Mya Mya Mu

The Inhibitory Action of Sodium Arsenite on Lipopolysaccharide-Induced Nitric Oxide Production in RAW 267.4 Macrophage Cells: A Role of Raf-1 in Lipopolysaccharide Signaling

The effect of sodium arsenite (SA) on LPS-induced NO production in RAW 267.4 murine macrophage cells was studied. SA pretreatment of LPS-stimulated RAW cells resulted in a striking reduction in NO production. No significant difference in LPS binding was observed between RAW cells pretreated with SA and control untreated RAW cells, suggesting that SA might impair the intracellular signal pathway for NO production. SA inhibited LPS-induced NF-κB activation by preventing loss of IκB-α and -β. Furthermore, SA blocked phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2), but not phosphorylation of p38 and c-Jun N-terminal kinase. SA treatment resulted in the disappearance of Raf-1, suggesting that it might cause the inhibition of the Erk1/2 mitogen-activated protein (MAP) kinase pathway. The SA-mediated loss of Raf-1 also abolished LPS-induced NF-κB activation as well as the Erk1/2 pathway. The dominant negative mutant of MAP kinase kinase 1 inhibited both NO production and NF-κB activation in LPS-stimulated RAW cells. Taken together, these results indicate that the inhibitory action of SA on NO production in LPS-stimulated macrophages might be due to abrogation of inducible NO synthase induction, and it might be closely related to inactivation of the NF-κB and Erk1/2 MAP kinase pathways through loss of Raf-1.

The inhibitory action of quercetin on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells

The effect of quercetin on lipopolysaccharide (LPS)-induced nitric oxide (NO) production was studied. Quercetin pretreatment significantly inhibited NO production in an LPS-stimulated RAW 264.7 murine macrophage cell line. Post-treatment with quercetin partially inhibited NO production. The inhibitory action of quercetin was due to neither the cytotoxic action nor altered LPS binding. The expression of inducible-type NO synthase (iNOS) was markedly down-regulated by quercetin. Quercetin suppressed the release of free nuclear factor (NF)-κB by preventing degradation of IκB-α and IκB-β. Moreover, quercetin blocked the phosphorylation of extracellular signal regulated kinase 1/2 (Erk1/2), p38, and c-Jun NH 2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and, further, the activity of tyrosine kinases in LPS-stimulated RAW cells. Quercetin also inhibited interferon (IFN)-γ-induced NO production. Taken together, these results indicate that the inhibitory action of quercetin on NO production in LPS- and/or IFN-γ-stimulated macrophages might be due to abrogation of iNOS protein induction by impairment of a series of intracellular signal pathways.

The inhibitory action of butyrate on lipopolysaccharide-induced nitric oxide production in RAW 264.7 murine macrophage cells

The effect of butyrate, a natural bacterial product of colonic bacterial flora, on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 murine macrophage cells was studied. Butyrate significantly reduced NO production in LPS-stimulated RAW cells. The inhibition was abolished by the removal of butyrate. Butyrate also inhibited the expression of inducible type NO synthase (iNOS) in LPS-stimulated RAW cells. Furthermore, butyrate prevented the activation of nuclear factor (NF)-κB through the stabilization of IκB-α and IκB-β. Butyrate did not affect the phosphorylation of mitogen-activated protein (MAP) kinases by LPS. It was, therefore, suggested that butyrate down-regulated LPS-induced NO production in RAW cells through preventing the expression of iNOS, and that it was due to the inhibitory action of butyrate on the activation of NF-κB.

Piceatannol Prevents Lipopolysaccharide (LPS)-Induced Nitric Oxide (NO) Production and Nuclear Factor (NF)-κB Activation by Inhibiting IκB Kinase (IKK)

The effect of piceatannol on lipopolysaccharide (LPS)‐induced nitric oxide (NO) production was examined. Piceatannol significantly inhibited NO production in LPS‐stimulated RAW 264.7 cells. The inhibition was due to the reduced expression of an inducible isoform of NO synthase (iNOS). The inhibitory effect of piceatannol was mediated by down‐regulation of LPS‐induced nuclear factor (NF)‐κB activation, but not by its cytotoxic action. Piceatannol inhibited IκB kinase (IKK)‐α and β phosphorylation, and subsequently IKB‐α phosphorylation in LPS‐stimulated RAW 264.7 cells. On the other hand, piceatannol did not affect activation of mitogen‐activated protein (MAP) kinases including extracellular signal regulated kinase 1/2 (Erk1/2), p38 and stress‐activated protein kinase/c‐Jun NH2‐terminal kinase (SAPK/JNK). Piceatannol inhibited the phosphorylation of Akt and Raf‐1 molecules, which regulated the activation of IKK‐α and β phosphorylation. The detailed mechanism of the inhibition of LPS‐induced NO production by piceatannol is discussed.

Inhibition of Caspase 3 Abrogates Lipopolysaccharide-Induced Nitric Oxide Production by Preventing Activation of NF-κB and c-Jun NH2-Terminal Kinase/Stress-Activated Protein Kinase in RAW 264.7 Murine Macrophage Cells

ABSTRACT The effect of caspase inhibitors on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 267.4 murine macrophage cells was investigated. Pretreatment of RAW cells with a broad caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), resulted in a striking reduction in LPS-induced NO production. Z-VAD-FMK inhibited LPS-induced NF-κB activation. Furthermore, it blocked phosphorylation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) but not that of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases. Similarly, a caspase 3-specific inhibitor, Z-Asp-Glu-Val-Asp-fluoromethylketone, inhibited NO production, NF-κB activation, and JNK/SAPK phosphorylation in LPS-stimulated RAW cells. The attenuated NO production was due to inhibition of the expression of an inducible-type NO synthase (iNOS). The overexpression of the dominant negative mutant of JNK/SAPK and the addition of a JNK/SAPK inhibitor blocked iNOS expression but did not block LPS-induced caspase 3 activation. It was therefore suggested that the inhibition of caspase 3 might abrogate LPS-induced NO production by preventing the activation of NF-κB and JNK/SAPK. The caspase family, especially caspase 3, is likely to play an important role in the signal transduction for iNOS-mediated NO production in LPS-stimulated mouse macrophages.

Lipopolysaccharide Prevents Doxorubicin-Induced Apoptosis in RAW 264.7 Macrophage Cells by Inhibiting p53 Activation

The effect of lipopolysaccharide on doxorubicin-induced cell death was studied by using mouse RAW 264.7 macrophage cells. Pretreatment with lipopolysaccharide at 10 ng/mL prevented doxorubicin-induced cell death and the inhibition was roughly dependent on the concentration of lipopolysaccharide. Posttreatment with lipopolysaccharide for 1 hour also prevented doxorubicin-induced cell death. Lipopolysaccharide inhibited DNA fragmentation and caspase-3 activation in doxorubicin-treated RAW 264.7 cells, suggesting the prevention of doxorubicin-induced apoptosis. Lipopolysaccharide did not significantly inhibit doxorubicin-induced DNA damage detected by single-cell gel electrophoresis (comet) assay. Lipopolysaccharide definitely inhibited the stabilization and nuclear translocation of p53 in doxorubicin-treated RAW 264.7 cells. Lipopolysaccharide, as well as being an inhibitor of p53, abolished doxorubicin-induced apoptosis. Therefore, p53 was suggested to play a pivotal role in the prevention of doxorubicin-induced apoptosis in RAW 264.7 cells by lipopolysaccharide.

Protective Effect of Wogonin on Endotoxin‐Induced Lethal Shock in D‐Galactosamine‐Sensitized Mice

The effects of wogonin, a major flavonoid from Scutellaria baicalensis Georgi, on lipopolysaccharide (LPS)‐induced lethal shock in mice was investigated. Wogonin pretreatment prevented the lethal shock in mice injected with D‐galactosamine (D‐GalN) and LPS, but not in mice injected with a high dose of LPS. Wogonin definitely inhibited the hepatic injury in mice injected with D‐GalN, and LPS and reduced the level of circulating tumor necrosis factor (TNF)‐α. The reduction was more marked in mice injected with D‐GalN and LPS compared with that in mice injected with a high dose of LPS. Wogonin pretreatment did not inhibit the lipid peroxidation in mice receiving either D‐GalN and LPS or a high dose of LPS. Wogonin inhibited the in vitro production of TNF‐α and nitric oxide in LPS‐stimulated RAW 264.7 cells. The mechanism of the protective effect of wogonin on the lethal shock in mice injected with D‐GalN and LPS is discussed.

C 2-ceramide inhibits LPS-induced nitric oxide production in RAW 264.7 macrophage cells through down-regulating the activation of Akt

The effect of C2-ceramide, a membrane-permeable ceramide analogue, on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was studied. The non-toxic concentration of C2-ceramide inhibited LPS-induced NO production. It was due to the attenuated expression of the inducible type of NO synthase (iNOS). C2-ceramide did not influence the phosphorylation of a series of mitogen-activated protein (MAP) kinases in response to LPS. On the other hand, C2-ceramide down-regulated the phosphorylation of Akt in LPS-stimulated RAW 264.7 cells, followed by the impairment of nuclear factor (NF)-κB activation. Moreover, the Akt dominant-negative mutant inhibited LPS-induced NO production. C2-ceramide was suggested to inhibit LPS-induced NO production through down-regulating the activation of Akt.

Gamma interferon-induced nitric oxide production in mouse CD5+ B1-like cell line and its association with apoptotic cell death

The in vitro effect of gamma interferon (IFN‐γ) on nitric oxide (NO) production in a mouse CD5+ B1‐like cell line, TH2.52, was studied. The TH2.52 cell line is the hybridoma line between mouse B lymphoma line and mouse splenic B cells and expresses a series of B1 markers. IFN‐γ induced a marked NO production in TH2.52 cells through the expression of an inducible type of NO synthase (iNOS). IFN‐γ‐induced NO production was triggered by the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) pathway since it was inhibited by AG490, a JAK2 inhibitor. The growth of TH2.52 cells significantly was inhibited in the presence of IFN‐γ. A significant number of cells underwent apoptotic cell death, accompanied by the DNA fragmentation, annexin V binding, and caspase 3 activation. N(G)‐monomethyl‐L‐arginine, an iNOS inhibitor, prevented IFN‐γ‐induced cell death. Therefore, IFN‐γ‐induced NO production was possible in causing cell death in TH2.52 cells. Further, IFN‐γ‐induced NO production and cell death significantly were prevented by interleukin‐4, a representative Th2 cytokine. The immunological significance of IFN‐γ‐induced NO production in a mouse B1‐like cell line is discussed.

Lipopolysaccharide enhances interferon-γ-induced nitric oxide (NO) production in murine vascular endothelial cells via augmentation of interferon regulatory factor-1 activation

Lipopolysaccharide (LPS) enhances the production of nitric oxide (NO) in interferon (IFN)-γstimulated vascular endothelial cells. We studied the mechanism by which LPS enhances IFN-γ-induced NO production by using the murine vascular endothelial cell line, END-D. LPS enhanced IFN-γinduced NO production via augmented expression of inducible type NO synthase (iNOS) mRNA. LPS significantly augmented the activation of interferon regulatory factor (IRF)-1 in IFN-γ-stimulated END-D cells, although it did not affect the activation of either MyD88-dependent nuclear factor (NF)-κB or MyD88-independent IRF-3. SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), prevented the nuclear translocation of IRF-1 in LPS and IFN-γ-stimulated END-D cells, and inhibited the iNOS expression and NO production in those cells. Therefore, it is proposed that LPS enhanced NO production in IFN-γ-stimulated END-D cells via augmenting p38 MAPKmediated IRF-1 activation.