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Sinularin from indigenous soft coral attenuates nociceptive responses and spinal neuroinflammation in carrageenan-induced inflammatory rat model.

Huang SY, Chen NF, Chen WF, Hung HC, Lee HP, Lin YY, Wang HM, Sung PJ, Sheu JH, Wen ZH - Mar Drugs (2012)

Bottom Line: We found that subcutaneous (s.c.) administration of sinularin (80 mg/kg) 1 h before carrageenan injection significantly inhibited carrageenan-induced nociceptive behaviors, including thermal hyperalgesia, mechanical allodynia, cold allodynia, and hindpaw weight-bearing deficits.The results of immunohistochemical studies indicate that s.c. sinularin (80 mg/kg) could upregulate production of TGF-β1 in carrageenan-induced inflamed paw tissue.The present results demonstrate that systemic sinularin exerts analgesic effects at the behavioral and spinal levels, which are associated with both inhibition of leukocyte infiltration and upregulation of TGF-β1.

View Article: PubMed Central - PubMed

Affiliation: Department of Marine Biotechnology and Resources, Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 80424, Taiwan. johnjohnkings@gmail.com

ABSTRACT
Three decades ago, the marine-derived compound sinularin was shown to have anti-edematous effects on paw edema induced by carrageenan or adjuvant. To the best of our knowledge, no new studies were conducted to explore the bioactivity of sinularin until we reported the analgesic properties of sinularin based on in vivo experiments. In the present study, we found that sinularin significantly inhibits the upregulation of proinflammatory proteins, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) and upregulates the production of transforming growth factor-β (TGF-β) in lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells according to western blot analysis. We found that subcutaneous (s.c.) administration of sinularin (80 mg/kg) 1 h before carrageenan injection significantly inhibited carrageenan-induced nociceptive behaviors, including thermal hyperalgesia, mechanical allodynia, cold allodynia, and hindpaw weight-bearing deficits. Further, s.c. sinularin (80 mg/kg) significantly inhibited carrageenan-induced microglial and astrocyte activation as well as upregulation of iNOS in the dorsal horn of the lumbar spinal cord. Moreover, s.c. sinularin (80 mg/kg) inhibited carrageenan-induced tissue inflammatory responses, redness and edema of the paw, and leukocyte infiltration. The results of immunohistochemical studies indicate that s.c. sinularin (80 mg/kg) could upregulate production of TGF-β1 in carrageenan-induced inflamed paw tissue. The present results demonstrate that systemic sinularin exerts analgesic effects at the behavioral and spinal levels, which are associated with both inhibition of leukocyte infiltration and upregulation of TGF-β1.

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Effect of sinularin on protein expression of pro-inflammatory inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and anti-inflammatory transforming growth factor-β (TGF-β) in RAW 264.7 cells induced by lipopolysaccharide (LPS; 0.01 μg/mL). (A) Western blots of iNOS, COX-2, TGF-β, and β-actin proteins from RAW 264.7 cells; (B) relative density of immunoblot of iNOS; (C) relative density of immunoblot of COX-2; (D) relative density of immunoblot of TGF-β. Relative intensity of the LPS-stimulated group was defined as 100%. Band intensities were quantified using densitometry and are indicated as the percentage change relative to that of the LPS-stimulated group. Western blotting using β-actin was performed to verify loading of equivalent amounts of protein in each lane. This experiment was repeated 3 times. * P < 0.05 compared with the LPS-stimulated group.
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marinedrugs-10-01899-f002: Effect of sinularin on protein expression of pro-inflammatory inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and anti-inflammatory transforming growth factor-β (TGF-β) in RAW 264.7 cells induced by lipopolysaccharide (LPS; 0.01 μg/mL). (A) Western blots of iNOS, COX-2, TGF-β, and β-actin proteins from RAW 264.7 cells; (B) relative density of immunoblot of iNOS; (C) relative density of immunoblot of COX-2; (D) relative density of immunoblot of TGF-β. Relative intensity of the LPS-stimulated group was defined as 100%. Band intensities were quantified using densitometry and are indicated as the percentage change relative to that of the LPS-stimulated group. Western blotting using β-actin was performed to verify loading of equivalent amounts of protein in each lane. This experiment was repeated 3 times. * P < 0.05 compared with the LPS-stimulated group.

Mentions: We used western blot analysis to evaluate expression of pro-inflammatory proteins (iNOS and COX-2) and TGF-β in the LPS-stimulated RAW 264.7 macrophage cells using whole cell lysates and 3 antibodies against mouse macrophage iNOS, COX-2, and TGF-β, respectively (Figure 2). Dose responses of sinularin for inhibition of LPS-induced 130-kDa iNOS and 71-kDa COX-2 and up-regulation of LPS-induced 13-kDa TGF-β are shown in Figure 2. At 0.1, 1, 10, and 20 μM doses of sinularin, the levels of iNOS protein were significantly reduced to 53.45 ± 3.27%, 36.45 ± 5.15%, 33.38 ± 4.61%, and 19.48 ± 3.95% of the control level, respectively (Figure 2B). At concentrations of 10 and 20 μM doses of sinularin, the level of COX-2 protein were also reduced significantly to 82.72 ± 6.17% and 66.23 ± 3.27% of the control level, respectively (Figure 2C. At 0.1, 1, 10, and 20 μM doses of sinularin, the level of TGF-β protein were increased significantly to 137.75 ± 5.97%, 149.82 ± 6.15%, 142.71 ± 4.57%, and 138.02 ± 5.15% of the control level, respectively (Figure 2D). Therefore, three changes in protein levels take place at 10 μM and 20 μM doses of sinularin, including inhibition of iNOS and of COX-2 as well as upregulation of TGF-β.


Sinularin from indigenous soft coral attenuates nociceptive responses and spinal neuroinflammation in carrageenan-induced inflammatory rat model.

Huang SY, Chen NF, Chen WF, Hung HC, Lee HP, Lin YY, Wang HM, Sung PJ, Sheu JH, Wen ZH - Mar Drugs (2012)

Effect of sinularin on protein expression of pro-inflammatory inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and anti-inflammatory transforming growth factor-β (TGF-β) in RAW 264.7 cells induced by lipopolysaccharide (LPS; 0.01 μg/mL). (A) Western blots of iNOS, COX-2, TGF-β, and β-actin proteins from RAW 264.7 cells; (B) relative density of immunoblot of iNOS; (C) relative density of immunoblot of COX-2; (D) relative density of immunoblot of TGF-β. Relative intensity of the LPS-stimulated group was defined as 100%. Band intensities were quantified using densitometry and are indicated as the percentage change relative to that of the LPS-stimulated group. Western blotting using β-actin was performed to verify loading of equivalent amounts of protein in each lane. This experiment was repeated 3 times. * P < 0.05 compared with the LPS-stimulated group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475263&req=5

marinedrugs-10-01899-f002: Effect of sinularin on protein expression of pro-inflammatory inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and anti-inflammatory transforming growth factor-β (TGF-β) in RAW 264.7 cells induced by lipopolysaccharide (LPS; 0.01 μg/mL). (A) Western blots of iNOS, COX-2, TGF-β, and β-actin proteins from RAW 264.7 cells; (B) relative density of immunoblot of iNOS; (C) relative density of immunoblot of COX-2; (D) relative density of immunoblot of TGF-β. Relative intensity of the LPS-stimulated group was defined as 100%. Band intensities were quantified using densitometry and are indicated as the percentage change relative to that of the LPS-stimulated group. Western blotting using β-actin was performed to verify loading of equivalent amounts of protein in each lane. This experiment was repeated 3 times. * P < 0.05 compared with the LPS-stimulated group.
Mentions: We used western blot analysis to evaluate expression of pro-inflammatory proteins (iNOS and COX-2) and TGF-β in the LPS-stimulated RAW 264.7 macrophage cells using whole cell lysates and 3 antibodies against mouse macrophage iNOS, COX-2, and TGF-β, respectively (Figure 2). Dose responses of sinularin for inhibition of LPS-induced 130-kDa iNOS and 71-kDa COX-2 and up-regulation of LPS-induced 13-kDa TGF-β are shown in Figure 2. At 0.1, 1, 10, and 20 μM doses of sinularin, the levels of iNOS protein were significantly reduced to 53.45 ± 3.27%, 36.45 ± 5.15%, 33.38 ± 4.61%, and 19.48 ± 3.95% of the control level, respectively (Figure 2B). At concentrations of 10 and 20 μM doses of sinularin, the level of COX-2 protein were also reduced significantly to 82.72 ± 6.17% and 66.23 ± 3.27% of the control level, respectively (Figure 2C. At 0.1, 1, 10, and 20 μM doses of sinularin, the level of TGF-β protein were increased significantly to 137.75 ± 5.97%, 149.82 ± 6.15%, 142.71 ± 4.57%, and 138.02 ± 5.15% of the control level, respectively (Figure 2D). Therefore, three changes in protein levels take place at 10 μM and 20 μM doses of sinularin, including inhibition of iNOS and of COX-2 as well as upregulation of TGF-β.

Bottom Line: We found that subcutaneous (s.c.) administration of sinularin (80 mg/kg) 1 h before carrageenan injection significantly inhibited carrageenan-induced nociceptive behaviors, including thermal hyperalgesia, mechanical allodynia, cold allodynia, and hindpaw weight-bearing deficits.The results of immunohistochemical studies indicate that s.c. sinularin (80 mg/kg) could upregulate production of TGF-β1 in carrageenan-induced inflamed paw tissue.The present results demonstrate that systemic sinularin exerts analgesic effects at the behavioral and spinal levels, which are associated with both inhibition of leukocyte infiltration and upregulation of TGF-β1.

View Article: PubMed Central - PubMed

Affiliation: Department of Marine Biotechnology and Resources, Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 80424, Taiwan. johnjohnkings@gmail.com

ABSTRACT
Three decades ago, the marine-derived compound sinularin was shown to have anti-edematous effects on paw edema induced by carrageenan or adjuvant. To the best of our knowledge, no new studies were conducted to explore the bioactivity of sinularin until we reported the analgesic properties of sinularin based on in vivo experiments. In the present study, we found that sinularin significantly inhibits the upregulation of proinflammatory proteins, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) and upregulates the production of transforming growth factor-β (TGF-β) in lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells according to western blot analysis. We found that subcutaneous (s.c.) administration of sinularin (80 mg/kg) 1 h before carrageenan injection significantly inhibited carrageenan-induced nociceptive behaviors, including thermal hyperalgesia, mechanical allodynia, cold allodynia, and hindpaw weight-bearing deficits. Further, s.c. sinularin (80 mg/kg) significantly inhibited carrageenan-induced microglial and astrocyte activation as well as upregulation of iNOS in the dorsal horn of the lumbar spinal cord. Moreover, s.c. sinularin (80 mg/kg) inhibited carrageenan-induced tissue inflammatory responses, redness and edema of the paw, and leukocyte infiltration. The results of immunohistochemical studies indicate that s.c. sinularin (80 mg/kg) could upregulate production of TGF-β1 in carrageenan-induced inflamed paw tissue. The present results demonstrate that systemic sinularin exerts analgesic effects at the behavioral and spinal levels, which are associated with both inhibition of leukocyte infiltration and upregulation of TGF-β1.

Show MeSH
Related in: MedlinePlus