Bee venom ameliorates lipopolysaccharide-induced memory loss by preventing NF-kappaB pathway

Abstract

Background: Accumulation of beta-amyloid and neuroinflammation trigger Alzheimer's disease. We previously found that lipopolysaccharide (LPS) caused neuroinflammation with concomitant accumulation of beta-amyloid peptides leading to memory loss. A variety of anti-inflammatory compounds inhibiting nuclear factor kappaB (NF-κB) activation have showed efficacy to hinder neuroinflammation and amyloidogenesis. We also found that bee venom (BV) inhibits NF-κB.

Methods: A mouse model of LPS-induced memory loss used administration of BV (0.8 and 1.6 μg/kg/day, i.p.) to ICR mice for 7 days before injection of LPS (2.5 mg/kg/day, i.p.). Memory loss was assessed using a Morris water maze test and passive avoidance test. For in vitro study, we treated BV (0.5, 1, and 2 μg/mL) to astrocytes and microglial BV-2 cells with LPS (1 μg/mL).

Results: We found that BV inhibited LPS-induced memory loss determined by behavioral tests as well as cell death. BV also inhibited LPS-induced increases in the level of beta-amyloid (Aβ), β-and γ-secretases activities, NF-κB and its DNA-binding activity and expression of APP, and BACE1 and neuroinflammation proteins (COX-2, iNOS, GFAP and IBA-1) in the brain and cultured cells. In addition, pull-down assay and molecular modeling showed that BV binds to NF-κB.

Conclusions: BV attenuates LPS-induced amyloidogenesis, neuroinflammation, and therefore memory loss via inhibiting NF-κB signaling pathway. Thus, BV could be useful for treatment of Alzheimer's disease.

Fig. 1

Timeline depicting the treatment of BV and assessments of cognitive functions of mice (a). Arrow heads represent days on which acquisition tests were conducted. Inhibitory effect of BV on LPS-induced memory defects. Mice were treated with BV (0.8 and 1.6 μg/kg, i.p.) after 20-min treatment of LPS (2.5 mg/kg, i.p.). The Morris water maze tests and passive avoidance tests were performed as described in the Methods section. LPS injection elongates escape distance (b) and time (c) without affecting average swimming speed (d). LPS decreases the latency to enter the dark compartment. The memory deficit induced by LPS was attenuated by BV treatment (e). Values are presented as mean ± S.E. from eight mice. ^# p < 0.05 compared to control, ^* p < 0.05 compared to LPS

Fig. 2

Inhibitory effect of BV on LPS-induced Aβ accumulation and amyloidogenesis in mice brain. Thioflavin S staining for detection of Aβ accumulation that represents the arrow in the graph (a). Immunostaining of Aβ protein in the hippocampus was performed in 30-μm-thick sections of mice brain with anti-Aβ_1-42 primary antibody and the biotinylated secondary antibody (b). The expression of APP and BACE1 were detected by western blotting using specific antibodies in the mice brain. Each blot is representative of three experiments (c). The levels of Aβ_1-42 in mice brain were measured by ELISA (d). Inhibitory effect of BV on LPS-induced alteration in secretase activity. The activity of β- and γ-secretase in mice brain were investigated using assay kit (e, f). Values are presented as mean ± S.E. from eight mice. ^# p < 0.05 compare to control, *p < 0.05 compared to LPS

Fig. 3

Inhibitory effect of BV on LPS-induced expression of Aβ_1-42 in both GFAP and IBA-1-positive mice brain. Staining was performed in 30-μm-thick sections of mice brain. Confocal microscope observation was performed as described in the Methods section. Immunostaining of GFAP (green) and Aβ₁₋₄₂ (red) protein in the hippocampus was performed with specific primary antibodies, and fluorescence was developed using Alexa 488-conjugated anti-goat and Alexa 568-conjugated anti-rabbit secondary antibodies (a). IBA-1 (red) and Aβ₁₋₄₂ (green) protein in the hippocampus was performed with specific primary antibodies, and fluorescence was developed using Alexa 488-conjugated anti-mouse and Alexa 568-conjugated anti-rabbit secondary antibodies (b). Values are presented as mean ± S.E. from eight mice. ^# p < 0.05 compared to control, *p < 0.05 compared to LPS

Fig. 4

Inhibitory effect of BV on LPS-induced cell death. DAPI/TUNEL assay for detection of apoptotic cell death in the hippocampal DG zone was performed in 30-μm-thick sections of mice brain (a). Apoptosis (%) was defined as the percentage of the number of TUNEL-positive cells per surface of unit (b). Values are mean ± S.E. (n = 8). ^# p < 0.05 compared to control, *p < 0.05 compared to LPS

Fig. 5

Inhibitory effect of BV on LPS-induced brain cell activation and neuroinflammation. Immunostaining of GFAP, IBA-1, COX-2, and iNOS proteins in the hippocampus were performed in 30-μm-thick sections of mice brain with specific primary antibodies and the biotinylated secondary antibodies (n = 8) (a). The expression of GFAP, IBA-1, COX-2, and iNOS were detected by western blotting using specific antibodies in the mice brain. Each blot is representative of three experiments (b)

Fig. 6

Inhibitory effect of BV on LPS-induced NF-κB DNA-binding activity and on NF-κB-related protein in mice brain. Activation of NF-κB was investigated using EMSA as described in the Methods section. Nuclear extracts were subjected to DNA-binding reaction with ³²P end-labeled oligonucleotide specific to NF-κB. Specific DNA binding of the NF-κB complex is indicated by an arrow (a). The expression of IκB, p-IκB, p50, and p65 were detected by western blotting using specific antibodies in the mice brain. Each blot is representative of three experiments (b). Structural interaction between melittin and p50 of NF-κB subunit. Melittin structure is c. Pull-down assay identifies an interaction between the melittin and p50. Melittin was conjugated with melittin-activated Sepharose 6B (d). The docking model of melittin with p50 is as described in the Materials and Methods section (e)

Fig. 7

The expression of APP and BACE1 in astrocyte primary cells (a) and microglia BV-2 cells (b) were detected by western blotting using specific antibodies. Inhibitory effect of BV on LPS-induced NF-κB DNA-binding activity and on NF-κB-related protein in astrocyte primary cells (c) and microglia BV-2 cells (d). Activation of NF-κB was investigated using EMSA as described in the Methods section. Nuclear extracts were subjected to DNA-binding reaction with ³²P end-labeled oligonucleotide specific to NF-κB. Specific DNA binding of the NF-κB complex is indicated by an arrow. The levels of Aβ_1-42 in astrocyte primary cells (e) and microglia BV-2 cells (f) were measured by ELISA (n = 3). Values are presented as mean ± S.E. of the three independent experiments performed in triplicate. ^# p < 0.05 compared to control, *p < 0.05 compared to LPS. The expression of IκB, p-IκB, p50, and p65 in astrocyte primary cells (g) and microglia BV-2 cells (h) was detected by western blotting using specific antibodies. The expression of GFAP, IBA-1, COX-2, and iNOS in astrocyte primary cells (i) and microglia BV-2 cells (j) were detected by western blotting using specific antibodies. Each blot is representative of three experiments

Fig. 8

The cultured astrocytes were incubated with anti-GFAP (green) and anti- Aβ₁₋₄₂ (red) primary antibodies, and microglial BV-2 cells were incubated with anti-IBA-1 (green) and anti- Aβ₁₋₄₂ (red) primary antibodies. Fluorescence was developed using Alexa 488-conjugated anti-mouse and goat and Alexa 568-conjugated anti-rabbit secondary antibodies (a) and (b). Values are presented as mean ± S.E. from three mice. ^# p < 0.05 compared to control, *p < 0.05 compared to LPS