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Anti-inflammatory effects of cordycepin in lipopolysaccharide-stimulated RAW 264.7 macrophages through Toll-like receptor 4-mediated suppression of mitogen-activated protein kinases and NF-κB signaling pathways.

Choi YH, Kim GY, Lee HH - Drug Des Devel Ther (2014)

Bottom Line: This compound possesses many pharmacological properties, such as an ability to enhance immune function, as well as antioxidant, antiaging, and anticancer effects.In addition, pretreatment with cordycepin significantly inhibited lipopolysaccharide (LPS)-induced phosphorylation of mitogen-activating protein kinases and attenuated nuclear translocation of NF-κB by LPS, which was associated with abrogation of inhibitor kappa B-alpha degradation.Taken together, the results suggest that the inhibitory effects of cordycepin on LPS-stimulated inflammatory responses in RAW 264.7 macrophages are associated with suppression of mitogen-activating protein kinases and activation of NF-κB by inhibition of the Toll-like receptor 4 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dongeui University College of Korean Medicine, Busan, Republic of Korea ; Anti-Aging Research Center and Blue-Bio Industry RIC, Dongeui University, Busan, Republic of Korea.

ABSTRACT
Cordycepin is the main functional component of the Cordyceps species, which has been widely used in traditional Oriental medicine. This compound possesses many pharmacological properties, such as an ability to enhance immune function, as well as antioxidant, antiaging, and anticancer effects. In the present study, we investigated the anti-inflammatory effects of cordycepin using a murine macrophage RAW 264.7 cell model. Our data demonstrated that cordycepin suppressed production of proinflammatory mediators such as nitric oxide (NO) and prostaglandin E2 by inhibiting inducible NO synthase and cyclooxygenase-2 gene expression. Cordycepin also inhibited the release of proinflammatory cytokines, including tumor necrosis factor-alpha and interleukin-1-beta, through downregulation of respective mRNA expression. In addition, pretreatment with cordycepin significantly inhibited lipopolysaccharide (LPS)-induced phosphorylation of mitogen-activating protein kinases and attenuated nuclear translocation of NF-κB by LPS, which was associated with abrogation of inhibitor kappa B-alpha degradation. Furthermore, cordycepin potently inhibited the binding of LPS to macrophages and LPS-induced Toll-like receptor 4 and myeloid differentiation factor 88 expression. Taken together, the results suggest that the inhibitory effects of cordycepin on LPS-stimulated inflammatory responses in RAW 264.7 macrophages are associated with suppression of mitogen-activating protein kinases and activation of NF-κB by inhibition of the Toll-like receptor 4 signaling pathway.

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Inhibition of nitric oxide and PGE2 production by cordycepin in LPS-stimulated RAW 264.7 macrophages.Notes: Cells were pretreated with different concentrations of cordycepin for 1 hour before a 24-hour incubation with LPS (100 ng/mL). Nitrite content was measured using the Griess reaction (A) and PGE2 concentration was measured in culture medium using a commercial enzyme-linked immunosorbent assay kit (B). Each value indicates the mean ± standard deviation and is representative of results obtained from three independent experiments. *P<0.05 indicates a significant difference from the value obtained for cells treated with LPS in the absence of cordycepin. (C) Total RNA was isolated and reverse-transcribed using iNOS and COX-2 primers after a 6-hour LPS treatment. The resulting complementary DNAs were then subjected to polymerase chain reaction. The reaction products were subjected to 1% agarose gel electrophoresis and visualized by ethidium bromide staining. (D) The cells were sampled and lysed after a 24-hour treatment, and equal proteins were then separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Western blotting was performed using anti-iNOS and anti-COX-2 antibodies and an enhanced chemiluminescence detection system. GAPDH and actin were used as internal controls for the reverse transcriptase polymerase chain reaction and Western blot assays, respectively.Abbreviations: COX-2, cyclooxygenase-2; LPS, lipopolysaccharide; iNOS, inducible nitric oxide synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGE2, prostaglandin E2.
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f1-dddt-8-1941: Inhibition of nitric oxide and PGE2 production by cordycepin in LPS-stimulated RAW 264.7 macrophages.Notes: Cells were pretreated with different concentrations of cordycepin for 1 hour before a 24-hour incubation with LPS (100 ng/mL). Nitrite content was measured using the Griess reaction (A) and PGE2 concentration was measured in culture medium using a commercial enzyme-linked immunosorbent assay kit (B). Each value indicates the mean ± standard deviation and is representative of results obtained from three independent experiments. *P<0.05 indicates a significant difference from the value obtained for cells treated with LPS in the absence of cordycepin. (C) Total RNA was isolated and reverse-transcribed using iNOS and COX-2 primers after a 6-hour LPS treatment. The resulting complementary DNAs were then subjected to polymerase chain reaction. The reaction products were subjected to 1% agarose gel electrophoresis and visualized by ethidium bromide staining. (D) The cells were sampled and lysed after a 24-hour treatment, and equal proteins were then separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Western blotting was performed using anti-iNOS and anti-COX-2 antibodies and an enhanced chemiluminescence detection system. GAPDH and actin were used as internal controls for the reverse transcriptase polymerase chain reaction and Western blot assays, respectively.Abbreviations: COX-2, cyclooxygenase-2; LPS, lipopolysaccharide; iNOS, inducible nitric oxide synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGE2, prostaglandin E2.

Mentions: To determine the level of NO production, we measured nitrite released into the culture medium using Griess reagent. As a result, LPS alone markedly induced NO production compared with that generated by the control. However, pretreatment with cordycepin significantly repressed the levels of NO produced in LPS-stimulated RAW 264.7 cells in a concentration-dependent manner up to 30 μg/mL (Figure 1A). As shown in Figure 1B, treatment of RAW 264.7 cells with LPS also resulted in a marked increase in PGE2 release compared with the untreated control. However, cordycepin inhibited LPS-mediated PGE2 production in a concentration-dependent manner.


Anti-inflammatory effects of cordycepin in lipopolysaccharide-stimulated RAW 264.7 macrophages through Toll-like receptor 4-mediated suppression of mitogen-activated protein kinases and NF-κB signaling pathways.

Choi YH, Kim GY, Lee HH - Drug Des Devel Ther (2014)

Inhibition of nitric oxide and PGE2 production by cordycepin in LPS-stimulated RAW 264.7 macrophages.Notes: Cells were pretreated with different concentrations of cordycepin for 1 hour before a 24-hour incubation with LPS (100 ng/mL). Nitrite content was measured using the Griess reaction (A) and PGE2 concentration was measured in culture medium using a commercial enzyme-linked immunosorbent assay kit (B). Each value indicates the mean ± standard deviation and is representative of results obtained from three independent experiments. *P<0.05 indicates a significant difference from the value obtained for cells treated with LPS in the absence of cordycepin. (C) Total RNA was isolated and reverse-transcribed using iNOS and COX-2 primers after a 6-hour LPS treatment. The resulting complementary DNAs were then subjected to polymerase chain reaction. The reaction products were subjected to 1% agarose gel electrophoresis and visualized by ethidium bromide staining. (D) The cells were sampled and lysed after a 24-hour treatment, and equal proteins were then separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Western blotting was performed using anti-iNOS and anti-COX-2 antibodies and an enhanced chemiluminescence detection system. GAPDH and actin were used as internal controls for the reverse transcriptase polymerase chain reaction and Western blot assays, respectively.Abbreviations: COX-2, cyclooxygenase-2; LPS, lipopolysaccharide; iNOS, inducible nitric oxide synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGE2, prostaglandin E2.
© Copyright Policy
Related In: Results  -  Collection

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f1-dddt-8-1941: Inhibition of nitric oxide and PGE2 production by cordycepin in LPS-stimulated RAW 264.7 macrophages.Notes: Cells were pretreated with different concentrations of cordycepin for 1 hour before a 24-hour incubation with LPS (100 ng/mL). Nitrite content was measured using the Griess reaction (A) and PGE2 concentration was measured in culture medium using a commercial enzyme-linked immunosorbent assay kit (B). Each value indicates the mean ± standard deviation and is representative of results obtained from three independent experiments. *P<0.05 indicates a significant difference from the value obtained for cells treated with LPS in the absence of cordycepin. (C) Total RNA was isolated and reverse-transcribed using iNOS and COX-2 primers after a 6-hour LPS treatment. The resulting complementary DNAs were then subjected to polymerase chain reaction. The reaction products were subjected to 1% agarose gel electrophoresis and visualized by ethidium bromide staining. (D) The cells were sampled and lysed after a 24-hour treatment, and equal proteins were then separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Western blotting was performed using anti-iNOS and anti-COX-2 antibodies and an enhanced chemiluminescence detection system. GAPDH and actin were used as internal controls for the reverse transcriptase polymerase chain reaction and Western blot assays, respectively.Abbreviations: COX-2, cyclooxygenase-2; LPS, lipopolysaccharide; iNOS, inducible nitric oxide synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGE2, prostaglandin E2.
Mentions: To determine the level of NO production, we measured nitrite released into the culture medium using Griess reagent. As a result, LPS alone markedly induced NO production compared with that generated by the control. However, pretreatment with cordycepin significantly repressed the levels of NO produced in LPS-stimulated RAW 264.7 cells in a concentration-dependent manner up to 30 μg/mL (Figure 1A). As shown in Figure 1B, treatment of RAW 264.7 cells with LPS also resulted in a marked increase in PGE2 release compared with the untreated control. However, cordycepin inhibited LPS-mediated PGE2 production in a concentration-dependent manner.

Bottom Line: This compound possesses many pharmacological properties, such as an ability to enhance immune function, as well as antioxidant, antiaging, and anticancer effects.In addition, pretreatment with cordycepin significantly inhibited lipopolysaccharide (LPS)-induced phosphorylation of mitogen-activating protein kinases and attenuated nuclear translocation of NF-κB by LPS, which was associated with abrogation of inhibitor kappa B-alpha degradation.Taken together, the results suggest that the inhibitory effects of cordycepin on LPS-stimulated inflammatory responses in RAW 264.7 macrophages are associated with suppression of mitogen-activating protein kinases and activation of NF-κB by inhibition of the Toll-like receptor 4 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dongeui University College of Korean Medicine, Busan, Republic of Korea ; Anti-Aging Research Center and Blue-Bio Industry RIC, Dongeui University, Busan, Republic of Korea.

ABSTRACT
Cordycepin is the main functional component of the Cordyceps species, which has been widely used in traditional Oriental medicine. This compound possesses many pharmacological properties, such as an ability to enhance immune function, as well as antioxidant, antiaging, and anticancer effects. In the present study, we investigated the anti-inflammatory effects of cordycepin using a murine macrophage RAW 264.7 cell model. Our data demonstrated that cordycepin suppressed production of proinflammatory mediators such as nitric oxide (NO) and prostaglandin E2 by inhibiting inducible NO synthase and cyclooxygenase-2 gene expression. Cordycepin also inhibited the release of proinflammatory cytokines, including tumor necrosis factor-alpha and interleukin-1-beta, through downregulation of respective mRNA expression. In addition, pretreatment with cordycepin significantly inhibited lipopolysaccharide (LPS)-induced phosphorylation of mitogen-activating protein kinases and attenuated nuclear translocation of NF-κB by LPS, which was associated with abrogation of inhibitor kappa B-alpha degradation. Furthermore, cordycepin potently inhibited the binding of LPS to macrophages and LPS-induced Toll-like receptor 4 and myeloid differentiation factor 88 expression. Taken together, the results suggest that the inhibitory effects of cordycepin on LPS-stimulated inflammatory responses in RAW 264.7 macrophages are associated with suppression of mitogen-activating protein kinases and activation of NF-κB by inhibition of the Toll-like receptor 4 signaling pathway.

Show MeSH
Related in: MedlinePlus