Nat Prod Sci.  2015 Dec;21(4):240-247. 10.20307/nps.2015.21.4.240.

Viridicatol from Marine-derived Fungal Strain Penicillium sp. SF-5295 Exerts Anti-inflammatory Effects through Inhibiting NF-kappaB Signaling Pathway on Lipopolysaccharide-induced RAW264.7 and BV2 Cells

• Ko W
• Sohn JH
• Kim YC
• Oh H

• KMID: 2312903
• DOI: http://doi.org/10.20307/nps.2015.21.4.240

Abstract

Viridicatol (1) has previously been isolated from the extract of the marine-derived fungus Penicillium sp. SF-5295. In the course of further biological evaluation of this quinolone alkaloid, anti-inflammatory effect of 1 in RAW264.7 and BV2 cells stimulated with lipopolysaccharide (LPS) was observed. In this study, our data indicated that 1 suppressed the expression of well-known pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, and consequently inhibited the production of iNOS-derived nitric oxide (NO) and COX-2-derived prostaglandin E2 (PGE2) in LPS stimulated RAW264.7 and BV2 cells. Compound 1 also reduced mRNA expression of pro-inflammatory cytokines such as interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha). In the further evaluation of the mechanisms of these anti-inflammatory effects, 1 was shown to inhibit nuclear factor-kappa B (NF-kappaB) pathway in LPS-stimulated RAW264.7 and BV2 cells. Compound 1 blocked the phosphorylation and degradation of inhibitor kappa B (IkappaB)-alpha in the cytoplasm, and suppressed the translocation of NF-kappaB p65 and p50 heterodimer in nucleus. In addition, viridicatol (1) attenuated the DNA-binding activity of NF-kappaB in LPS-stimulated RAW264.7 and BV2 cells.

Keyword

Viridicatol; Penicillium sp.; Marine fungus; Anti-neuroinflammation; Nuclear factor-kappa B (NF-kappaB)

MeSH Terms

Cytokines
Cytoplasm
Dinoprostone
Fungi
Interleukin-1beta
Interleukin-6
NF-kappa B*
Nitric Oxide
Nitric Oxide Synthase Type II
Penicillium*
Phosphorylation
Prostaglandin-Endoperoxide Synthases
RNA, Messenger
Tumor Necrosis Factor-alpha
Cytokines
Dinoprostone
Interleukin-1beta
Interleukin-6
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase Type II
Prostaglandin-Endoperoxide Synthases
RNA, Messenger
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1. Chemical structure of viridicatol (1).

• 

  Fig. 2. Effects of 1 on cell viability. RAW264.7 (A) and BV2 cells (B) were incubated for 24 h with various concentrations of 1 (1 - 160μ M). Cell viability was determined by MTT assay. Each bar represents the mean ± S.D. of three independent experiments.

• 

  Fig. 3. Effects of 1 on the protein expression of nitrite (A), PGE2 (B) iNOS (C) and COX-2 (D) in RAW 264.7 cells stimulated with LPS. The cells were pre-treated for 3 h with indicated concentrations of 1, and then stimulated for 24 h with LPS (1 μ g/mL). The concentrations of nitrite, PGE2 and Western blot analysis were performed as described in the experimental section, and representative data of three independent experiments are shown. ∗p < 0.05 compared with the LPS.

• 

  Fig. 4. Effects of 1 on the protein expression of nitrite (A), PGE2 (B) iNOS (C) and COX-2 (D) in BV2 cells stimulated with LPS. The cells were pre-treated for 3 h with indicated concentrations of 1, and then stimulated for 24 h with LPS (1 μg/mL). The concentrations of nitrite, PGE2 and Western blot analysis were performed as described in the experimental section, and representative data of three independent experiments are shown. ∗p < 0.05 compared with the LPS.

• 

  Fig. 5. Effects of 1 on IL-1β mRNA (A), IL-6 mRNA (B), and TNF-α mRNA (C) expression in RAW264.7 cells. Cells were incubated with the indicated concentrations of 1 for 3 h, and then treated for 6 h with LPS (1 μg/mL). RNA quantification for IL-1β, IL-6 and TNF-α expression were performed as described in the experimental section. Data shown represent the mean values of three independent experiments. ∗p < 0.05 compared with the LPS.

• 

  Fig. 6. Effects of 1 on IL-1β mRNA (A), IL-6 mRNA (B), and TNF-α mRNA (C) expression in BV2 cells. Cells were incubated with the indicated concentrations of 1 for 3 h, and then treated for 6 h with LPS (1 μg/mL). RNA quantification for IL-1β, IL-6 and TNF-α expression were performed, as described in the experimental section. Data shown represent the mean values of three independent experiments. ∗p < 0.05 compared with the LPS.

• 

  Fig. 7. Effects of 1 on the Iκ B-α phosphorylation and degradation (A), NF-κ B activation (p65 and p50), and NF-κ B DNA-binding activity (C). RAW264.7 cells were pre-treated with the indicated concentrations of 1 for 3 h, and then stimulated with LPS (1 μg/mL) for 1 h. The western blot analysis of IκBα and p-IκBα in the cytoplasm and NF-κB in the nucleus was performed as described in the experimental section. The representative blots of three independent experiments are shown. A commercially available NF-κB ELISA (Active Motif) was then used to test nuclear extracts and to determine the degree of NF-κB binding. The data represent the mean values of 3 experiments ± SD. ∗P < 0.05 compared with the group treated with LPS.

• 

  Fig. 8. Effects of 1 on the Iκ B-α phosphorylation and degradation (A), NF-κ B activation (p65 and p50), and NF-κ B DNA-binding activity (C). BV2 cells were pre-treated with the indicated concentrations of 1 for 3 h, and then stimulated with LPS (1 μg/mL) for 1 h. The western blot analysis of Iκ Bα and p-Iκ Bα in the cytoplasm and NF-κ B in the nucleus was performed as described in the experimental section. The representative blots of three independent experiments are shown. A commercially available NF-κB ELISA (Active Motif) was then used to test nuclear extracts and to determine the degree of NF-κB binding. The data represent the mean values of 3 experiments ± SD. ∗P < 0.05 compared with the group treated with LPS.

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