EGb761 Provides a Protective Effect against Aβ1-42 Oligomer-Induced Cell Damage and Blood-Brain Barrier Disruption in an In Vitro bEnd.3 Endothelial Model

Alzheimer’s disease (AD) is the most common form of senile dementia which is characterized by abnormal amyloid beta (Aβ) accumulation and deposition in brain parenchyma and cerebral capillaries, and leads to blood-brain barrier (BBB) disruption. Despite great progress in understanding the etiology of AD, the underlying pathogenic mechanism of BBB damage is still unclear, and no effective treatment has been devised. The standard Ginkgo biloba extract EGb761 has been widely used as a potential cognitive enhancer for the treatment of AD. However, the cellular mechanism underlying the effect remain to be clarified. In this study, we employed an immortalized endothelial cell line (bEnd.3) and incubation of Aβ1–42 oligomer, to mimic a monolayer BBB model under conditions found in the AD brain. We investigated the effect of EGb761 on BBB and found that Aβ1–42 oligomer-induced cell injury, apoptosis, and generation of intracellular reactive oxygen species (ROS), were attenuated by treatment with EGb761. Moreover, treatment of the cells with EGb761 decreased BBB permeability and increased tight junction scaffold protein levels including ZO-1, Claudin-5 and Occludin. We also found that the Aβ1–42 oligomer-induced upregulation of the receptor for advanced glycation end-products (RAGE), which mediates Aβ cytotoxicity and plays an essential role in AD progression, was significantly decreased by treatment with EGb761. To our knowledge, we provide the first direct in vitro evidence of an effect of EGb761 on the brain endothelium exposed to Aβ1–42 oligomer, and on the expression of tight junction (TJ) scaffold proteins and RAGE. Our results provide a new insight into a possible mechanism of action of EGb761. This study provides a rational basis for the therapeutic application of EGb761 in the treatment of AD.


Introduction
Ginkgo biloba leaves are a type of medicinal herb and their extract has been shown to have neuroprotective properties and enhance cognitive functions [1,2]. EGb761 is the standardized extract of Ginkgo biloba produced by Dr. Willar Schwabe Pharmaceuticals, which contains 22-27% flavonol glycosides, 5.4-6.6% terpene trilactones, 2.8-3.4% ginkgolides (A, B and C), 2.6-3.2% bilobalide, and less than 5 ppm ginkgolic acids [1]. Recently, EGb761 has received significant attention as a potential cognitive enhancer for the treatment of Alzheimer's disease (AD) [1][2][3][4]. Substantial clinical and preclinical evidence indicates that EGb761 limits vascular and neural damage and has many beneficial effects that support its use in treating AD individuals [5][6][7]. However, the cellular and molecular mechanisms underlying these effects remain to be elucidated.
AD is the most common neurodegenerative disease that causes progressive cognitive and behavioral deterioration in the elderly [8,9]. Extracellular deposition of the amyloid beta (Ab) is widely accepted as an important event in the pathogenesis of AD [10,11]. Ab is considered to be one of the most acute neurotoxins in the central nervous system [10][11][12]. Very recently, cerebrovascular changes leading to blood-brain barrier (BBB) leakiness have been associated with Ab deposition in the brains of AD individuals, and this may be involved in AD progression [13][14][15]. Despite great progress in understanding the etiology of AD, the process of deposition of Ab aggregates in cerebral capillaries and the brain is still poorly understood and the underlying pathogenic mechanisms of BBB leakage remain unclear. Furthermore, no effective treatment has been devised.
The receptor for advanced glycation end-products (RAGE) is an essential transmembrane cell-signaling receptor, which binds free Ab and mediates pathophysiological cellular responses, including oxidative stress, neurodegeneration, transport of circulating plasma Ab across the BBB into the brain, and brain endothelial cell (EC) damage [16][17][18][19]. RAGE expression is increased in cells of the neurovascular unit in the brains of AD individuals, and in disease models of AD both in vivo and in vitro [19,20]. This is particularly the case in models associated with an Ab-rich environment [21]. More importantly, antagonizing RAGE expression, or RAGE-knockout studies, show that blocking the RAGE-Ab interaction at the BBB suppresses the accumulation of Ab in brain parenchyma [22], prevents Ab-induced BBB disruption and ameliorates tight junction (TJ) scaffold protein expression [20]. These data suggest that RAGE is related to Ab accumulation as well as disruption of BBB integrity, and that RAGE might be a potential therapeutic target for AD.
Recently, an in vitro study in a cell monolayer BBB model reported that EGb761 diminished cell injury induced by chronic hypoxia and hypoglycemia (CHH), and significantly reversed CHH-induced upregulation of RAGE expression [23]. Considering the protective properties of EGb761 and its therapeutic potential, we speculated that EGb761 treatment might have a protective effect on Ab-induced BBB disruption by inhibition of RAGE. To testify our hypothesis, we employed an in vitro BBB model comprising an immortalized mouse brain capillary endothelial cell line (bEnd.3). Our study assessed the effects of Ab 1-42 oligomer treatment of bEnd.3 endothelial cells with respect to changes in the expression of RAGE, and TJ scaffold proteins including ZO-1, Claudin-5 and Occludin. Finally, we investigated the effect of EGb761 on Ab 1-42 oligomer treatment of bEnd.3 endothelial cells.

Reagents preparation
Lyophilized human Ab 1-42 was used to prepare Ab 1-42 oligomer as described previously [24,25]. Ab 1-42 was initially dissolved to 1 mM in hexafluoroisopropanol (HFIP, Sigma, USA) and aliquoted into sterile microcentrifuge tubes. Then, HFIP was removed under vacuum in a Speed Vac, and the peptide stored at 220uC. For oligomer preparation, 2 mM Ab 1-42 peptide that dissolved in dry dimethyl sulfoxide (DMSO, Sigma, USA) was subsequently diluted into ice-cold Opti-MEM (Gibco, USA) to bring the peptide to a final concentration of 100 mM. The solution was vortexed for 30 seconds, centrifuged for 1 minute, and incubated at 4uC for 24 h before use. EGb761 was dissolved in DMSO at a concentration of 200 mg/ml and stored at room temperature. The required concentrations of EGb761 were made by further dilution of the concentrated stock solution with Opti-MEM.
Cells were grown to 70-80% confluence prior to treatment. Before the treatments were applied, cells were rinsed in PBS and then the medium was replaced with Opti-MEM (Invitrogen, USA). For treatment of the cells exposed to Ab 1-42 oligomer and EGb761, the cells were pretreated with EGb761 for 2 h and then treated with Ab 1-42 oligomer.

Measurement of cell viability
Cell viability was measured the using MTT assay. bEnd.3 cells were seeded onto 96-well plates and treated with EGb761 at different concentrations. MTT (20 mL of a 5 mg/ml stock, diluted in PBS) was added to each cell culture well containing 100 mL of medium. After 4 h incubation at 37uC, the medium was gently aspirated. Deposited formazan crystals were lysed in 100 mL DMSO by gently shaking the plate. Absorbance at 570 nm was measured using a micro plate reader (Bio-Rad). The cell viability (%) was expressed as a percentage relative to the untreated control cells.

Detection of cell apoptosis
Apoptosis was observed by Hoechst-33258 staining (Apoptosis Hoechst staining kit; Beyotime, China). Briefly, cells were fixed in 0.5 mL of methanol for 15 min, followed by two washes with PBS. Cells were stained with 1 mg/mL Hoechst 33258 in a dark chamber at room temperature for 10 min and again washed twice in PBS. Cells were analyzed by fluorescence microscopy using excitation at 350 nm and emission at 460 nm. Apoptotic cells were identified on the basis of nuclear morphology changes such as chromatin condensation and fragmentation. In each group, ten fields of view were selected randomly and counted.

Detection of intracellular ROS
The level of intracellular reactive oxygen species (ROS) was quantified using the Reactive Oxygen Species Assay Kit (Beyotime, China). DCFH-DA is oxidized by reactive oxygen species in viable cells to 29,79-dichlorofluorescein (DCF) which is highly fluorescent at 530 nm. Cells were washed three times with PBS and then DCFH-DA, diluted to a final concentration of 10 mM, was added and the cells were incubated for 30 min at 37uC in the dark. After washing three times with PBS, the stained cells in the 6-well plate were analyzed by inverted fluorescence microscopy (CKX41, OLYMPUS, Japan). The relative levels of fluorescence in cells were quantified by a multi-detection microplate reader (Bio-Rad) with excitation at 488 nm and emission at 525 nm. The level of intracellular ROS was expressed as the percentage of the control cells.

BBB permeability assay
Transendothelial permeability was measured using Na-F as described previously [26,27] with the following modifications. bEnd.3 cells (5610 4 cells/cm 2 ) were cultured in the apical compartment, on a 0.4 mm pore size, 6.5 mm diameter polycarbonate membrane Transwell permeable insert (Corning). After the cells achieved confluence, 1.5 ml HHBS assay buffer (136 mM NaCl, 0.9 mM CaCl 2 , 0.5 mM MgCl 2 , 2.7 mM KCl, 1.5 mM KH 2 PO 4 , 10 mM Na H 2 PO 4 , 25 mM glucose, and 10 mM HEPES, pH 7.4) was added to the basolateral compartment. Culture medium in the apical compartment was replaced by 0.5 ml HHBS assay buffer containing 10 mg/ml Na-F (Kayon Biology). After 30 min, the medium from the basolatera compartments was removed and fluorescence in this medium was determined by a multiwell plate reader (Bio-Rad) at the wavelengths of 485 nm (excitation) and 535 nm (emission).

Western blotting
Cells extracts were prepared by washing cells twice with PBS and resuspending in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 7.4), 20 mM NaF, 20 mM EGTA, 1 mM DTT, 1 mM Na 3 VO 4 ) with PMSF containing protease and phosphatase inhibitors. The extracts were then subjected to ultrasonication. Western blotting was performed to measure the change in tight junction protein levels including ZO-1, Claudin-5 and Occludin, and RAGE. Protein samples (30 mg total protein per lane) were subjected to 10% SDS-PAGE. After electrophoresis, protein was transferred onto a nitrocellulose (NC) blotting membrane (Millipore). Membranes were blocked with 5% fat-free milk for 1 h at room temperature, and then incubated overnight at 4uC with the following rabbit primary antibodies diluted to 1:1000; anti-ZO-1 (Invitrogen, USA), anti-Claudin-5 (Invitrogen, USA), anti-Occludin (Invitrogen, USA), anti-RAGE (Millipore, USA) and anti-GAPDH (Santa Cruz, USA). Secondary goat anti-rabbit antibody (LI-COR, USA) was incubated with the filters for 1 h at room temperature. The images were captured using Odyssey infrared fluorescence imaging system (LI-COR, USA).

Statistical analysis
All results are expressed as the mean 6 S.E.M. Statistical analysis was performed using GraphPad Prism 5.0 software (GraphPad Software, Inc.). All experiments were repeated three times independently. Statistical significance of differences among different groups was analyzed by one-way analysis of variance (ANOVA) or student t test. A p-value,0.05 was considered statistically significant.

EGb761 diminished Ab 1-42 oligomer-induced cell injury of bEnd.3 cells
In this study, we first investigated whether EGb761 influenced the cell viability of bEnd.3 cells by MTT analysis. The results showed that incubation with various concentrations of EGb761 (25-200 mg/ml) in Opti-MEM did not lead to any significant changes in cell viability (Fig. 1A). However, at a concentration of 300 mg/ml, EGb761-treatment resulted in a significant decrease in cell viability (p = 0.0008, Fig. 1A). Therefore, concentration of EGb761 between 25-200 mg/ml was used in the subsequent experiments. This concentration range of EGb761 includes the 100 mg/ml concentration, which was showed to be effective in bEnd.3 cells in a related study [23].
The viability of bEnd.3 cells, pretreated with 25-200 mg/ml EGb761 and then incubated with Ab 1-42 oligomer was determined. The concentration of Ab 1-42 oligomer (10 mM) was based on the optimization data as described previously [20,28] with some modifications. The results showed that cells treated with Ab 1-42 oligomer alone had significantly reduced viability compared with untreated controls. Pretreatment with EGb761 for 2 h prior to addition of Ab 1-42 oligomer resulted in a significant increase in cell viability in a dose-dependent manner from 25 mg/ ml to 100 mg/ml EGb761. Fold changes in cell viability following EGb761 and Ab 1-42 oligomer treatment, relative to Ab 1-42 oligomer alone, were 1.07, 1.19, 1.48 and 1.41-fold at 25, 50, 100 and 200 mg/ml EGb761 respectively (#p,0.01, Fig. 1B).

EGb761 prevented Ab 1-42 oligomer-triggered apoptosis in bEnd.3 cells
To investigate the effect of EGb761 on bEnd.3 cell apoptosis, cells were incubated with or without EGb761 for 2 h, followed by treatment with 10 mM Ab 1-42 oligomer for another 24 h. We used a concentration of 100 mg/ml EGb761 since this was most effective in the MTT assay (Fig. 1B). In the untreated (Control) group, cell nuclei were uniformly stained with the Hoechst-33258 dye ( Fig. 2A, Control), whilst in the group treated with Ab 1-42 oligomer alone, bright chromatin condensation and nuclear fragmentation were observed, which is typical of apoptotic nuclei ( Fig. 2A, Ab). In the EGb761 and Ab 1-42 treated group, the nuclei were stained uniformly and the intensity of staining matched the untreated (Control) group ( Fig. 2A, EGb761+Ab). Apoptotic nuclei were quantitated and the results showed a significant increase in the percentage of apoptotic cells following treatment with Ab 1-42 oligomer alone (p,0.01, Ab versus Control, Fig. 2B). Treatment with EGb761 prior to addition of Ab 1-42 oligomer significantly reduced the percentage of apoptotic cells (p,0.01, EGb761+Ab versus Ab, Fig. 2B).

EGb761 attenuated Ab 1-42 oligomer-induced ROS generation in bEnd.3 cells
Oxidative stress plays an important role in Ab-induced cytotoxicity. Therefore, we examined the effect of EGb761 on Ab 1-42 oligomer-induced ROS generation in bEnd.3 endothelial cells. A marked increase in ROS generation was detected after treatment with Ab 1-42 oligomer alone, with 4.05-fold higher levels of oxidized DCF detected compared with untreated control cells (*p,0.01, Ab versus Control, Fig. 3A). Treatment with EGb761 prior to addition of Ab 1-42 oligomer significantly reduced ROS formation induced by the Ab 1-42 oligomer (#p,0.01, EGb761+ Ab versus Ab, Fig. 3A). These data suggest that EGb761 attenuated Ab 1-42 oligomer-induced ROS generation in bEnd.3 cells.

EGb761 reduced BBB leakage induced by the Ab 1-42 oligomer
The BBB is a specialized barrier that controls the transport of various molecules and maintains the integrity of brain by restricting permeability across the brain endothelium [17]. We found that Ab 1-42 oligomer increased permeability in cultured bEnd.3 cells (*p,0.01, Fig. 4). Pretreatment with EGb761 reversed the barrier permeability damaged induced by Ab 1-42 oligomer (#p,0.01, Fig. 4), and the effect was detected in a dosedependent manner from 25 mg/ml to 100 mg/ml.

EGb761 increased protein levels of ZO-1, Claudin-5 and Occludin in Ab 1-42 oligomer-induced bEnd.3 cells
TJs are the most prominent feature of the brain endothelium and are key structures that ensure the integrity of the BBB [28,29]. On the basis of the above results, we determined the effect of EGb761-pretreatment of bEnd.3 cells on the expression of TJ scaffold proteins ZO-1, Claudin-5 and Occludin. Cells were pretreated with or without EGb761 for 2 h, at concentrations from 25 mg/ml to 200 mg/ml, then exposed to 10 mM Ab 1-42 oligomer. Western blot and semi-quantitative analysis showed that the treatment with Ab 1-42 oligomer alone significantly decreased the levels of ZO-1, Claudin-5 and Occludin in bEnd.3 cells relative to the control (Ctrl) (*p,0.01, Fig. 5). Pretreatment with EGb761significantly increased the levels of those proteins (#p,0.01, Fig. 5). The protective effect of EGb761 on ZO-1 and Claudin-5 was in a concentration dependent manner from 25 mg/ml to 100 mg/ml, whereas Occludin levels increased in a concentration dependent manner from 25 mg/ml to 200 mg/ml.    (Fig. 3A, Control) and assessed by inverted fluorescent microscopy (1006). Following treatment for 24 h with 10 mM Ab 1-42 oligomer, an increase in fluorescence was detected (Fig. 3A, Ab). Cells treated with 100 mg/mL EGb761 for 2 h prior Ab 1-42 oligomer treatment for 24 h, showed a decrease in fluorescence (Fig. 3A, EGb761+Ab)  In this study, we hypothesized that EGb761 would protect against Ab-induced BBB disruption through inhibition of RAGE. To test the hypothesis, we determined the effect on the expression of RAGE in Ab 1-42 oligomer-induced bEnd.3 cells. Western blot and semi-quantitative analysis revealed that after incubation with Ab 1-42 oligomer for 24 h, the expression of RAGE was significantly increased by 1.97-fold when compared with the unexposed Control bEnd.3 cells (*p,0.01, Fig. 6). Whereas, treatment of Ab 1-42 oligomer-induced bEnd.3 cells with various concentrations of EGb761 led to a significant decrease in the expression of RAGE (#p,0.01, Fig. 6). Furthermore, the findings suggest that the protective effect of EGb761 on RAGE was in a dose-dependent manner from 25 mg/ml to 100 mg/ml. A further decrease in RAGE expression after pretreated with  200 mg/ml EGb761 was not detectable, when compared with 100 mg/ml EGb761 (Fig. 6).

Discussion
According to the vascular hypothesis of AD, initial vascular damage plays a critical role in the disease development [30]. The origin of BBB dysfunction during AD is not known. However, in a number of AD transgenic animal models, accumulation of Ab in blood vessels results in the disruption of the BBB [15,20,31]. The hypothesis is that BBB breakdown leads to accumulation in the brain of multiple vasculotoxic and neurotoxic macromolecules, and this can initiate functional and structural changes in neurons before Ab deposition occurs [30]. More importantly, BBB damage impairs vascular clearance of brain Ab and increases RAGEmediated influx of blood Ab into the brain [22,30]. In this study, we treated cultured immortalized mouse cerebral microvessel endothelial cells with Ab to model the conditions of the BBB in AD, and subsequently observed the effect of EGb761 on this cell monolayer model of BBB. bEnd.3 cell viability was significantly decreased in response to incubation with Ab 1-42 oligomer (Fig. 1). There was also a qualitative increase in the number of apoptotic bEnd.3 cells (Fig. 2) and an increase in ROS generation (Fig. 3). Treatment of EGb761 restored cell viability and reduced both Ab 1-42 oligomer-induced cell apoptosis and ROS production in vitro.
Intercellular TJs are the most prominent feature of brain endothelium and are responsible for BBB integrity [32]. The physical seal of the BBB is maintained by several different interendothelial TJ complexes that are composed of connecting transmembrane proteins (Occludin and Claudins). These proteins form the primary seal and are linked to accessory cytoplasmic proteins of Zona Occludens family members (ZO-1/2/3 etc), which can also independently link other types of transmembrane proteins to the actin cytoskeleton [33,34]. Studies have shown that TJ breakdown contributes to the deficiency in BBB function, and abnormal expression of TJ scaffold proteins results in loss of TJ integrity and increased BBB permeability [35,36]. In this study, we demonstrated that treatment with Ab 1-42 oligomer caused significant BBB leakage (Fig. 4) and downregulations of ZO-1, Claudin-5 and Occludin (Fig. 5). These effects were reduced by EGb761 treatment.
RAGE is a pattern recognition receptor that binds to number of ligands including Ab [37]. With the exception of the lungs, the basal expression of RAGE is low in physiological conditions but increases with the levels of its ligands [37,38]. Further, RAGEligand interaction and the subsequent up-regulation of RAGE through a positive feedback loop are associated with various diseases, including AD [39]. Accumulating evidence suggests that Ab plays an essential role in BBB disruption, however, the exact mechanism leading to BBB alteration has not been determined. Recently, Ab treatment was shown to induce RAGE expression in an in vitro study, and furthermore, interaction between Ab and RAGE triggered an intercellular cascade that disrupted TJ leading to the breakdown of BBB integrity [20,33]. When pathogenic Ab species accumulated in the AD brain, either in transgenic models of b-amyloidosis or in the human brain, RAGE expression was increased in affected cerebral vessels, neurons or microglia [40]. This mechanism provides the potential for exacerbating cellular dysfunction due to RAGE-Ab interactions. The activation of RAGE expressed in neuronal cells promotes synaptic dysfunction and as well leads to neurodegeneration by inducing inflammation in glial cells [9,41]. Moreover, RAGE-Ab interaction is implicated in the development of Alzheimer's neurovascular disorder through various mechanisms. These include mediation of transcytosis of circulating Ab across the BBB, induction of inflammatory responses in the endothelium, brain endothelial nuclear factor-kB (NF-kB) dependent apoptosis and suppression of cerebral blood flow (CBF), all of which culminate in BBB disruption [19,42]. In our present study we demonstrated that Ab 1-42 oligomer exposure led to a significant increase in the expression level of RAGE in bEnd.3 cells (Fig. 6).
Accumulating evidence suggests that RAGE is a potential target for therapies to lower brain Ab burden, prevent BBB damage, and improve both CBF and behavioral performance [19,20]. These data suggest RAGE is a potential therapeutic target for AD. A recent study showed that EGb761 markedly reversed the upregulation of RAGE induced by a CHH condition in a BBB in vitro model at both the RAGE mRNA and protein level [23]. These data suggest a rational basis for the therapeutic application of EGb761 in the treatment of AD [23]. Thus, we hypothesized that EGb761 would protect brain ECs against Ab toxicity via inhibition of RAGE expression. The results indicated that the upregulation of RAGE expression induced by Ab 1-42 oligomer was reversed by treatment with EGb761 (Fig. 6).
EGb761 has received a great many attentions because it exerts beneficial effects in conditions which are associated with impaired cognitive function [1,3,7]. In the present study, we found that 100 mg/ml of EGb61 showed maximal protection in mainly detection indexes including cell viability, apoptosis, ROS, and the expression levels of ZO-1 and Claudin-5. However, the results also showed that 200 mg/ml of EGb761 resulted in maximal protection with regard to the expression of Occludin. Furthermore, the data indicated that the difference was not significant between 100 mg/ ml and 200 mg/ml of EGb761 at the BBB permeability and the expression level of RAGE after incubation with Ab.
In conclusion, we have presented novel evidence to show that EGb761 effectively prevented Ab 1-42 oligomer-induced brain EC damage, which was characterized by reduced cell viability injury, increased cell apoptosis and increased intracellular ROS generation. Furthermore, we found that EGb761 reduced BBB leakage, reversed Ab 1-42 oligomer-induced down-regulation of TJ scaffold proteins and prevented the Ab 1-42 oligomer-induced up-regulation of RAGE in bEnd.3 cells. To our knowledge, this is the first direct evidence for an effect of EGb761 on brain endothelial cells, and for an effect of EGb761 on the expression of RAGE and TJ scaffold proteins exposed to Ab 1-42 oligomer. Our results provide a rational basis for the therapeutic application of EGb761 in the treatment of AD.