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Phorbaketal A, Isolated from the Marine Sponge Phorbas sp., Exerts Its Anti-Inflammatory Effects via NF-κB Inhibition and Heme Oxygenase-1 Activation in Lipopolysaccharide-Stimulated Macrophages.

Seo YJ, Lee KT, Rho JR, Choi JH - Mar Drugs (2015)

Bottom Line: We found that phorbaketal A significantly inhibited the LPS-induced production of nitric oxide (NO), but not prostaglandin E₂, in RAW 264.7 cells.In addition, phorbaketal A reduced the LPS-induced production of inflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and monocyte chemotactic protein-1.These data suggest that phorbaketal A, isolated from the marine sponge Phorbas sp., inhibits the production of inflammatory mediators via down-regulation of the NF-κB pathway and up-regulation of the HO-1 pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Life & Nanopharmaceutical Sciences, Kyung Hee University, Seoul 130-701, Korea. syg9108@khu.ac.kr.

ABSTRACT
Marine sponges harbor a range of biologically active compounds. Phorbaketal A is a tricyclic sesterterpenoid isolated from the marine sponge Phorbas sp.; however, little is known about its biological activities and associated molecular mechanisms. In this study, we examined the anti-inflammatory effects and underlying molecular mechanism of phorbaketal A in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We found that phorbaketal A significantly inhibited the LPS-induced production of nitric oxide (NO), but not prostaglandin E₂, in RAW 264.7 cells. Further, phorbaketal A suppressed the expression of inducible NO synthase at both the mRNA and protein levels. In addition, phorbaketal A reduced the LPS-induced production of inflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and monocyte chemotactic protein-1. Treatment with phorbaketal A inhibited the transcriptional activity of nuclear factor-kappaB (NF-κB), a crucial signaling molecule in inflammation. Moreover, phorbaketal A up-regulated the expression of heme oxygenase-1 (HO-1) in LPS-stimulated RAW 264.7 cells. These data suggest that phorbaketal A, isolated from the marine sponge Phorbas sp., inhibits the production of inflammatory mediators via down-regulation of the NF-κB pathway and up-regulation of the HO-1 pathway.

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Effects of phorbaketal A on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 cells. (A) RAW 264.7 cells were pretreated with different concentrations of phorbaketal A (2.5, 5, and 10 μM) for 1 h and then stimulated with LPS (1 μg/mL) for 24 h. Protein levels of iNOS, COX-2, and β-actin were determined by Western blot analysis. β-Actin was used as an internal control. The data shown are representative of three separate experiments; (B–C) RAW 264.7 cells were pretreated with different concentrations of phorbaketal A (2.5, 5, and 10 μM) for 1 h and then stimulated with LPS (1 μg/mL) for 8 h. The mRNA levels of iNOS (B) and COX-2 (C) were measured by real-time RT-PCR. Data are presented as the means ± SD of three independent experiments. Statistical analysis was carried out using the one-way ANOVA followed by the Tukey’s test. #p < 0.05 vs. CTRL group. * p < 0.05 vs. LPS-stimulated group. #p < 0.05 vs. CON group. * p < 0.05 vs. LPS-stimulated group.
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marinedrugs-13-07005-f002: Effects of phorbaketal A on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 cells. (A) RAW 264.7 cells were pretreated with different concentrations of phorbaketal A (2.5, 5, and 10 μM) for 1 h and then stimulated with LPS (1 μg/mL) for 24 h. Protein levels of iNOS, COX-2, and β-actin were determined by Western blot analysis. β-Actin was used as an internal control. The data shown are representative of three separate experiments; (B–C) RAW 264.7 cells were pretreated with different concentrations of phorbaketal A (2.5, 5, and 10 μM) for 1 h and then stimulated with LPS (1 μg/mL) for 8 h. The mRNA levels of iNOS (B) and COX-2 (C) were measured by real-time RT-PCR. Data are presented as the means ± SD of three independent experiments. Statistical analysis was carried out using the one-way ANOVA followed by the Tukey’s test. #p < 0.05 vs. CTRL group. * p < 0.05 vs. LPS-stimulated group. #p < 0.05 vs. CON group. * p < 0.05 vs. LPS-stimulated group.

Mentions: We first evaluated the inhibitory effects of phorbaketal A (Figure 1A), isolated from the marine sponge Phorbas sp., on the production of two key inflammatory mediators, NO and PGE2, in macrophages. Based on MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay data (Figure 1B), concentrations of phorbaketal A that would not affect cell viability (2.5, 5, and 10 μM) were used for following experiments. Phorbaketal A significantly and dose-dependently suppressed NO production in LPS-stimulated RAW 264.7 cells (Figure 1C). In contrast, treatment with phorbaketal A did not modify PGE2 production (Figure 1D). l-N6-(1-iminoehyl)lysine (10 μM) and NS398 (10 μM) were used as inhibitors of NO and PGE2 production, respectively. We next investigated whether the inhibitory effects of phorbaketal A on NO production were associated with regulation of the expression of inducible NO synthase (iNOS). Western blotting revealed that phorbaketal A significantly suppressed LPS-induced iNOS expression at the protein level (Figure 2A). In addition, real-time RT-PCR analysis revealed that phorbaketal A markedly decreased the mRNA expression of iNOS (Figure 2B). Notably, phorbaketal A did not induce a significant change in either the mRNA or protein level of cyclooxygenase-2 (COX-2), an enzyme involved in the synthesis of PGE2 (Figure 2A,C). These observations suggest that phorbaketal A significantly suppressed NO production by inhibiting iNOS expression at the transcriptional level, and that it had little effect on PGE2 and COX-2.


Phorbaketal A, Isolated from the Marine Sponge Phorbas sp., Exerts Its Anti-Inflammatory Effects via NF-κB Inhibition and Heme Oxygenase-1 Activation in Lipopolysaccharide-Stimulated Macrophages.

Seo YJ, Lee KT, Rho JR, Choi JH - Mar Drugs (2015)

Effects of phorbaketal A on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 cells. (A) RAW 264.7 cells were pretreated with different concentrations of phorbaketal A (2.5, 5, and 10 μM) for 1 h and then stimulated with LPS (1 μg/mL) for 24 h. Protein levels of iNOS, COX-2, and β-actin were determined by Western blot analysis. β-Actin was used as an internal control. The data shown are representative of three separate experiments; (B–C) RAW 264.7 cells were pretreated with different concentrations of phorbaketal A (2.5, 5, and 10 μM) for 1 h and then stimulated with LPS (1 μg/mL) for 8 h. The mRNA levels of iNOS (B) and COX-2 (C) were measured by real-time RT-PCR. Data are presented as the means ± SD of three independent experiments. Statistical analysis was carried out using the one-way ANOVA followed by the Tukey’s test. #p < 0.05 vs. CTRL group. * p < 0.05 vs. LPS-stimulated group. #p < 0.05 vs. CON group. * p < 0.05 vs. LPS-stimulated group.
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marinedrugs-13-07005-f002: Effects of phorbaketal A on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 cells. (A) RAW 264.7 cells were pretreated with different concentrations of phorbaketal A (2.5, 5, and 10 μM) for 1 h and then stimulated with LPS (1 μg/mL) for 24 h. Protein levels of iNOS, COX-2, and β-actin were determined by Western blot analysis. β-Actin was used as an internal control. The data shown are representative of three separate experiments; (B–C) RAW 264.7 cells were pretreated with different concentrations of phorbaketal A (2.5, 5, and 10 μM) for 1 h and then stimulated with LPS (1 μg/mL) for 8 h. The mRNA levels of iNOS (B) and COX-2 (C) were measured by real-time RT-PCR. Data are presented as the means ± SD of three independent experiments. Statistical analysis was carried out using the one-way ANOVA followed by the Tukey’s test. #p < 0.05 vs. CTRL group. * p < 0.05 vs. LPS-stimulated group. #p < 0.05 vs. CON group. * p < 0.05 vs. LPS-stimulated group.
Mentions: We first evaluated the inhibitory effects of phorbaketal A (Figure 1A), isolated from the marine sponge Phorbas sp., on the production of two key inflammatory mediators, NO and PGE2, in macrophages. Based on MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay data (Figure 1B), concentrations of phorbaketal A that would not affect cell viability (2.5, 5, and 10 μM) were used for following experiments. Phorbaketal A significantly and dose-dependently suppressed NO production in LPS-stimulated RAW 264.7 cells (Figure 1C). In contrast, treatment with phorbaketal A did not modify PGE2 production (Figure 1D). l-N6-(1-iminoehyl)lysine (10 μM) and NS398 (10 μM) were used as inhibitors of NO and PGE2 production, respectively. We next investigated whether the inhibitory effects of phorbaketal A on NO production were associated with regulation of the expression of inducible NO synthase (iNOS). Western blotting revealed that phorbaketal A significantly suppressed LPS-induced iNOS expression at the protein level (Figure 2A). In addition, real-time RT-PCR analysis revealed that phorbaketal A markedly decreased the mRNA expression of iNOS (Figure 2B). Notably, phorbaketal A did not induce a significant change in either the mRNA or protein level of cyclooxygenase-2 (COX-2), an enzyme involved in the synthesis of PGE2 (Figure 2A,C). These observations suggest that phorbaketal A significantly suppressed NO production by inhibiting iNOS expression at the transcriptional level, and that it had little effect on PGE2 and COX-2.

Bottom Line: We found that phorbaketal A significantly inhibited the LPS-induced production of nitric oxide (NO), but not prostaglandin E₂, in RAW 264.7 cells.In addition, phorbaketal A reduced the LPS-induced production of inflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and monocyte chemotactic protein-1.These data suggest that phorbaketal A, isolated from the marine sponge Phorbas sp., inhibits the production of inflammatory mediators via down-regulation of the NF-κB pathway and up-regulation of the HO-1 pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Life & Nanopharmaceutical Sciences, Kyung Hee University, Seoul 130-701, Korea. syg9108@khu.ac.kr.

ABSTRACT
Marine sponges harbor a range of biologically active compounds. Phorbaketal A is a tricyclic sesterterpenoid isolated from the marine sponge Phorbas sp.; however, little is known about its biological activities and associated molecular mechanisms. In this study, we examined the anti-inflammatory effects and underlying molecular mechanism of phorbaketal A in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We found that phorbaketal A significantly inhibited the LPS-induced production of nitric oxide (NO), but not prostaglandin E₂, in RAW 264.7 cells. Further, phorbaketal A suppressed the expression of inducible NO synthase at both the mRNA and protein levels. In addition, phorbaketal A reduced the LPS-induced production of inflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and monocyte chemotactic protein-1. Treatment with phorbaketal A inhibited the transcriptional activity of nuclear factor-kappaB (NF-κB), a crucial signaling molecule in inflammation. Moreover, phorbaketal A up-regulated the expression of heme oxygenase-1 (HO-1) in LPS-stimulated RAW 264.7 cells. These data suggest that phorbaketal A, isolated from the marine sponge Phorbas sp., inhibits the production of inflammatory mediators via down-regulation of the NF-κB pathway and up-regulation of the HO-1 pathway.

Show MeSH
Related in: MedlinePlus