Cilostazol is anti-inflammatory in BV2 microglial cells by inactivating nuclear factor-kappaB and inhibiting mitogen-activated protein kinases

Abstract

Background and purpose: Cilostazol is a specific inhibitor of 3'-5'-cyclic adenosine monophosphate (cAMP) phosphodiesterase, which is widely used to treat ischemic symptoms of peripheral vascular disease. Although cilostazol has been shown to exhibit vasodilator properties as well as antiplatelet and anti-inflammatory effects, its cellular mechanism in microglia is unknown. In the present study, we assessed the anti-inflammatory effect of cilostazol on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated murine BV2 microglia.

Experimental approach: We examined the effects of cilostazol on LPS-induced nuclear factor-kappaB (NF-kappaB) activation and phosphorylation of mitogen-activated protein kinases (MAPKs).

Key results: Cilostazol suppressed production of nitric oxide (NO), prostaglandin E(2) (PGE(2)) and the proinflammatory cytokines, interleukin-1 (IL-1), tumour necrosis factor-alpha, and monocyte chemoattractant protein-1 (MCP-1), in a concentration-dependent manner. Inhibitory effects of cilostazol were not affected by treatment with an adenylate cyclase inhibitor, SQ 22536, indicating that these actions of cilostazol were cAMP-independent. Cilostazol significantly inhibited the DNA binding and transcriptional activity of NF-kappaB. Moreover, cilostazol blocked signalling upstream of NF-kappaB activation by inhibiting extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinase (JNK), but without affecting the activity of p38 MAPK.

Conclusion and implications: Our results demonstrate that suppression of the NF-kappaB, ERK, JNK signalling pathways may inhibit LPS-induced NO and PGE(2) production. Therefore, cilostazol may have therapeutic potential for neurodegenerative diseases by inhibiting pro-inflammatory mediators and cytokine production in activated microglia.

Figure 1

Inhibition of NO and PGE₂ production by cilostazol in LPS-stimulated BV2 microglia. BV2 microglia were pretreated with various concentrations of cilostazol (10, 20 and 30 µM) for 6 h before incubation with LPS (1 µg·mL⁻¹) for 24 h. Nitrite content was measured using the Griess reaction (A) and the PGE₂ concentration in culture media was measured using a commercial ELISA kit (B). Each value indicates the mean ± SEM, and is representative of results obtained from four independent experiments. *P < 0.05 indicates a significant difference from cells treated with LPS in the absence of cilostazol. ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide; NO, nitric oxide; PGE₂, prostaglandin E₂.

Figure 2

Effect of cilostazol on the viability of BV2 microglia. Cells were treated with the indicated concentrations of cilostazol (10, 20 and 30 µM) for 6 h prior to LPS (1 µg·mL⁻¹) treatment for 24 h. Cell viability was assessed by MTT reduction assays, and the results are expressed as the percentage of surviving cells over control cells (no addition of cilostazol). Each value indicates the mean ± SEM and is representative of results obtained from three independent experiments. LPS, lipopolysaccharide.

Figure 3

Inhibition of iNOS and COX-2 protein (A) and mRNA (B) expression by cilostazol in LPS-stimulated BV2 microglia. (A) BV2 microglia were pretreated with cilostazol (10, 20 and 30 µM) 6 h prior to incubation with LPS (1 µg·mL⁻¹) for 24 h. Cell lysates were then prepared and Western blots were performed using an antibodies specific for murine iNOS or COX-2. (B) After LPS treatment for 6 h, total RNA was prepared for RT-PCR analysis of iNOS and COX-2 gene expression in LPS-stimulated BV2 microglia. β-actin and GAPDH were used as internal controls for Western blot analysis and RT-PCR assays respectively. The experiment was repeated three times and similar results were obtained. Each value indicates the mean ± SEM and is representative of results obtained from five independent experiments. *P < 0.05 indicates a significant difference from cells treated with LPS in the absence of cilostazol. COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide.

Figure 4

Effect of cilostazol induced-intracellular cAMP on iNOS and COX-2 expression in LPS-stimulated BV2 microglia. (A) Intracellular cAMP levels. Cells were treated with LPS and cilostazol (Cilo) for 10 min. Each value indicates the mean ± SEM, and is representative of results obtained from three independent experiments. *P < 0.05 indicates a significant difference from untreated cells (MED). (B) Effect of cAMP-elevating agents on iNOS and COX-2 expression. Cells were pretreated with cilostazol (Cilo, 30 µM, 6 h), forskolin (FSK, 10 µM, 30 min) and dibutyryl-cAMP (dbC, 100 µM, 30 min) and then stimulated with LPS (1 µg·mL⁻¹) for another 24 h. (C) Effect of cilostazol on iNOS and COX-2 expression by SQ 22536, cAMP antagonist (SQ) in LPS-stimulated microglia. Cells were pretreated with SQ (30 µM) for 10 min, followed by cilostazol (30 µM) for 6 h, and then stimulated with LPS (1 µg·mL⁻¹) for another 24 h. Cell lysates were prepared for the determination of protein levels of iNOS and COX-2. Each value indicates the mean ± SEM and is representative of results obtained from three independent experiments. *P < 0.05 indicates a significant difference from cells treated with LPS in the absence of cilostazol. cAMP, 3′-5′-cyclic adenosine monophosphate; COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide.

Figure 5

Effect of cilostazol on proinflammatory cytokines in LPS-stimulated BV2 microglia. Cells were pretreated with the indicated concentrations of cilostazol for 6 h before LPS treatment (1 µg·mL⁻¹), and total RNA and the supernatants were isolated at 6 h or 24 h after LPS treatment respectively. (A) After incubation for 24 h, the levels of IL-1β and TNF-α present in the supernatants were measured. (B) After incubation for 6 h, the levels of IL-1β and TNF-α mRNA were determined by RT-PCR. Each value indicates the mean ± SEM and is representative of results obtained from three independent experiments. *P < 0.05 indicates a significant difference from cells treated with LPS in the absence of cilostazol. IL, interleukin; LPS, lipopolysaccharide; TNF, tumour necrosis factor.

Figure 6

Effect of cilostazol on MCP-1 production in LPS-stimulated BV2 microglia. BV2 microglia were pretreated with cilostazol (10, 20 and 30 µM) 6 h prior to incubation with LPS (1 µg·mL⁻¹), and total RNA and the supernatants were isolated at 6 h and 24 h after LPS treatment respectively. (A) After LPS treatment for 6 h, total RNA was prepared for RT-PCR analysis of MCP-1 mRNA gene expression in LPS-stimulated BV2 microglia. Results are representative of those obtained from three independent experiments, and the densitometric data below the RT-PCR results are presented as fold changes as compared with their respective controls. (B) Extracellular levels of MCP-1 were measured in culture media using commercial ELISA kits. Experiments were repeated three times and similar results were obtained. *P < 0.05 indicates a significant difference from cells treated with LPS in the absence of cilostazol. ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1.

Figure 7

Effect of cilostazol on NF-κB activity in LPS-stimulated BV2 microglia. (A) Nuclear extracts (4 µg) were prepared and analysed for the DNA binding activity of NF-κB by EMSA. Binding specificity was determined using the unlabelled probe (100-fold in excess; shown as ‘cold’) to compete with the labelled oligonucleotide. BV2 microglial cells were pretreated with vehicle or the indicated concentrations of cilostazol for 6 h before stimulation with LPS (1 µg·mL⁻¹) for another 1 h. The results shown are representative of three independent experiments. *P < 0.05 indicates a significant difference from LPS alone group. (B) BV2 microglia cells were pretreated with 30 µM cilostazol for 6 h prior to stimulation with LPS (1 µg·mL⁻¹) for 1 h. p65 protein localization was determined using an anti-p65 antibody and a FITC-labelled anti-rabbit IgG antibody, and cells were visualized using laser confocal scanning microscopy. A representative result from three to five independent experiments is shown. EMSA, electrophoretic mobility shift assay; LPS, lipopolysaccharide; NF-κB, nuclear factor-κB.

Figure 8

Effect of cilostazol on LPS-induced phosphorylation of ERK-1/2, SAPK/JNK and p38 MAP kinase in BV-2 microglia. BV-2 microglia were treated with vehicle or the indicated concentrations of cilostazol for 6 h before being incubated with LPS (1 µg·mL⁻¹) for 30 min. Cell extracts were then prepared and subjected to Western blotting with antibodies specific for phosphorylated forms of ERK-1/2, SAPK/JNK and p38. The results presented are representative of three independent experiments. *P < 0.05 indicates a significant difference from cells treated with LPS in the absence of cilostazol. ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MAP, mitogen-activated protein; SAPK, stress-activated protein kinase.