Bee Venom Soluble Phospholipase A2 Exerts Neuroprotective Effects in a Lipopolysaccharide-Induced Mouse Model of Alzheimer's Disease via Inhibition of Nuclear Factor-Kappa B

Abstract

Neuroinflammation is important in the pathogenesis and development of Alzheimer's disease (AD). In the AD brain, microglial activation and upregulation of pro-inflammatory mediators both induce amyloid beta (Aβ) accumulation. Regulatory T cells (Tregs) and nuclear factor-kappa B (NF-κB) signaling have been implicated in AD development through their effects on neuroinflammation and microglial activation. The bee venom soluble phospholipase A2 (bv-sPLA2) enzyme is known to exert anti-inflammatory and anti-immune effects. Here, we investigated the inhibitory effects of bv-sPLA2 on memory deficiency in a lipopolysaccharide (LPS)-induced mouse model of AD. We examined whether bv-sPLA2 (0.02, 0.2, and 2 mg/kg by i.p. injection three times for 1 week) could inhibit neuroinflammation and memory impairment in LPS-treated mice (250 μg/kg by i.p. injection daily for 1 week). We also assessed the effects of bv-sPLA2 administration (0.01, 0.1, and 1 μg/ml) on LPS (1 μg/ml)-treated microglial BV-2 cells. In the LPS-injected mouse brain, sPLA2 treatment rescued memory dysfunction and decreased Aβ levels, through the downregulation of amyloidogenic proteins, and decreased the expression of inflammatory proteins and pro-inflammatory cytokines. Moreover, the LPS-mediated increase in inflammatory protein expression was attenuated bv-sPLA2 treatment in BV-2 cells. Treatment with bv-sPLA2 also downregulated signaling by NF-κB, which is considered to be an important factor in the regulation of neuroinflammatory and amyloidogenic responses, both in vivo and in vitro. Additionally, co-treatment with NF-κB (5 μM) and bv-sPLA2 (0.1 μg/ml) exerted more marked anti-inflammatory effects, compared to bv-sPLA2 treatment alone. These results indicate that bv-sPLA2 inhibits LPS-induced neuroinflammation and amyloidogenesis via inhibition of NF-κB.

Keywords: Alzheimer’s disease; NF-κB; bee venom phospholipase A2; neuroinflammation; regulatory T cells.

Figure 1

Effects of bv-sPLA2 on lipopolysaccharide (LPS)-induced improvement of memory impairment in the mice. (A) The mice (n = 8) were daily treated bv-sPLA2 by i.p. injection at dose of 0.02, 0.2 and 2 mg/kg for three times. Intraperitoneal injection of LPS (250 μg/kg) was treated except for control group for 7 days, and they were evaluated for learning and memory of spatial information using the water maze. (B) Escape latency, the time required to find the platform and (C) escape distance, the distance swam to find the platform was measured. After the water maze test, (D) probe test to measure maintenance of memory was performed. The time spent in the target quadrant and target site crossing within 60 s was represented. (E) A passive avoidance test was performed by step-through method. n = 8 per group. The data are shown as the means ± standard deviation (SD) of the mean. ^#p < 0.05, ^##p < 0.005, ^###p < 0.001 control group vs. LPS group, *p < 0.05, **p < 0.005 LPS-group vs. LPS with bv-sPLA2 group.

Figure 2

Effects of bv-sPLA2 on LPS-induced amyloid β (Aβ) deposition and expression of amyloidogenic protein in the mice brain. (A) The levels of Aβ_1–42 in the brain of mice were assessed using a specific Aβ ELISA, n = 4 per group. (B) The β-secretase activity in the brain of mice was measured using assay kit, n = 4 per group. (C) The expression of amyloid precursor protein (APP) and BACE1 were detected by western blot using specific antibodies in the brain of mice. β-actin protein was used as an internal control. The data are shown as the means ± SD of the mean. ^#p < 0.05, ^##p < 0.005, ^###p < 0.001 control group vs. LPS group, *p < 0.05, **p < 0.005, ***p < 0.001 LPS-group vs. LPS with bv-sPLA2 group.

Figure 3

Effects of bv-sPLA2 on LPS-induced neuroinflammation in the mice brain. Immunohistochemical analysis of (A) GFAP, IBA-1 (B) iNOS and COX-2 antibodies were investigated two different regions (CA3; cornu ammonis 3 and DG; dentate gyrus) in 10-μm-thick sections of the brain hippocampus of mice with specific primary antibodies and the biotinylated secondary antibodies (scale bars, 100 μm). ^###p < 0.001 control group vs. LPS group, *p < 0.05, **p < 0.005 LPS-group vs. LPS with bv-sPLA2 group.

Figure 4

Effects of bv-sPLA2 on LPS-induced inflammatory responses and pro-inflammatory cytokines in the mice brain. (A) The expression of GFAP, IBA-1, iNOS and COX-2 were detected by western blot using specific antibodies in the brain of mice. (B) The expression of p-IκB-α, IκB-α, p50 and p65 were detected by western blot using specific antibodies in the brain of mice. β-actin protein was used as an internal control. (C) mRNA expression levels of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, IL-1β and IL-6 in the brain of mice were measured using quantitative real-time RT-PCR. The data are shown as the means ± SD of the mean. ^#p < 0.05, ^##p < 0.005, ^###p < 0.001 control group vs. LPS group, *p < 0.05, **p < 0.005, ***p < 0.001 LPS-group vs. LPS with bv-sPLA2 group.

Figure 5

Effects of bv-sPLA2 on modulation of Tregs in the mice brain. (A) Immunohistochemical analysis of Foxp3 antibodies was investigated in two different regions (CA3; cornu ammonis 3 and DG; dentate gyrus) in 10-μm-thick sections of the brain hippocampus of mice with specific primary antibodies and the biotinylated secondary antibodies (scale bars, 100 μm). (B) Graph represented the relative intensity of Foxp3-reactive cells, n = 3 per group. (C) mRNA expression levels of Foxp3 in the brain of mice were measured using quantitative real-time RT-PCR. The data are shown as the means ± SD of the mean. ^#p < 0.05 control group vs. LPS group, *p < 0.05, **p < 0.005, ***p < 0.001 LPS-group vs. LPS with bv-sPLA2 group. (D) The expression of Foxp3 was detected by western blot using specific antibody in the brain of mice. β-actin protein was used as an internal control.

Figure 6

Effects of bv-sPLA2 on LPS-induced amyloidogenesis and neuroinflammation in the microglial cells. Microglia BV-2 cells were treated with LPS (1 μg/ml) and bv-sPLA2 (0.01, 0.1 and 1 μg/ml). (A) The expression of APP and BACE1 were detected by western blot using specific antibodies in the microglia BV-2 cells. (B) The levels of β-secretase activity in the microglia BV-2 cells were assessed using assay kit. (C) The expression of iNOS, COX-2 and IBA-1 were detected by western blot using specific antibodies in the microglia BV-2 cells. (D) The expression of p-IκB-α, IκB-α, p50 and p65 were detected by western blot using specific antibodies in the microglia BV-2 cells. β-actin protein was used as an internal control. (E) mRNA expression levels of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6 in the microglia BV-2 cells were measured using quantitative real-time RT-PCR. The data are shown as the means ± SD of the mean. ^##p < 0.005, ^###p < 0.001 control group vs. LPS group, *p < 0.05, **p < 0.005, ***p < 0.001 LPS-group vs. LPS with bv-sPLA2 group.

Figure 7

Combination effects of bv-sPLA2 and NF-κB inhibitor. Microglia BV-2 cells were treated with LPS (1 μg/ml), bv-sPLA2 (0.1 μg/ml) and Bay 11-7082 (5 μM). (A) The expression of iNOS and COX-2 was detected by western blot using specific antibodies in the microglia BV-2 cells. (B) The expression of p-IκB-α, IκB-α, p50 and p65 were detected by western blot using specific antibodies in the microglia BV-2 cells. β-actin protein was used as an internal control. (C) mRNA expression levels of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6 in the microglia BV-2 cells were measured using quantitative real-time RT-PCR. The data are shown as the means ± SD of the mean. ^###p < 0.001 control group vs. LPS group, **p < 0.005, ***p < 0.001 LPS with bv-sPLA2 vs. LPS with bv-sPLA2 and Bay 11-7082 group.