Compound FLZ inhibits lipopolysaccharide-induced inflammatory effects via down-regulation of the TAK-IKK and TAK-JNK/p38MAPK pathways in RAW264.7 macrophages

Aim:

The aim of this study was to investigate the effect of the squamosamide derivative FLZ (N-2-(4-hydroxy-phenyl)-ethyl-2-(2,5-dimethoxy-phenyl)-3-(3-methoxy-4-hydroxy-phenyl)-acrylamide) on lipopolysaccharide (LPS)-induced inflammatory mediator production and the underlying mechanism in RAW264.7 macrophages.

Methods:

RAW264.7 cells were preincubated with non-toxic concentrations of compound FLZ (1, 5, and 10 μmol/L) for 30 min and then stimulated with 10 μg/L LPS. The production of nitric oxide (NO), the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), and the activation of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways were examined.

Results:

FLZ significantly inhibited the LPS-induced production of NO, as well as the expression of iNOS and COX-2 at both the RNA and the protein levels in RAW264.7 cells. The LPS-induced increase in the DNA binding activity of NF-κB and activator protein 1 (AP-1), the nuclear translocation of NF-κB p65, the degradation of the inhibitory κBα protein (IκBα) and the phosphorylation of IκBα, IκB kinase (IKK) α/β, c-Jun NH₂-terminal kinase (JNK) and p38 MAPKs were all suppressed by FLZ. However, the phosphorylation of extracellular signal-regulated kinase (ERK) was not affected. Further study revealed that FLZ inhibited the phosphorylation of transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1), which is an upstream signaling molecule required for IKKα/β, JNK and p38 activation.

Conclusion:

FLZ inhibited the LPS-induced production of inflammatory mediators at least partly through the downregulation of the TAK-IKK and TAK-JNK/p38MAPK pathways.     

Curcumin inhibits LPS-induced inflammation in rat vascular smooth muscle cells in vitro via ROS-relative TLR4-MAPK/NF-κB pathways

Aim:

To investigate whether curcumin (Cur) suppressed lipopolysaccharide (LPS)-induced inflammation in vascular smooth muscle cells (VSMCs) of rats, and to determine its molecular mechanisms.

Methods:

Primary rat VSMCs were treated with LPS (1 μg/L) and Cur (5, 10, or 30 μmol/L) for 24 h. The levels of MCP-1, TNF-α, and iNOS were measured using ELISA and real-time RT-PCR. NO level was analyzed with the Griess reaction. Western-blotting was used to detect the activation of TLR4, MAPKs, IκBα, NF-κB p65, and the p47^phox subunit of NADPH oxidase in the cells.

Results:

Treatment of VSMCs with LPS dramatically increased expression of inflammatory cytokines MCP-1 and TNF-α, expression of TLR4 and iNOS, and NO production. LPS also significantly increased phosphorylation of IκBα, nuclear translocation of NF-κB (p65) and phosphorylation of MAPKs in VSMCs. Furthermore, LPS significantly increased production of intracellular ROS, and decreased expression of p47^phox subunit of NADPH oxidase. Pretreatment with Cur concentration-dependently attenuated all the aberrant changes in LPS-treated VSMCs. The LPS-induced overexpression of MCP-1 and TNF-α, and NO production were attenuated by pretreatment with the ERK inhibitor PD98059, the p38 MAPK inhibitor SB203580, the NF-κB inhibitor PDTC or anti-TLR4 antibody, but not with the JNK inhibitor SP600125.

Conclusion:

Cur suppresses LPS-induced overexpression of inflammatory mediators in VSMCs in vitro via inhibiting the TLR4-MAPK/NF-κB pathways, partly due to block of NADPH-mediated intracellular ROS production.     

Anti-Inflammatory Effect of Mangostenone F in Lipopolysaccharide-Stimulated RAW264.7 Macrophages by Suppressing NF-κB and MAPK Activation

Mangostenone F (MF) is a natural xanthone isolated from Garcinia mangostana. However, little is known about the biological activities of MF. This study was designed to investigate the anti-inflammatory effect and underlying molecular mechanisms of MF in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. MF dose-dependently inhibited the production of NO, iNOS, and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in LPS-stimulated RAW264.7 macrophages. Moreover, MF decreased the NF-κB luciferase activity and NF-κB DNA binding capacity in LPS-stimulated RAW264.7 macrophages. Furthermore, MF suppressed the NF-κB activation by inhibiting the degradation of IκBα and nuclear translocation of p65 subunit of NF-κB. In addition, MF attenuated the AP-1 luciferase activity and phosphorylation of ERK, JNK, and p38 MAP kinases. Taken together, these results suggest that the anti-inflammatory effect of MF is associated with the suppression of NO production and iNOS expression through the down-regulation of NF-κB activation and MAPK signaling pathway in LPS-stimulated RAW264.7 macrophages.     

WMJ–S–001, a novel aliphatic hydroxamate derivative,exhibits anti-inflammatory properties via MKP-1 in LPS-stimulated RAW264.7 macrophages

Background and Purpose

Hydroxamate derivatives have attracted considerable attention because of their broad pharmacological properties. Recent studies reported their potential use in the treatment of cardiovascular diseases, arthritis and infectious diseases. However, the mechanisms of the anti-inflammatory effects of hydroxamate derivatives remain to be elucidated. In an effort to develop a novel pharmacological agent that could suppress abnormally activated macrophages, we investigated a novel aliphatic hydroxamate derivative, WMJ-S-001, and explored its anti-inflammatory mechanisms.

Experimental Approach

RAW264.7 macrophages were exposed to LPS in the absence or presence of WMJ-S-001. COX-2 expression and signalling molecules activated by LPS were assessed.

Key Results

LPS-induced COX-2 expression was suppressed by WMJ-S-001. WMJ-S-001 inhibited p38MAPK, NF-κB subunit p65 and CCAAT/enhancer-binding protein (C/EBP)β phosphorylation in cells exposed to LPS. Treatment of cells with a p38MAPK inhibitor (p38MAPK inhibitor III) markedly inhibited LPS-induced p65 and C/EBPβ phosphorylation and COX-2 expression. LPS-increased p65 and C/EBPβ binding to the COX-2 promoter region was suppressed in the presence of WMJ-S-001. In addition, WMJ-S-001 suppression of p38MAPK, p65 and C/EBPβ phosphorylation, and subsequent COX-2 expression were restored in cells transfected with a dominant-negative (DN) mutant of MAPK phosphatase-1 (MKP-1). WMJ-S-001 also caused an increase in MKP-1 activity in RAW264.7 macrophages.

Conclusions and Implications

WMJ-S-001 may activate MKP-1, which then dephosphorylates p38MAPK, resulting in a decrease in p65 and C/EBPβ binding to the COX-2 promoter region and COX-2 down-regulation in LPS-stimulated RAW264.7 macrophages. The present study suggests that WMJ-S-001 may be a potential drug candidate for alleviating LPS-associated inflammatory diseases.     

Withaferin A inhibits iNOS expression and nitric oxide production by Akt inactivation and down-regulating LPS-induced activity of NF-kappaB in RAW 264.7 cells

Induction of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production is thought to have beneficial immunomodulatory effects in acute and chronic inflammatory disorders. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, withaferin A inhibited LPS-induced expression of both iNOS protein and mRNA in a dose-dependent manner. To investigate the mechanism by which withaferin A inhibits iNOS gene expression, we examined activation of mitogen-activated protein kinases (MAPKs) and Akt in Raw 264.7 cells. We did not observe any significant changes in the phosphorylation of p38 MAPK in cells treated with LPS alone or LPS plus withaferin A. However, LPS-induced Akt phosphorylation was markedly inhibited by withaferin A, while the phosphorylation of p42/p44 extracellular signal-regulated kinases (ERKs) was slightly inhibited by withaferin A treatment. Withaferin A prevented IkappaB phosphorylation, blocking the subsequent nuclear translocation of nuclear factor-kappaB (NF-kappaB) and inhibiting its DNA binding activity. LPS-induced p65 phosphorylation, which is mediated by extracellular signal-regulated kinase (ERK) and Akt pathways, was attenuated by withaferin A treatment. Moreover, LPS-induced NO production and NF-kappaB activation were inhibited by SH-6, a specific inhibitor of Akt. Taken together, these results suggest that withaferin A inhibits inflammation through inhibition of NO production and iNOS expression, at least in part, by blocking Akt and subsequently down-regulating NF-kappaB activity.     

Neocryptotanshinone inhibits lipopolysaccharide-induced inflammation in RAW264.7 macrophages by suppression of NF-κB and iNOS signaling pathways

Neocryptotanshinone (NCTS) is a natural product isolated from traditional Chinese herb Salvia miltiorrhiza Bunge. In this study, we investigated its anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW264.7) cells. MTT results showed that NCTS partly reversed LPS-induced cytotoxicity. Real-time PCR results showed that NCTS suppressed LPS-induced mRNA expression of inflammatory cytokines, including tumor necrosis factor α (TNFα), interleukin-6 (IL-6) and interleukin-1β (IL-1β). Moreover, NCTS could decrease LPS-induced nitric oxide (NO) production. Western blotting results showed that NCTS could down-regulate LPS-induced expression of inducible nitric oxide synthase (iNOS), p-IκBα, p-IKKβ and p-NF-κB p65 without affecting cyclooxygenase-2 (COX-2). In addition, NCTS inhibited LPS-induced p-NF-κB p65 nuclear translocation. In conclusion, these data demonstrated that NCTS showed anti-inflammatory effect by suppression of NF-κB and iNOS signaling pathways.KEY WORDS: Neocryptotanshinone, Inflammation, NF-κB, Inducible nitric oxide synthase     

The Pore-Forming α-Toxin from Clostridium septicum Activates the MAPK Pathway in a Ras-c-Raf-Dependent and Independent Manner

Clostridium septicum is the causative agent of atraumatic gas gangrene, with α-toxin, an extracellular pore-forming toxin, essential for disease. How C. septicum modulates the host’s innate immune response is poorly defined, although α-toxin-intoxicated muscle cells undergo cellular oncosis, characterised by mitochondrial dysfunction and release of reactive oxygen species. Nonetheless, the signalling events that occur prior to the initiation of oncosis are poorly characterised. Our aims were to characterise the ability of α-toxin to activate the host mitogen activated protein kinase (MAPK) signalling pathway both in vitro and in vivo. Treatment of Vero cells with purified α-toxin activated the extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 arms of the MAPK pathway and stimulated the release of TNF-α in a dose-dependent manner. Studies using inhibitors of all three MAPK components suggested that activation of ERK occurred in a Ras-c-Raf dependent manner, whereas activation of JNK and p38 occurred by a Ras-independent mechanism. Toxin-mediated activation was dependent on efficient receptor binding and pore formation and on an influx of extracellular calcium ions. In the mouse myonecrosis model we showed that the MAPK pathway was activated in tissues of infected mice, implying that it has an important role in the disease process.     

Kaempferol acts through mitogen-activated protein kinases and protein kinase B/AKT to elicit protection in a model of neuroinflammation in BV2 microglial cells

BACKGROUND AND PURPOSE

Kaempferol, a dietary flavonoid and phyto-oestrogen, is known to have anti-inflammatory properties. Microglial activation has been implicated in various neurodegenerative diseases. Anti-inflammatory effects of kaempferol and the underlying mechanisms were investigated by using LPS-stimulated microglial BV2 cells.

EXPERIMENTAL APPROACH

Cell viability was measured using MTT and neutral red assays. elisa, Western blot, immunocytochemistry and electrophoretic mobility-shift assay were used to analyse NO, PGE₂, TNF-α and IL-1β production, inducible NOS (iNOS), COX-2 expression and the involvement of signalling pathways such as toll-like receptor-4 (TLR4), MAPK cascades, PKB (AKT) and NF-κB. Accumulation of reaction oxygen species (ROS) was measured by nitroblue tetrazolium and 2′7′-dichlorofluorescein diacetate assay. Matrix metalloproteinase activity was investigated by zymography and immunoblot assay. Phagocytotic activity was assessed by use of latex beads.

KEY RESULTS

Kaempferol significantly attenuated LPS-induced NO, PGE₂, TNF-α, IL-1β and ROS production and phagocytosis in a concentration-dependent manner. Kaempferol suppressed the expression of iNOS, COX-2, MMP-3 and blocked the TLR4 activation. Moreover, kaempferol inhibited LPS-induced NF-κB activation and p38 MAPK, JNK and AKT phosphorylation.

CONCLUSION AND IMPLICATIONS

Kaempferol was able to reduce LPS-induced inflammatory mediators through the down-regulation of TLR4, NF-κB, p38 MAPK, JNK and AKT suggesting that kaempferol has therapeutic potential for the treatment of neuroinflammatory diseases.     

Vitisin A suppresses LPS-induced NO production by inhibiting ERK,p38, and NF-κB activation in RAW 264.7 cells

Vitisin A, a resveratrol tetramer isolated from Vitis vinifera roots, exhibits antioxidative, anticancer, antiapoptotic, and anti-inflammatory effects. It also inhibits nitric oxide (NO) production. Here, we examined the mechanism by which vitisin A inhibits NO production in lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells. Vitisin A dose dependently inhibited LPS-induced NO production and inducible NO synthase (iNOS) expression. In contrast, the production of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) was not altered by vitisin A. To investigate the signaling pathway for NO inhibition by vitisin A, we examined nuclear factor-κB (NF-κB) activation in the mitogen-activated protein kinase (MAPK) pathway, an inflammation-induced signal pathway in RAW 264.7 cells. Vitisin A inhibited LPS-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 phosphorylation and suppressed LPS-induced NF-κB activation in RAW 264.7 cells. This suggests that vitisin A decreased NO production via downregulation of ERK1/2 and p38 and the NF-κB signal pathway in RAW 264.7 cells.     

Inhibition of lipopolysaccharide-induced expression of inducible nitric oxide synthase by phenolic (3E)-4-(2-hydroxyphenyl)but-3-en-2-one in RAW 264.7 macrophages

The large amount of nitric oxide (NO) produced by inducible NO synthase (iNOS) contributes to cellular injury in inflammatory disease. In the present study, a novel synthetic compound (3E)-4-(2-hydroxyphenyl)but-3-en-2-one (HPB) was found to inhibit lipopolysaccharide (LPS)-induced NO generation, but not through the inhibition of iNOS activity, in RAW 264.7 macrophages. Administration of HPB into mice also inhibited the LPS-induced increase in serum nitrite/nitrate levels. To evaluate the underlying mechanisms of HPB inhibition of NO generation, the expression of the iNOS gene in RAW 264.7 macrophages was examined. HPB abolished the LPS-induced expression of iNOS protein, iNOS mRNA and iNOS promoter activity in a similar concentration-dependent manner. LPS-induced nuclear factor-κB (NF-κB) DNA binding and NF-κB-dependent reporter gene activity were both significantly inhibited by HPB. This effect was mediated through the inhibition of inhibitory factor-κBα (IκBα) phosphorylation and degradation, and of p65 nuclear translocation. HPB had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinases (MAPK), and c-Jun NH₂-terminal kinase (JNK). However, HPB suppressed the LPS-induced intracellular reactive oxygen species (ROS) production. These results indicate that HPB down-regulates iNOS gene expression probably through the inhibition of LPS-induced intracellular ROS production, which has been implicated in the activation of NF-κB.     

Nafamostat Mesilate Inhibits TNF-α-Induced Vascular Endothelial Cell Dysfunction by Inhibiting Reactive Oxygen Species Production

Nafamostat mesilate (NM) is a serine protease inhibitor with anticoagulant and anti-inflammatory effects. NM has been used in Asia for anticoagulation during extracorporeal circulation in patients undergoing continuous renal replacement therapy and extra corporeal membrane oxygenation. Oxidative stress is an independent risk factor for atherosclerotic vascular disease and is associated with vascular endothelial function. We investigated whether NM could inhibit endothelial dysfunction induced by tumor necrosis factor-α (TNF-α). Human umbilical vein endothelial cells (HUVECs) were treated with TNF-α for 24 h. The effects of NM on monocyte adhesion, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) protein expression, p38 mitogen-activated protein kinase (MAPK) activation, and intracellular superoxide production were then examined. NM (0.01~100 µg/mL) did not affect HUVEC viability; however, it inhibited the increases in reactive oxygen species (ROS) production and p66shc expression elicited by TNF-α (3 ng/mL), and it dose dependently prevented the TNF-α-induced upregulation of endothelial VCAM-1 and ICAM-1. In addition, it mitigated TNF-α-induced p38 MAPK phosphorylation and the adhesion of U937 monocytes. These data suggest that NM mitigates TNF-α-induced monocyte adhesion and the expression of endothelial cell adhesion molecules, and that the anti-adhesive effect of NM is mediated through the inhibition of p66shc, ROS production, and p38 MAPK activation.     

Signalling pathways regulating inducible nitric oxide synthase expression in human kidney epithelial cells

The purpose of this study was to elucidate the signalling pathways involved in the cytokine-activated inducible nitric oxide synthase (iNOS) response in a human kidney epithelial cell line, A498. Unstimulated cells did not express iNOS. Exposure of A498 cells to a cytokine mixture consisting of interferon gamma, interleukin-1 beta and tumor necrosis factor-alpha (TNF-alpha) increased nitrite production, iNOS mRNA and protein expression. Pharmacological inhibition of tyrosine kinases, including janus kinase (JAK2), and protein kinase C (PKC) inhibited cytokine-mediated nitrite production and iNOS protein expression. The involvement of mitogen-activated protein kinases (MAPKs) was investigated. Inhibition of p38 MAPK, but not of an upstream activator of extracellular signal-regulated kinase (ERK), caused a decrease in iNOS expression and nitrite production in response to cytokines. Electrophoretic mobility shift assay of nuclear extract from cytokine-stimulated cells demonstrated a pronounced binding to a nuclear factor kappa B (NF-kappa B) sequence present in the human iNOS promoter. Furthermore, the NF-kappa B inhibitor pyrrolidinedithiocarbamate (PDTC) decreased cytokine-activated iNOS protein expression and nitrite production. The present study has demonstrated that cytokine-stimulated iNOS expression in human kidney epithelial cells involves activation of tyrosine kinases, including JAK2, PKC, p38 MAPK and NF-kappa B.     

p38 but not p44/42 mitogen-activated protein kinase is required for nitric oxide synthase induction mediated by lipopolysaccharide in RAW 264.7 macrophages

Protein kinase C (PKC)-alpha, -betaI, and -delta are known to be involved in the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages. The role of mitogen-activated protein kinases (MAPK) p44/42 and p38 in the LPS effect was studied further. LPS-mediated NO release and the inducible form of NO synthase expression were inhibited by the p38 inhibitor, SB 203580, but not by the MAPK kinase inhibitor, PD 98059. Ten-minute treatment of cells with LPS resulted in the activation of p44/42 MAPK, p38, and c-Jun NH2-terminal kinase. Marked or slight activation, respectively, of p44/42 MAPK or p38 was also seen after 10-min treatment with 12-O-tetradecanoylphorbol-13-acetate, but c-Jun NH2-terminal kinase activation did not occur. Tyrosine kinase inhibitor, genestein, attenuated the LPS-induced activation of both p44/42 MAPK and p38, whereas the PKC inhibitors, Ro 31-8220 and calphostin C, or long-term treatment with 12-O-tetradecanoylphorbol-13-acetate resulted in inhibition of p44/42 MAPK activation, but had only a slight effect on p38 activation, indicating that LPS-mediated PKC activation resulted in the activation of p44/42 MAPK. Nuclear factor-kappaB (NF-kappaB)-specific DNA-protein-binding activity in the nuclear extracts was enhanced by 10-min, 1-h, or 24-h treatment with LPS. Analysis of the proteins involved in NF-kappaB binding showed translocation of p65 from the cytosol to the nucleus after 10-min treatment with LPS. The onset of NF-kappaB activation correlated with the cytosolic degradation of both inhibitory proteins of NF-kappaB, IkappaB-alpha and IkappaB-beta. IkappaB-alpha was resynthesized rapidly after loss (1-h LPS treatment), whereas IkappaB-beta levels were not restored until after 24-h treatment. SB 203580 but not PD 98059 inhibited the LPS-induced stimulation of NF-kappaB DNA-protein binding. Thus, activation of p38 but not p44/42 MAPK by LPS resulted in the stimulation of NF-kappaB-specific DNA-protein binding and the subsequent expression of inducible form of NO synthase and NO release in RAW 264.7 macrophages.     

N-(p-Coumaryol)-Tryptamine Suppresses the Activation of JNK/c-Jun Signaling Pathway in LPS-Challenged RAW264.7 Cells

N-(p-Coumaryol) tryptamine (CT), a phenolic amide, has been reported to exhibit anti-oxidant and anti-inflammatory activities. However, the underlying mechanism by which CT exerts its pharmacological properties has not been clearly demonstrated. The objective of this study is to elucidate the anti-inflammatory mechanism of CT in lipopolysaccharide (LPS)-challenged RAW264.7 macrophage cells. CT significantly inhibited LPS-induced extracellular secretion of pro-inflammatory mediators such as nitric oxide (NO) and PGE₂, and protein expressions of iNOS and COX-2. In addition, CT significantly suppressed LPS-induced secretion of pro-inflammatory cytokines such as TNF-α and IL-1β. To elucidate the underlying anti-inflammatory mechanism of CT, involvement of MAPK and Akt signaling pathways was examined. CT significantly attenuated LPS-induced activation of JNK/c-Jun, but not ERK and p38, in a concentration-dependent manner. Interestingly, CT appeared to suppress LPS-induced Akt phosphorylation. However, JNK inhibition, but not Akt inhibition, resulted in the suppression of LPS-induced responses, suggesting that JNK/c-Jun signaling pathway significantly contributes to LPS-induced inflammatory responses and that LPS-induced Akt phosphorylation might be a compensatory response to a stress condition. Taken together, the present study clearly demonstrates CT exerts anti-inflammatory activity through the suppression of JNK/c-Jun signaling pathway in LPS-challenged RAW264.7 macrophage cells.