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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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Effects of cordycepin on LPS-induced nuclear translocation of NF-κB and degradation of IκBα in RAW 264.7 macrophages.Notes: Cells were treated with the indicated concentrations of cordycepin for 1 hour before LPS treatment (100 ng/mL) for the indicated times. (A) Nuclear and cytosolic proteins were resolved on 10% sodium dodecyl sulfate-polyacrylamide gels followed by Western blotting using anti-NF-κB/p65 and anti-IκBα antibodies. Nucleolin and actin were used as internal controls for the nuclear and cytosolic fractions, respectively. (B) Cells were pretreated with 30 μg/mL cordycepin for 1 hour prior to stimulation with LPS for 1 hour. Localization of NF-κB/p65 was visualized with a fluorescence microscope after immunofluorescence staining with anti-NF-κB/p65 antibody and fluorescein isothiocyanate-labeled anti-rabbit immunoglobulin G antibody (green). Nuclei of the corresponding cells were visualized with 4,6-diamidino-2-phenylindole (DAPI, blue). The cells were visualized using a fluorescence microscope.Abbreviations: LPS, lipopolysaccharide; IκBα, inhibitor kappa B-alpha; NF-κB, nuclear factor kappa B.
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f3-dddt-8-1941: Effects of cordycepin on LPS-induced nuclear translocation of NF-κB and degradation of IκBα in RAW 264.7 macrophages.Notes: Cells were treated with the indicated concentrations of cordycepin for 1 hour before LPS treatment (100 ng/mL) for the indicated times. (A) Nuclear and cytosolic proteins were resolved on 10% sodium dodecyl sulfate-polyacrylamide gels followed by Western blotting using anti-NF-κB/p65 and anti-IκBα antibodies. Nucleolin and actin were used as internal controls for the nuclear and cytosolic fractions, respectively. (B) Cells were pretreated with 30 μg/mL cordycepin for 1 hour prior to stimulation with LPS for 1 hour. Localization of NF-κB/p65 was visualized with a fluorescence microscope after immunofluorescence staining with anti-NF-κB/p65 antibody and fluorescein isothiocyanate-labeled anti-rabbit immunoglobulin G antibody (green). Nuclei of the corresponding cells were visualized with 4,6-diamidino-2-phenylindole (DAPI, blue). The cells were visualized using a fluorescence microscope.Abbreviations: LPS, lipopolysaccharide; IκBα, inhibitor kappa B-alpha; NF-κB, nuclear factor kappa B.

Mentions: Previous reports suggested that NF-κB is an important transcription factor regulating the expression of iNOS, COX-2, and inflammatory cytokines; thus, we explored whether cordycepin could block the NF-κB signaling pathway. Western blot analyses using cytosolic and nuclear fractions showed that the amount of NF-κB/p65 in the nucleus was markedly increased after 30 minutes of exposure to LPS alone, concomitant with degradation of Iκ B- alpha (IκBα) in the cytosol. However, LPS-induced NF-κB/p65 levels in the nuclear fraction were concentration-dependently reduced by cordycepin pretreatment, and LPS-induced IκBα degradation was obviously blocked by pretreatment with cordycepin (Figure 3A). Furthermore, the shift of NF-κB to the nucleus in RAW 264.7 cells was analyzed using immunofluorescence staining to clearly determine the influence of cordycepin on NF-κB/p65 nuclear translocation by LPS treatment (Figure 3B). The immunofluorescence images revealed that NF-κB/p65 was normally sequestered in the cytoplasm (Figure 3B, control panel), and that nuclear accumulation of NF-κB/p65 was strongly induced after stimulating RAW 264.7 cells with LPS (Figure 3B, LPS panel). However, nuclear translocation of NF-κB/p65 was not induced in the cells after pretreatment with cordycepin alone (Figure 3B, cordycepin panel), and the LPS-induced translocation of NF-κB/p65 was completely abolished after pretreating the cells with cordycepin (Figure 3B, LPS + cordycepin panel).


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)

Effects of cordycepin on LPS-induced nuclear translocation of NF-κB and degradation of IκBα in RAW 264.7 macrophages.Notes: Cells were treated with the indicated concentrations of cordycepin for 1 hour before LPS treatment (100 ng/mL) for the indicated times. (A) Nuclear and cytosolic proteins were resolved on 10% sodium dodecyl sulfate-polyacrylamide gels followed by Western blotting using anti-NF-κB/p65 and anti-IκBα antibodies. Nucleolin and actin were used as internal controls for the nuclear and cytosolic fractions, respectively. (B) Cells were pretreated with 30 μg/mL cordycepin for 1 hour prior to stimulation with LPS for 1 hour. Localization of NF-κB/p65 was visualized with a fluorescence microscope after immunofluorescence staining with anti-NF-κB/p65 antibody and fluorescein isothiocyanate-labeled anti-rabbit immunoglobulin G antibody (green). Nuclei of the corresponding cells were visualized with 4,6-diamidino-2-phenylindole (DAPI, blue). The cells were visualized using a fluorescence microscope.Abbreviations: LPS, lipopolysaccharide; IκBα, inhibitor kappa B-alpha; NF-κB, nuclear factor kappa B.
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f3-dddt-8-1941: Effects of cordycepin on LPS-induced nuclear translocation of NF-κB and degradation of IκBα in RAW 264.7 macrophages.Notes: Cells were treated with the indicated concentrations of cordycepin for 1 hour before LPS treatment (100 ng/mL) for the indicated times. (A) Nuclear and cytosolic proteins were resolved on 10% sodium dodecyl sulfate-polyacrylamide gels followed by Western blotting using anti-NF-κB/p65 and anti-IκBα antibodies. Nucleolin and actin were used as internal controls for the nuclear and cytosolic fractions, respectively. (B) Cells were pretreated with 30 μg/mL cordycepin for 1 hour prior to stimulation with LPS for 1 hour. Localization of NF-κB/p65 was visualized with a fluorescence microscope after immunofluorescence staining with anti-NF-κB/p65 antibody and fluorescein isothiocyanate-labeled anti-rabbit immunoglobulin G antibody (green). Nuclei of the corresponding cells were visualized with 4,6-diamidino-2-phenylindole (DAPI, blue). The cells were visualized using a fluorescence microscope.Abbreviations: LPS, lipopolysaccharide; IκBα, inhibitor kappa B-alpha; NF-κB, nuclear factor kappa B.
Mentions: Previous reports suggested that NF-κB is an important transcription factor regulating the expression of iNOS, COX-2, and inflammatory cytokines; thus, we explored whether cordycepin could block the NF-κB signaling pathway. Western blot analyses using cytosolic and nuclear fractions showed that the amount of NF-κB/p65 in the nucleus was markedly increased after 30 minutes of exposure to LPS alone, concomitant with degradation of Iκ B- alpha (IκBα) in the cytosol. However, LPS-induced NF-κB/p65 levels in the nuclear fraction were concentration-dependently reduced by cordycepin pretreatment, and LPS-induced IκBα degradation was obviously blocked by pretreatment with cordycepin (Figure 3A). Furthermore, the shift of NF-κB to the nucleus in RAW 264.7 cells was analyzed using immunofluorescence staining to clearly determine the influence of cordycepin on NF-κB/p65 nuclear translocation by LPS treatment (Figure 3B). The immunofluorescence images revealed that NF-κB/p65 was normally sequestered in the cytoplasm (Figure 3B, control panel), and that nuclear accumulation of NF-κB/p65 was strongly induced after stimulating RAW 264.7 cells with LPS (Figure 3B, LPS panel). However, nuclear translocation of NF-κB/p65 was not induced in the cells after pretreatment with cordycepin alone (Figure 3B, cordycepin panel), and the LPS-induced translocation of NF-κB/p65 was completely abolished after pretreating the cells with cordycepin (Figure 3B, LPS + cordycepin panel).

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