Anti-Inflammatory Effects of Miyako Bidens pilosa in a Mouse Model of Amyotrophic Lateral Sclerosis and Lipopolysaccharide-Stimulated BV-2 Microglia

Abstract

Neuroinflammation is a fundamental feature in the pathogenesis of amyotrophic lateral sclerosis (ALS) and arises from the activation of astrocytes and microglial cells. Previously, we reported that Miyako Bidens pilosa extract (MBP) inhibited microglial activation and prolonged the life span in a human ALS-linked mutant superoxide dismutase-1 (SOD1^G93A) transgenic mouse model of ALS (G93A mice). Herein, we evaluated the effect of MBP on microglial activation in the spinal cord of G93A mice and lipopolysaccharide-stimulated BV-2 microglial cells. The administration of MBP inhibited the upregulation of the M1-microglia/macrophage marker (interferon-γ receptor (IFN-γR)) and pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) in G93A mice. However, MBP did not affect the increase in the M2-microglia/macrophage marker (IL-13R) and anti-inflammatory cytokines (transforming growth factor (TGF)-β and IL-10) in G93A mice. BV-2 cell exposure to MBP resulted in a decrease in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) reduction activity and bromodeoxyuridine incorporation, without an increase in the number of ethidium homodimer-1-stained dead cells. Moreover, MBP suppressed the production of lipopolysaccharide-induced pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in BV-2 cells. These results suggest that the selective suppression of M1-related pro-inflammatory cytokines is involved in the therapeutic potential of MBP in ALS model mice.

Keywords: Bidens pilosa; M1-microglia/macrophages; amyotrophic lateral sclerosis; pro-inflammatory cytokines; spinal cord.

Figure 1

Miyako Bidens pilosa (MBP) selectively inhibited the induction of pro-inflammatory cytokines in the lumber spinal cord of G93A mice. Mice were orally administered injection water (vehicle) or MBP (2 g/kg/day), starting at 15 weeks old (asymptomatic stage). One week after the start of treatment, the lumbar spinal cords were analyzed via real-time polymerase chain reaction (PCR). The graphs show the mRNA expression profiles of pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 (a)) and anti-inflammatory cytokines (TGF-β and IL-10 (b)). Data are shown as the average ± standard error, calculated from 4–6 independent biological replicates, each with 2 technical replicates.

Figure 2

Miyako Bidens pilosa (MBP) regulated microglia/macrophage M1–M2 polarization in the lumber spinal cord of WT and G93A mice. Mice were orally administered injection water (vehicle) or MBP (2 g/kg/day), starting at 15 weeks old (asymptomatic stage). One week after the start of treatment, the lumbar spinal cords were analyzed via real-time polymerase chain reaction (PCR). The graphs show the mRNA expression profiles of the M1-like microglia/macrophage marker (interferon (IFN)-γR (a) and M2-like microglia/macrophage marker (interleukin (IL)-13R (b)) in the spinal cord. Data are shown as the average ± standard error, calculated from 4–6 independent biological replicates, each with 2 technical replicates.

Figure 3

Miyako Bidens pilosa (MBP) suppressed the proliferation of BV-2 microglia cells. (a) The research protocol conducted in this study is shown. BV-2 cells were treated with the indicated concentrations of MBP for 24 h, and cell proliferation was determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) reduction assay and bromodeoxyuridine (BrdU) incorporation assay. (b) The graph shows the effect of MBP on MTT reduction activity in the cells. Values are indicated as percentages relative to untreated cells. Data are shown as the average ± standard error (SE), calculated from four independent biological replicates, each with four technical replicates. (c) The graph shows the effect of MBP on BrdU incorporation into newly synthesized DNA of proliferating cells. Values are indicated as percentages relative to untreated cells. Data are shown as the average ± SE, calculated from four independent biological replicates, each with four technical replicates. (d) Cell morphology was observed with an optical microscope; photographs show typical phase-contrast microscopy images of BV-2 cells treated with indicated concentrations of MBP. The scale bar represents 100 μm.

Figure 4

Miyako Bidens pilosa (MBP) did not induce cell death in BV-2 cells. (a) The research protocol conducted in this study is shown. BV-2 cells were treated with indicated concentrations of MBP for 24 h. (b) The photographs show typical fluorescence images of calcein-AM (green, live cells) and ethidium homodimer-1 (EthD-1) (red, dead cells) double staining in each treatment group. The scale bar indicates 100 μm. The left graph shows the percentage of EthD-1-positive dead cells in these cells. The right graph shows the number of EthD-1-positive dead cells (red bar) and calcein-AM-positive live cells (pale green bar) in these cells. n.s.: no significance. Data are shown as the average ± standard error, calculated from four independent biological replicates, each with eight technical replicates.

Figure 5

Miyako Bidens pilosa (MBP) inhibited the lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines in BV-2 cells. Cells incubated at different concentrations of MBP with or without LPS (1 μg/mL) for 4 h. The expression level of pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) was analyzed via real-time polymerase chain reaction (PCR). The graphs show the relative expression of mRNA of LPS-induced pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6). Data are shown as the average ± standard error, calculated from six independent biological replicates, each with two technical replicates.

Figure 6

A proposed mechanism of treatment with Miyako Bidens pilosa (MBP) in the spinal cord of G93A mice. Red arrows indicate increase of cells proliferation or upregulation of cytokines. Red triangles and sky blue squares indicate pro-inflammatory cytokines and anti-inflammatory cytokines respectively.