Limits...
SM905, an artemisinin derivative, inhibited NO and pro-inflammatory cytokine production by suppressing MAPK and NF-kappaB pathways in RAW 264.7 macrophages.

Wang JX, Hou LF, Yang Y, Tang W, Li Y, Zuo JP - Acta Pharmacol. Sin. (2009)

Bottom Line: Pretreatment with SM905 (0, 0.1, 1, and 10 micromol/L) suppressed LPS-induced NO, TNF-alpha, IL-1beta, and IL-6 production, and decreased both protein and mRNA levels of iNOS and COX-2.The mRNA expression of LPS receptor Toll-like receptor 4 (TLR4) and myeloid differentiation protein-2 (MD-2) was not changed, while LPS-induced CD14 expression was slightly reduced after SM905 treatment.SM905 markedly decreased the activation of ERK1/2, p38 and JNK suppressed the degradation of IkappaBalpha, but did not modify the expression of interferon regulatory factor-1 (IRF-1), signal transducer and activator of transcription 1 (STAT1) or interferon-inducible protein-10 (IP-10).

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunopharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

ABSTRACT

Aim: To elucidate the anti-inflammatory potentials and underlying mechanisms of SM905, a novel artemisinin derivative, in lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells.

Methods: Nitric oxide (NO) generation, cytokine production, and the protein expression levels of inducible nitric-oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were examined using a Griess assay, an enzyme-linked immunosorbent assay (ELISA) and a Western blotting assay, respectively. The mRNA expression was measured using real-time PCR. The phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), p38, c-jun N-terminal kinase (JNK), and the degradation of IkappaBalpha were assessed by Western blotting analysis. The nuclear translocation of nuclear factor-kappaB (NF-kappaB) was observed using confocal microscopy.

Results: Pretreatment with SM905 (0, 0.1, 1, and 10 micromol/L) suppressed LPS-induced NO, TNF-alpha, IL-1beta, and IL-6 production, and decreased both protein and mRNA levels of iNOS and COX-2. The mRNA expression of LPS receptor Toll-like receptor 4 (TLR4) and myeloid differentiation protein-2 (MD-2) was not changed, while LPS-induced CD14 expression was slightly reduced after SM905 treatment. SM905 markedly decreased the activation of ERK1/2, p38 and JNK suppressed the degradation of IkappaBalpha, but did not modify the expression of interferon regulatory factor-1 (IRF-1), signal transducer and activator of transcription 1 (STAT1) or interferon-inducible protein-10 (IP-10). By using confocal microscopy, we further observed that NF-kappaB was correspondingly inhibited in SM905-treated cells.

Conclusion: SM905 inhibited NO and pro-inflammatory cytokine production in LPS-stimulated RAW 264.7 cells and these effects are at least partially mediated through suppression of the MAPK and NF-kappaB signaling pathways.

Show MeSH

Related in: MedlinePlus

Effect of SM905 on LPS-induced MAPK activation, IκBα degradation and expression of IRF-1, STAT1, and IP-10 mRNA. (A) SM905 inhibited activation of ERK, p38, and JNK MAPKs in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells (2×106cells/mL) were pretreated with the indicated concentrations of SM905 for 1 h followed by stimulation with LPS (1 μg/mL) for 15 min. Cells were lysed and assayed for phospho-ERK, phospho-p38, and phospho-JNK by Western blotting. The blots were probed with β-actin antibody to confirm equal protein levels. (B) SM905 inhibited IκBα degradation in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with SM905 for 1 h followed by stimulation with LPS (1 μg/mL) for 15 min. Cells were lysed and assayed for IκBα and β-actin by Western blotting. The results presented are from one experiment, which is representative of two others performed. (C) SM905 had no apparent effect on IRF-1, STAT1, and IP-10 expression. RAW 264.7 cells were pretreated with SM905 for 1 h followed by stimulation with LPS (1 μg/mL) for 3 h. Total RNA was isolated and analyzed for mRNA expression with real-time PCR. The relative gene expression was obtained after normalization with HPRT. Results were presented as mean±SEM, n=3.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4007329&req=5

fig4: Effect of SM905 on LPS-induced MAPK activation, IκBα degradation and expression of IRF-1, STAT1, and IP-10 mRNA. (A) SM905 inhibited activation of ERK, p38, and JNK MAPKs in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells (2×106cells/mL) were pretreated with the indicated concentrations of SM905 for 1 h followed by stimulation with LPS (1 μg/mL) for 15 min. Cells were lysed and assayed for phospho-ERK, phospho-p38, and phospho-JNK by Western blotting. The blots were probed with β-actin antibody to confirm equal protein levels. (B) SM905 inhibited IκBα degradation in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with SM905 for 1 h followed by stimulation with LPS (1 μg/mL) for 15 min. Cells were lysed and assayed for IκBα and β-actin by Western blotting. The results presented are from one experiment, which is representative of two others performed. (C) SM905 had no apparent effect on IRF-1, STAT1, and IP-10 expression. RAW 264.7 cells were pretreated with SM905 for 1 h followed by stimulation with LPS (1 μg/mL) for 3 h. Total RNA was isolated and analyzed for mRNA expression with real-time PCR. The relative gene expression was obtained after normalization with HPRT. Results were presented as mean±SEM, n=3.

Mentions: Next, the action of SM905 on intracellular signaling transduction pathways downstream of the LPS receptor was investigated. The MAPK pathway has been well recognized to regulate pro-inflammatory cytokine production13. We examined the phosphorylation of ERK, p38, and JNK MAPKs in RAW 264.7 cells after LPS stimulation for 15 min. The result showed that the levels of activated ERK, p38, and JNK MAPKs were very low in control cells without LPS stimulation, and LPS activated all three major MAPKs within 15 min. Treatment with SM905 exerted an apparent inhibitory effect on LPS-triggered phosphorylation of ERK, p38, and JNK MAPKs in RAW 264.7 cells (Figure 4A).


SM905, an artemisinin derivative, inhibited NO and pro-inflammatory cytokine production by suppressing MAPK and NF-kappaB pathways in RAW 264.7 macrophages.

Wang JX, Hou LF, Yang Y, Tang W, Li Y, Zuo JP - Acta Pharmacol. Sin. (2009)

Effect of SM905 on LPS-induced MAPK activation, IκBα degradation and expression of IRF-1, STAT1, and IP-10 mRNA. (A) SM905 inhibited activation of ERK, p38, and JNK MAPKs in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells (2×106cells/mL) were pretreated with the indicated concentrations of SM905 for 1 h followed by stimulation with LPS (1 μg/mL) for 15 min. Cells were lysed and assayed for phospho-ERK, phospho-p38, and phospho-JNK by Western blotting. The blots were probed with β-actin antibody to confirm equal protein levels. (B) SM905 inhibited IκBα degradation in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with SM905 for 1 h followed by stimulation with LPS (1 μg/mL) for 15 min. Cells were lysed and assayed for IκBα and β-actin by Western blotting. The results presented are from one experiment, which is representative of two others performed. (C) SM905 had no apparent effect on IRF-1, STAT1, and IP-10 expression. RAW 264.7 cells were pretreated with SM905 for 1 h followed by stimulation with LPS (1 μg/mL) for 3 h. Total RNA was isolated and analyzed for mRNA expression with real-time PCR. The relative gene expression was obtained after normalization with HPRT. Results were presented as mean±SEM, n=3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4007329&req=5

fig4: Effect of SM905 on LPS-induced MAPK activation, IκBα degradation and expression of IRF-1, STAT1, and IP-10 mRNA. (A) SM905 inhibited activation of ERK, p38, and JNK MAPKs in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells (2×106cells/mL) were pretreated with the indicated concentrations of SM905 for 1 h followed by stimulation with LPS (1 μg/mL) for 15 min. Cells were lysed and assayed for phospho-ERK, phospho-p38, and phospho-JNK by Western blotting. The blots were probed with β-actin antibody to confirm equal protein levels. (B) SM905 inhibited IκBα degradation in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with SM905 for 1 h followed by stimulation with LPS (1 μg/mL) for 15 min. Cells were lysed and assayed for IκBα and β-actin by Western blotting. The results presented are from one experiment, which is representative of two others performed. (C) SM905 had no apparent effect on IRF-1, STAT1, and IP-10 expression. RAW 264.7 cells were pretreated with SM905 for 1 h followed by stimulation with LPS (1 μg/mL) for 3 h. Total RNA was isolated and analyzed for mRNA expression with real-time PCR. The relative gene expression was obtained after normalization with HPRT. Results were presented as mean±SEM, n=3.
Mentions: Next, the action of SM905 on intracellular signaling transduction pathways downstream of the LPS receptor was investigated. The MAPK pathway has been well recognized to regulate pro-inflammatory cytokine production13. We examined the phosphorylation of ERK, p38, and JNK MAPKs in RAW 264.7 cells after LPS stimulation for 15 min. The result showed that the levels of activated ERK, p38, and JNK MAPKs were very low in control cells without LPS stimulation, and LPS activated all three major MAPKs within 15 min. Treatment with SM905 exerted an apparent inhibitory effect on LPS-triggered phosphorylation of ERK, p38, and JNK MAPKs in RAW 264.7 cells (Figure 4A).

Bottom Line: Pretreatment with SM905 (0, 0.1, 1, and 10 micromol/L) suppressed LPS-induced NO, TNF-alpha, IL-1beta, and IL-6 production, and decreased both protein and mRNA levels of iNOS and COX-2.The mRNA expression of LPS receptor Toll-like receptor 4 (TLR4) and myeloid differentiation protein-2 (MD-2) was not changed, while LPS-induced CD14 expression was slightly reduced after SM905 treatment.SM905 markedly decreased the activation of ERK1/2, p38 and JNK suppressed the degradation of IkappaBalpha, but did not modify the expression of interferon regulatory factor-1 (IRF-1), signal transducer and activator of transcription 1 (STAT1) or interferon-inducible protein-10 (IP-10).

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunopharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

ABSTRACT

Aim: To elucidate the anti-inflammatory potentials and underlying mechanisms of SM905, a novel artemisinin derivative, in lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells.

Methods: Nitric oxide (NO) generation, cytokine production, and the protein expression levels of inducible nitric-oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were examined using a Griess assay, an enzyme-linked immunosorbent assay (ELISA) and a Western blotting assay, respectively. The mRNA expression was measured using real-time PCR. The phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), p38, c-jun N-terminal kinase (JNK), and the degradation of IkappaBalpha were assessed by Western blotting analysis. The nuclear translocation of nuclear factor-kappaB (NF-kappaB) was observed using confocal microscopy.

Results: Pretreatment with SM905 (0, 0.1, 1, and 10 micromol/L) suppressed LPS-induced NO, TNF-alpha, IL-1beta, and IL-6 production, and decreased both protein and mRNA levels of iNOS and COX-2. The mRNA expression of LPS receptor Toll-like receptor 4 (TLR4) and myeloid differentiation protein-2 (MD-2) was not changed, while LPS-induced CD14 expression was slightly reduced after SM905 treatment. SM905 markedly decreased the activation of ERK1/2, p38 and JNK suppressed the degradation of IkappaBalpha, but did not modify the expression of interferon regulatory factor-1 (IRF-1), signal transducer and activator of transcription 1 (STAT1) or interferon-inducible protein-10 (IP-10). By using confocal microscopy, we further observed that NF-kappaB was correspondingly inhibited in SM905-treated cells.

Conclusion: SM905 inhibited NO and pro-inflammatory cytokine production in LPS-stimulated RAW 264.7 cells and these effects are at least partially mediated through suppression of the MAPK and NF-kappaB signaling pathways.

Show MeSH
Related in: MedlinePlus