Elsevier

Experimental Gerontology

Volume 81, August 2016, Pages 92-100
Experimental Gerontology

EGb761 improves cognitive function and regulates inflammatory responses in the APP/PS1 mouse

https://doi.org/10.1016/j.exger.2016.05.007Get rights and content

Highlights

  • Chronic treatment of EGb761 improves learning and memory in the APP/SP1 mouse.

  • Regulation of inflammation is involved in EGb761-alleviated Aβ plaque deposition.

  • EGb761 increased microglial around Aβ plaques in the APP/PS1 mouse.

  • EGb761 inhibits pro-inflammatory responses while increasing anti-inflammatory responses in microglia.

Abstract

There is accumulating evidence that the Ginkgo biloba extract EGb761 may help to prevent Alzheimer's disease (AD). However, the underlying mechanism of its action remains to be elaborated. In this study, we examined the effects of EGb761 using the APP/PS1 transgenic mouse model of AD. Two-month-old APP/PS1 mice were supplemented with EGb761 daily for 6 months. We found that this chronic treatment with EGb761 improved the cognitive function of these mice and also significantly alleviated amyloid plaque deposition. Although the level of insoluble amyloid beta (Aβ) was decreased, the soluble content of Aβ was not changed after administration of EGb761. We then determined the changes in central inflammation and observed that the activated microglia around amyloid plaque was increased in these treated mice. We also found that chronic EGb761 treatment downregulated pro-inflammatory cytokines and inducible nitric oxide synthase (iNOS), and upregulated anti-inflammatory cytokines and Arginase-1 (Arg-1), suggesting that EGb761 regulated the phenotype of activated microglia in the APP/PS1 mouse brain. In support of this, pretreatment of the BV2 microglial cell line with EGb761 inhibited the inflammatory reaction to Aβ. Furthermore, the addition of conditioned media derived from BV2 cells that were co-treated with Aβ and EGb761, protected neurons against treatment of Aβ and inhibited apoptotic damage. Taken together, our results demonstrated that EGb761 provided a protective effect in APP/PS1 mouse. This protection was correlated with an inhibition of the pro-inflammatory effects of microglia and an induction of anti-inflammatory effects. These results strongly suggest that EGb761 provides a protective effect in APP/PS1 mouse via regulation of inflammation in the brain.

Introduction

Alzheimer's disease (AD) is a progressive disease characterized by amyloid beta (Aβ) accumulation, intraneuronal paired helical filaments, and neuronal dysfunction and loss (Selkoe, 1989). Although AD has been intensively studied, there is no known therapy that can halt or reverse its progression.

Increasing evidence suggests that neuroinflammation is prominent in AD as supported by activated microglia (Dheen et al., 2007, Orre et al., 2014, Varnum and Ikezu, 2012) and increased levels of inflammatory cytokines in the brain (Hanzel et al., 2014, Millington et al., 2014). Microglia, the resident immune cells in the central nervous system (CNS), are ubiquitously distributed in the brain (Heneka et al., 2015). Microglial activation leads to significant and highly detrimental neurotoxic effects by producing cytotoxic and inflammatory factors such as Interleukin-1β (IL-1β), IL-6, Tumor Necrosis Factor α (TNFα), Monocyte Chemoattractant Protein (MCP-1) and inducible Nitric Oxide Synthase (iNOS) (Roodveldt et al., 2013, Yang et al., 2014). The inflammatory process mediated by microglia in the CNS is believed to play an essential role in the pathway of neuron death in various neurodegenerative diseases including AD (Dheen et al., 2007, Millington et al., 2014, Varnum and Ikezu, 2012, Yang et al., 2014). Paradoxically, studies indicate that neuroinflammation may also be beneficial in neuronal repair since anti-inflammatory factors such as IL-4, IL-10 and Transforming Growth Factor β (TGF β) are also released by activated microglia. These factors may help to remove aggregated Aβ and attenuate disease progression (Tang and Le, 2015). Thus, neuroinflammation is a double-edged sword that executes both detrimental and beneficial effects on neurons. Therefore, therapeutic strategies designed to regulate the activation status of microglia may lead to significant advancements in the treatment of AD.

EGb761 is a standardized extract from the leaves of the Ginkgo biloba, that contains 24% flavonoid glycosides, 6% terpenoids, and 5–10% organic acids (Montes et al., 2015, Yan et al., 2008). Preclinical studies have demonstrated neuroprotective effects of EGb761 and suggest that it is a promising agent for the treatment and prevention of AD (Ihl, 2013, Lautenschlager et al., 2012, Rainer et al., 2013). Previous studies have shown that EGb761 inhibits Aβ peptide aggregation and destabilizes fibrils in a transgenic mouse model of AD (Augustin et al., 2009, Garcia-Alloza et al., 2010, Yan et al., 2008). In humans, Amieva et al. found that cognitive decline in a non-demented elderly population was lower in individuals who received EGb761 for 20 years than those who did not (Amieva et al., 2013). Furthermore, Vellas and his collaborators found that this reduction in the risk for development to AD could be detected after 3 years of treatment with the extract (Vellas et al., 2012) suggesting a relatively fast acting protective effect of EGb761.

The present study investigated the effects and the possible mechanisms of action of chronic EGb761 treatment in APP/PS1 (APPswe/PS1-ΔE9) transgenic mice. Furthermore, we employed an in vitro study using both microglial BV2 tissue culture cells and primary neurons, and evaluated the potential role of EGb761 pretreatment in Aβ-induced toxicity.

Section snippets

Study approval

All animal studies were approved by the Medical Experimental Animal Administrative Committee of Huadong Hospital, Fudan University.

Animals and treatment

APP/PS1 transgenic mice and littermate controls (WT) were purchased from the Model Animal Research Centre of Nanjing University. These mice were maintained by cross-breeding with the C57BL/6 breeders. Genotype was confirmed by polymerase chain reaction (PCR) analysis of tail biopsies. The mice were housed at 22˚C with free access to food and water and maintained on

Chronic EGb761 treatment improved cognitive function in APP/PS1 mice

It has been shown in previous studies that treatment with EGb761 for 3 months improves learning and memory ability in a transgenic mouse model of AD (Rainer et al., 2013). However, little is known about the long term effects of EGb761 administration in this disease model. In the current study, we first evaluated the learning and memory ability of APP/PS1 transgenic mice after chronic EGb761 treatment for 6 months (Fig. 1.A). Mice were treated with or without EGb761 for 6 months. They were then

Discussion

Although AD has been intensively studied for decades, the underlying mechanisms of the disease are still unclear and it remains incurable (Nazem et al., 2015). A hallmark of the disease is amyloid beta (Aβ) accumulation (Sagare et al., 2012). In the past few years, several promising disease-modifying agents that target Aβ have been proposed for the treatment of AD. Many of these agents did have efficacy in rodent models of AD (Mangialasche et al., 2010, Nazem et al., 2015). However, almost all

Founding source

This study was funded by National Natural Science Foundation of China (Grant No. 81473739).

Disclosure

The authors declare that they have no conflict of interest in the current study.

Acknowledgements

None.

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