Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of LPS-induced TLR4 and MyD88 expression, and interaction between LPS and TLR4 by cordycepin in LPS-stimulated RAW 264.7 macrophages.Notes: (A) Cells were pretreated with different cordycepin concentrations for 1 hour prior to LPS treatment, and total proteins were isolated at 6 hours after LPS treatment. The levels of TLR4 and MyD88 proteins were assessed by Western blot analyses using the anti-TLR4 and anti-MyD88 antibodies and an enhanced chemiluminescence detection system. Actin was used as the internal control. (B) Cells were incubated with AF-LPS for 1 hour in the absence or presence of cordycepin (30 μg/mL), and the LPS binding in the surface of RAW 264.7 cells was then measured by flow cytometry. (C) Cells were incubated with 100 ng/mL AF-LPS for 30 minutes in the absence or presence of cordycepin (30 μg/mL), and the interaction between AF-LPS and TLR4 was then detected by fluorescence microscopy using an anti-TLR4 antibody.Abbreviations: LPS, lipopolysaccharide; AF-LPS, Alexa Fluor 594-conjugated LPS; TLR4, Toll-like receptor 4; MyD88, myeloid differentiation factor 88.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4206205&req=5

f5-dddt-8-1941: Inhibition of LPS-induced TLR4 and MyD88 expression, and interaction between LPS and TLR4 by cordycepin in LPS-stimulated RAW 264.7 macrophages.Notes: (A) Cells were pretreated with different cordycepin concentrations for 1 hour prior to LPS treatment, and total proteins were isolated at 6 hours after LPS treatment. The levels of TLR4 and MyD88 proteins were assessed by Western blot analyses using the anti-TLR4 and anti-MyD88 antibodies and an enhanced chemiluminescence detection system. Actin was used as the internal control. (B) Cells were incubated with AF-LPS for 1 hour in the absence or presence of cordycepin (30 μg/mL), and the LPS binding in the surface of RAW 264.7 cells was then measured by flow cytometry. (C) Cells were incubated with 100 ng/mL AF-LPS for 30 minutes in the absence or presence of cordycepin (30 μg/mL), and the interaction between AF-LPS and TLR4 was then detected by fluorescence microscopy using an anti-TLR4 antibody.Abbreviations: LPS, lipopolysaccharide; AF-LPS, Alexa Fluor 594-conjugated LPS; TLR4, Toll-like receptor 4; MyD88, myeloid differentiation factor 88.

Mentions: We next assessed the effects of cordycepin on the LPS-activated TLR4 signaling pathway to further determine the mechanisms underlying the anti-inflammatory effects of cordycepin. As indicated in Figure 5A, TLR4 protein expression increased significantly in RAW 264.7 cells treated with LPS compared with that in untreated cells, which was accompanied by up-regulation of MyD88 expression. However, treatment with cordycepin before LPS stimulation almost completely blocked the LPS-induced induction of TLR4 and MyD88. In addition, treatment with LPS in the presence of cordycepin significantly prevented the binding of LPS to the BV2 cell surface (Figure 5B). We further tested whether cordycepin could inhibit the interaction between LPS and TLR4 in RAW 264.7 cells using AF-LPS (Figure 5C). When cells were treated with AF-LPS alone, the fluorescence intensities of LPS and TLR4 were observed outside the cell membrane by immunofluorescence assay (Figure 5B, LPS panel). However, in the presence of cordycepin, the fluorescence intensity of TLR4 was markedly inhibited (Figure 5C, LPS + cordycepin panel), suggesting LPS-stimulated activation of TLR4 signaling pathway was potently blocked by cordycepin.


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 LPS-induced TLR4 and MyD88 expression, and interaction between LPS and TLR4 by cordycepin in LPS-stimulated RAW 264.7 macrophages.Notes: (A) Cells were pretreated with different cordycepin concentrations for 1 hour prior to LPS treatment, and total proteins were isolated at 6 hours after LPS treatment. The levels of TLR4 and MyD88 proteins were assessed by Western blot analyses using the anti-TLR4 and anti-MyD88 antibodies and an enhanced chemiluminescence detection system. Actin was used as the internal control. (B) Cells were incubated with AF-LPS for 1 hour in the absence or presence of cordycepin (30 μg/mL), and the LPS binding in the surface of RAW 264.7 cells was then measured by flow cytometry. (C) Cells were incubated with 100 ng/mL AF-LPS for 30 minutes in the absence or presence of cordycepin (30 μg/mL), and the interaction between AF-LPS and TLR4 was then detected by fluorescence microscopy using an anti-TLR4 antibody.Abbreviations: LPS, lipopolysaccharide; AF-LPS, Alexa Fluor 594-conjugated LPS; TLR4, Toll-like receptor 4; MyD88, myeloid differentiation factor 88.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4206205&req=5

f5-dddt-8-1941: Inhibition of LPS-induced TLR4 and MyD88 expression, and interaction between LPS and TLR4 by cordycepin in LPS-stimulated RAW 264.7 macrophages.Notes: (A) Cells were pretreated with different cordycepin concentrations for 1 hour prior to LPS treatment, and total proteins were isolated at 6 hours after LPS treatment. The levels of TLR4 and MyD88 proteins were assessed by Western blot analyses using the anti-TLR4 and anti-MyD88 antibodies and an enhanced chemiluminescence detection system. Actin was used as the internal control. (B) Cells were incubated with AF-LPS for 1 hour in the absence or presence of cordycepin (30 μg/mL), and the LPS binding in the surface of RAW 264.7 cells was then measured by flow cytometry. (C) Cells were incubated with 100 ng/mL AF-LPS for 30 minutes in the absence or presence of cordycepin (30 μg/mL), and the interaction between AF-LPS and TLR4 was then detected by fluorescence microscopy using an anti-TLR4 antibody.Abbreviations: LPS, lipopolysaccharide; AF-LPS, Alexa Fluor 594-conjugated LPS; TLR4, Toll-like receptor 4; MyD88, myeloid differentiation factor 88.
Mentions: We next assessed the effects of cordycepin on the LPS-activated TLR4 signaling pathway to further determine the mechanisms underlying the anti-inflammatory effects of cordycepin. As indicated in Figure 5A, TLR4 protein expression increased significantly in RAW 264.7 cells treated with LPS compared with that in untreated cells, which was accompanied by up-regulation of MyD88 expression. However, treatment with cordycepin before LPS stimulation almost completely blocked the LPS-induced induction of TLR4 and MyD88. In addition, treatment with LPS in the presence of cordycepin significantly prevented the binding of LPS to the BV2 cell surface (Figure 5B). We further tested whether cordycepin could inhibit the interaction between LPS and TLR4 in RAW 264.7 cells using AF-LPS (Figure 5C). When cells were treated with AF-LPS alone, the fluorescence intensities of LPS and TLR4 were observed outside the cell membrane by immunofluorescence assay (Figure 5B, LPS panel). However, in the presence of cordycepin, the fluorescence intensity of TLR4 was markedly inhibited (Figure 5C, LPS + cordycepin panel), suggesting LPS-stimulated activation of TLR4 signaling pathway was potently blocked by cordycepin.

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