Ganoderma lucidum ethanol extract inhibits the inflammatory response by suppressing the NF-κB and toll-like receptor pathways in lipopolysaccharide-stimulated BV2 microglial cells

Abstract

Ganoderma lucidum is a traditional Oriental medicine that has been widely used as a tonic to promote longevity and health in Korea and other Asian countries. Although a great deal of work has been carried out on the therapeutic potential of this mushroom, the pharmacological mechanisms of its anti-inflammatory actions remain unclear. In this study, we evaluated the inhibitory effects of G. lucidum ethanol extract (EGL) on the production of inflammatory mediators and cytokines in lipopolysaccharide (LPS)-stimulated murine BV2 microglia. We also investigated the effects of EGL on the LPS-induced activation of nuclear factor kappaB (NF-κB) and upregulation of toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). Elevated levels of nitric oxide (NO), prostaglandin E(2) (PGE(2)) and pro-inflammatory cytokine production were detected in BV2 microglia following LPS stimulation. We identifed that EGL significantly inhibits the excessive production of NO, PGE(2) and pro-inflammatory cytokines, including interleukin (IL)-1β and tumor necrosis factor-α in a concentration-dependent manner without causing cytotoxicity. In addition, EGL suppressed NF-κB translocation and transcriptional activity by blocking IκB degradation and inhibiting TLR4 and MyD88 expression in LPS-stimulated BV2 cells. Our results indicate that the inhibitory effects of EGL on LPS-stimulated inflammatory responses in BV2 microglia are associated with the suppression of the NF-κB and TLR signaling pathways. Therefore, EGL may be useful in the treatment of neurodegenerative diseases by inhibiting inflammatory mediator responses in activated microglia.

Keywords: BV2 microglia; Ganoderma lucidum; inflammation; nuclear factor κB; toll-like receptor.

Figure 1.

Inhibition of NO and PGE₂ production by EGL in LPS-stimulated BV2 microglia. BV2 cells were pre-treated with various concentrations of EGL (0.1, 0.5 and 1.0 mg/ml) for 1 h before incubation with LPS (0.5 μg/ml) for 24 h. (A) Nitrite content was measured using the Griess reaction and (B) PGE₂ concentration was measured in culture media using a commercial ELISA kit. Each value indicates the mean ± standard deviation (SD) of three independent experiments. ^*P<0.05 vs. cells treated with LPS in the absence of EGL. NO, nitric oxide; PGE₂, prostaglandin E₂; EGL, ethanol extract of Ganoderma lucidum; LPS, lipopolysaccharide; ELISA, enzyme-linked immunosorbent assay.

Figure 2.

Effects of EGL and LPS on the cell viability of BV2 microglia. Cells were treated with the indicated concentrations of EGL or pre-treated with EGL for 1 h prior to LPS (0.5 μg/ml) treatment, After 24 h, cell viability was assessed using an MTT reduction assay. The results are expressed as the percentage of surviving cells vs. control cells (no addition of EGL and LPS). Each value is the mean ± SD of three independent experiments. EGL, ethanol extracts of Ganoderma lucidum; LPS, lipopolysaccharide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SD, standard deviation.

Figure 3.

Inhibition of iNOS and COX-2 expression by EGL in LPS-stimulated BV2 microglia. (A) BV2 microglia were pre-treated with the indicated concentrations of EGL 1 h prior to incubation with LPS (0.5 μg/ml) for 24 h. Cell lysates were then prepared and western blotting was performed using antibodies specific for murine iNOS and COX-2. (B) After LPS treatment for 6 h, total RNA was prepared for RT-PCR analysis of iNOS and COX-2 gene expression. Actin and GAPDH were used as internal controls for the western blot analysis and RT-PCR assays, respectively. iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; EGL, ethanol extract of Ganoderma lucidum; LPS, lipopolysaccharide; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 4.

Effects of EGL on LPS-stimulated IL-1β and TNF-α production in BV2 microglia. BV2 cells were pre-treated with various concentrations of EGL for 1 h prior to LPS treatment (0.5 μg/ml). After 24 h incubation, the levels of (A) IL-1β and (B) TNF-α present in the supernatants were measured. Each value indicates the mean ± SD of three independent experiments. ^*P<0.05 vs. cells treated with LPS in the absence of EGL. EGL, ethanol extract of Ganoderma lucidum; LPS, lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor; SD, standard deviation.

Figure 5.

Effects of EGL on LPS-stimulated IL-1β and TNF-α expression in BV2 microglia. BV2 cells were pre-treated with the indicated concentrations of EGL for 1 h prior to LPS treatment (0.5 μg/ml) and total RNA was isolated 6 h after LPS treatment. The levels of IL-1β and TNF-α mRNA were determined using RT-PCR. GAPDH was used as an internal control. EGL, ethanol extract of Ganoderma lucidum; LPS, lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 6.

Effects of EGL on LPS-induced NF-κB translocation, IκB degradation and NF-κB activation in BV2 microglia. (A) Cells were treated with EGL (1 mg/ml) for 1 h prior to LPS treatment (0.5 μg/ml) for the indicated times. Nuclear and cytosolic proteins were subjected to 10% SDS-polyacrylamide gel electrophoresis followed by western blotting using anti-NF-κB p65 and anti-IκB-α antibodies. Nucleolin and actin were used as internal controls for the nuclear and cytosolic fractions, respectively. (B) Transfected BV2 microglia with NF-κB-luciferase reporter plasmids were pre-treated with EGL (1.0 mg/ml) for 0.5 or 1 h and then stimulated with LPS (0.5 μg/ml) for 0.5 or 1 h. NF-κB activity was expressed as luciferase activity. Each value is the mean ± SD of three independent experiments. ^*P<0.05 vs. cells treated with LPS in the absence of EGL. EGL, ethanol extract of Ganoderma lucidum; LPS, lipopolysaccharide; NF, nuclear factor; SDS, sodium dodecyl sulfate; SD, standard deviation.

Figure 7.

Effects of EGL on LPS-induced TLR4 and MyD88 expression in BV2 microglia. (A) Cells were treated with 0.5 μg/ml LPS for the indicated times. The cells were sampled, lysed and 50 μg proteins were separated by 10% SDS-polyacrylamide gel electrophoresis. (B) BV2 cells were pre-treated with 1.0 mg/ml EGL for 1 h prior to LPS treatment (0.5 μg/ml) and total proteins were isolated 24 h after LPS treatment. The proteins were subjected to 10% SDS-polyacrylamide gel electrophoresis followed by western blotting using anti-TLR4 and anti-MyD88 antibodies and an ECL detection system. Actin was used as an internal control. EGL, ethanol extract of Ganoderma lucidum; LPS, lipopolysaccharide; TLR, toll-like receptor; SDS, sodium dodecyl sulfate; ECL, enhanced chemiluminescence; MyD88, myeloid differentiation factor 88.