Steppogenin Isolated from Cudrania tricuspidata Shows Antineuroinflammatory Effects via NF-κB and MAPK Pathways in LPS-Stimulated BV2 and Primary Rat Microglial Cells

Abstract

Excessive microglial stimulation has been recognized in several neurodegenerative diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), amyotropic lateral sclerosis (ALS), HIV-associated dementia (HAD), multiple sclerosis (MS), and stroke. When microglia are stimulated, they produce proinflammatory mediators and cytokines, including nitric oxide (NO) derived from inducible NO synthase (iNOS), prostaglandin E2 (PGE₂) derived from cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-12 (IL-12), and interleukin-6 (IL-6). These inflammatory reactions are related to the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Therefore, the modulation of NF-κB and MAPK is vital to prevent microglial activation and confer resistance against neuronal injury. In this study, steppogenin (1) isolated from Cudrania tricuspidata suppressed the neuroinflammatory responses to lipopolysaccharide (LPS). Steppogenin (1) inhibited the production of proinflammatory mediators and cytokines in LPS-challenged BV2 and rat primary microglial cells. Moreover, western blot analysis and immunofluorescence revealed that the nuclear translocation of NF-κB was inhibited in LPS-induced BV2 and rat primary microglial cells. The LPS-stimulated activation of BV2 and rat primary microglial cells was inhibited by steppogenin (1) through the suppression of c-Jun NH2-terminal kinase (JNK) and p38 MAPK signaling. These results suggested that steppogenin (1) exerted antineuroinflammatory effects against acute neuroinflammation in BV2 and rat primary microglial cells by suppressing the activation of NF-κB and MAPK signaling and the production of proinflammatory mediators and cytokines.

Keywords: Cudrania tricuspidata; mitogen-activated protein kinase (MAPK); neuroinflammation; nuclear factor-kappa B (NF-κB); steppogenin.

Figure 1

Chemical structure of steppogenin (1).

Figure 2

The effects of steppogenin (1) on the cell viability of BV2 microglial cells. BV2 microglial cells were incubated for 24 h with steppogenin in the range from 10.0 to 80.0 μM. The data are presented as the mean ± SD of three experiments.

Figure 3

The effects of steppogenin (1) on the mRNA expression of tumor necrosis factor (TNF)-α (A), interleukin (IL)-1β (B), IL-12 (C), and IL-6 (D) in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. (A–D) The cells were pretreated for 3 h with the indicated concentrations of 1 and then stimulated for 12 h with LPS (1 μg/mL). The data are presented as the mean ± SD of three experiments. * p < 0.05; ** p < 0.01; *** p < 0.001 compared with the LPS-treated group.

Figure 4

The effects of steppogenin (1) on nitrite (A) and prostaglandin E2 (PGE₂) (B) production and iNOS and COX-2 expression (C) in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. (A–C) The cells were pretreated for 3 h with the indicated concentrations of 1 and then stimulated for 24 h with LPS (1 μg/mL). The data are presented as the mean ± SD of three experiments. The band intensity was quantified by densitometry and normalized to the intensity of the β-actin band; the normalized values are presented below each band. * p < 0.05; ** p < 0.01; *** p < 0.001 compared with the LPS-treated group.

Figure 5

The effects of steppogenin (1) on IκB-α phosphorylation and degradation (A) NF-κB activation (B,C), NF-κB DNA binding activity (D), and NF-κB localization (E) in LPS-stimulated BV2 microglial cells. (A–E) The cells were pretreated for 3 h with the indicated concentrations of 1 and then stimulated for 1 h with LPS (1 μg/mL). The data are presented as the mean ± SD of three experiments. The band intensity was quantified by densitometry and normalized to the intensity of the β-actin or proliferating cell nuclear antigen (PCNA) band; the normalized values are presented below each band. ** p < 0.01; *** p < 0.001 compared with the LPS-treated group.

Figure 6

The effects of steppogenin (1) on extracellular signal–regulated kinase (ERK) (A), c-Jun N-terminal kinase (JNK) (B), and p38 (C) MAPK phosphorylation and protein expression. (A–C) The cells were pretreated for 3 h with the indicated concentrations of 1 and stimulated for 1 h with LPS (1 μg/mL). The levels of phosphorylated ERK (p-ERK), phosphorylated JNK (p-JNK), and phosphorylated-p38 MAPK (p-p38 MAPK) were determined by western blot analysis. Representative blots from three independent experiments with similar results and densitometric evaluations are shown. The band intensities were quantified by densitometry and normalized to the density of the β-actin band; the normalized values are presented below each band.

Figure 7

The effects of steppogenin (1) on nitrite (A) production and iNOS and COX-2 expression (B) in lipopolysaccharide (LPS)-stimulated primary rat microglial cells. (A,B) The cells were pretreated for 3 h with the indicated concentrations of 1 and then stimulated for 24 h with LPS (1 μg/mL). The data are presented as the mean ± SD of three experiments. The band intensities were quantified by densitometry and normalized to the intensities of the β-actin band; the normalized values are presented below each band. ** p < 0.01; *** p < 0.001 compared with the LPS-treated group.

Figure 8

The effects of steppogenin (1) on the mRNA expression of TNF-α (A), IL-1β (B), IL-6 (C), and IL-12 (D) in LPS-stimulated primary rat microglial cells. (A–D) The cells were pretreated for 3 h with the indicated concentrations of 1 and then stimulated for 12 h with LPS (1 μg/mL). The data are presented as the mean ± SD of three experiments. * p < 0.05; ** p < 0.01; *** p < 0.001 compared with the LPS-treated group.

Figure 9

The effects of steppogenin (1) on IκB-α phosphorylation and degradation (A), NF-κB activation (B,C), and NF-κB localization (D) in LPS-stimulated primary rat microglial cells. (A–D) The cells were pretreated for 3 h with the indicated concentrations of 1 and then stimulated for 1 h with LPS (1 μg/mL). Total proteins were prepared and the western blot analysis was performed using specific IκB-α, p-IκB-α p65, and p50 antibodies. A commercially available NF-κB ELISA (Active Motif) was used to test the nuclear extracts and determine the degree of NF-κB binding. The data are presented as the mean ± SD of three experiments. The band intensities were quantified by densitometry and normalized to the intensity of β-actin or PCNA; the normalized values are presented below each band.

Figure 10

Schematic diagram showing the relationship of steppogenin (1) and NF-κB/inflammation/MAPK damage after LPS in BV2 microglial and rat primary microglial cells.