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Aciculatin inhibits lipopolysaccharide-mediated inducible nitric oxide synthase and cyclooxygenase-2 expression via suppressing NF-κB and JNK/p38 MAPK activation pathways.

Hsieh IN, Chang AS, Teng CM, Chen CC, Yang CR - J. Biomed. Sci. (2011)

Bottom Line: Aciculatin remarkably decreased the LPS (1 μg/mL)-induced mRNA and protein expression of iNOS and COX-2 as well as their downstream products, NO and PGE2 respectively, in a concentration-dependent manner (1-10 μM).Such inhibition was found, via immunoblot analyses, reporter gene assays, and confocal microscope observations that aciculatin not only acts through significant suppression of LPS-induced NF-κB activation, an effect highly correlated with its inhibitory effect on LPS-induced IκB kinase (IKK) activation, IκB degradation, NF-κB phosphorylation, nuclear translocation and binding of NF-κB to the κB motif of the iNOS and COX-2 promoters, but also suppressed phosphorylation of JNK/p38 mitogen-activated protein kinases (MAPKs).Our results demonstrated that aciculatin exerts potent anti-inflammatory activity through its dual inhibitory effects on iNOS and COX-2 by regulating NF-κB and JNK/p38 MAPK pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT

Objectives: Natural products have played a significant role in drug discovery and development. Inflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) have been suggested to connect with various inflammatory diseases. In this study, we explored the anti-inflammatory potential of aciculatin (8-((2R,4S,5S,6R)-tetrahydro-4,5-dihydroxy-6-methyl-2H-pyran-2-yl)-5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4H-chromen-4-one), one of main components of Chrysopogon aciculatis, by examining its effects on the expression and activity of iNOS and COX-2 in lipopolysaccharide (LPS)-activated macrophages.

Methods: We used nitrate and prostaglandin E2 (PGE2) assays to examine inhibitory effect of aciculatin on nitric oxide (NO) and PGE2 levels in LPS-activated mouse RAW264.7 macrophages and further investigated the mechanisms of aciculatin suppressed LPS-mediated iNOS/COX-2 expression by western blot, RT-PCR, reporter gene assay and confocal microscope analysis.

Results: Aciculatin remarkably decreased the LPS (1 μg/mL)-induced mRNA and protein expression of iNOS and COX-2 as well as their downstream products, NO and PGE2 respectively, in a concentration-dependent manner (1-10 μM). Such inhibition was found, via immunoblot analyses, reporter gene assays, and confocal microscope observations that aciculatin not only acts through significant suppression of LPS-induced NF-κB activation, an effect highly correlated with its inhibitory effect on LPS-induced IκB kinase (IKK) activation, IκB degradation, NF-κB phosphorylation, nuclear translocation and binding of NF-κB to the κB motif of the iNOS and COX-2 promoters, but also suppressed phosphorylation of JNK/p38 mitogen-activated protein kinases (MAPKs).

Conclusion: Our results demonstrated that aciculatin exerts potent anti-inflammatory activity through its dual inhibitory effects on iNOS and COX-2 by regulating NF-κB and JNK/p38 MAPK pathways.

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Aciculatin suppresses JNK, p38 MAPKs and MKK4, MKK3/6 phosphorylation in LPS-activated macrophages. RAW264.7 cells (1 × 106) were treated with (A) 1 μg/mL LPS for the indicated time periods or (B, C) 1 μg/mL LPS with or without aciculatin (1-10 μM) for 30 min, and cell lysates were then subjected to western blot analysis for the indicated proteins.
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Figure 6: Aciculatin suppresses JNK, p38 MAPKs and MKK4, MKK3/6 phosphorylation in LPS-activated macrophages. RAW264.7 cells (1 × 106) were treated with (A) 1 μg/mL LPS for the indicated time periods or (B, C) 1 μg/mL LPS with or without aciculatin (1-10 μM) for 30 min, and cell lysates were then subjected to western blot analysis for the indicated proteins.

Mentions: MAPKs pathways are also involved in the regulation of proinflammatory mediator expression [21]. Treatment with LPS for 30 min resulted in a significant increase in the phosphorylation of JNK, p38, and ERK compared to the control group (Figure 6A). Aciculatin (1-10 μM) markedly prevented LPS-induced increase of JNK and p38 phosphorylation in a concentration-dependent manner, but not phosphorylation of ERK (Figure 6B). Furthermore, activation of MAPKs (JNK, p38, and ERK) is known to require both tyrosine and threonine phosphorylation by the activated MAPKKs (MKK4, MKK3/6, and MEK1/2), therefore we next to investigate whether aciculatin has inhibitory effect on the activation of MAPKKs. As shown in Figure 6C, LPS treatment mediated a significant increase in the phosphorylation of MKK4, MKK3/6, and MEK1/2. Interestingly, consistent with the inhibitory effect on MAPKs, aciculatin concentration-dependently inhibited LPS-mediated increase of MKK4 and MKK3/6 phosphorylation, but not phosphorylation of MEK1/2.


Aciculatin inhibits lipopolysaccharide-mediated inducible nitric oxide synthase and cyclooxygenase-2 expression via suppressing NF-κB and JNK/p38 MAPK activation pathways.

Hsieh IN, Chang AS, Teng CM, Chen CC, Yang CR - J. Biomed. Sci. (2011)

Aciculatin suppresses JNK, p38 MAPKs and MKK4, MKK3/6 phosphorylation in LPS-activated macrophages. RAW264.7 cells (1 × 106) were treated with (A) 1 μg/mL LPS for the indicated time periods or (B, C) 1 μg/mL LPS with or without aciculatin (1-10 μM) for 30 min, and cell lysates were then subjected to western blot analysis for the indicated proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113733&req=5

Figure 6: Aciculatin suppresses JNK, p38 MAPKs and MKK4, MKK3/6 phosphorylation in LPS-activated macrophages. RAW264.7 cells (1 × 106) were treated with (A) 1 μg/mL LPS for the indicated time periods or (B, C) 1 μg/mL LPS with or without aciculatin (1-10 μM) for 30 min, and cell lysates were then subjected to western blot analysis for the indicated proteins.
Mentions: MAPKs pathways are also involved in the regulation of proinflammatory mediator expression [21]. Treatment with LPS for 30 min resulted in a significant increase in the phosphorylation of JNK, p38, and ERK compared to the control group (Figure 6A). Aciculatin (1-10 μM) markedly prevented LPS-induced increase of JNK and p38 phosphorylation in a concentration-dependent manner, but not phosphorylation of ERK (Figure 6B). Furthermore, activation of MAPKs (JNK, p38, and ERK) is known to require both tyrosine and threonine phosphorylation by the activated MAPKKs (MKK4, MKK3/6, and MEK1/2), therefore we next to investigate whether aciculatin has inhibitory effect on the activation of MAPKKs. As shown in Figure 6C, LPS treatment mediated a significant increase in the phosphorylation of MKK4, MKK3/6, and MEK1/2. Interestingly, consistent with the inhibitory effect on MAPKs, aciculatin concentration-dependently inhibited LPS-mediated increase of MKK4 and MKK3/6 phosphorylation, but not phosphorylation of MEK1/2.

Bottom Line: Aciculatin remarkably decreased the LPS (1 μg/mL)-induced mRNA and protein expression of iNOS and COX-2 as well as their downstream products, NO and PGE2 respectively, in a concentration-dependent manner (1-10 μM).Such inhibition was found, via immunoblot analyses, reporter gene assays, and confocal microscope observations that aciculatin not only acts through significant suppression of LPS-induced NF-κB activation, an effect highly correlated with its inhibitory effect on LPS-induced IκB kinase (IKK) activation, IκB degradation, NF-κB phosphorylation, nuclear translocation and binding of NF-κB to the κB motif of the iNOS and COX-2 promoters, but also suppressed phosphorylation of JNK/p38 mitogen-activated protein kinases (MAPKs).Our results demonstrated that aciculatin exerts potent anti-inflammatory activity through its dual inhibitory effects on iNOS and COX-2 by regulating NF-κB and JNK/p38 MAPK pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT

Objectives: Natural products have played a significant role in drug discovery and development. Inflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) have been suggested to connect with various inflammatory diseases. In this study, we explored the anti-inflammatory potential of aciculatin (8-((2R,4S,5S,6R)-tetrahydro-4,5-dihydroxy-6-methyl-2H-pyran-2-yl)-5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4H-chromen-4-one), one of main components of Chrysopogon aciculatis, by examining its effects on the expression and activity of iNOS and COX-2 in lipopolysaccharide (LPS)-activated macrophages.

Methods: We used nitrate and prostaglandin E2 (PGE2) assays to examine inhibitory effect of aciculatin on nitric oxide (NO) and PGE2 levels in LPS-activated mouse RAW264.7 macrophages and further investigated the mechanisms of aciculatin suppressed LPS-mediated iNOS/COX-2 expression by western blot, RT-PCR, reporter gene assay and confocal microscope analysis.

Results: Aciculatin remarkably decreased the LPS (1 μg/mL)-induced mRNA and protein expression of iNOS and COX-2 as well as their downstream products, NO and PGE2 respectively, in a concentration-dependent manner (1-10 μM). Such inhibition was found, via immunoblot analyses, reporter gene assays, and confocal microscope observations that aciculatin not only acts through significant suppression of LPS-induced NF-κB activation, an effect highly correlated with its inhibitory effect on LPS-induced IκB kinase (IKK) activation, IκB degradation, NF-κB phosphorylation, nuclear translocation and binding of NF-κB to the κB motif of the iNOS and COX-2 promoters, but also suppressed phosphorylation of JNK/p38 mitogen-activated protein kinases (MAPKs).

Conclusion: Our results demonstrated that aciculatin exerts potent anti-inflammatory activity through its dual inhibitory effects on iNOS and COX-2 by regulating NF-κB and JNK/p38 MAPK pathways.

Show MeSH
Related in: MedlinePlus