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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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Effect of aciculatin on LPS-induced NF-κB translocation into the nucleus. RAW264.7 cells (1 × 105 cells) were pretreated with aciculatin (10 μM) for 1 h followed by stimulation with LPS (1 μg/mL) for 1 h. Samples were stained by anti-p65 antibody (BioVision) and DAPI, then prepared for confocal microscopy analysis. The results shown are representative of those obtained in four independent experiments. Scale bar = 10 μm.
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Figure 5: Effect of aciculatin on LPS-induced NF-κB translocation into the nucleus. RAW264.7 cells (1 × 105 cells) were pretreated with aciculatin (10 μM) for 1 h followed by stimulation with LPS (1 μg/mL) for 1 h. Samples were stained by anti-p65 antibody (BioVision) and DAPI, then prepared for confocal microscopy analysis. The results shown are representative of those obtained in four independent experiments. Scale bar = 10 μm.

Mentions: It has been reported that NF-κB signals regulate the transcription of a wide array of genes, including pro-inflammatory enzymes iNOS and COX-2 in macrophages [18,19]. However, the precise role of aciculatin on regulating NF-κB activation is still unclear. To examine whether aciculatin regulates NF-κB pathways, RAW264.7 macrophages were treated with LPS (1 μg/mL) for 24 h in the presence or absent of aciculatin (3, 10 μM) and levels of the phosphorylated and total forms of IKK(/(, I(B(, and p65 were also examined. LPS treatment not only mediated significant phosphorylation of IKK(/( at serine 180/181, the phosphorylation of IκBa at serine 32, and IκBaα degradation, but also increased the phosphorylation of p65 (Figure 4A). However, 3 μM aciculatin treatment remarkably prevented IKK/IκB/p65 phosphorylation and IκB degradation; 10 μM aciculatin even more significantly rescued to reach basal level. The result of promoter activity assay also showed that aciculatin markedly inhibited LPS-mediated NF-κB promoter activation in a concentration-dependent manner (Figure 4B). Furthermore, the nuclear translocation of NF-κB/p65 was observed under a laser confocal microscope. RAW264.7 macrophages stimulated with LPS showed a dramatic increase in the translocation of NF-κB into the nucleus (Figure 5). In contrast, the LPS-induced NF-κB nuclear translocation was markedly impaired after aciculatin (10 μM) treatment. These results demonstrate that aciculatin significantly inhibited IKK/IκB/NF- κB pathways and NF-κB nuclear translocation.


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)

Effect of aciculatin on LPS-induced NF-κB translocation into the nucleus. RAW264.7 cells (1 × 105 cells) were pretreated with aciculatin (10 μM) for 1 h followed by stimulation with LPS (1 μg/mL) for 1 h. Samples were stained by anti-p65 antibody (BioVision) and DAPI, then prepared for confocal microscopy analysis. The results shown are representative of those obtained in four independent experiments. Scale bar = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Effect of aciculatin on LPS-induced NF-κB translocation into the nucleus. RAW264.7 cells (1 × 105 cells) were pretreated with aciculatin (10 μM) for 1 h followed by stimulation with LPS (1 μg/mL) for 1 h. Samples were stained by anti-p65 antibody (BioVision) and DAPI, then prepared for confocal microscopy analysis. The results shown are representative of those obtained in four independent experiments. Scale bar = 10 μm.
Mentions: It has been reported that NF-κB signals regulate the transcription of a wide array of genes, including pro-inflammatory enzymes iNOS and COX-2 in macrophages [18,19]. However, the precise role of aciculatin on regulating NF-κB activation is still unclear. To examine whether aciculatin regulates NF-κB pathways, RAW264.7 macrophages were treated with LPS (1 μg/mL) for 24 h in the presence or absent of aciculatin (3, 10 μM) and levels of the phosphorylated and total forms of IKK(/(, I(B(, and p65 were also examined. LPS treatment not only mediated significant phosphorylation of IKK(/( at serine 180/181, the phosphorylation of IκBa at serine 32, and IκBaα degradation, but also increased the phosphorylation of p65 (Figure 4A). However, 3 μM aciculatin treatment remarkably prevented IKK/IκB/p65 phosphorylation and IκB degradation; 10 μM aciculatin even more significantly rescued to reach basal level. The result of promoter activity assay also showed that aciculatin markedly inhibited LPS-mediated NF-κB promoter activation in a concentration-dependent manner (Figure 4B). Furthermore, the nuclear translocation of NF-κB/p65 was observed under a laser confocal microscope. RAW264.7 macrophages stimulated with LPS showed a dramatic increase in the translocation of NF-κB into the nucleus (Figure 5). In contrast, the LPS-induced NF-κB nuclear translocation was markedly impaired after aciculatin (10 μM) treatment. These results demonstrate that aciculatin significantly inhibited IKK/IκB/NF- κB pathways and NF-κB nuclear translocation.

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