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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 nuclear transcription factor erythroid 2-related factor 2 (Nrf2)/heme oxyganase-1 (HO-1) pathway and ROS production in 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 HO-1 and β-actin were determined in the whole protein lysate by Western blot analysis. β-Actin was used as an internal control. The data shown are representative of three separate experiments; (B) 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 16 h. Western blot analysis was performed to detect Nrf2 in the nuclear fraction of the cell lysate. Nuclear protein PARP was used as an internal control. The data shown are representative of three separate experiments; (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 24 h. DCFH-DA (10 μM) was added and incubated for 30 min at 37 °C. DCF fluorescence by reactive oxygen species (ROS) was measured by flow cytometry. 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.
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marinedrugs-13-07005-f005: Effects of phorbaketal A on the nuclear transcription factor erythroid 2-related factor 2 (Nrf2)/heme oxyganase-1 (HO-1) pathway and ROS production in 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 HO-1 and β-actin were determined in the whole protein lysate by Western blot analysis. β-Actin was used as an internal control. The data shown are representative of three separate experiments; (B) 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 16 h. Western blot analysis was performed to detect Nrf2 in the nuclear fraction of the cell lysate. Nuclear protein PARP was used as an internal control. The data shown are representative of three separate experiments; (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 24 h. DCFH-DA (10 μM) was added and incubated for 30 min at 37 °C. DCF fluorescence by reactive oxygen species (ROS) was measured by flow cytometry. 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.

Mentions: In addition to inhibition of the NF-κB pathway, up-regulation of the Nrf2/HO-1 pathway has been suggested as a key mechanism of various anti-inflammatory activities [7,8]. HO-1 and its products exert anti-inflammatory effects via attenuation of the expression of pro-inflammatory mediators such as NO, PGE2, TNFα, IL-1β, IL-6, and MCP-1 in activated macrophages [10,11,12,13]. In addition, HO-1 has been demonstrated to inhibit pro-inflammatory responses in activated macrophages through modulation of NF-κB activation [11,13,45]. Thus, we next examined whether phorbaketal A induces HO-1 expression in LPS-stimulated RAW 264.7 cells. Phorbaketal A markedly increased HO-1 protein expression in RAW 264.7 cells (Figure 5A). Considering that HO-1 is induced by the transcription factor Nrf2 [46], we investigated whether phorbaketal A is able to induce the nuclear translocation of Nrf2. Phorbaketal A increased the nuclear translocation of Nrf2 (Figure 5B), suggesting that the induction of HO-1 by phorbaketal A is associated with Nrf2 activation. It is well known that the Nrf2/HO-1 pathway involves ROS generation. ROS have been proposed to be key signaling molecules in LPS-induced inflammation [47,48]. In fact, during inflammation, activated macrophages show massive ROS accumulation. ROS can stimulate cellular activities such as cytokine secretion and cell death [49]. Therefore, we examined the effect of phorbaketal A on LPS-induced ROS accumulation in RAW 264.7 cells. As shown in Figure 5C, phorbaketal A significantly reduced ROS accumulation in a dose-dependent manner. Thus, the anti-inflammatory effects of phorbaketal may be associated with its anti-oxidant property. Additional studies are needed to reveal the definitive molecular mechanisms.


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 nuclear transcription factor erythroid 2-related factor 2 (Nrf2)/heme oxyganase-1 (HO-1) pathway and ROS production in 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 HO-1 and β-actin were determined in the whole protein lysate by Western blot analysis. β-Actin was used as an internal control. The data shown are representative of three separate experiments; (B) 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 16 h. Western blot analysis was performed to detect Nrf2 in the nuclear fraction of the cell lysate. Nuclear protein PARP was used as an internal control. The data shown are representative of three separate experiments; (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 24 h. DCFH-DA (10 μM) was added and incubated for 30 min at 37 °C. DCF fluorescence by reactive oxygen species (ROS) was measured by flow cytometry. 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.
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marinedrugs-13-07005-f005: Effects of phorbaketal A on the nuclear transcription factor erythroid 2-related factor 2 (Nrf2)/heme oxyganase-1 (HO-1) pathway and ROS production in 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 HO-1 and β-actin were determined in the whole protein lysate by Western blot analysis. β-Actin was used as an internal control. The data shown are representative of three separate experiments; (B) 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 16 h. Western blot analysis was performed to detect Nrf2 in the nuclear fraction of the cell lysate. Nuclear protein PARP was used as an internal control. The data shown are representative of three separate experiments; (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 24 h. DCFH-DA (10 μM) was added and incubated for 30 min at 37 °C. DCF fluorescence by reactive oxygen species (ROS) was measured by flow cytometry. 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.
Mentions: In addition to inhibition of the NF-κB pathway, up-regulation of the Nrf2/HO-1 pathway has been suggested as a key mechanism of various anti-inflammatory activities [7,8]. HO-1 and its products exert anti-inflammatory effects via attenuation of the expression of pro-inflammatory mediators such as NO, PGE2, TNFα, IL-1β, IL-6, and MCP-1 in activated macrophages [10,11,12,13]. In addition, HO-1 has been demonstrated to inhibit pro-inflammatory responses in activated macrophages through modulation of NF-κB activation [11,13,45]. Thus, we next examined whether phorbaketal A induces HO-1 expression in LPS-stimulated RAW 264.7 cells. Phorbaketal A markedly increased HO-1 protein expression in RAW 264.7 cells (Figure 5A). Considering that HO-1 is induced by the transcription factor Nrf2 [46], we investigated whether phorbaketal A is able to induce the nuclear translocation of Nrf2. Phorbaketal A increased the nuclear translocation of Nrf2 (Figure 5B), suggesting that the induction of HO-1 by phorbaketal A is associated with Nrf2 activation. It is well known that the Nrf2/HO-1 pathway involves ROS generation. ROS have been proposed to be key signaling molecules in LPS-induced inflammation [47,48]. In fact, during inflammation, activated macrophages show massive ROS accumulation. ROS can stimulate cellular activities such as cytokine secretion and cell death [49]. Therefore, we examined the effect of phorbaketal A on LPS-induced ROS accumulation in RAW 264.7 cells. As shown in Figure 5C, phorbaketal A significantly reduced ROS accumulation in a dose-dependent manner. Thus, the anti-inflammatory effects of phorbaketal may be associated with its anti-oxidant property. Additional studies are needed to reveal the definitive molecular mechanisms.

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