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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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Anti-redness and anti-edematous effects of subcutaneous (s.c.) sinularin in intraplantar (i.p.l.) carrageenan-induced paw edema model. Photographic images show the gross pathology of paws from i.p.l. saline plus s.c. vehicle (dimethyl sulfoxide [DMSO]) (A, E), i.p.l. carrageenan plus s.c. vehicle (B, F), i.p.l. carrageenan plus s.c. sinularin (20 mg/kg) (C, G), and i.p.l. carrageenan plus s.c. sinularin (80 mg/kg) (D, H) groups. The lateral view of paws are shown in A, B, C, and D; top view of paws are shown in E, F, G, and H. The images (A–H) were taken 6 h after i.p.l. saline or carrageenan injection (as well as 7 h after s.c. vehicle or sinularin injection). The unit of scale bars shown in images A–H is in centimeter. S.c. sinularin of 20 mg/kg and 80 mg/kg reduced levels of carrageenan-induced edema of paw, and s.c. sinularin 80 mg/kg also clearly inhibited redness induced by carrageenan in rats than that by 20 mg/kg sinularin. We used 20 mg/kg indomethacin as a positive control. The time course of the percentage change of the increase in paw volume induced by i.p.l. injection of carrageenan in rats pretreated with vehicle, sinularin (80 mg/kg), or indomethacin (20 mg/kg) (I), administrated 1 h before carrageenan injection. Basal volume of each rat paw before i.p.l. saline or carrageenan injection was considered to be 100%, and the increase in paw volume is shown as the percentage change from the basal values by subtracting the basal paw volume from the paw volume measured at each time point. Area under the edematous effect-time curve (J), which was transformed from image I, for s.c. vehicle, sinularin (80 mg/kg), or indomethacin (20 mg/kg) injection, extending from 3 to 24 h after i.p.l. carrageenan injection. Carrageenan-induced paw edema was well-developed by nearly 3 h and persisted for at least 24 h. This phenomenon was significantly reduced by s.c. sinularin (80 mg/kg) or indomethacin (20 mg/kg). The duration of the anti-edematous effects of sinularin (80 mg/kg) or indomethacin (20 mg/kg) are shown as the area under the curve (AUC). Each point or bar in images I and J represent the mean ± standard error of the mean (SEM) of 6 rats per group. * P < 0.05 compared with the same time points in i.p.l. carrageenan plus s.c. vehicle group.
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marinedrugs-10-01899-f003: Anti-redness and anti-edematous effects of subcutaneous (s.c.) sinularin in intraplantar (i.p.l.) carrageenan-induced paw edema model. Photographic images show the gross pathology of paws from i.p.l. saline plus s.c. vehicle (dimethyl sulfoxide [DMSO]) (A, E), i.p.l. carrageenan plus s.c. vehicle (B, F), i.p.l. carrageenan plus s.c. sinularin (20 mg/kg) (C, G), and i.p.l. carrageenan plus s.c. sinularin (80 mg/kg) (D, H) groups. The lateral view of paws are shown in A, B, C, and D; top view of paws are shown in E, F, G, and H. The images (A–H) were taken 6 h after i.p.l. saline or carrageenan injection (as well as 7 h after s.c. vehicle or sinularin injection). The unit of scale bars shown in images A–H is in centimeter. S.c. sinularin of 20 mg/kg and 80 mg/kg reduced levels of carrageenan-induced edema of paw, and s.c. sinularin 80 mg/kg also clearly inhibited redness induced by carrageenan in rats than that by 20 mg/kg sinularin. We used 20 mg/kg indomethacin as a positive control. The time course of the percentage change of the increase in paw volume induced by i.p.l. injection of carrageenan in rats pretreated with vehicle, sinularin (80 mg/kg), or indomethacin (20 mg/kg) (I), administrated 1 h before carrageenan injection. Basal volume of each rat paw before i.p.l. saline or carrageenan injection was considered to be 100%, and the increase in paw volume is shown as the percentage change from the basal values by subtracting the basal paw volume from the paw volume measured at each time point. Area under the edematous effect-time curve (J), which was transformed from image I, for s.c. vehicle, sinularin (80 mg/kg), or indomethacin (20 mg/kg) injection, extending from 3 to 24 h after i.p.l. carrageenan injection. Carrageenan-induced paw edema was well-developed by nearly 3 h and persisted for at least 24 h. This phenomenon was significantly reduced by s.c. sinularin (80 mg/kg) or indomethacin (20 mg/kg). The duration of the anti-edematous effects of sinularin (80 mg/kg) or indomethacin (20 mg/kg) are shown as the area under the curve (AUC). Each point or bar in images I and J represent the mean ± standard error of the mean (SEM) of 6 rats per group. * P < 0.05 compared with the same time points in i.p.l. carrageenan plus s.c. vehicle group.

Mentions: According to the results in LPS-stimulated RAW 264.7 macrophages, we considered that 10–20 μM doses of sinularin both inhibit LPS-induced iNOS and COX-2 and upregulates LPS-induced TGF-β. To estimate an effective in vivo dosage of sinularin, we used a previous formula developed by Caraci et al. to calculate in vivo dosage for central administration based on in vitro dosage [23], considering that the CSF volume of a 300 g rat equals about 580 μL [24]. Hence, we estimated that the dosage of sinularin for central administration to a 300 g rat should be approximately 1.94–3.87 μg. Systemic dosage (mg/kg level) is well-known to be approximately equivalent to 1000 times of central dosage (μg/kg level), so we supposed that the sinularin dosage for systemic administration to a rat was at least about 6.47–12.9 mg/kg. Therefore, we selected two dosages of sinularin, 20 mg/kg and 80 mg/kg, administrated 1 h before carrageenan injection, for following preliminary tests. We prepared the following 4 groups of rats: intraplantar (i.p.l.) saline plus subcutaneous (s.c.) vehicle (dimethyl sulfoxide [DMSO]), i.p.l. carrageenan plus s.c. vehicle, i.p.l. carrageenan plus s.c. sinularin (20 mg/kg), and i.p.l. carrageenan plus s.c. sinularin (80 mg/kg) groups. At 6 h after i.p.l. carrageenan injection, carrageenan caused visible redness and swelling of rat paw (Figure 3B,F) compared with the i.p.l. saline plus s.c. vehicle group ( 3A,E B,F), both s.c. 20 mg/kg ( 3C,G D,H) sinularin inhibited carrageenan-induced paw edema; moreover, s.c. 80 mg/kg sinularin also clearly reduced redness induced by carrageenan in rats that received 20 mg/kg of sinularin. Next, we chose a dose of 80 mg/kg for evaluating the anti-edematous effect of sinularin on i.p.l. carrageenan-induced paw edema. We used 20 mg/kg indomethacin as a positive control [25,26]. The time course of percentage change of increased paw volume induced by i.p.l. injection of carrageenan was inhibited by sinularin (80 mg/kg) or indomethacin (20 mg/kg) (Figure 3I). Analysis of AUC of the edematous effect-time curve also supports the anti-edematous effect of sinularin (Figure 3J). Based on the above findings, we selected a dose of 80 mg/kg of sinularin for following nociceptive behavioral testing.


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)

Anti-redness and anti-edematous effects of subcutaneous (s.c.) sinularin in intraplantar (i.p.l.) carrageenan-induced paw edema model. Photographic images show the gross pathology of paws from i.p.l. saline plus s.c. vehicle (dimethyl sulfoxide [DMSO]) (A, E), i.p.l. carrageenan plus s.c. vehicle (B, F), i.p.l. carrageenan plus s.c. sinularin (20 mg/kg) (C, G), and i.p.l. carrageenan plus s.c. sinularin (80 mg/kg) (D, H) groups. The lateral view of paws are shown in A, B, C, and D; top view of paws are shown in E, F, G, and H. The images (A–H) were taken 6 h after i.p.l. saline or carrageenan injection (as well as 7 h after s.c. vehicle or sinularin injection). The unit of scale bars shown in images A–H is in centimeter. S.c. sinularin of 20 mg/kg and 80 mg/kg reduced levels of carrageenan-induced edema of paw, and s.c. sinularin 80 mg/kg also clearly inhibited redness induced by carrageenan in rats than that by 20 mg/kg sinularin. We used 20 mg/kg indomethacin as a positive control. The time course of the percentage change of the increase in paw volume induced by i.p.l. injection of carrageenan in rats pretreated with vehicle, sinularin (80 mg/kg), or indomethacin (20 mg/kg) (I), administrated 1 h before carrageenan injection. Basal volume of each rat paw before i.p.l. saline or carrageenan injection was considered to be 100%, and the increase in paw volume is shown as the percentage change from the basal values by subtracting the basal paw volume from the paw volume measured at each time point. Area under the edematous effect-time curve (J), which was transformed from image I, for s.c. vehicle, sinularin (80 mg/kg), or indomethacin (20 mg/kg) injection, extending from 3 to 24 h after i.p.l. carrageenan injection. Carrageenan-induced paw edema was well-developed by nearly 3 h and persisted for at least 24 h. This phenomenon was significantly reduced by s.c. sinularin (80 mg/kg) or indomethacin (20 mg/kg). The duration of the anti-edematous effects of sinularin (80 mg/kg) or indomethacin (20 mg/kg) are shown as the area under the curve (AUC). Each point or bar in images I and J represent the mean ± standard error of the mean (SEM) of 6 rats per group. * P < 0.05 compared with the same time points in i.p.l. carrageenan plus s.c. vehicle group.
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marinedrugs-10-01899-f003: Anti-redness and anti-edematous effects of subcutaneous (s.c.) sinularin in intraplantar (i.p.l.) carrageenan-induced paw edema model. Photographic images show the gross pathology of paws from i.p.l. saline plus s.c. vehicle (dimethyl sulfoxide [DMSO]) (A, E), i.p.l. carrageenan plus s.c. vehicle (B, F), i.p.l. carrageenan plus s.c. sinularin (20 mg/kg) (C, G), and i.p.l. carrageenan plus s.c. sinularin (80 mg/kg) (D, H) groups. The lateral view of paws are shown in A, B, C, and D; top view of paws are shown in E, F, G, and H. The images (A–H) were taken 6 h after i.p.l. saline or carrageenan injection (as well as 7 h after s.c. vehicle or sinularin injection). The unit of scale bars shown in images A–H is in centimeter. S.c. sinularin of 20 mg/kg and 80 mg/kg reduced levels of carrageenan-induced edema of paw, and s.c. sinularin 80 mg/kg also clearly inhibited redness induced by carrageenan in rats than that by 20 mg/kg sinularin. We used 20 mg/kg indomethacin as a positive control. The time course of the percentage change of the increase in paw volume induced by i.p.l. injection of carrageenan in rats pretreated with vehicle, sinularin (80 mg/kg), or indomethacin (20 mg/kg) (I), administrated 1 h before carrageenan injection. Basal volume of each rat paw before i.p.l. saline or carrageenan injection was considered to be 100%, and the increase in paw volume is shown as the percentage change from the basal values by subtracting the basal paw volume from the paw volume measured at each time point. Area under the edematous effect-time curve (J), which was transformed from image I, for s.c. vehicle, sinularin (80 mg/kg), or indomethacin (20 mg/kg) injection, extending from 3 to 24 h after i.p.l. carrageenan injection. Carrageenan-induced paw edema was well-developed by nearly 3 h and persisted for at least 24 h. This phenomenon was significantly reduced by s.c. sinularin (80 mg/kg) or indomethacin (20 mg/kg). The duration of the anti-edematous effects of sinularin (80 mg/kg) or indomethacin (20 mg/kg) are shown as the area under the curve (AUC). Each point or bar in images I and J represent the mean ± standard error of the mean (SEM) of 6 rats per group. * P < 0.05 compared with the same time points in i.p.l. carrageenan plus s.c. vehicle group.
Mentions: According to the results in LPS-stimulated RAW 264.7 macrophages, we considered that 10–20 μM doses of sinularin both inhibit LPS-induced iNOS and COX-2 and upregulates LPS-induced TGF-β. To estimate an effective in vivo dosage of sinularin, we used a previous formula developed by Caraci et al. to calculate in vivo dosage for central administration based on in vitro dosage [23], considering that the CSF volume of a 300 g rat equals about 580 μL [24]. Hence, we estimated that the dosage of sinularin for central administration to a 300 g rat should be approximately 1.94–3.87 μg. Systemic dosage (mg/kg level) is well-known to be approximately equivalent to 1000 times of central dosage (μg/kg level), so we supposed that the sinularin dosage for systemic administration to a rat was at least about 6.47–12.9 mg/kg. Therefore, we selected two dosages of sinularin, 20 mg/kg and 80 mg/kg, administrated 1 h before carrageenan injection, for following preliminary tests. We prepared the following 4 groups of rats: intraplantar (i.p.l.) saline plus subcutaneous (s.c.) vehicle (dimethyl sulfoxide [DMSO]), i.p.l. carrageenan plus s.c. vehicle, i.p.l. carrageenan plus s.c. sinularin (20 mg/kg), and i.p.l. carrageenan plus s.c. sinularin (80 mg/kg) groups. At 6 h after i.p.l. carrageenan injection, carrageenan caused visible redness and swelling of rat paw (Figure 3B,F) compared with the i.p.l. saline plus s.c. vehicle group ( 3A,E B,F), both s.c. 20 mg/kg ( 3C,G D,H) sinularin inhibited carrageenan-induced paw edema; moreover, s.c. 80 mg/kg sinularin also clearly reduced redness induced by carrageenan in rats that received 20 mg/kg of sinularin. Next, we chose a dose of 80 mg/kg for evaluating the anti-edematous effect of sinularin on i.p.l. carrageenan-induced paw edema. We used 20 mg/kg indomethacin as a positive control [25,26]. The time course of percentage change of increased paw volume induced by i.p.l. injection of carrageenan was inhibited by sinularin (80 mg/kg) or indomethacin (20 mg/kg) (Figure 3I). Analysis of AUC of the edematous effect-time curve also supports the anti-edematous effect of sinularin (Figure 3J). Based on the above findings, we selected a dose of 80 mg/kg of sinularin for following nociceptive behavioral testing.

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