Korean J Physiol Pharmacol.  2013 Jun;17(3):217-222. 10.4196/kjpp.2013.17.3.217.

2-(4-Hydroxyphenyl)-5-(3-Hydroxypropenyl)-7-Methoxybenzofuran, a Novel Ailanthoidol Derivative, Exerts Anti-Inflammatory Effect through Downregulation of Mitogen-Activated Protein Kinase in Lipopolysaccharide-Treated RAW 264.7 Cells

• Kim HJ
• Jun JG
• Kim JK

• KMID: 1429301
• DOI: http://doi.org/10.4196/kjpp.2013.17.3.217

Abstract

We reported that ailanthoidol, a neolignan from Zanthoxylum ailanthoides and Salvia miltiorrhiza Bunge, inhibited inflammatory reactions by macrophages and protected mice from endotoxin shock. We examined the anti-inflammatory activity of six synthetic ailanthoidol derivatives (compounds 1-6). Among them, compound 4, 2-(4-hydroxyphenyl)-5-(3-hydroxypropenyl)-7-methoxybenzofuran, had the lowest IC50 value concerning nitric oxide (NO) release from lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Compound 4 suppressed the generation of prostaglandin (PG) E2 and the expression of inducible NO synthase and cyclooxygenase (COX)-2 induced by LPS, and inhibited the release of LPS-induced pro-inflammatory cytokines from RAW264.7 cells. The underlying mechanism of compound 4 on anti-inflammatory action was correlated with the down-regulation of mitogen-activated protein kinase and activator protein-1 activation. Compound 4 is potentially an effective functional chemical candidate for the prevention of inflammatory diseases.

Keyword

Ailanthoidol derivatives; AP-1; Cytokines; Inflammation; Macrophage

MeSH Terms

Animals
Benzofurans
Cytokines
Down-Regulation
Inflammation
Inhibitory Concentration 50
Macrophages
Mice
Nitric Oxide
Nitric Oxide Synthase
Prostaglandin-Endoperoxide Synthases
Protein Kinases
Salvia miltiorrhiza
Shock
Transcription Factor AP-1
Zanthoxylum
Benzofurans
Cytokines
Nitric Oxide
Nitric Oxide Synthase
Prostaglandin-Endoperoxide Synthases
Protein Kinases
Transcription Factor AP-1

Figure

• 

  Fig. 1 Chemical structure of ailanthoidol and its derivatives, compounds 1-6.

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  Fig. 2 Effects of compound 4 on murine macrophage viability. RAW264.7 cells were treated with indicated concentrations of compound 4 for 24 h, and proliferation was determined. The results are reported as mean±SEM of three independent experiments in triplicate.

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  Fig. 3 Effects of compound 4 on LPS-induced NO and PGE2 release. RAW264.7 cells were treated with 0~10 µM of compound in the presence of 100 ng/ml of LPS or with LPS alone for 24 h, and (A) NO and (B) PGE2 release was determined. The results are reported as mean±SEM of three independent experiments in triplicate. Statistical significance is based on the difference when compared with LPS-stimulated cells (**p<0.01, ***p<0.001). Thirty micrograms of protein obtained from each cell lysate was resolved by 10% SDS-PAGE for (C) iNOS and (D) COX-2 determination. β-actin expression is shown as a loading control. The bands were quantified using image analysis software and their relative intensity was expressed as fold against the image of the LPS-stimulated RAW264.7 cells.

• 

  Fig. 4 Effects of compound 4 on LPS-induced inflammatory cytokine production in murine macrophages. RAW264.7 cells were treated with 0~10 µM of compound 4 in the presence of 100 ng/ml LPS or with LPS alone for 24 h. The cell culture media were then collected, and the amount of (A) IL-1β and (B) IL-6 released was measured. The results are reported as mean±SEM of three independent experiments in triplicate. Statistical significance is based on the difference when compared with LPS-stimulated cells (**p<0.01, ***p<0.001).

• 

  Fig. 5 Effect of compound 4 on LPS-induced MAPK and AP-1 activation. RAW264.7 cells were plated in 100 mm-diameter dishes. After 12 h of seeding, cells were treated with different doses of compound 4 for 1 h, followed by stimulation with 500 ng/ml of LPS for 30 min. Cell lysates (30 µg protein) were prepared and subjected to Western blot analysis by using antibodies specific for (A) IκB-α, phosphorylated forms of ERK1/2, JNK, p38 MAPK and (B) phosphorylated forms of c-Jun. Equivalent loading of cell lysates was determined by reprobing the blots with anti-β-actin, total ERK1/2, JNK or p38 MAPK antibody. (C) Nuclear protein (30 µg protein) was prepared and subjected to Western blot analysis by using antibodies specific for c-Jun and equivalent loading of nuclear protein was determined by reprobing the blots with anti-PARP. The bands were quantified using image analysis software and their relative intensity was expressed as fold against the image of the LPS-stimulated RAW264.7 cells.

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