furthermore, we revealed that KPG significantly abrogated the LPS-induced phosphorylation of signal transducer and activator of transcription (STAT) 1 (Ser 727, Tyr 701) and STAT3 (Tyr 705) through inhibiting the phosphorylation of Janus kinase (JAK) 1 and JAK2, its upstream activating proteins. Taken together, our data suggest that KPG induces anti-inflammatory activity by blocking NF-κB, AP-1, and JAK-STAT signaling pathways in LPS-treated RAW 264.7 macrophages, thus suppressing inflammatory mediators.

Sodium azide suppresses LPS-induced expression MCP-1 through regulating IκBζ and STAT1 activities in macrophages

Sodium azide (NaN3) is a chemical compound with multiple toxic effects on vascular and neuronal systems, causing hypotension and neurotoxicity, respectively. In order to test its effects on the immune system, human and mouse macrophage-like cell lines were treated with nontoxic doses of NaN3 and the changes in LPS-induced inflammatory activation was measured. Interestingly, the LPS-induced expression of monocyte chemoattractant protein (MCP)-1 was suppressed by NaN3 without affecting the expression of IL-8 and TNF-α. Further analysis of cellular signaling mediators involved in the expression of these cytokines revealed that NaN3 suppressed the LPS-induced activation of signal transducers and activator of transcription (STAT)1 and inhibitor of κB (IκB) ς, which are involved in the LPS-induced expression of MCP-1, while the LPS-induced activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) was not affected. The LPS-induced expression of MCP-2 and CXCL10, which are also regulated by STAT1, was suppressed by NaN3. Similarly, the LPS-induced expression of IL-6, which is regulated by IκBζ, was suppressed by NaN3. These results demonstrate that NaN3 selectively suppresses the LPS-induced expression of pro-inflammatory mediators through the suppression of STAT1 and IκBζ activation. These new findings about the activity of NaN3 may contribute to the development of specific regulators of macrophage activity during acute and chronic inflammation.

Galectin-3 Inhibition Is Associated with Neuropathic Pain Attenuation after Peripheral Nerve Injury.

Neuropathic pain remains a prevalent and persistent clinical problem because it is often poorly responsive to the currently used analgesics. It is very urgent to develop novel drugs to alleviate neuropathic pain. Galectin-3 (gal3) is a multifunctional protein belonging to the carbohydrate-ligand lectin family, which is expressed by different cells. Emerging studies showed that gal3 elicits a pro-inflammatory response by recruiting and activating lymphocytes, macrophages and microglia. In the study we investigated whether gal3 inhibition could suppress neuroinflammation and alleviate neuropathic pain following peripheral nerve injury. We found that L5 spinal nerve ligation (SNL) increases the expression of gal3 in dorsal root ganglions at the mRNA and protein level. Intrathecal administration of modified citrus pectin (MCP), a gal3 inhibitor, reduces gal3 expression in dorsal root ganglions. MCP treatment also inhibits SNL-induced gal3 expression in primary rat microglia. SNL results in an increased activation of autophagy that contributes to microglial activation and subsequent inflammatory response. Intrathecal administration of MCP significantly suppresses SNL-induced autophagy activation. MCP also inhibits lipopolysaccharide (LPS)-induced autophagy in cultured microglia in vitro. MCP further decreases LPS-induced expression of proinflammatory mediators including IL-1β, TNF-α and IL-6 by regulating autophagy. Intrathecal administration of MCP results in adecreased mechanical and cold hypersensitivity following SNL. These results demonstrated that gal3 inhibition is associated with the suppression of SNL-induced inflammatory process andneurophathic pain attenuation.

Anti-Inflammatory Effects of 3-(4'-Hydroxyl-3',5'-Dimethoxyphenyl)Propionic Acid, an Active Component of Korean Cabbage Kimchi, in Lipopolysaccharide-Stimulated BV2 Microglia.

We investigated the protective ability of 3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid (HDMPPA), an active principle in Korean cabbage kimchi, against the production of proinflammatory mediators and cytokines, and the mechanisms involved in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. HDMPPA significantly suppressed the production of nitric oxide (NO) and prostaglandin E2, along with the expression of inducible NO synthase and cyclooxygenase-2 in LPS-stimulated BV2 cells, at concentrations with no cytotoxicity. HDMPPA also attenuated the LPS-induced expression and secretion of proinflammatory cytokines, such as tumor necrosis factor-α and interleukin-1β. Furthermore, HDMPPA inhibited LPS-induced nuclear factor-κB (NF-κB) activation, which was associated with the abrogation of IκB-α degradation and phosphorylation, and subsequent decreases in NF-κB p65 levels. Moreover, the phosphorylation of mitogen-activated protein kinases (MAPKs) and Akt, a downstream molecule of phosphatidylinositol-3-kinase (PI3K), in LPS-stimulated BV2 cells was suppressed markedly by HDMPPA. This effect was associated with a significant reduction in the formation of intracellular reactive oxygen species. The findings in this study suggest that HDMPPA may exert anti-inflammatory responses by suppressing LPS-induced expression of proinflammatory mediators and cytokines through blockage of NF-κB, MAPKs, and PI3K/Akt signaling pathways and oxidative stress in microglia.

Anti-inflammatory effect of garlic 14-kDa protein on LPS-stimulated-J774A.1 macrophages

Context: Garlic 14-kDa protein is purified from garlic (Allium sativum L.) which is used in traditional medicine and exerts various immunomodulatory activities.Objective: The present study investigated the suppressive effect of garlic 14-kDa protein on LPS-induced expression of pro-inflammatory mediators and underlying mechanism in inflammatory macrophages.Materials and methods: J774A.1 macrophages were treated with 14-kDa protein (5–30 μg/ml) with/without LPS (1 μg/ml) and the production of inflammatory mediators such as prostaglandin E2 (PGE2), TNF-α, and IL-1β released were measured using ELISA. Nitric oxide (NO) production was determined using the Griess method. The anti-inflammatory activity of 14-kDa protein was examined by measuring inducible nitric oxide synthase and cyclooxygenase-2 proteins using western blot. The expression of nuclear NF-κB p65 subunit was assessed by western blot.Results: Garlic 14-kDa protein significantly inhibited the excessive production of NO, PGE, TNF-α, and IL-1β in lipopolysaccharide (LPS)-activated J774A.1 macrophages in a concentration-related manner without cytotoxic effect. Western blot analysis demonstrated that garlic 14-kDa protein suppressed corresponding inducible NO synthase expression and activated cyclooxygenase-2 protein expression. The inhibitory effect was mediated partly by a reduction in the activity and expression of transcription factor NF-κB protein.Conclusion: Our results suggested, for the first time, garlic 14-kDa protein exhibits anti-inflammatory properties in macrophages possibly by suppressing the inflammatory mediators via the inhibition of transcription factor NF-κB signaling pathway. The traditional use of garlic as anti-inflammatory remedy could be ascribed partly to 14-kDa protein content. This protein might be a useful candidate for controlling inflammatory diseases and further investigations in vivo.

Anti-inflammatory effect of enzymatic hydrolysates from Styela clava flesh tissue in lipopolysaccharide-stimulated RAW 264.7 macrophages and in vivo zebrafish model.

BACKGROUND/OBJECTIVESIn this study, potential anti-inflammatory effect of enzymatic hydrolysates from Styela clava flesh tissue was assessed via nitric oxide (NO) production in lipopolysaccahride (LPS) induced RAW 264.7 macrophages and in vivo zebrafish model.MATERIALS/METHODSWe investigated the ability of enzymatic hydrolysates from Styela clava flesh tissue to inhibit LPS-induced expression of pro-inflammatory mediators in RAW 264.7 macrophages, and the molecular mechanism through which this inhibition occurred. In addition, we evaluated anti-inflammatory effect of enzymatic hydrolysates against a LPS-exposed in in vivo zebrafish model.RESULTSAmong the enzymatic hydrolysates, Protamex-proteolytic hydrolysate exhibited the highest NO inhibitory effect and was fractionated into three ranges of molecular weight by using ultrafiltration (UF) membranes (MWCO 5 kDa and 10 kDa). The above 10 kDa fraction down-regulated LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), thereby reducing production of NO and prostaglandin E2 (PGE2) in LPS-activated RAW 264.7 macrophages. The above 10 kDa fraction suppressed LPS-induced production of pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. In addition, the above 10 kDa fraction inhibited LPS-induced phosphorylation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK), and p38. Furthermore, NO production in live zebrafish induced by LPS was reduced by addition of the above 10 kDa fraction from S. clava enzymatic hydrolysate.CONCLUSIONThe results of this study suggested that hydrolysates derived from S. clava flesh tissue would be new anti-inflammation materials in functional resources.

Salicortin suppresses lipopolysaccharide-stimulated inflammatory responses via blockade of NF-κB and JNK activation in RAW 264.7 macrophages

We isolated the phenolic glucoside salicortin from a Populus euramericana bark extract, and examined its ability to suppress inflammatory responses as well as the molecular mechanisms underlying these abilities, using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Salicortin inhibited iNOS expression and the subsequent production of NO in a dose-dependent manner in the LPS-stimulated RAW 264.7 cells. Salicortin significantly suppressed LPS-induced signal cascades of NF-κB activation, such as IKK activation, IκBα phosphorylation and p65 phosphorylation in RAW 264.7 cells. In addition, salicortin inhibited the LPS-induced activation of JNK, but not ERK or p38 MAPK. Furthermore, salicortin significantly inhibited production of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6 in the LPS-stimulated RAW 264.7 cells. These findings suggest that salicortin may show its anti-inflammatory activity by suppressing the LPS-induced expression of pro-inflammatory mediators through inhibition of NF-κB and JNK MAPK signaling cascades in macrophages. [BMB Reports 2014; 47(6): 318-323]

Protective effect of Ginkgo biloba leaves extract, EGb761, on endotoxin-induced acute lung injury via a JNK- and Akt-dependent NFκB pathway.

Acute lung injury (ALI) is a clinical syndrome mainly caused by Gram-negative bacteria which is still in need of an effective therapeutic medicine. EGb761, an extract of Ginkgo biloba leaves, has several bioeffects including anti-inflammation, cardioprotection, neuroprotection, and free radical scavenging. Preadministration of EGb761 inhibited lipopolysaccharide (LPS)-induced histopathological changes and exchange of arterial blood gas. In addition, LPS-induced expression of proinflammatory mediators, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, macrophage inflammatory protein (MIP)-2, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), were suppressed by EGb761. The activation of nuclear factor (NF)κB, a transcription factor of proinflammatory mediators, and phosphorylation of IκB, an inhibitor of NFκB, were also reduced by EGb761. Furthermore, we found the inhibitory concentration of EGb761 on phosphorylation of JNK and Akt was less than those of ERK and p38 MAPK. In conclusion, EGb761 is a potential protective agent for ALI, possibly via downregulating the JNK- and Akt-dependent NFκB activation pathway.

Suppression of iNOS and COX-2 expression by flavokawain A via blockade of NF-κB and AP-1 activation in RAW 264.7 macrophages

Flavokawain A, a major constituent of chalcones derived from kava extracts, exerts various biological activities such as anti-tumor activities. In this study, we examined the suppressive effect of flavokawain A on LPS-induced expression of pro-inflammatory mediators and the molecular mechanisms responsible for these activities in the murine macrophages. Flavokawain A significantly suppressed expression of iNOS and COX-2, as well as the subsequent production of NO and PGE2 in the LPS-stimulated RAW 264.7 cells. Flavokawain A significantly inhibited LPS-induced activation of NF-κB and AP-1 signaling pathways. In addition, flavokawain A inhibited activation of JNK and p38 MAPK which was responsible for expression of iNOS and COX-2 in the LPS-stimulated RAW 264.7 cells. Furthermore, flavokawain A suppressed LPS-induced expression of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6. These results suggest that flavokawain A may exert anti-inflammatory responses by suppressing LPS-induced expression of pro-inflammatory mediators via blockage of NF-κB-AP-1-JNK/p38 MAPK signaling pathways in the murine macrophages.

Water extract of Cynanchi atrati Radix regulates inflammation and apoptotic cell death through suppression of IKK-mediated NF-κB signaling

Cynanchi atrati Radix has been traditionally used as an anti-inflammatory agent to treat febrile diseases, acute urinary infection or subcutaneous pyogenic infection with invasion of the pathogenic factors. Nuclear factor (NF)-κB is a pleiotropic transcriptional factor of many genes involved in inflammatory and anti-apoptotic responses. To identify a novel, potent inhibitor of NF-κB signaling pathway, a plant extract library of traditional oriental medicine was screened for the capability to block the NF-κB activity in cells overexpressing toll-like receptor 4 (TLR4), and then evaluated the anti-inflammatory and pro-apoptotic functions of water extract of Cynanchi atrati Radix (WECR) in macrophages and cancer cells, respectively. The effect of WECR on the proinflammatory mediators (inducible NO synthase [iNOS], cyclooxygenase [COX]-2), IκB-α degradation, RelA/p65 phosphorylation and caspase cleavages were measured by immunblotting. NF-κB transcriptional activity, IκB kinase (IKK) activity and nitric oxide (NO) production was measured using the luciferase assay, in vitro kinase assay and Griess reaction. WECR efficiently inhibited LPS-induced expression of proinflammatory mediators including iNOS and COX-2. IKK kinase activity, IκB-α degradation, nuclear translocation of RelA/p65 and NF-κB transcriptional activity induced by LPS were suppressed by WECR. Furthermore, WECR dramatically enhances the apoptotic response, as evident by the combination with tumor necrosis factor (TNF) was able to induce the cytotoxic action through caspase-dependent pathway. These results indicate that WECR has a potential to inhibit IKK-mediated NF-κB activation, and is a valuable compound for modulating inflammatory or cancerous conditions.

Fra-1 negatively regulates lipopolysaccharide-mediated inflammatory responses

Stimulation of macrophages with a Toll-like receptor ligand, LPS, facilitates gene expression. The activator protein-1 (AP-1) family of transcription factors mediates these responses. However, c-Fos, a member of the AP-1 family, has been shown to inhibit LPS-induced gene expression in macrophages. In this study, we analyzed the role of Fos-related antigen-1 (Fra-1), another member of the AP-1 family of transcription factors, in LPS-induced responses in RAW264.7 macrophages. Fra-1 was induced in LPS-stimulated macrophages with delayed time kinetics compared with c-Fos. Lentiviral introduction of Fra-1 blocked LPS-induced expression of pro-inflammatory mediators at the protein and mRNA levels. A Fra-1 mutant, which lacks the basic leucine zipper domain required for heterodimer formation and DNA binding, did not inhibit LPS-induced responses. c-Fos bound to the AP-1-binding site early, but afterward it was replaced by Fra-1 in LPS-stimulated macrophages. Over-expression of Fra-1 induced its association with Jun proteins and stable DNA binding from an early time point following LPS stimulation. These findings indicate that Fra-1 suppresses LPS-induced mRNA expression by binding to the AP-1-binding site. RNAi-mediated knockdown of Fra-1 in RAW264.7 macrophages resulted in enhanced LPS-induced expression of a subset of genes. Thus, like c-Fos, Fra-1 negatively regulates LPS-induced responses in RAW264.7 macrophages.

Monocyte deactivation by interleukin 10 via inhibition of tyrosine kinase activity and the Ras signaling pathway.

Activation of monocytes by bacterial lipopolysaccharides (LPSs) is a central component in the pathogenesis of septic shock syndrome. Interleukin 10 (IL-10) is a potent monocyte-deactivating factor and transcriptionally inhibits LPS-induced expression of proinflammatory mediators. The intracellular signaling pathways of LPS have been only partially characterized and mechanisms of IL-10 signaling remain unknown. We show that LPS activates the protein tyrosine kinase (PTK) p56lyn and that this is associated with tyrosine phosphorylation of the protooncogene product Vav. These events are completely blocked by the tyrosine kinase inhibitor herbimycin A. LPS also increases Ras activation in monocytes. LPS-triggered phosphorylation of mitogen-activated protein kinase is a downstream activation event that is also reduced by herbimycin A. Analysis of the IL-10 effects shows that it completely inhibits the p56lyn tyrosine kinase activation and all other subsequent events in this pathway including Ras activation. The IL-10 effects are selective since it reduced PTK-dependent cytokine mRNA expression but not the PTK independent induction of c-jun and c-fos mRNA in LPS-activated monocytes. These results identify the Ras signaling pathway as a component of intracellular signaling in LPS-stimulated monocytes and define early events in this response as targets of monocyte deactivation by IL-10.