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

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.

Keywords: NF-κB; Toll-like receptor 4; anti-inflammation; cordycepin; mitogen-activated protein kinases.

Figure 1

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.

Figure 2

Inhibitory effects of cordycepin on TNF-α and IL-1β release induced by LPS in RAW 264.7 macrophages. Notes: Cells were treated with cordycepin following 1 hour of LPS treatment. The supernatants were prepared following 24 hours of treatment, and the amounts of TNF-α (A) and IL-1β (B) were measured by enzyme-linked immunosorbent assay. Data are shown as the mean ± standard deviation of three independent experiments. (*P<0.05 between the treated and the untreated control group). (C) Levels of IL-1β and TNF-α mRNA were assessed by reverse transcriptase polymerase chain reaction after 6 hours of treatment. (D) IL-1β and TNF-α protein expression was determined by Western blot analysis after 24 hours of treatment. GAPDH and actin were used as internal controls for the reverse transcriptase polymerase chain reaction and Western blot assays, respectively. Abbreviations: LPS, lipopolysaccharide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; TNF-α, tumor necrosis factor-alpha.

Figure 3

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.

Figure 4

Effects of cordycepin on LPS-induced mitogen-activated protein kinase phosphorylation in RAW 264.7 macrophages. Notes: Cells were treated with 100 ng/mL LPS for the indicated times (A) or treated with different concentrations of cordycepin 1 hour before LPS treatment for 30 minutes (B). Total proteins were prepared and separated on 10% sodium dodecyl sulfate-polyacrylamide gels, followed by Western blotting using the indicated antibodies. Abbreviations: ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide.

Figure 5

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.

Figure 6

Effects of the TLR4 inhibitor CLI-095 on nitric oxide and PGE₂ production in LPS-stimulated RAW 264.7 macrophages. Notes: (A, B) Cells were treated with 30 μg/mL cordycepin alone or in combination with 15 μM CLI-095 for 1 hour before LPS treatment. Following 24 hours of treatment, the amounts of nitric oxide and PGE₂ production were measured with the supernatants. The data are shown as the mean ± standard deviation of three independent experiments (*P<0.05 versus LPS treated cells; ^#P<0.05 versus cells treated with LPS plus cordycepin). The levels of iNOS and COX-2 mRNA (C) and protein (D) were assessed by reverse transcriptase polymerase chain reaction and Western blot assays after 6 hours and 24 hours of treatment, respectively. GAPDH and actin were used as internal controls, respectively. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LPS, lipopolysaccharide; iNOS, inducible nitric oxide synthase; PGE₂, prostaglandin E₂;COX-2, cyclooxygenase-2.

Figure 7

Effects of the TLR4 inhibitor CLI-095 on production of TNF-α and IL-1β in LPS-stimulated RAW 264.7 macrophages. Notes: (A, B) Cells were treated with cordycepin alone or in combination with CLI-095 for 1 hour before LPS treatment. Following 24 hours of treatment, the amounts of TNF-α and IL-1β production were measured in the supernatants. The data are shown as the mean ± standard deviation of three independent experiments (*P<0.05 versus LPS-treated cells; ^#P<0.05 versus cells treated with LPS plus cordycepin). TNF-α and IL-1β mRNA (C) and protein (D) levels were assessed by reverse transcriptase polymerase chain reaction and Western blot assays after 6 hours and 24 hours of treatment, respectively. GAPDH and actin were used as internal controls, respectively. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LPS, lipopolysaccharide; TLR4, Toll-like receptor 4; IL, interleukin; TNF-α, tumor necrosis factor-alpha.

Figure 8

Effects of cordycepin, CLI-095, and LPS on viability of RAW 264.7 macrophages. The cells were treated with the indicated concentrations of cordycepin, CLI-095, or LPS alone, or pretreated with cordycepin or CLI-095 for 1 hour before LPS treatment. Cell viability was assessed after 24 hours using MTT reduction assays. The data are shown as the mean ± standard deviation of three independent experiments. Abbreviation: LPS, lipopolysaccharide.

Figure 9

Schematic figure of possible signaling mechanisms of cordycepin in inhibition of the LPS-induced inflammatory response. Abbreviations: LPS, lipopolysaccharide; TLR4, Toll-like receptor 4; IL, interleukin; TNF-α, tumor necrosis factor-alpha; MyD88, myeloid differentiation factor 88; MAPKs, mitogen-activated protein kinases; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; IKK, inhibitor kappa B kinase; IRAK, IL-1R-associated kinase.