Ginkgolide B Alleviates Learning and Memory Impairment in Rats With Vascular Dementia by Reducing Neuroinflammation via Regulating NF-κB Pathway

Abstract

Ginkgobalide B (GB) as the main active ingredient of traditional Chinese medicine Ginkgo biloba extract is reported to reduce neuroinflammation, protect neurons and promote cognitive learning ability. To explore that GB can reduce neuroinflammation through regulating nuclear factor-kappaB (NF-κB) signaling pathway and overcome cognitive dysfunction in rats with vascular dementia (VD), we aim at investigating the potential effect of GB on enhancing cognitive function in rats with VD. It was found that GB improved survival of oxygen-glucose deprivation (OGD) treated SH-SY5Y cells by attenuating inflammatory response via Toll-like Receptor 4 (TLR4)/NF-κB pathway. When rats were treated with bilateral common carotid artery occlusion (BCCAO) for 24 h, saline and GB were administered in Sprague-Dawley (SD) rats via a single intraperitoneal injection for consecutive 14 days. The behavioral changes of VD like rats treated with GB were observed through open field test, Morris water maze (MWM) and Y-maze electric maze. Nissl staining and immunofluorescence were used to observe changes of neurons in the hippocampus of rats. Western blot analysis was performed by detecting NF-κB pathway related inflammatory factors. The results found that GB can significantly improve the learning and memory ability of VD rats by reducing TLR4/NF-κB mediated neuroinflammation. In conclusion, GB seemed to be a potential drug for amelioration of learning and memory impairment in rats with VD.

Keywords: NF-κB; TLR4; ginkgobalide B; inflammatory; vascular dementia.

FIGURE 1

Graphical Abstract: Ginkgolide B alleviates learning and memory impairment in rats with vascular dementia by reducing TLR4/NF-κB mediated neuroinflammation.

FIGURE 2

(A) The cell viability rate of Oxygen-Glucose Deprivation (OGD) cells treated with Ginkgobalide B (GB) and normal cells treated with Lipopolysaccharides (LPS) and the combination of LPS and GB. Data represented the mean ± SD (n = 5), ***p < 0.001 (B) Quantitative analysis on the number of TUNEL-positive of OGD cells treated with GB and normal cells treated with LPS and the combination of LPS and GB. Data represented the mean ± SD (n = 3), ***p < 0.001 (C) The images of TUNEL positive cells in different groups, the scale bar is 50 μm and applies to all figure parts.

FIGURE 3

Detection of the protein levels of NF-κB p65, NF-κB p-p65, IκBα, p-IκBα, TLR4, MyD88, TRAF6, TNF-α and IL-6 by western blotting. Data represented the mean ± SD (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

Effects of GB on movement activity and memory ability in BCCAO rats. In open field test, the change of the total distance (A) and mean speed (B) in sham group, BCCAO group and GB treated BCCAO group. Data represented the mean ± SD (n = 5), *p < 0.05, ***p < 0.001 (C) Track diagram of rats movement in the Sham, BCCAO model and GB treated BCCAO groups. Y-maze test was used to analyze working memory in rats (D) Escape latencies analysis. Data represented the mean ± SD (n = 5), *p < 0.05, ***p < 0.001 (E) Active avoidance response number analysis. Data represented the mean ± SD (n = 5), *p < 0.05, ***p < 0.001.

FIGURE 5

Effects of GB on improving spatial learning and memory in BCCAO rats (A) Escape latencies analysis. Data represented the mean ± SD (n = 5), *p < 0.05, **p < 0.01, ***p < 0.001 (B) The platform crossing number in the spatial probe test. Data represented the mean ± SD (n = 5), *p < 0.05 (C) Mapping of swimming location of rats in water maze.

FIGURE 6

Nissl staining of CA1, CA3, DG region of rat hippocampus of sham group, BCCAO group and GB treated BCCAO group. The Nissl body was stained purple-blue. (A) Representative Nissl staining images in hippocampus of sham group, BCCAO group and GB treated BCCAO group. (B) Quantitative analysis of the number of Nissl bodies in hippocampus of sham group, BCCAO group and GB treated BCCAO group. Data are expressed as means ± SD (n = 3), *p < 0.05, **p < 0.01. Scale bar: 100 and 50 μm.

FIGURE 7

Representative immunofluorescence staining for NeuN positive cells in hippocampus of sham group, BCCAO group and GB treated BCCAO group. NeuN antibodies were used to stain neurons in the hippocampus. DAPI (blue) was used as a nuclear marker. Data are expressed as means ± SD (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001. The scale bar is 200 μm and applies to figure 7.

FIGURE 8

Detection of the protein levels of NFκB p-p65, p-IκBα, Caspase9 and TNF-α in brain tissue by western blotting. Data represented the mean ± SD (n = 3), *p < 0.05,**p < 0.01, ***p < 0.001.