An Optimized Combination of Ginger and Peony Root Effectively Inhibits Amyloid-β Accumulation and Amyloid-β-Mediated Pathology in AβPP/PS1 Double-Transgenic Mice

Abstract

The progressive aggregation of amyloid-β protein (Aβ) into senile plaques is a major pathological factor of Alzheimer’s disease (AD) and is believed to result in memory impairment. We aimed to investigate the effect of an optimized combination of ginger and peony root (OCGP), a standardized herbal mixture of ginger and peony root, on Aβ accumulation and memory impairment in amyloid-β protein precursor (AβPP)/presenilin 1 (PS1) double-transgenic mice. In an in vitro thioflavin T fluorescence assay, 100 μg/ml OCGP inhibited Aβ accumulation to the same extent as did 10 μM curcumin. Furthermore, AβPP/PS1 double-transgenic mice treated with OCGP (50 or 100 mg/kg/day given orally for 14 weeks) exhibited reduced Aβ plaque accumulation in the hippocampus and lower levels of glial fibrillary acid protein and cyclooxygease-2 expression compared with vehicle-treated controls. These results suggest that OCGP may prevent memory impairment in AD by inhibiting Aβ accumulation and inflammation in the brain.

Keywords

Aβ accumulation AβPP/PS1 double-transgenic mice neuroinflammation optimized combination of ginger and peony root

INTRODUCTION

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by memory and cognition loss, as well as behavioral and occupational instability in old age. Although the precise mechanism of AD pathogenesis has not yet been determined, progressive aggregation of insoluble amyloid-β protein (Aβ) in the form of senile plaques is thought to be a major pathological factor in the development of AD [1, 2]. Aggregated forms of Aβ such as small soluble oligomers, protofibrils, diffuse plaques, and fibrils act as neurotoxins by inducing filamentous aggregates, synaptic loss, neuroinflammation, and neuronal cell loss. These effects culminate in memory impairment [3 –5]. Therefore, inhibition of Aβ aggregation in the brain is an important strategy for treating AD.

Mutations in genes encoding amyloid-β protein precursor (AβPP) have been linked to autosomal dominant forms of familial AD. These mutations have been shown to increase the production of AβPP-derived Aβ peptides, both total Aβ and Aβ_1 - 42. The production of Aβ is enhanced by mutations in presenilin 1 (PS1) [6 –9]. It exhibits remarkable elevation of Aβ production associated with more certain behavioral abnormalities as well as neuropathological features compared with artificial AD mice such as Aβ stereotaxic injected model [10, 11]. Previous studies have reported that AβPP/PS1 double-transgenic mice expressing a chimeric mouse/human version of AβPP (Mo/HuAβPP695swe) and a mutant human version of PS1 (PS1-dE9) exhibit age-dependent increases in Aβ deposition and cognitive deficits [12 –14]. Moreover, Aβ aggregates and neuroinflammatory markers [e.g., nuclear transcription factor kappa B (NF-κB), cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and prostaglandin E2 (PGE2)] have been reported to be highly correlated with memory impairment in AβPP/PS1 mice [15 –17].

Herbal extracts have been widely used as supplements in traditional and alternative medicines [18]. Since herbal extracts often work in multimodal manners, interest in these extracts as pharmacological remedies for central nervous system diseases has increased [18]. Many herbal extracts are combined into multi-herbal prescriptions, thus creating novel therapeutic agents that are more effective than the individual herbal extracts themselves [19]. These combinations can be more effective because many herbal mixtures have synergistic effects and can neutralize toxic or adverse effects of their individual constituent herbs [20]. For example, Danggui-Shaoyao-San, a mixture composed of six medicinal herbs, has been shown to improve Aβ-induced spatial memory deficits by preventing Aβ aggregation and fibrillation in mice [21]. Similarly, Yokukan-San, a mixture composed of five medicinal herbs, has been reported to have neuroprotective effects against Aβ aggregation and glutamate [22, 23]. Moreover, Hachimi-Jio-Gan, composed of eight medicinal herbs, has been used to ameliorate cognitive deficits in the elderly [24].

We previously showed that ginger, the rhizome of Zingiber officinale Roscoe (Zingiberaceae), exerts potent synaptogenic effects by activating nerve growth factor (NGF)-induced extracellular signal-regulated kinase (ERK)/cAMP response element-binding protein (CREB) signaling. This activation results in enhanced memory function in normal mice. Moreover, 6-shogaol, a major compound of ginger, inhibits glial cell activation and reduces cognitive deficits in animal models of dementia [25, 26]. We first evaluated the effects of five different combinations of ginger with other herbs on scopolamine-induced memory impairment (Supplementary Figure 1A). We found that the mixture of ginger and peony root was most effective in preventing memory impairment out of all the mixtures tested. Next, we evaluated the efficacy differences by the ratio of ginger and peony root in scopolamine-treated mice (Supplementary Figure 1B). These experimental results indicated that a mixture was most effective when the rate of ginger and peony root was 3:1. An optimized combination of ginger and peony root (OCGP) was thus chosen in this study. We next investigated the effects of OCGP on Aβ accumulation in vitro using a thioflavin T (Th T) assay.

Th T preferentially binds in the channels formed between adjacent side-chains running across β-strands within a β-sheet layer and parallel to the long axis of fibril [27]. Th T fluorescence enhancement upon fibril binding could contain useful information on fibril formation by random coil structured changes [28]. For these reasons, Th T fluorescence in vitro assay that could reflect Aβ biophysical conformation was performed in this study. To validate the effects of OCGP in vivo, we evaluated the effects of OCGP on memory impairment in AβPP/PS1 mice using a standard behavior test. We also used immunohistochemistry to measure Aβ plaque accumulation, inflammatory marker levels, and astrocyte activation in the brain.

MATERIALS AND METHODS

Chemicals

Rabbit monoclonal anti-gilal fibrillary acid protein (GFAP) antibody was purchased from Millipore Bioscience Research (Bedford, MA, USA). Goat and rabbit polyclonal anti-COX-2 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal antibody anti-Aβ_17–24 (4G8) was purchased from Covance (Emeryville, CA, USA). Biotinylated horse anti-goat antibody, goatanti-rabbit antibody, normal goat serum, normal horse serum, and avidin-biotin complex were purchased from Vector Laboratories (Burlingame, CA, USA). Aβ_1–40 peptide was purchased from American Peptide (Sunnyvale, CA, USA). Donepezil hydrochloride was supplied by Eisai Korea Co. Ltd. (Aricept^®; Seoul, Korea). All other reagents including gallic acid, albiflorin, paeoniflorin, benzoic acid, paeonol, 6-gingerol and 6-shogaol were purchased from Sigma-Aldrich (St. Louis, MO, USA) and solvents used of guaranteed or analytical grade.

Preparation of the OCGP

The raw material mixture (100 g) composed of the rhizome of Paeonia lactiflora Pall. (75 g) and the radix of Zingiber officinale Roscoe (25 g) was ground and extracted with 1000 ml of 95% ethanol for 3 h at 79°C. Then, the extract was filtered, evaporated on a rotary vacuum evaporator, and freeze dry (yield; 23.25%). The powder was kept at 4°C before use.

HPLC fingerprinting of OCGP extract

Chromatographic analyses were carried out on an Agilent 1260 HPLC system (Agilent Technologies Inc.,), equipped with Chemstation software, a quaternary pump G1311C, a thermostat G1330B, an autosampler G1329B, a thermostatic column compartment G1316A, and a diode array detector G1315D. An analytical Xselect CSH C18 reversed-phase column (250 mm × 4.6 mm ID) with a particle size of 5 μm from Waters (Milford, MA, USA) was used for chromatographic separation at 35°C. The mobile phase consisted of (A) 0.1% formic acid in water and (B) acetonitrile. The total running time was 66 min, with the mobile phase gradient of 4 min at10% B, 10 min at 15% B, 32 min 20% B, 40 min at 45% B, 50 min at 4% B, 52 min at 70% B, 60 min at 70% B, 61 min at 10% B, and 66 min at 10% B. The mobile phase flow rate was 1.0 ml/min, and the injection volume was 10 μl. The effluent was monitored at 254 nm. We observed that OCGP extract contained 0.52% gallic acid, 5.71% albiflorin, 6.77% paeoniflorin, 0.61% benzoic acid, 0.04% paeonol, 0.30% 6-gingerol and 0.12% 6-shogaol (Fig. 1).

Thioflavin T measurement

Briefly, the effect of OCGP on accumulation of monomeric Aβ was examined using Th T assay as described previously [29]. The solutions of monomeric Aβ_1–40 (5 μl of 100 mM in DMSO) and 5 μl of samples or phosphate buffered saline (PBS) were added to 40 ml PBS at pH 7.4. The resulting mixture was incubated for 2 h at room temperature. To the resulting mixture, 150 μl of Th T solution (5 mM in 50 mM glycine–NaOH at pH 8.5) was added and incubated for 30 min. The Th T fluorescence was measured at 520 nm with excitation at 470 nm in FLUOstar Omega multi-mode microplate reader (BMG LABTECH GmbH, Ortenberg, Germany).

Animals and drug administration

All experiments with mice were performed according to the protocols approved by the Institutional Animal Care and Use Committee of Kyung Hee University (KHP-2012-01-1). Male AβPP/PS1 mice (AβPPswe, PSEN1dE9, 6 weeks, 25–28 g) were purchased from Central Lab. Animal Inc. (Seoul, Korea). The mice were housed five per cage, had free access to water and food and maintained under a constant temperature (23 ± 1°C), humidity (60 ± 10%) and a 12 h light–12 h dark cycle. After mice were seven months old, the treatments had been proceeding for 14 weeks. OCGP was dissolved 0.5% carboxymethyl cellulose (CMC) and administered orally once per day for 14 weeks. The wild-type (WT) group and AβPP/PS1 control group were administered with an equal volume of 0.5% CMC. The mice were randomly divided into four groups as follows (n = 5 in each group): (1) WT group (C57/BL mice plus intraorally 0.5% CMC-treated group), (2) AβPP/PS1 control group (AβPP/PS1 mice plus intraorally 0.5% CMC-treated group), (3) Donepezil group (AβPP/PS1 mice plus intraorally donepezil 2 mg/kg/day treated group), (4) OCGP 50 group (AβPP/PS1 mice plus intraorally OCGP 50 mg/kg/day treated group), (5) OCGP 100 group (AβPP/PS1 mice plus intraorally OCGP 100 mg/kg/day treated group). Donepezil was used as a positive control that plays a role of scientific controlled experiments rather than comparison with effects of OCGP extract. Recent pre-clinical and clinical studies have demonstrated that donepezil was also proved to protect against the Aβ toxicity in AβPP/PS1 mice [30 –32].

Behavior test

Novel object recognition test

The novel object recognition test was performed according to the method described previously [33]. The experiments were carried out in a white open field box (45 × 45 × 50 cm). Prior to the test, mice were habituated to the test box for 5 min without objects. After a habituation period, mice were placed into the test box with two identical objects and allowed to explore for 3 min. The objects used in this study were wooden blocks of the same size but different shape. The time spent by the animal exploring each object was measured (defined as the training session). Twenty-four hours after training session, mice were allowed to explore the objects for 3 min, in which familiar object used in the previous training session was placed with a novel object. The time that the animals spent exploring the novel and the familiar objects were recorded (defined as the test session). The animals were regarded to be exploring when they were facing, sniffing or biting the object. Results were expressed as percentage of novel object recognition time (time percentage = _tnovel/_tnovel + _tfamiliar] × 100).

Y-maze test

The Y-maze test was performed according to the method described previously [34]. The Y-maze apparatus is composed of a three-arm with equal angles between all arms. Mice were placed in the one arm, and were to explore freely through the maze. The spontaneous alternation and total entries were manually measured for each mouse over an 8-min period. An actual alternation was defined as entries into all three arms consecutively (i.e., ABC, CAB, or BAC but not ABA). The percentage of alternations was calculated as shown by the following equation: [(the number of alternations)/(the total number of arm entries - 2)] × 100.

Brain tissue preparation

At 24 h after examination of memory behavior, mice were perfused transcardially with 0.05 M PBS, and then fixed with cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. Brains were removed and post fixed in 0.1 M phosphate buffer containing 4% PFA overnight at 4°C and then immersed in a solution containing 30% sucrose in 0.05 M PBS for cryoprotection. Serial 30 μm-thick coronal sections were cut on a freezing microtome (Leica, Nussloch, Germany) and stored in cryoprotectant (25% ethylene glycol, 25% glycerol, and 0.05 M phosphate buffer) at 4°C until use.

Immunohistochemistry

For immunohistochemical study, brain sections were briefly rinsed in PBS and treated with 1% hydrogen peroxide for 15 min. The sections were incubated with a mouse 4G8 antibody (1:1000 dilution), a rabbit anti-GFAP antibody (1:3000 dilution) and a goat polyclonal anti-COX-2 (1:1000 dilution) overnight at 4°C in the presence of 0.3% triton X-100 and NHS or NGS. After rinsing in PBS, the sections were then incubated with biotinylated anti-rabbit and anti-goat IgG (1:200 dilution) for 90 min and with ABC (1:100 dilution) for 1 h at room temperature. Peroxidase activity was visualized by incubating sections with DAB in 0.05 M tris–buffered saline (pH 7.6). After several rinses with PBS, sections were mounted on gelatin-coated slices, dehydrated, and cover-slipped using histomount medium. The optical densities of GFAP immunoreactivities in the hippocampus, those of COX-2 immunoreactivities of CA3 region in the hippocampus and those of Aβ in the hippocampus were analyzed with ImageJ software (Bethesda, MD, USA). For measurement of the optical density of GFAP, COX-2 and Aβ, the total region of interest was manually outlined and averaged optical densities were acquired in images with converted eight-bit indexed color. The images were photographed at 200 × or 400 × magnification using an optical light microscope (Olympus Microscope System BX51; Olympus, Tokyo, Japan) equipped with a 20 × objective lens.

Statistical analysis

All statistical parameters were calculated using Graphpad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA) Values were expressed as the mean ± standard error of the mean (S.E.M.). Th T assay was evaluated by one-way analysis of variance (ANOVA) analysis followed by the Tukey’s post hoc test. Behavior tests and immunohistochemistry were evaluated by the Student’s t-test. Differences with a p-value less than 0.05 were considered statistically significant.

RESULTS

Effects of OCGP on Aβ accumulation in vitro

As an initial investigation into the effect of OCGP on Aβ accumulation, we performed a Th T assay. Th T, a cationic benzothiazole dye, is widely used for the identification and quantification of amyloid fibrils in vitro because it selectively localizes to amyloid deposits and shows enhanced fluorescence upon binding toamyloid [35]. OCGP (1, 10, and 100 μg/ml) treatment inhibited the aggregation of monomeric Aβ_1–40 (10 mM) in a dose-dependent manner, as demonstrated by the reduced Th T fluorescence intensity in the presence of OCGP (Fig. 2). These results suggest that OCGP interacts with Aβ_1–40 monomers and may be able to reduce the accumulation of Aβ oligomers and fibrils.

Effects of OCGP on Aβ accumulation in the hippocampus of AβPP/PS1 mice

To assess the effect of OCGP on Aβ accumulation in the hippocampus of AβPP/PS1 mice, we treated AβPP/PS1 mice with OCGP (50 or 100 mg/kg/day for 14 weeks). We then determined the percentage of 4G8-immunoreactivity in each hippocampus. These data were expressed as a rate of 4G8-immunoreactive area to the entire hippocampal area. The Aβ-positive percentage in the WT group (0.33 ± 0.02%) was significantly increased compared with that in the AβPP/PS1 control group (0.03 ± 0.01%). However, in the OCGP 50 mg/kg/day and OCGP 100 mg/kg/day groups, the Aβ-positive percentages were significantly decreased (0.17 ± 0.00% and 0.15 ± 0.01% , respectively) compared with that in the AβPP/PS1 control group (Fig. 3).

Effect of OCGP on neuroinflammation in the hippocampus

To investigate the effect of OCGP on neuroinflammation in AβPP/PS1 mice, we performed immunohistochemical staining of GFAP and COX-2 in hippocampal slices from AβPP/PS1 mice. These data were expressed as percentages of WT values. Rapid synthesis of GFAP, which is characteristic of astrogliosis, can be detected by looking for increased protein content or by immunostaining with anti-GFAP antibodies [36]. COX-2 is also rapidly induced during inflammation [37]. We found that the GFAP-positive area in the AβPP/PS1 control group (167.32 ± 14.36%) was significantly increased compared with that in the WT group. However, the GFAP-positive area was significantly decreased in the 100 mg/kg OCGP group (97.91 ± 18.37%) compared with that in the AβPP/PS1 control group (Fig. 4A). The GFAP-positive area was also significantly decreased in the 100 mg/kg OCGP group compared with that in the donepezil group (155.42 ± 7.31%). In addition, the intensity of the COX-2-positive area was significantly increased in the AβPP/PS1 control group (150.79 ± 8.39%) compared with that in the WT group. Moreover, the COX-2-positive area in the 100 mg/kg OCGP group was significantly decreased (102.64 ± 13.59%) compared with that in the AβPP/PS1 control group. It was also significantly decreased in the 100 mg/kg OCGP group compared with that in the donepezil group (149.96 ± 10.03% ; Fig. 4B). These results indicate that OCGP inhibits astrocyte activation and downregulates COX-2 expression. These results also suggest that OCGP prevents the inflammatory response associated with Aβ aggregates, thereby reducing their neurotoxicity.

Effects of OCGP on cognitive behavior-related memory impairment

To determine whether OCGP ameliorates the cognitive deficits induced by soluble Aβ aggregates, we performed a novel object recognition test and the Y-maze test. The novel object recognition test was performed on weeks 8 and 12. The recognition index (%) of the AβPP/PS1 control group on weeks 8 and 12 were decreased (35.64 ± 4.54% and 52.66 ± 8.59% , respectively) compared with those of the WT group (48.58 ± 3.44% and 67.83 ± 4.04% , respectively). In contrast, the recognition index of the 100 mg/kg OCGP group on week 8 and 12 were increased (48.54 ± 3.21% and 64.06 ± 4.30% , respectively) compared with that of the AβPP/PS1 control group (Fig. 5A, B). Thus, OCGP treatment tended to ameliorate cognitive deficits. To confirm the effect of OCGP on spatial memory, the Y-maze test was performed on weeks 12 and 14. The spontaneous alternations (%) of the AβPP/PS1 control group were decreased (52.90 ± 3.24% and 46.78 ± 3.56% , respectively) compared with those of the WT group (55.72 ± 5.09% and 59.74 ± 2.25% , respectively). In contrast, the spontaneous alternations (%) of the OCGP 100 group (70.80 ± 2.77% and 65.84 ± 5.26% , respectively) were significantly increased compared with those of the AβPP/PS1 control group (Fig. 5C, D). These data show that OCGP treatment can significantly improve spatial memory.

DISCUSSION

The aim of the present study was to assess the effect of OCGP on memory impairment in AβPP/PS1 mice and to investigate the mechanisms underlying this effect. To evaluate the effect of OCGP on Aβ accumulation, we performed a Th T assay. To evaluate the effect of OCGP on memory impairment in AβPP/PS1 mice, we performed a memory-related behavior test. Finally, to measure Aβ plaque accumulation, inflammatory marker levels, and astrocyte activation in the brain, we performed immunohistochemical analyses. We found that OCGP could prevent memory impairment by inhibiting Aβ accumulation and neuroinflammation.

We found that OCGP inhibited Aβ accumulation both in vitro (Fig. 2) and in vivo (Fig. 3). The Aβ peptide and its aggregates are believed to be central to the pathology of AD [38]. The neurotoxic forms of Aβ are oligomeric, not monomeric; moreover, they act rapidly and with high potency, though their effects are temporary [39]. The neurotoxic forms also lead to neurotoxicity, oxidative stress, and neuroinflammation [40]. Thus, inhibiting Aβ accumulation is a promising therapeutic strategy for treating AD. We found that OCGP inhibited Aβ accumulation and reduced the Aβ-positive area in AβPP/PS1 mice. This finding suggests that OCGP may protect the brain from Aβ-induced toxicity by reducing Aβ accumulation using at least in mice due to difficulties for keeping a breeding system or genotyping of transgene. This protection may result from decreased production of Aβ, increased enzymatic degradation of Aβ, or increased clearance of Aβ across the blood-brain barrier.

Neuroinflammation has been shown to be linked with amyloidogenesis [41]. Some studies have shown that the in vitro neurotoxicity of Aβ is enhanced by microglia [42, 43] and astrocytes [44, 45]. Additionally, neuroinflammation has been shown to upregulate AβPP expression and the accumulation of intracellular Aβ [46]. COX-2 is stimulated by Aβ, glutamate, and inflammatory cytokines [47]; moreover, several studies have reported that COX-2 expression is increased in the early stages of AD [48 –50]. Previous studies have suggested that COX-1 and COX-2 expression may affect the generation of Aβ through mechanisms that involve potentiation of γ-secretase activity [51]. Moreover, ibuprofen, a nonselective COX inhibitor, has been shown to decrease Aβ aggregates in an AD-type mouse model [52, 53]. Some studies using transgenic mice have shown that the numbers of brain cells positive for GFAP, a specific marker for astrocytes, are associated with memory impairment in AβPP/tau double-transgenic mice [54] and AβPP/PS1 mice [55]. The levels of COX-2 have also been reported to be significantly elevated in AβPP/PS1 mice compared with their wild-type counterparts [16]. Thus, we performed immunohistochemical analysis of GFAP and COX-2 expression in the hippocampus to investigate the effect of OCGP on neuroinflammation in AβPP/PS1 mice (Fig. 4). Our data are consistent with previous studies demonstrating a link between astrocyte activation and COX-2 expression [16, 55]. Our data also indicate that OCGP inhibits astrocyte activation and that COX-2 expression is upregulated by Aβ aggregates. These results suggest that OCGP inhibits the inflammatory response associated with Aβ-mediated neurotoxicity.

Memory impairment is a major symptom of AD [56]. Some studies have revealed a significant correlation between Aβ plaques and cognitive deficits in patients with AD [57 –59] and in AβPP transgenic mice [60, 61]. In addition, some studies have reportedcorrelations between soluble Aβ and cognitive deficits in human patients with AD [62 –64], as well as in AβPP transgenic mice [65, 66]. Previous studies have shown that memory is impaired in AβPP/PS1 mice, which are a well-established transgenic mouse model of AD [67]. Therefore, we used AβPP/PS1 mice to evaluate the effect of OCGP on cognitive deficits in an AD-type animal model. We found that OCGP ameliorated Aβ aggregate-induced cognitive deficits (Fig. 5).

The search for novel AD treatments has gradually changed focus from single-targeted organic compound synthesis to the development of natural compounds from herbs with multiple actions [68]. In this study, OCGP showed multiple therapeutic effects against both amyloidogenesis and neuroinflammation. Ginger, one of the components of OCGP, has been shown to exhibit anti-AD activities by regulating acetylcholinesterase, protecting neuronal cells, and exerting anti-inflammatory and nootropic effects [69 –72]. We also previously showed that ginger exhibited potent synaptogenic effects via NGF-induced ERK/CREB activation, resulting in memory enhancement in vivo. Moreover, 6-shogaol, a major compound of ginger, inhibited glial cell activation and reduced cognitive deficits in animal models of dementia [25, 73]. Peony root, which is another herb present in OCGP, has been shown to exert neuroprotective and anti-convulsant effects and to improve memory impairment [74 –78]. Paeoniflorin, which is a major compound of peony root, has also been reported to attenuate Aβ peptide-induced neurotoxicity [79]. These effects of ginger and peony root may be responsible for the beneficial effects of OCGP against Aβ aggregate-induced toxicity and memory deficits.

In conclusion, the present study showed that OCGP ameliorated memory impairment in AβPP/PS1 mice due to its effects by inhibiting the Aβ accumulation, reducing astrocyte activation, and dampening neuroinflammation. As a result of these effects, OCGP is potentially a novel therapeutic agent for treating other related neuroinflammatory diseases as well as AD.

Footnotes

ACKNOWLEDGMENTS

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (Grant No.: NRF-2015R1A2A2A01004341).

Authors’ disclosures available online ().

Appendix

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