Anti-Inflammatory Activity of Bee Venom in BV2 Microglial Cells: Mediation of MyD88-Dependent NF-κB Signaling Pathway

Abstract

Bee venom has long been used as a traditional folk medicine in Korea. It has been reportedly used for the treatment of arthritis, cancer, and inflammation. Although its anti-inflammatory activity in lipopolysaccharide- (LPS-) stimulated inflammatory cells has been reported, the exact mechanism of its anti-inflammatory action has not been fully elucidated. Therefore, the aim of this study was to investigate the anti-inflammatory mechanism of bee venom in BV2 microglial cells. We first investigated whether NO production in LPS-activated BV2 cells was inhibited by bee venom, and further iNOS mRNA and protein expressions were determined. The mRNA and protein levels of proinflammatory cytokines were examined using semiquantitative RT-PCR and immunoblotting, respectively. Moreover, modulation of the transcription factor NF-κB by bee venom was also investigated using a luciferase assay. LPS-induced NO production in BV2 microglial cells was significantly inhibited in a concentration-dependent manner upon pretreatment with bee venom. Bee venom markedly reduced the mRNA expression of COX-2, TNF-α, IL-1β, and IL-6 and suppressed LPS-induced activation of MyD88 and IRAK1 and phosphorylation of TAK1. Moreover, NF-κB translocation by IKKα/β phosphorylation and subsequent IκB-α degradation were also attenuated. Thus, collectively, these results indicate that bee venom exerts its anti-inflammatory activity via the IRAK1/TAK1/NF-κB signaling pathway.

Figure 1

HPLC analysis of bee venom extract used in this experiment.

Figure 2

Bee venom inhibits lipopolysaccharide- (LPS-) induced nitric oxide (NO) production in BV2 microglia. (a) The effect of bee venom on the LPS-induced NO production in BV2 cells. (b) The effect of bee venom on the cell viability of LPS-stimulated BV2 cells. BV2 microglial cells were pretreated with different concentrations of bee venom extract (0.625 μg/mL–2.5 μg/mL) for 30 min and then incubated with LPS (0.1 μg/mL) for 24 h. NO production was determined in the cell supernatants as described in Section 2. After determination of NO production, the viability was immediately examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described in Section 2. The data are presented as mean ± standard error (SEM), and the experiments were repeated three to five times. ^∗ p < 0.05  and  ^∗∗∗ p < 0.001 versus LPS alone. ^# p < 0.05 versus basal.

Figure 3

Bee venom inhibits the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA and protein in LPS-activated BV2 microglia. (a) The effect of bee venom on iNOS mRNA expression in LPS-stimulated BV2 cells. (b) The effect of bee venom on COX-2 mRNA expression in LPS-stimulated BV2 cells. (c) The effect of bee venom on iNOS and COX-2 protein expression in LPS-stimulated BV2 cells. BV2 microglial cells were pretreated with bee venom (0.625–2.5 μg/mL) or vehicle for 30 min and then stimulated with LPS (0.1 μg/mL) for 24 h. Then, total RNA was prepared (to assess mRNA expression), or protein was extracted as described in Section 2. ((a) and (b)) The levels of iNOS and COX-2 mRNA expression were determined using quantitative real-time polymerase chain reaction (PCR). The protein concentration of the cell extracts was determined with PRO-MEASURE (iNtRON Biotechnology, Korea). The protein separation and immunoblot procedures are described in Section 2. The data are presented as mean ± SEM, and experiments were performed three to five times. Representative images of experiments performed at least in triplicate are shown. ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001 versus LPS alone.

Figure 4

Bee venom inhibits the expression level of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA in LPS-stimulated BV2 microglia. Dose-dependent inhibition of TNF-α (a) and IL-6 (b) mRNA expression were assessed using quantitative real-time PCR. BV2 cells were pretreated with bee venom for 30 min, and then 0.1 μg/mL LPS was added and the cells were incubated for an additional 24 h. The total RNA preparation and real-time PCR were performed as described in Section 2. GAPDH was used as an internal control, and relative expression levels of TNF-α and IL-6 mRNA were calculated by normalization to GAPDH. The data are presented as mean ± SEM of three to five experiments. ^∗∗ p < 0.01 versus LPS alone.

Figure 5

Inhibition of degradation of IκB-α, phosphorylation of IKKα/β, nuclear translocation of the p65 subunit of NF-κB/Rel, and NF-κB transcriptional activity by bee venom in LPS-stimulated BV2 microglia. The inhibitory effect of bee venom on the nuclear translocation of the p65 subunit of NF-κB/Rel, degradation of IκB-α (a), phosphorylation of IKKα/β (b), and NF-κB transcriptional activity (c). BV2 cells were pretreated with bee venom or vehicle for 30 min and then stimulated with 0.1 μg/mL LPS for the indicated times. The protein extraction and SDS-PAGE methods are described in Section 2. The β-actin was used as an internal loading control for the immunoblot analysis. The effect of bee venom on NF-κB transcriptional activity was determined by firefly luciferase activity by using a luminometer. ^∗ p < 0.05 and ^∗∗∗ p < 0.001 versus LPS alone.

Figure 6

Bee venom modulates MAPK and TAK1 phosphorylation in LPS-stimulated BV2 microglia. The inhibitory effects of bee venom on the phosphorylation of extracellular-signal regulated kinase 1/2 (ERK1/2), c-jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38-MAPK) (a), and TGF-β-activated kinase 1 (TAK1) (b) in LPS-activated microglial cells. BV2 cells were pretreated with bee venom or vehicle for 30 min and then incubated with LPS for the indicated times. The protein extraction and protein separation by SDS-PAGE are described in Section 2. β-actin was used as an internal loading control for the immunoblot analysis. Representative images of experiments performed at least in triplicate are shown. ^∗ p < 0.05 versus LPS alone.

Figure 7

The interaction of MyD88 with either TRAF6 and IKKα/β or MKK4 and TAK1 was diminished by bee venom treatment in LPS-stimulated BV2 microglia. The inhibitory effects of bee venom on the interaction of MyD88 with TRAF6 (a) and IKKα/β or MKK4 and TAK1 (b) in LPS-activated microglial cells are shown. BV2 cells were pretreated with bee venom or vehicle for 30 min and then incubated with LPS for the indicated times. The protein extraction, immunoprecipitation with MyD88 antibody, and protein separation by SDS-PAGE are described in Section 2. Representative images of experiments performed at least in triplicate are shown.

Figure 8

Graphical summary of anti-inflammatory activity of bee venom in LPS-activated BV2 glial cells.