Resveratrol inhibits inflammation induced by heat-killed Listeria monocytogenes

Abstract

Resveratrol is a polyphenolic compound in red wine that has antioxidant and cardioprotective effects in animal models. Listeria monocytogenes is a pathogen that mainly affects immunocompromised individuals and is initially detected at the cell surface or in phagosomes by toll-like receptor 2. Many antioxidants also exert anti-inflammatory activities; therefore, we evaluated the anti-inflammatory properties of resveratrol by studying the various inflammatory responses induced by heat-killed L. monocytogenes (HKLM). Resveratrol strongly blocked HKLM-induced NADPH oxidase-1 mRNA and reactive oxygen species production by macrophages. Resveratrol also suppressed monocyte chemotactic protein-1 expression, cyclooxygenase-2 expression, prostaglandin production, inducible nitric oxide (NO) synthase expression, and NO production induced by HKLM. We investigated the signaling pathway involved in the resveratrol effect. HKLM stimulated glycogen synthase kinase 3β (GSK3β) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. The involvement of GSK3β and ERK1/2 was tested using inhibitors. While the GSK3β inhibitor LiCl potentiated the effect of HKLM, the MEK inhibitor U0126 blocked these responses. Additionally, pretreatment with resveratrol blocked phosphorylation of both kinases induced by HKLM. These results suggest that HKLM is strong inducer of inflammatory mediators, and that the inhibitory effect of resveratrol may be mediated by the GSK3β and ERK1/2 pathways.

FIG. 1.

Resveratrol suppresses heat-killed Listeria monocytogenes (HKLM)-induced NADPH oxidase-1 (Nox-1) expression and reactive oxygen species generation. (A) Raw264.7 cells were incubated for 1–6 h with 10⁸ cells/mL HKLM. Total mRNAs were isolated at the end of incubation. Nox-1 mRNA expression was determined by reverse transcription–polymerase chain reaction (RT-PCR) and was normalized to that of β-actin. (B) Cells were pretreated with 30 μM resveratrol for 1 h and stimulated with 10⁸ cells/mL HKLM for the indicated times. Nox-1 mRNA expression was determined by real-time PCR or RT-PCR. The Nox-1 mRNA level was normalized to that of β-actin. (C) Cells were changed to a fresh serum-free DMEM containing dichlorofluorescein diacetate (DCF-DA) (50 μM) for 40 min. The loaded cells were then incubated with 10⁸ cells/mL HKLM in the absence or presence of 30 μM resveratrol, and changes in fluorescence intensity were monitored by flow cytometry. Each data point represents the mean±standard deviation of three independent experiments (*P<.05 and **P<.01).

FIG. 2.

Resveratrol inhibits HKLM-induced monocyte chemotactic protein-1 (MCP-1) expression. (A) Raw264.7 cells were incubated for 1–6 h with 10⁸ cells/mL HKLM. Total mRNAs were isolated at the end of incubation. MCP-1 mRNA expression was determined by RT-PCR and was normalized to that of β-actin. (B) Cells were pretreated with 30 μM resveratrol for 1 h and stimulated with 10⁸ cells/mL HKLM for the indicated times. MCP-1 mRNA expression was determined by real-time PCR or RT-PCR. MCP-1 mRNA was normalized to that of β-actin. Each data point represents the mean±standard deviation of three independent experiments (*P<.05 and **P<.01).

FIG. 3.

Resveratrol suppress cyclooxygenase-2 (COX-2) expression induced by HKLM. (A) Cells were treated with the indicated concentrations of resveratrol for 1 h before stimulation with 10⁸ cells/mL HKLM. Total lysates were isolated at the end of incubation, and COX-2 proteins were analyzed by Western blot. (B) Cells were pretreated with the indicated concentrations of resveratrol for 1 h and stimulated with 10⁸ cells/mL HKLM for 6 h. Prostaglandin E₂ (PGE₂) and PGI₂ levels in cell supernatants were analyzed with an enzyme immunoassay kit. Each data point represents the mean±standard deviation of three independent experiments (**P<.01).

FIG. 4.

Resveratrol suppresses inducible nitric oxide synthase (iNOS) expression induced by HKLM. (A) Cells were treated with resveratrol at the indicated concentrations for 1 h before stimulation with 10⁸ cells/mL HKLM. Total lysates were isolated at the end of incubation, and the iNOS protein was analyzed by Western blot. (B) Cells were pretreated with the indicated concentrations of resveratrol for 1 h and stimulated with 10⁸ cells/mL HKLM for 24 h. NO amounts in cell supernatants were analyzed with the Griess reagent. Each data point represents the mean±standard deviation of three independent experiments (*P<.05 and **P<.01).

FIG. 5.

Extracellular signal-regulated kinase 1/2 (ERK1/2) and glycogen synthase kinase 3β (GSK3β) are targets for the effects of resveratrol. (A) Raw264.7 cells were stimulated with 10⁸ cells/mL HKLM for the indicated times. ERK1/2 and GSK3β phosphorylation was determined by Western blot using phosphoprotein antibodies and was normalized to the total protein. (B) Cells were treated with 10⁸ cells/mL HKLM in the absence or presence of 30 μM resveratrol for the indicated times. ERK1/2 or GSK3β phosphorylation was determined by Western blot using phosphoprotein antibodies and was normalized to the total protein. (C) Cells were treated with 10⁸ cells/mL HKLM in the absence or presence of 1 μM U0126. MCP-1 mRNA expression was determined by RT-PCR and was normalized to that of β-actin. iNOS and COX-2 proteins were analyzed by Western blot. (D) Cells were treated with 10⁸ cells/mL HKLM in the absence or presence of 20 mM LiCl. MCP-1 mRNA expression was determined by RT-PCR and was normalized to that of β-actin. iNOS and COX-2 proteins were analyzed by Western blot.