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Journal of Alzheimer’s Disease 41 (2014) 599–613 DOI 10.3233/JAD-140270 IOS Press
Bombycis excrementum Reduces Amyloid- Oligomer-Induced Memory Impairments, Neurodegeneration, and Neuroinflammation in Mice Minho Moona,1,2 , Jin Gyu Choib,d,1 , Sun Yeou Kimc and Myung Sook Ohb,d,∗ a School
of Medicine, Kyung Hee University, Dongdaemun-gu, Seoul, Republic of Korea of Pharmacy, Kyung Hee University, Dongdaemun-gu, Seoul, Republic of Korea c College of Pharmacy, Gachon University, Incheon, Republic of Korea d Department of Life and Nanopharmaceutical Science and Kyung Hee East-West Pharmaceutical Research Institute, Kyung Hee University, Dongdaemun-gu, Seoul, Republic of Korea b College
Accepted 9 February 2014
Abstract. Alzheimer’s disease (AD) is the most common cause of progressive dementia and is characterized by memory impairments, neuronal death, and neuroinflammation. AD-related pathophysiology is caused primarily by the presence of amyloid- oligomers (AO). Recently, an increased focus has been directed toward natural compounds or medicinal extracts for the treatment of AD. Extracts from Bombycis excrementum (BE), which is composed of various bioactive constituents and mulberry leaves (the preferred food of silkworms), have been shown to possess anti-inflammatory, anti-diabetic, and antioxidative effects. Additionally, mulberry leaves exert anti-amyloidogenic action and neuroprotective effects against A peptides but it is unknown whether BE has a therapeutic effect on AD-related pathologies. Therefore, the present study examined whether BE inhibits AO-induced memory loss, neuronal death, and inflammation. Behavioral tests revealed that BE significantly ameliorated AO-induced memory impairments and inhibited AO-induced neuronal loss in cultured cells and the brains of mice. BE also significantly inhibited microgliosis and astrogliosis following intra-hippocampal AO injections in mice. Furthermore, BE significantly attenuated the release of nitric oxide from microglia and reduced AO-induced S100- cytokine release from activated astrocytes. These results suggest that BE may be a candidate agent for the treatment of AD. Keywords: Alzheimer’s disease, amyloid- oligomer, Bombycis excrementum, cognitive impairment, neuroinflammation, neuronal death, silkworm droppings
INTRODUCTION Alzheimer’s disease (AD) is one of the most common age-related neurological disorders. Although the 1 These
authors contributed equally to this study. address: Molecular Neurobiology Laboratory, McLean Hospital/Harvard Medical School, Belmont, MA, USA. ∗ Correspondence to: Myung Sook Oh, OMD, PhD, Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, #1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea. Tel.: +82 2 961 9436; Fax: +82 2 963 9436; E-mail: [email protected]. 2 Present
exact mechanisms underlying the pathogenesis of this disease remain unclear, it is believed that intracellular and extracellular accumulations of amyloid- (A) peptides play a central role [1, 2]. The progression of AD pathology occurs early in hippocampal formation. This brain region is critical for learning and memory behaviors and is one of the most susceptible to A-mediated degeneration [3–6]. Several studies have demonstrated that AD-mediated damage is caused primarily by A oligomers (AO) that may lead to the induction of long-term synaptic depression and inhibition of pro-synaptic long-term potentiation in the
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hippocampus [7, 8]. Additionally, AO are correlated with the degree of hippocampal cell loss and cognitive impairment [9], and activated astrocytes and microglial cells are characteristically found in abundance near neurons damaged by AO in brains with AD [10, 11]. Because neurodegenerative disorders such as AD are a result of multiple pathogenic factors, single-target therapeutic agents have proved inefficient for their treatment [12, 13]. Based on such evidence, the development of therapeutic agents with various functions that target multiple pathological pathways has attracted much attention [12, 13]. In recent years, an increased focus has been placed on the use of natural compounds or medicinal herbal extracts that appear to function in a multimodal manner for the treatment of AD [14, 15]. For example, Ginkgo biloba extract inhibits A fibril formation, modulates cholinergic neurotransmission, and stimulates cholinergic activity in the brain [16]. Huperzine A, an alkaloid extracted from Lycopodium serratum, exerts neuroprotective effects against Ainduced death [17] and Jangwonhwan, an oriental medicine used for amnesia, has been shown to reduce neural A deposition in a Tg-APPswe/PS1dE9 mouse model of AD [18]. Additionally, vitamin D treatment exhibits the protective effects against A toxicity in cortical neuronal cultures [19]. In clinical trial, AD patients receiving vitamin E showed a slowing in functional decline [20]. Thus, it is suggested that naturally occurring agents with multi-functional effects should be examined for their efficacy when treating AD. Bombycis excrementum (BE) is also known as silkworm (Bombyx mori L) excrement, silkworm feces, silkworm excreta, silkworm droppings, and can sha (Chinese name). BE acts as a raw material for a variety of products including chlorophyll, carotenoids, sodium copper chlorophyllin, pectin, and phytol [21, 22]. Chlorophyll extracted from BE has been used as a therapy for gastric ulcers and hepatitis [23], and pectin extracted from BE has been shown to reduce blood triglycerides and cholesterol levels [21]. 1-Deoxynojirimycin, a typical natural alkaloid component of BE, exerts anti-diabetic effects in a mouse model of Sandhoff disease via marked inhibition of ␣glucosidase activity [24] and a synergistic effect with non-steroidal anti-inflammatory drugs [25]. Furthermore, it has been demonstrated that megastigmane sesquiterpenes and flavonoids from BE increase the expression and promote the activity of the antioxidant enzymes heme oxygenase-1 and SIRT1, which are involved in the suppression of inflammatory medi-
ators [26, 27]. Studies show that more than half of the mulberry leaves used to feed silkworms are excreted without being digested [26] and that these leaves exert anti-amyloidogenic action and neuroprotective effects against A peptides [28, 29]. However, it is unknown whether BE treatment has protective effects against AO-mediated pathology and the subsequent cognitive deficits. Thus, this study evaluated whether BE extract (BEE) exerts protective effects against AO-induced memory impairments, neuronal death, and neuroinflammation in a mouse model utilizing intra-hippocampal injections of AO and AO-treated cultured cells.
MATERIALS AND METHODS Materials Roswell Park Memorial Institute (RPMI) medium, Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), horse serum (HS), and penicillin–streptomycin (P/S) were purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). Rabbit monoclonal anti-glial fibrillary acid protein (GFAP) and rat monoclonal anti-GFAP were purchased from Millipore Bioscience Research (Bedford, MA, USA). Rat monoclonal anti-CD11b (Mac-1) was purchased from Chemicon International (Temecula, CA, USA). Rabbit polyclonal anti-S100- was purchased from Abcam (Cambridge, UK). Biotinylated goat antirabbit antibody, rabbit anti-rat antibody, normal goat serum (NGS), normal horse serum (NHS), goat anti-rat IgG Cy3 conjugate Alexa 594 antibody, Streptavidin Alexa 488 antibody, Vectashield Mounting Medium with 4 ,6-diamidino-2-phenylindole (DAPI), and avidin-biotin complex (ABC) were purchased from Vector Lab (Burlingame, CA, USA). Zoletil 50® and Rompun® were purchased from Virbac (Carros, France) and Bayer Korea (Seoul, Korea), respectively. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), cresyl violet, paraformaldehyde (PFA), 3,3-diaminobenzidine (DAB), dimethyl sulfoxide (DMSO), sodium bicarbonate, sodium chloride, sucrose, ethanol, phosphate buffered saline (PBS), collagen, Griess reagent, DMSO anhydrous form, and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A1-42 peptide was purchased from American Peptide (Sunnyvale, CA, USA). Protein assay was purchased from Bio-Rad Laboratories (Hercules, CA, USA).
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Preparation of oligomeric Aβ1-42 solution
Culture of PC12 and BV-2 cell and treatment
Soluble oligomers were generated by previously described methods with slight modifications [30, 31]. Briefly, A1-42 was dissolved in HFIP to the final concentration of 1 mg/ml at room temperature for 3 days. The peptide was aliquoted and dried under vacuum for 1 h. The aliquoted peptide was dissolved in DMSO anhydrous form to the final concentration of 1 mM. Protein determination was performed by Bradford assay to calculate molarities of solution. The A stock in DMSO anhydrous form was diluted directly into PBS at 10 M and incubated at 4◦ C for 24 h to make oligomeric form of A.
Rat pheochromocytoma PC12 cells were maintained in RPMI media, supplemented with 5% heat-inactivated FBS, 10% HS, and 1% P/S in watersaturated atmosphere of 5% CO2 at 37◦ C. Mouse BV-2 microglial cells were maintained in DMEM, supplemented with 10% heat-inactivated FBS, and 1% P/S in the same condition. All experiments were carried out 12 h after PC12 and BV-2 cells had been seeded in 96well plates at a density of 2.0 × 105 cells/ml. After the cells were about 70% confluent, various concentrations (0.1–100 g/ml) of BEE in serum free media added to the cells for 24 h at 37◦ C with or without 1 M AO. The equal volume of vehicles was given to the control and toxin groups, for each.
Preparation of standard and bombycis excrementum extract
Measurement of cell viability Standard compounds, Pheophorbide a and pheophytin a were purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). The standard stock solutions of two components were prepared by dissolving 1 mg of each compound in 1 ml acetone and stored at −20◦ C. The BE was purchased from Jungdo Herbal Drug Co. Ltd (Seoul, Korea). The 100 g of samples was extracted with 500 ml acetone, and then left under ultrasonic bath for 60 min. After the extract was evaporated and freeze-dried, the dried extract was dissolved in acetone at concentration of 30 mg/ml. The extract was stored at −20◦ C for further use. HPLC analysis The HPLC analysis was carried out on a Waters system (Waters Corp., Milford, MA, USA), consisting of separation module (e2695) with an integrated column heater, autosampler, and a photodiode array detector (2998). UV absorbance was monitored from 200 to 700 nm. Quantification was carried out by integration of the peak areas at 660 nm. Injection volume was 10 l. A column (YMC-Triart C18, 250 × 4.6 mm; particle size, 5 m; YMC Co. Ltd., Japan) was installed in a column oven, and then maintained at 40◦ C. The separation method was developed based on a method previously reported [32]. The mobile phase consisted of ethyl acetate-methanol-water (15 : 65 : 20, v/v/v %, solvent A) and ethyl acetate-methanolwater (60 : 30 : 10, v/v/v %, solvent B). The flow rate was 0.6 ml/min. The gradient was 0.0–5 min, 100% A; 24.0–32.0 min, 100% B; 33.0–40.0 min, 100% A.
Cell viability was measured by MTT assay. In brief, PC12 cells were seeded on the 96-well plates and were treated with BEE at doses of 0.1–100 g/ml for 24 h or pre-treated with BEE for 1 h, then stimulated with 1 M AO for additional 23 h (pre-treatment) or added with 1 M AO for 1 h before treatment with BEE for additional 23 h (post-treatment). After the treatment, supernatants were removed, and 1 mg/ml of MTT was added to the cells for 3 h. MTT medium was carefully removed from the wells, and the MTT formazan dye was eluted using DMSO. Absorbance was measured at a wavelength of 570 nm using a spectrophotometer (Versamax microplate reader, Molecular Device; Sunnyvale, CA, USA), and then data was expressed as a percentage of the value in control. Measurement of extracellular nitric oxide The accumulated level of nitric oxide (NO) in culture supernatants was measured using the colorimetric reaction with the Griess reagent. The supernatants (100 l) were transferred to a separate plate and reacted with 100 l of Griess reagent in the dark for 10 min at room temperature. Absorbance at 550 nm was measured. For each experiment, freshly prepared NaNO2 that had been serially diluted was used as a standard, in parallel with culture supernatants. Animals and surgery procedure It is known that administration of soluble A peptide into rodents’ brain is a useful and efficient tool to test whether some compounds act at early stages
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of AD pathology [33]. Male ICR (Institute of Cancer Research) mice (8 weeks, 27–30 g) were purchased from the Daehan Biolink Co. Ltd (Eumseong, Korea). The ICR mice are outbred stocks derived from CD1 strain [34]. This mouse strain is widely employed in studies for neuroscience, pharmacology, and cognition [35]. In the present study, all animal experiments were performed using ICR mice. Animals were housed 5 or 6 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/dark cycle. Animal treatment and maintenance were carried out in accordance with the Principle of Laboratory Animal Care (NIH publication No. 85–23, revised 1985) and the Animal Care and Use Guidelines of Kyung Hee University, Seoul, Korea. The mice were immediately anesthetized by mixture of Zoletil 50® and Rompun® solution (3 : 1 ratio, 1 ml/kg, i.p.) and mounted in a stereotaxic apparatus (myNeuroLab, St. Louis, MO, USA). Each mouse were unilaterally injected (at rate 0.5 l/min) with 3 l of AO (10 M) into the granule cell layer (GCL) of hippocampus (coordinates with respect to bregma in mm: AP –2.0, ML 1.5, DV 2.0), according to the stereotaxic atlas of mouse brain [36]. The sham-operated mice were injected with the same volume of saline alone. The accuracy of stereotaxic injection to the targeted region was monitored in all animals by examination of the needle tract within brain sections. Drug administration The mice were randomly divided into 4 groups (n = 6 in each group): (1) Sham group (sham-operated plus intraorally saline-treated group), (2) AO group (AO-lesioned plus intraorally saline-treated group), (3) AO + BEE 20 mg/kg/day group (AO-lesioned plus intraorally BEE 20 mg/kg/day treated group), (4) AO+BEE 100 mg/kg/day group (AO-lesioned plus intraorally BEE 100 mg/kg/day treated group). BEE dissolved in saline was totally administered once per day for 14 days (for 5 days before surgery and for 9 days after surgery).
a guillotine door. The bright compartment contained an 50 W electric lamp, and the floor of the dark compartment was composed of 2 mm stainless steel rods spaced 1 cm apart. Mice were treated with BEE or vehicle 1 h before the acquisition trial and initially placed in the bright compartment for the acquisition trial. The door between the two compartments was opened 10 s later. Then, when the hind legs of the mice entered into the dark chamber, the guillotine door was closed and electrical foot shock (0.6 mA) was delivered through the grid floor for 3 s. The mice were again placed in the bright chamber for the retention trial 24 h after the acquisition trial. The time taken for a mouse to enter the dark chamber after the door opening was defined as the latency time. The latency time was recorded for up to 300 s. Novel object recognition test The novel object recognition test was performed according to the method described previously [37, 38]. The experiments were carried out in a grey open field box (45 × 45 × 50 cm). Prior to the test, mice were habituated to the test box for 5 min without objects. At 24 h after a habituation period, the mice were placed into the same 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). One day 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. The test box and objects were cleaned with 70% ethanol between sessions. Results were expressed as percentage of novel object recognition time (time percentage = t novel/[t novel + t familiar] × 100). Brain tissue preparation
The step-through passive avoidance test The step-through passive avoidance test was performed according to the method described previously [30]. Learning and memory test was performed using a two-compartment step-through passive avoidance apparatus. The box was divided into bright (21 × 21 × 21 cm) and dark (21 × 21 × 21 cm) compartments by
At 24 h after the examination of memory behavior, mice were perfused transcardially with 0.05 M PBS, and then fixed with cold 4% 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
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30 m-thick coronal sections were cut on a freezing microtome (Leica, Nussloch, Germany) and stored in cryoprotectant (25% ethylene glycol, 25% glycerol, 0.05 M phosphate buffer) at 4◦ C until use for immunohistochemical study.
are presented as the percentages of sham group value.
Cresyl violet staining and immunohistochemistry
The linear length of GCL and the number of Nisslstained cells in GCL were measured by Image-Pro Plus Version 6.0. (Media Cybernetics, Bethesda, USA) to estimate the number of cells per 1 mm length in each group. The clearly survival neuron-appearing cells were counted under a light microscope (Olympus Microscope System BX51; Olympus, Tokyo, Japan). Data were presented as percentages of the value in the vehicle-treated sham group. To quantify the immunoreactivity of GFAP and Mac-1 in the DG, the images were photographed at 200× magnification using an optical light microscope. The average area fraction of GFAP and Mac-1-staied areas was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA). Selection of GFAP and Mac1-positive area was conducted by manual threshold adjustment. To acquire the fraction of immunoreactive areas, the selected area was divided by total region of interest (captured total area). Data were presented as percentages of the value in the vehicle-treated sham group.
For histological assessment of cell loss, the free floating sections of the brains were prepared and processed for cresyl violet staining. After mounting sections onto gelatin-coated slides, they were stained with 0.5% cresyl violet, dehydrated through graded alcohols (70%, 80%, 90% and 100%), placed in xylene, and coverslipped using histomount medium. For immunohistochemistry, brain sections were briefly rinsed in PBS buffer and treated with 1% hydrogen peroxide for 15 min. The sections were incubated with a rabbit anti-GFAP antibody (1 : 3000 dilution) and a rat anti-Mac-1 (1 : 1000 dilution) overnight at 4◦ C in the presence of 0.3% triton X-100 and NGS or NHS. After rinsing in PBS buffer, they were then incubated with biotinylated anti-rabbit IgG and antirat 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 slides, dehydrated, and coverslipped in histomount medium. For immunofluorescence (IF) study for GFAP and S100- colocalization, the sections were washed in PBS, and then incubated with a rat anti-GFAP antibody (1 : 500 dilution) overnight at 4◦ C. The sections were incubated with a goat anti-rat IgG Cy3 conjugate Alexa 594 (1 : 500 dilution) for 2 h and then incubated with a rabbit anti-S100- (1 : 1000 dilution) overnight at 4◦ C. After rinsing in PBS buffer, they were incubated with biotinylated anti-rabbit IgG (1 : 200 dilution) for 90 min, and ABC (1 : 100 dilution) for 1 h, and then with Streptavidin-Alexa 488 (1 : 500 dilution) for 1 h at room temperature. Sequentially, they were washed in PBS and mounted using Vectashield Mounting Medium with DAPI. The area fraction of GFAP and Mac-1-stained areas in the dentate gyrus (DG) of hippocampus was analyzed with ImageJ software (Bethesda, MD, USA). The images were photographed at 400× magnification using an optical light microscope (Olympus Microscope System BX51; Olympus, Tokyo, Japan) equipped with a 20× objective lens. Confocal immunofluorescent images were carried out using Zeiss LSM700 confocal microscope (Zeiss, Thornwood, NY). Data
Quantification of Nissl, Mac-1, and GFAP-stained cells
Statistical analysis All statistical parameters were calculated using GraphPad Prism 5.0 software. Values are expressed as the mean ± S.E.M. Results were analyzed by oneway ANOVA analysis followed by the Tukey’s post hoc test. Differences with a p value less than 0.05 were considered statistically significant. RESULTS Using the HPLC, we measured contents of pheophorbide a and pheophytin a from BE. Figure 1 shows the HPLC chromatogram of pheophytin a, pheophorbide a, and BE. The linearity of each compound was calculated by three concentrations of each compound. Content of pheophytin a in silkworm feces was 3.635 g/mg, and a peak of pheophorbide a was detected at this HPLC condition (Table 1). Through the passive avoidance test using scopolamine-treated animals, we determined the effective doses for treatment of BEE in the AOinjected mice (Fig. 2). A passive avoidance test was also employed to investigate whether BEE improved
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Fig. 1. HPLC chromatogram of pheophytin a (A), pheophorbide a (B), and bombycis excrementum (C).
memory impairment in mice receiving intrahippocampal injection of AO. The retention time of the AO-injected vehicle-treated group (65.50 ± 7.73 s)
was significantly shorter than that of the shamoperated group (266.20 ± 11.18 s). In contrast, mice treated with BEE (20 and 100 mg/kg/day) exhibited
M. Moon et al. / Therapeutic Effects of BE in Model of AD Table 1 Contents (g/mg) of pheophorbide a and pheophytin a Pheophorbide a Pheophytin a
r2
Bombycis excrementum
0.9987 0.9993
– 3.635 ± 0.091
Fig. 2. Using a passive avoidance test, we found that 20 and 100 mg/kg of BE exhibit significant memory-enhancing effect. Donepezil (1 mg/kg), is a well known anti-dementia agent, was used as a positive control. Values are indicated as the mean ± S.E.M. ### p < 0.001 compared to the control group, ∗∗∗ p < 0.001 and ∗∗ p < 0.01 compared to the scopolamine-only treated group. BEE, Bombycis excrementum extract.
significant recovery in this test (151.48 ± 14.94 s, 176.62 ± 18.19 s; Fig. 3A). No difference was observed in passive avoidance latencies during the acquisition trial in any other groups. To confirm
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the protective effect of BEE on cognitive deficit, a novel-object recognition test was also utilized. Sham-operated mice (66.93 ± 2.59%) spent more time exploring the novel object than the familiar object during the test session. In contrast, the AO-injected mice (54.90 ± 0.78%) spent similar amount of time exploring the novel object and the familiar object. Treatment with BEE (20 and 100 mg/kg/day) significantly improved AO-induced cognitive deficits in this test (64.56 ± 2.18%, 61.68 ± 1.09%; Fig. 3B). During the training session, no significant difference in exploratory preferences was found for any group. Neuronal atrophy, a well known hallmark of AD in humans, is thought to be a primary component of memory deficits associated with this condition [39]. To further understand the mechanisms underlying the recovery of memory function by BEE, the inhibition of AO-triggered neuronal cell death by BEE was investigated using a Nissl staining. Brain tissue samples stained with cresyl violet exhibited an AO-induced reduction of neuronal density in the GCL of hippocampus (Fig. 4A, B). Compared with the sham-operated group, the AO-treated group showed a partial or complete loss of neurons. This loss was prevented by the concomitant administration of BEE (20 and 100 mg/kg/day, 5 days). To evaluate the direct protective effect of BEE on the neurotoxicity induced by AO, MTT assay was performed in PC12 cells. The treatment with AO induced death of PC12 cells in a dose-dependent manner (Fig. 4C). BEE treatment at doses of 0.1–100 g/ml for 24 h showed no significant toxicity in their viability compared to the control group (Fig. 4D). After incubation with 1 M AO for
Fig. 3. Protective effect of BEE on AO-induced cognitive impairment in a mouse model of AD model. Mice were treated with vehicle or BEE for 14 days and with stereotaxic injection of AO (10 M) on the fifth day. Passive avoidance test was conducted at 9 (acquisition) and 10 (retention) days after AO injection (A). Novel object recognition test was carried out at 7 (training) and 8 (test) days after AO injection (B). The group treated with AO (10 m) and BEE (20 and 100 mg/kg) showed significantly amelioating effect on behavioral impairment induced by AO injection, compared to the AO and saline-treated group. Values are indicated as the mean ± S.E.M. ### p < 0.001 compared to the sham group, ∗∗∗ p < 0.001, ∗∗ p < 0.01 and ∗ p < 0.05 compared to the AO and saline-injected group. BEE, Bombycis excrementum extract.
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B
A *
#
BEE (mg/kg) AβO (10 µM)
** ***
***
***
***
BEE (µg/ml)
AβO (µM)
E Cell viability (% of control)
Cell viability (% of control)
D
F
** * ###
BEE (µg/ml)
Cell viability (% of control)
Cell viability (% of control)
C
*
**
###
BEE (µg/ml) AβO (1 µM)
AβO (1 µM)
Fig. 4. Protective effect of BEE on AO-induced neuronal cell loss in the hippocampal granule cell layers (GCL) and PC12 cells culture. A, B) Mice were treated with vehicle or BEE for 14 days and with stereotaxic injection of AO (10 M) on the fifth day. Hippocampal cell loss was determined using nissl staining staining. B) Quantification of the cresyl violet-stained cells was performed by measuring the cell density in the GCL. A) Representative photomicrographs are shown for the GCL from each group (400× magnification). Scale bar = 50 m. Values are indicated as the mean ± S.E.M. compared to the control group. # p < 0.05 compared to the sham group, and ∗ p < 0.05 compared to the AO and saline-treated group. C) The treatment with AO for 24 h induced death of PC12 cells in a dose-dependent manner. Values are indicated as the mean ± S.E.M. ∗∗∗ p < 0.001 and ∗∗ p < 0.01. D) The PC21 cells were treated with AO for 24 h and BEE for 24 h. Cell viabilites were measured using by MTT assay. BEE treatment did not change their viability. Values are indicated as the mean ± S.E.M. E) PC12 cells were preincubated with BEE for 1 h and then treated with 1 M AO for 23 h. F) PC12 cells are also incubated with 1 M AO 1 h before BEE post-treatment. Cell viabilites were measured using by MTT assay. BEE pre-treatment or post-treatment protected PC12 cells against AO-induced toxicity. Values are indicated as the mean ± S.E.M. ### p < 0.001 compared to the control group, ∗∗ p < 0.01, and ∗ p < 0.05 compared to the AO-only treated group. BEE, Bombycis excrementum extract.
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Fig. 5. Anti-inflammatory effects of BEE on AO-induced gliosis in the DG region of hippocampus. Mice were treated with vehicle or BEE for 14 days and with AO (10 M) stereotaxic injection on the fifth day. Astrogliosis and microgliosis were determined using GFAP (A, C–F) and Mac-1 (B, G–J) immunostaining, respectively. Quantification of the immunoreactivity of GFAP and Mac-1 was performed by measuring the area fraction of GFAP- (A) and Mac-1 (B)-immunoreactive areas in the DG. Representative photomicrographs are shown for the GFAP (C–F) and Mac-1 (G–J)-positive cells from each group (400× magnification). Scale bar = 50 m. (C, G) sham group; (D, H) AO and saline-treated group; (E, I) AO + BEE 20 mg/kg/day group; (F, J) AO + BEE 100 mg/kg/day group. Values are indicated as the mean ± S.E.M. compared to the sham group, ### p < 0.001 compared to the sham group, ∗∗∗ p < 0.001 and ∗∗ p < 0.01 compared to the AO and saline-treated group. BEE, Bombycis excrementum extract.
23 h, the percentage of viable cells compared to controls decreased to 49.03%, while pre-treatment with BEE at a concentration of 0.1–100 g/ml prevented the cell loss by 55.30–82.73% compared with the control group (Fig. 4E). Incubation with 1 M AO for 24 h results in the reduction of cell viability by 55.40%, and post-treatment with BEE at a concentration of 0.1–100 g/ml protected the cell damage by 62.01–71.38% compared with the control group after AO treatment for 1 h (Fig. 4F). It is known that the activation of astrocytes and microglia, evidence of neuroinflammatory response,
is involved in the cognitive dysfunction and neuronal loss associated with AD [40, 41]. To determine the mechanism by which BEE restores AO-associated memory deficits and neurodegeneration, the effect of on AO-mediated neuroinflammation around the AO injection site was examined. Using GFAP antibody, a specific marker for astrocytes, GFAP-IR in the DG was analyzed (Fig. 5A, C). Quantification of the data revealed a significant increase in the GFAPpositive areas in the AO-injected group (190.37%), compared with the saline-injected group. However, in the AO-injected group treated with BEE (20
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Fig. 6. Inhibitory effects of BEE on AO-induced production of NO in BV-2 cells culture. The cells were pre-treated with BEE for 1 h before 1 M AO stimulation (A) or post-treated with BEE after 1 M AO stimulation (B). NO generation was determined by the nitrite level in the supernatant using the Griess reagent colorimetric reaction. The pre- or post-treatment with BEE inhibited overproduction of NO level by AO stress. Values are indicated as the mean ± S.E.M. ### p < 0.001 compared to the control group, ∗∗∗ p < 0.001, ∗∗ p < 0.01 and ∗ p < 0.05 compared to the AO-only treated group. BEE, Bombycis excrementum extract.
and 100 mg/kg/day), the GFAP immunoreactivity was significantly decreased (144.84% and 134.18%), compared with the AO-injected vehicle-treated group. Mac-1 immunoreactivity was also investigated to measure microgliosis in the DG (Fig. 5B, D). The mac-1-positive area exhibited a significant increase in the AO-injected group (188.56%) compared with the vehicle-injected group. In contrast, the mac-1-stained area of AO-injected mice treated with BEE (20 and 100 mg/kg/day) significantly decreased (166.32% and 151.49%) compared with the AO-injected vehicletreated group. Our data indicate that BEE significantly inhibits the reactive astrogliosis and microgliosis induced by AO. To examine the direct anti-inflammatory effects of BEE against AO, we examined the release or production of inflammatory mediators from activated microglial cells and astrocytes. First, we measured the NO production in BV-2 cells using Griess reagents. NO has been shown to act as a critical inflammatory factor released from microglia, and it contributes to A-mediated neurodegeneration [41, 42]. Incubation with 1 M AO increased NO production up to 696.02%, while pre-treatment with 0.1–10 g/ml BEE significantly inhibited the NO generation by 356.27–216.03% compared with the control group (Fig. 6A). Incubation with 1 M AO increased NO production by 542.65%, post-treatment with 0.1–10 g/ml BEE significantly inhibited the NO generation by 357.35–337.63% compared with the control group after AO treatment for 1 h (Fig. 6B). It is reported that activated astrocytes overproduce a proinflammatory cytokine, S100 in the brains of AD [43],
and S100- is involved in neuroinflammatory processes of AD [44]. To evaluate whether BEE reduces AO-induced release of S100- from astrocytes, the immunofluorescence staining for GFAP and S100 was carried out. Representative photomicrographs of double-labeled immunofluorescence staining with anti-GFAP and anti-S100- antibodies in the DG are shown in Fig. 7. The number of merged cells with GFAP and S100- in the DG increased in the AO-injected group compared with the vehicleinjected group, while the extent of GFAP and S100- colocalization was evidently declined in the BEE administration group (20 and 100 mg/kg/day).
DISCUSSION It is well established that AO, rather than amyloid plaques, are the major players underlying AD pathogenesis. Although several lines of evidence indicate that BE is an effective therapeutic agent for a variety of diseases, there is a lack of evidence demonstrating that BE directly influences AO-induced brain dysfunction. The current data showed that systemically administered BEE prevented memory impairments, neuronal loss, gliosis, and cytokine (S100-) production in an AO-injection mouse model of AD. Additionally, the direct inhibitory effects of BE on neurodegeneration in PC12 cells and NO generation in BV-2 cells (Supplementary Figure 1) were confirmed. It has been suggested that AD is a result of altered activity in multiple pathological pathways that are responsible for various functions [12, 13]. Thus, new
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Fig. 7. Inhibitory effect of BEE on AO-induced S100- production from astrocytes in the DG region. Mice were treated with vehicle or BEE for 14 days and with stereotaxic injection of AO (10 M) on the fifth day. The of S100- cytokine expression in activated astrocytes was examined using double-staining with GFAP of S100-. Representative photomicrographs are shown for the immunoreactivity of GFAP (A, D, G, J), S100- (B, E, H, K) from each group. The number of GFAP and S100--immunoreactive cells in the DG were elevated by AO injection, while BEE treatment inhibited the increase in number of GFAP and S100--stained cells in the DG. White arrow indicate the clearly merged cells in the DG. Scale bar = 50 m. (A–C) sham group; (D–F) AO and saline-treated group; (G–I) AO + BEE (20 mg/kg/day) group; (J–L) AO + BEE (100 mg/kg/day) group.
multi-functional pharmacological therapeutic strategies are needed to act on the multiple neural and biochemical targets underlying the pathogenesis of AD. Taking this into consideration, natural compounds or medicinal herbal extracts composed of multiple factors represent promising drug candidates because they possess multi-functional effects. The current findings demonstrate that BE is not neurotoxic (Fig. 4D). BE is known to contain a number of biogenic components or physiologically active substances, including chlorophyll, sodium copper chlorophyllin, pectin,
phytol, 1-deoxynojirimycin, megastigmane sesquiterpenes, flavonoids, and mulberry leaves [20–26]. These components have been reported to exert antioxidant effects in the brain and have multiple activities, including antiviral, anti-cancer, and blood-glucose-lowering properties [20–26]. Therefore, it is proposed here that BE is an excellent pharmacological candidate for the treatment of neurodegenerative disorders due to its effects on multiple targets. Previous studies have confirmed the central role of AO and its fibrils in the pathogenesis of AD [45].
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Recent findings have demonstrated that soluble AO, which is stable and can lead to synaptic dysfunction, are the most toxic of the A species to neurons and can ultimately result in memory deficits [46, 47]. AO also induce several markers of a neuroinflammatory condition, such as glial cell-driven pathological materials, and AO-induced toxicity plays a role in this cascade and ultimately accelerates neuronal death via various actions in the brain [48, 49]. To date, no studies have examined the effects of BE on the brain or neuronal function. Therefore, to determine the effective doses of BE for the treatment of AD-like pathologies, a scopolamine-treatment model was used to examine the effects of BE on cognitive function (Fig. 2). Scopolamine, an anticholinergic agent, inhibits central cholinergic neuron activity, and impairs learning and memory acutely [50]. Thus, scopolamine-treatment models are useful for the investigation of cognitive function and cognitionenhancing agents. It has been shown that unilateral intra-hippocampal injections of A induce cognitive impairments in rodents [51, 52] and that AO impairs long-term potentiation-mediated memory formation [53]. The present study demonstrated that BE attenuated memory impairments in both the AO-injection mouse model of AD and the scopolamine-treatment cognitive impairment model. These data suggest that BE can directly regulate or enhance cognitive functions following AD-like damage. Significant neuronal degeneration has been observed in the brains of AD patients [54]. Likewise, the intra-hippocampal administration of AO in rats induces apoptosis-mediated cell death [55]. Nissl staining data from the current study revealed that BEE treatment significantly ameliorated AO-induced cell loss and atrophy in a mouse model of AD. To examine the direct protective effects of BEE against AO-induced toxicity, cell viability was examined using the MTT assay in PC12 cells. Pre-treatment and post-treatment with BEE significantly protected PC12 cells against AO-induced stress and resulted in no toxicity to the cells. These data suggest that BE can act as a neuroprotectant against AO toxicity. Moreover, because neuronal death has been shown to result in the release of microglial activators such as laminin, MMP3, and ␣-synuclein (which all cause memory deficits) [41, 56], it is possible that the memory-enhancing and anti-inflammatory effects of BE might be, at least in part, mediated by the prevention of AO-induced neuronal death. In the present study, BEE treatment resulted in a significant reduction of AO-induced glial hyperac-
tivation. BE significantly inhibited the AO-induced release of NO in a dose-dependent manner as well as the production of S100- in a mouse model of AD. One of the characteristic pathological features of AD is a strong inflammatory response that is associated with the extracellular deposition of A proteins [57]. The activation of glial cells, which is the result of acute neuronal inflammation, is abundantly observed following the accumulation of A peptides in the AD brain [58, 59]. An examination of the postmortem brains of AD patients reveals the heavy presence of proinflammatory cytokines and chemokines such as interlukin-1 (IL1), tumor necrosis factor-␣, and oxygen radicals [48, 60]. Inflammatory processes are also evident in transgenic AD mice that exhibit the accumulation of human A peptides that develop into AO [61, 62]. Moreover, previous studies have identified the presence of astrogliosis and microgliosis in an AD mouse model that utilized intra-hippocampal injections of AO [55]. It has been assumed that the inflammatory response is detrimental and contributes to neuronal degeneration [63] and, because activated glial cells produce several toxic molecules such as superoxide, NO, and cytokines that all directly result in cognitive impairments [40, 64, 65], it is possible that the cognition-enhancing effects of BE are, in part, mediated via anti-inflammatory action. It is known that the astrocytic overexpression of S100- is an important pathogenic factor in the generation of neuritic plaques in AD [66]. S100- that is released from activated astrocytes causes further microglial activation, which culminates in an aggravation of inflammatory responses and the stimulation of neuropathological processes thought to underlie AD [67]. Additionally, NO has been reported to mediate the regulation of A action and A clearance, which indicates that elevated levels of NO may contribute to the maintenance, self-perpetuation, and progression of AD [68, 69]. During the early stages of AD progression, the presence of A, S100-, and NO could result in a vicious perpetuating cycle of neuronal death and A generation between glial cells and neurons, which, in turn, leads to progressive and sustained inflammation [41, 44]. The current data demonstrate that BEE can inhibit the AO-stimulated release of NO and S100- from glial cells, which suggests that BE can regulate A plaque deposits and neuronal death via the modulation of these compounds. The current data show that BE is a multi-functional compound that exerts a variety of activities, including neuroprotective, anti-inflammatory, and cognitionenhancing effects. Based on the present findings
M. Moon et al. / Therapeutic Effects of BE in Model of AD
and previous reports, it may be speculated that BE (1) directly enhances cognitive function, (2) attenuates memory loss via the inhibition of AO-induced cell death and inflammation, (3) inhibits memory impairments and neuroinflammation by means of its neuroprotective effects against AO toxicity and (4) reduces cognitive deficits and neuronal death through its inhibition of the release of inflammatory mediators such as NO and S100- (Supplementary Figure 1). Further detailed examination of BE is required to elucidate the molecular mechanisms underlying its influence on the deactivation of glial cells, cognition-enhancing effects, protection versus neuronal death, and the suppression of inflammatory mediators. In summary, a water extract of BE may directly protect against the cognitive deficits induced by AO. This action is likely mediated via the inhibition of neuronal loss and inflammation. BE significantly reduced the release and/or production of proinflammatory factors, such as NO and S100-, from microglial cells and astrocytes, suggesting it to be a potential candidate agent for AD treatment.
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[9] [10] [11]
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ACKNOWLEDGMENTS [14]
This study was supported by a Grant of the Oriental Medicine R&D Project, Ministry for Health & Welfare & Family Affairs, Republic of Korea (B090039). The authors thank Soohyun Kwak (Newton South High School, MA, USA) for drawing the illustration. Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=2149).
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SUPPLEMENTARY MATERIAL
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The supplementary figure is available in the electronic version of this article: http://dx.doi.org/ 10.3233/JAD-140270.
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Journal of Alzheimer’s Disease 41 (2014) 599–613 DOI 10.3233/JAD-140270 IOS Press
Bombycis excrementum Reduces Amyloid- Oligomer-Induced Memory Impairments, Neurodegeneration, and Neuroinflammation in Mice Minho Moona,1,2 , Jin Gyu Choib,d,1 , Sun Yeou Kimc and Myung Sook Ohb,d,∗ a School
of Medicine, Kyung Hee University, Dongdaemun-gu, Seoul, Republic of Korea of Pharmacy, Kyung Hee University, Dongdaemun-gu, Seoul, Republic of Korea c College of Pharmacy, Gachon University, Incheon, Republic of Korea d Department of Life and Nanopharmaceutical Science and Kyung Hee East-West Pharmaceutical Research Institute, Kyung Hee University, Dongdaemun-gu, Seoul, Republic of Korea b College
Accepted 9 February 2014
Abstract. Alzheimer’s disease (AD) is the most common cause of progressive dementia and is characterized by memory impairments, neuronal death, and neuroinflammation. AD-related pathophysiology is caused primarily by the presence of amyloid- oligomers (AO). Recently, an increased focus has been directed toward natural compounds or medicinal extracts for the treatment of AD. Extracts from Bombycis excrementum (BE), which is composed of various bioactive constituents and mulberry leaves (the preferred food of silkworms), have been shown to possess anti-inflammatory, anti-diabetic, and antioxidative effects. Additionally, mulberry leaves exert anti-amyloidogenic action and neuroprotective effects against A peptides but it is unknown whether BE has a therapeutic effect on AD-related pathologies. Therefore, the present study examined whether BE inhibits AO-induced memory loss, neuronal death, and inflammation. Behavioral tests revealed that BE significantly ameliorated AO-induced memory impairments and inhibited AO-induced neuronal loss in cultured cells and the brains of mice. BE also significantly inhibited microgliosis and astrogliosis following intra-hippocampal AO injections in mice. Furthermore, BE significantly attenuated the release of nitric oxide from microglia and reduced AO-induced S100- cytokine release from activated astrocytes. These results suggest that BE may be a candidate agent for the treatment of AD. Keywords: Alzheimer’s disease, amyloid- oligomer, Bombycis excrementum, cognitive impairment, neuroinflammation, neuronal death, silkworm droppings
INTRODUCTION Alzheimer’s disease (AD) is one of the most common age-related neurological disorders. Although the 1 These
authors contributed equally to this study. address: Molecular Neurobiology Laboratory, McLean Hospital/Harvard Medical School, Belmont, MA, USA. ∗ Correspondence to: Myung Sook Oh, OMD, PhD, Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, #1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea. Tel.: +82 2 961 9436; Fax: +82 2 963 9436; E-mail: [email protected]. 2 Present
exact mechanisms underlying the pathogenesis of this disease remain unclear, it is believed that intracellular and extracellular accumulations of amyloid- (A) peptides play a central role [1, 2]. The progression of AD pathology occurs early in hippocampal formation. This brain region is critical for learning and memory behaviors and is one of the most susceptible to A-mediated degeneration [3–6]. Several studies have demonstrated that AD-mediated damage is caused primarily by A oligomers (AO) that may lead to the induction of long-term synaptic depression and inhibition of pro-synaptic long-term potentiation in the
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hippocampus [7, 8]. Additionally, AO are correlated with the degree of hippocampal cell loss and cognitive impairment [9], and activated astrocytes and microglial cells are characteristically found in abundance near neurons damaged by AO in brains with AD [10, 11]. Because neurodegenerative disorders such as AD are a result of multiple pathogenic factors, single-target therapeutic agents have proved inefficient for their treatment [12, 13]. Based on such evidence, the development of therapeutic agents with various functions that target multiple pathological pathways has attracted much attention [12, 13]. In recent years, an increased focus has been placed on the use of natural compounds or medicinal herbal extracts that appear to function in a multimodal manner for the treatment of AD [14, 15]. For example, Ginkgo biloba extract inhibits A fibril formation, modulates cholinergic neurotransmission, and stimulates cholinergic activity in the brain [16]. Huperzine A, an alkaloid extracted from Lycopodium serratum, exerts neuroprotective effects against Ainduced death [17] and Jangwonhwan, an oriental medicine used for amnesia, has been shown to reduce neural A deposition in a Tg-APPswe/PS1dE9 mouse model of AD [18]. Additionally, vitamin D treatment exhibits the protective effects against A toxicity in cortical neuronal cultures [19]. In clinical trial, AD patients receiving vitamin E showed a slowing in functional decline [20]. Thus, it is suggested that naturally occurring agents with multi-functional effects should be examined for their efficacy when treating AD. Bombycis excrementum (BE) is also known as silkworm (Bombyx mori L) excrement, silkworm feces, silkworm excreta, silkworm droppings, and can sha (Chinese name). BE acts as a raw material for a variety of products including chlorophyll, carotenoids, sodium copper chlorophyllin, pectin, and phytol [21, 22]. Chlorophyll extracted from BE has been used as a therapy for gastric ulcers and hepatitis [23], and pectin extracted from BE has been shown to reduce blood triglycerides and cholesterol levels [21]. 1-Deoxynojirimycin, a typical natural alkaloid component of BE, exerts anti-diabetic effects in a mouse model of Sandhoff disease via marked inhibition of ␣glucosidase activity [24] and a synergistic effect with non-steroidal anti-inflammatory drugs [25]. Furthermore, it has been demonstrated that megastigmane sesquiterpenes and flavonoids from BE increase the expression and promote the activity of the antioxidant enzymes heme oxygenase-1 and SIRT1, which are involved in the suppression of inflammatory medi-
ators [26, 27]. Studies show that more than half of the mulberry leaves used to feed silkworms are excreted without being digested [26] and that these leaves exert anti-amyloidogenic action and neuroprotective effects against A peptides [28, 29]. However, it is unknown whether BE treatment has protective effects against AO-mediated pathology and the subsequent cognitive deficits. Thus, this study evaluated whether BE extract (BEE) exerts protective effects against AO-induced memory impairments, neuronal death, and neuroinflammation in a mouse model utilizing intra-hippocampal injections of AO and AO-treated cultured cells.
MATERIALS AND METHODS Materials Roswell Park Memorial Institute (RPMI) medium, Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), horse serum (HS), and penicillin–streptomycin (P/S) were purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). Rabbit monoclonal anti-glial fibrillary acid protein (GFAP) and rat monoclonal anti-GFAP were purchased from Millipore Bioscience Research (Bedford, MA, USA). Rat monoclonal anti-CD11b (Mac-1) was purchased from Chemicon International (Temecula, CA, USA). Rabbit polyclonal anti-S100- was purchased from Abcam (Cambridge, UK). Biotinylated goat antirabbit antibody, rabbit anti-rat antibody, normal goat serum (NGS), normal horse serum (NHS), goat anti-rat IgG Cy3 conjugate Alexa 594 antibody, Streptavidin Alexa 488 antibody, Vectashield Mounting Medium with 4 ,6-diamidino-2-phenylindole (DAPI), and avidin-biotin complex (ABC) were purchased from Vector Lab (Burlingame, CA, USA). Zoletil 50® and Rompun® were purchased from Virbac (Carros, France) and Bayer Korea (Seoul, Korea), respectively. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), cresyl violet, paraformaldehyde (PFA), 3,3-diaminobenzidine (DAB), dimethyl sulfoxide (DMSO), sodium bicarbonate, sodium chloride, sucrose, ethanol, phosphate buffered saline (PBS), collagen, Griess reagent, DMSO anhydrous form, and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A1-42 peptide was purchased from American Peptide (Sunnyvale, CA, USA). Protein assay was purchased from Bio-Rad Laboratories (Hercules, CA, USA).
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Preparation of oligomeric Aβ1-42 solution
Culture of PC12 and BV-2 cell and treatment
Soluble oligomers were generated by previously described methods with slight modifications [30, 31]. Briefly, A1-42 was dissolved in HFIP to the final concentration of 1 mg/ml at room temperature for 3 days. The peptide was aliquoted and dried under vacuum for 1 h. The aliquoted peptide was dissolved in DMSO anhydrous form to the final concentration of 1 mM. Protein determination was performed by Bradford assay to calculate molarities of solution. The A stock in DMSO anhydrous form was diluted directly into PBS at 10 M and incubated at 4◦ C for 24 h to make oligomeric form of A.
Rat pheochromocytoma PC12 cells were maintained in RPMI media, supplemented with 5% heat-inactivated FBS, 10% HS, and 1% P/S in watersaturated atmosphere of 5% CO2 at 37◦ C. Mouse BV-2 microglial cells were maintained in DMEM, supplemented with 10% heat-inactivated FBS, and 1% P/S in the same condition. All experiments were carried out 12 h after PC12 and BV-2 cells had been seeded in 96well plates at a density of 2.0 × 105 cells/ml. After the cells were about 70% confluent, various concentrations (0.1–100 g/ml) of BEE in serum free media added to the cells for 24 h at 37◦ C with or without 1 M AO. The equal volume of vehicles was given to the control and toxin groups, for each.
Preparation of standard and bombycis excrementum extract
Measurement of cell viability Standard compounds, Pheophorbide a and pheophytin a were purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). The standard stock solutions of two components were prepared by dissolving 1 mg of each compound in 1 ml acetone and stored at −20◦ C. The BE was purchased from Jungdo Herbal Drug Co. Ltd (Seoul, Korea). The 100 g of samples was extracted with 500 ml acetone, and then left under ultrasonic bath for 60 min. After the extract was evaporated and freeze-dried, the dried extract was dissolved in acetone at concentration of 30 mg/ml. The extract was stored at −20◦ C for further use. HPLC analysis The HPLC analysis was carried out on a Waters system (Waters Corp., Milford, MA, USA), consisting of separation module (e2695) with an integrated column heater, autosampler, and a photodiode array detector (2998). UV absorbance was monitored from 200 to 700 nm. Quantification was carried out by integration of the peak areas at 660 nm. Injection volume was 10 l. A column (YMC-Triart C18, 250 × 4.6 mm; particle size, 5 m; YMC Co. Ltd., Japan) was installed in a column oven, and then maintained at 40◦ C. The separation method was developed based on a method previously reported [32]. The mobile phase consisted of ethyl acetate-methanol-water (15 : 65 : 20, v/v/v %, solvent A) and ethyl acetate-methanolwater (60 : 30 : 10, v/v/v %, solvent B). The flow rate was 0.6 ml/min. The gradient was 0.0–5 min, 100% A; 24.0–32.0 min, 100% B; 33.0–40.0 min, 100% A.
Cell viability was measured by MTT assay. In brief, PC12 cells were seeded on the 96-well plates and were treated with BEE at doses of 0.1–100 g/ml for 24 h or pre-treated with BEE for 1 h, then stimulated with 1 M AO for additional 23 h (pre-treatment) or added with 1 M AO for 1 h before treatment with BEE for additional 23 h (post-treatment). After the treatment, supernatants were removed, and 1 mg/ml of MTT was added to the cells for 3 h. MTT medium was carefully removed from the wells, and the MTT formazan dye was eluted using DMSO. Absorbance was measured at a wavelength of 570 nm using a spectrophotometer (Versamax microplate reader, Molecular Device; Sunnyvale, CA, USA), and then data was expressed as a percentage of the value in control. Measurement of extracellular nitric oxide The accumulated level of nitric oxide (NO) in culture supernatants was measured using the colorimetric reaction with the Griess reagent. The supernatants (100 l) were transferred to a separate plate and reacted with 100 l of Griess reagent in the dark for 10 min at room temperature. Absorbance at 550 nm was measured. For each experiment, freshly prepared NaNO2 that had been serially diluted was used as a standard, in parallel with culture supernatants. Animals and surgery procedure It is known that administration of soluble A peptide into rodents’ brain is a useful and efficient tool to test whether some compounds act at early stages
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of AD pathology [33]. Male ICR (Institute of Cancer Research) mice (8 weeks, 27–30 g) were purchased from the Daehan Biolink Co. Ltd (Eumseong, Korea). The ICR mice are outbred stocks derived from CD1 strain [34]. This mouse strain is widely employed in studies for neuroscience, pharmacology, and cognition [35]. In the present study, all animal experiments were performed using ICR mice. Animals were housed 5 or 6 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/dark cycle. Animal treatment and maintenance were carried out in accordance with the Principle of Laboratory Animal Care (NIH publication No. 85–23, revised 1985) and the Animal Care and Use Guidelines of Kyung Hee University, Seoul, Korea. The mice were immediately anesthetized by mixture of Zoletil 50® and Rompun® solution (3 : 1 ratio, 1 ml/kg, i.p.) and mounted in a stereotaxic apparatus (myNeuroLab, St. Louis, MO, USA). Each mouse were unilaterally injected (at rate 0.5 l/min) with 3 l of AO (10 M) into the granule cell layer (GCL) of hippocampus (coordinates with respect to bregma in mm: AP –2.0, ML 1.5, DV 2.0), according to the stereotaxic atlas of mouse brain [36]. The sham-operated mice were injected with the same volume of saline alone. The accuracy of stereotaxic injection to the targeted region was monitored in all animals by examination of the needle tract within brain sections. Drug administration The mice were randomly divided into 4 groups (n = 6 in each group): (1) Sham group (sham-operated plus intraorally saline-treated group), (2) AO group (AO-lesioned plus intraorally saline-treated group), (3) AO + BEE 20 mg/kg/day group (AO-lesioned plus intraorally BEE 20 mg/kg/day treated group), (4) AO+BEE 100 mg/kg/day group (AO-lesioned plus intraorally BEE 100 mg/kg/day treated group). BEE dissolved in saline was totally administered once per day for 14 days (for 5 days before surgery and for 9 days after surgery).
a guillotine door. The bright compartment contained an 50 W electric lamp, and the floor of the dark compartment was composed of 2 mm stainless steel rods spaced 1 cm apart. Mice were treated with BEE or vehicle 1 h before the acquisition trial and initially placed in the bright compartment for the acquisition trial. The door between the two compartments was opened 10 s later. Then, when the hind legs of the mice entered into the dark chamber, the guillotine door was closed and electrical foot shock (0.6 mA) was delivered through the grid floor for 3 s. The mice were again placed in the bright chamber for the retention trial 24 h after the acquisition trial. The time taken for a mouse to enter the dark chamber after the door opening was defined as the latency time. The latency time was recorded for up to 300 s. Novel object recognition test The novel object recognition test was performed according to the method described previously [37, 38]. The experiments were carried out in a grey open field box (45 × 45 × 50 cm). Prior to the test, mice were habituated to the test box for 5 min without objects. At 24 h after a habituation period, the mice were placed into the same 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). One day 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. The test box and objects were cleaned with 70% ethanol between sessions. Results were expressed as percentage of novel object recognition time (time percentage = t novel/[t novel + t familiar] × 100). Brain tissue preparation
The step-through passive avoidance test The step-through passive avoidance test was performed according to the method described previously [30]. Learning and memory test was performed using a two-compartment step-through passive avoidance apparatus. The box was divided into bright (21 × 21 × 21 cm) and dark (21 × 21 × 21 cm) compartments by
At 24 h after the examination of memory behavior, mice were perfused transcardially with 0.05 M PBS, and then fixed with cold 4% 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
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30 m-thick coronal sections were cut on a freezing microtome (Leica, Nussloch, Germany) and stored in cryoprotectant (25% ethylene glycol, 25% glycerol, 0.05 M phosphate buffer) at 4◦ C until use for immunohistochemical study.
are presented as the percentages of sham group value.
Cresyl violet staining and immunohistochemistry
The linear length of GCL and the number of Nisslstained cells in GCL were measured by Image-Pro Plus Version 6.0. (Media Cybernetics, Bethesda, USA) to estimate the number of cells per 1 mm length in each group. The clearly survival neuron-appearing cells were counted under a light microscope (Olympus Microscope System BX51; Olympus, Tokyo, Japan). Data were presented as percentages of the value in the vehicle-treated sham group. To quantify the immunoreactivity of GFAP and Mac-1 in the DG, the images were photographed at 200× magnification using an optical light microscope. The average area fraction of GFAP and Mac-1-staied areas was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA). Selection of GFAP and Mac1-positive area was conducted by manual threshold adjustment. To acquire the fraction of immunoreactive areas, the selected area was divided by total region of interest (captured total area). Data were presented as percentages of the value in the vehicle-treated sham group.
For histological assessment of cell loss, the free floating sections of the brains were prepared and processed for cresyl violet staining. After mounting sections onto gelatin-coated slides, they were stained with 0.5% cresyl violet, dehydrated through graded alcohols (70%, 80%, 90% and 100%), placed in xylene, and coverslipped using histomount medium. For immunohistochemistry, brain sections were briefly rinsed in PBS buffer and treated with 1% hydrogen peroxide for 15 min. The sections were incubated with a rabbit anti-GFAP antibody (1 : 3000 dilution) and a rat anti-Mac-1 (1 : 1000 dilution) overnight at 4◦ C in the presence of 0.3% triton X-100 and NGS or NHS. After rinsing in PBS buffer, they were then incubated with biotinylated anti-rabbit IgG and antirat 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 slides, dehydrated, and coverslipped in histomount medium. For immunofluorescence (IF) study for GFAP and S100- colocalization, the sections were washed in PBS, and then incubated with a rat anti-GFAP antibody (1 : 500 dilution) overnight at 4◦ C. The sections were incubated with a goat anti-rat IgG Cy3 conjugate Alexa 594 (1 : 500 dilution) for 2 h and then incubated with a rabbit anti-S100- (1 : 1000 dilution) overnight at 4◦ C. After rinsing in PBS buffer, they were incubated with biotinylated anti-rabbit IgG (1 : 200 dilution) for 90 min, and ABC (1 : 100 dilution) for 1 h, and then with Streptavidin-Alexa 488 (1 : 500 dilution) for 1 h at room temperature. Sequentially, they were washed in PBS and mounted using Vectashield Mounting Medium with DAPI. The area fraction of GFAP and Mac-1-stained areas in the dentate gyrus (DG) of hippocampus was analyzed with ImageJ software (Bethesda, MD, USA). The images were photographed at 400× magnification using an optical light microscope (Olympus Microscope System BX51; Olympus, Tokyo, Japan) equipped with a 20× objective lens. Confocal immunofluorescent images were carried out using Zeiss LSM700 confocal microscope (Zeiss, Thornwood, NY). Data
Quantification of Nissl, Mac-1, and GFAP-stained cells
Statistical analysis All statistical parameters were calculated using GraphPad Prism 5.0 software. Values are expressed as the mean ± S.E.M. Results were analyzed by oneway ANOVA analysis followed by the Tukey’s post hoc test. Differences with a p value less than 0.05 were considered statistically significant. RESULTS Using the HPLC, we measured contents of pheophorbide a and pheophytin a from BE. Figure 1 shows the HPLC chromatogram of pheophytin a, pheophorbide a, and BE. The linearity of each compound was calculated by three concentrations of each compound. Content of pheophytin a in silkworm feces was 3.635 g/mg, and a peak of pheophorbide a was detected at this HPLC condition (Table 1). Through the passive avoidance test using scopolamine-treated animals, we determined the effective doses for treatment of BEE in the AOinjected mice (Fig. 2). A passive avoidance test was also employed to investigate whether BEE improved
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Fig. 1. HPLC chromatogram of pheophytin a (A), pheophorbide a (B), and bombycis excrementum (C).
memory impairment in mice receiving intrahippocampal injection of AO. The retention time of the AO-injected vehicle-treated group (65.50 ± 7.73 s)
was significantly shorter than that of the shamoperated group (266.20 ± 11.18 s). In contrast, mice treated with BEE (20 and 100 mg/kg/day) exhibited
M. Moon et al. / Therapeutic Effects of BE in Model of AD Table 1 Contents (g/mg) of pheophorbide a and pheophytin a Pheophorbide a Pheophytin a
r2
Bombycis excrementum
0.9987 0.9993
– 3.635 ± 0.091
Fig. 2. Using a passive avoidance test, we found that 20 and 100 mg/kg of BE exhibit significant memory-enhancing effect. Donepezil (1 mg/kg), is a well known anti-dementia agent, was used as a positive control. Values are indicated as the mean ± S.E.M. ### p < 0.001 compared to the control group, ∗∗∗ p < 0.001 and ∗∗ p < 0.01 compared to the scopolamine-only treated group. BEE, Bombycis excrementum extract.
significant recovery in this test (151.48 ± 14.94 s, 176.62 ± 18.19 s; Fig. 3A). No difference was observed in passive avoidance latencies during the acquisition trial in any other groups. To confirm
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the protective effect of BEE on cognitive deficit, a novel-object recognition test was also utilized. Sham-operated mice (66.93 ± 2.59%) spent more time exploring the novel object than the familiar object during the test session. In contrast, the AO-injected mice (54.90 ± 0.78%) spent similar amount of time exploring the novel object and the familiar object. Treatment with BEE (20 and 100 mg/kg/day) significantly improved AO-induced cognitive deficits in this test (64.56 ± 2.18%, 61.68 ± 1.09%; Fig. 3B). During the training session, no significant difference in exploratory preferences was found for any group. Neuronal atrophy, a well known hallmark of AD in humans, is thought to be a primary component of memory deficits associated with this condition [39]. To further understand the mechanisms underlying the recovery of memory function by BEE, the inhibition of AO-triggered neuronal cell death by BEE was investigated using a Nissl staining. Brain tissue samples stained with cresyl violet exhibited an AO-induced reduction of neuronal density in the GCL of hippocampus (Fig. 4A, B). Compared with the sham-operated group, the AO-treated group showed a partial or complete loss of neurons. This loss was prevented by the concomitant administration of BEE (20 and 100 mg/kg/day, 5 days). To evaluate the direct protective effect of BEE on the neurotoxicity induced by AO, MTT assay was performed in PC12 cells. The treatment with AO induced death of PC12 cells in a dose-dependent manner (Fig. 4C). BEE treatment at doses of 0.1–100 g/ml for 24 h showed no significant toxicity in their viability compared to the control group (Fig. 4D). After incubation with 1 M AO for
Fig. 3. Protective effect of BEE on AO-induced cognitive impairment in a mouse model of AD model. Mice were treated with vehicle or BEE for 14 days and with stereotaxic injection of AO (10 M) on the fifth day. Passive avoidance test was conducted at 9 (acquisition) and 10 (retention) days after AO injection (A). Novel object recognition test was carried out at 7 (training) and 8 (test) days after AO injection (B). The group treated with AO (10 m) and BEE (20 and 100 mg/kg) showed significantly amelioating effect on behavioral impairment induced by AO injection, compared to the AO and saline-treated group. Values are indicated as the mean ± S.E.M. ### p < 0.001 compared to the sham group, ∗∗∗ p < 0.001, ∗∗ p < 0.01 and ∗ p < 0.05 compared to the AO and saline-injected group. BEE, Bombycis excrementum extract.
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B
A *
#
BEE (mg/kg) AβO (10 µM)
** ***
***
***
***
BEE (µg/ml)
AβO (µM)
E Cell viability (% of control)
Cell viability (% of control)
D
F
** * ###
BEE (µg/ml)
Cell viability (% of control)
Cell viability (% of control)
C
*
**
###
BEE (µg/ml) AβO (1 µM)
AβO (1 µM)
Fig. 4. Protective effect of BEE on AO-induced neuronal cell loss in the hippocampal granule cell layers (GCL) and PC12 cells culture. A, B) Mice were treated with vehicle or BEE for 14 days and with stereotaxic injection of AO (10 M) on the fifth day. Hippocampal cell loss was determined using nissl staining staining. B) Quantification of the cresyl violet-stained cells was performed by measuring the cell density in the GCL. A) Representative photomicrographs are shown for the GCL from each group (400× magnification). Scale bar = 50 m. Values are indicated as the mean ± S.E.M. compared to the control group. # p < 0.05 compared to the sham group, and ∗ p < 0.05 compared to the AO and saline-treated group. C) The treatment with AO for 24 h induced death of PC12 cells in a dose-dependent manner. Values are indicated as the mean ± S.E.M. ∗∗∗ p < 0.001 and ∗∗ p < 0.01. D) The PC21 cells were treated with AO for 24 h and BEE for 24 h. Cell viabilites were measured using by MTT assay. BEE treatment did not change their viability. Values are indicated as the mean ± S.E.M. E) PC12 cells were preincubated with BEE for 1 h and then treated with 1 M AO for 23 h. F) PC12 cells are also incubated with 1 M AO 1 h before BEE post-treatment. Cell viabilites were measured using by MTT assay. BEE pre-treatment or post-treatment protected PC12 cells against AO-induced toxicity. Values are indicated as the mean ± S.E.M. ### p < 0.001 compared to the control group, ∗∗ p < 0.01, and ∗ p < 0.05 compared to the AO-only treated group. BEE, Bombycis excrementum extract.
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Fig. 5. Anti-inflammatory effects of BEE on AO-induced gliosis in the DG region of hippocampus. Mice were treated with vehicle or BEE for 14 days and with AO (10 M) stereotaxic injection on the fifth day. Astrogliosis and microgliosis were determined using GFAP (A, C–F) and Mac-1 (B, G–J) immunostaining, respectively. Quantification of the immunoreactivity of GFAP and Mac-1 was performed by measuring the area fraction of GFAP- (A) and Mac-1 (B)-immunoreactive areas in the DG. Representative photomicrographs are shown for the GFAP (C–F) and Mac-1 (G–J)-positive cells from each group (400× magnification). Scale bar = 50 m. (C, G) sham group; (D, H) AO and saline-treated group; (E, I) AO + BEE 20 mg/kg/day group; (F, J) AO + BEE 100 mg/kg/day group. Values are indicated as the mean ± S.E.M. compared to the sham group, ### p < 0.001 compared to the sham group, ∗∗∗ p < 0.001 and ∗∗ p < 0.01 compared to the AO and saline-treated group. BEE, Bombycis excrementum extract.
23 h, the percentage of viable cells compared to controls decreased to 49.03%, while pre-treatment with BEE at a concentration of 0.1–100 g/ml prevented the cell loss by 55.30–82.73% compared with the control group (Fig. 4E). Incubation with 1 M AO for 24 h results in the reduction of cell viability by 55.40%, and post-treatment with BEE at a concentration of 0.1–100 g/ml protected the cell damage by 62.01–71.38% compared with the control group after AO treatment for 1 h (Fig. 4F). It is known that the activation of astrocytes and microglia, evidence of neuroinflammatory response,
is involved in the cognitive dysfunction and neuronal loss associated with AD [40, 41]. To determine the mechanism by which BEE restores AO-associated memory deficits and neurodegeneration, the effect of on AO-mediated neuroinflammation around the AO injection site was examined. Using GFAP antibody, a specific marker for astrocytes, GFAP-IR in the DG was analyzed (Fig. 5A, C). Quantification of the data revealed a significant increase in the GFAPpositive areas in the AO-injected group (190.37%), compared with the saline-injected group. However, in the AO-injected group treated with BEE (20
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Fig. 6. Inhibitory effects of BEE on AO-induced production of NO in BV-2 cells culture. The cells were pre-treated with BEE for 1 h before 1 M AO stimulation (A) or post-treated with BEE after 1 M AO stimulation (B). NO generation was determined by the nitrite level in the supernatant using the Griess reagent colorimetric reaction. The pre- or post-treatment with BEE inhibited overproduction of NO level by AO stress. Values are indicated as the mean ± S.E.M. ### p < 0.001 compared to the control group, ∗∗∗ p < 0.001, ∗∗ p < 0.01 and ∗ p < 0.05 compared to the AO-only treated group. BEE, Bombycis excrementum extract.
and 100 mg/kg/day), the GFAP immunoreactivity was significantly decreased (144.84% and 134.18%), compared with the AO-injected vehicle-treated group. Mac-1 immunoreactivity was also investigated to measure microgliosis in the DG (Fig. 5B, D). The mac-1-positive area exhibited a significant increase in the AO-injected group (188.56%) compared with the vehicle-injected group. In contrast, the mac-1-stained area of AO-injected mice treated with BEE (20 and 100 mg/kg/day) significantly decreased (166.32% and 151.49%) compared with the AO-injected vehicletreated group. Our data indicate that BEE significantly inhibits the reactive astrogliosis and microgliosis induced by AO. To examine the direct anti-inflammatory effects of BEE against AO, we examined the release or production of inflammatory mediators from activated microglial cells and astrocytes. First, we measured the NO production in BV-2 cells using Griess reagents. NO has been shown to act as a critical inflammatory factor released from microglia, and it contributes to A-mediated neurodegeneration [41, 42]. Incubation with 1 M AO increased NO production up to 696.02%, while pre-treatment with 0.1–10 g/ml BEE significantly inhibited the NO generation by 356.27–216.03% compared with the control group (Fig. 6A). Incubation with 1 M AO increased NO production by 542.65%, post-treatment with 0.1–10 g/ml BEE significantly inhibited the NO generation by 357.35–337.63% compared with the control group after AO treatment for 1 h (Fig. 6B). It is reported that activated astrocytes overproduce a proinflammatory cytokine, S100 in the brains of AD [43],
and S100- is involved in neuroinflammatory processes of AD [44]. To evaluate whether BEE reduces AO-induced release of S100- from astrocytes, the immunofluorescence staining for GFAP and S100 was carried out. Representative photomicrographs of double-labeled immunofluorescence staining with anti-GFAP and anti-S100- antibodies in the DG are shown in Fig. 7. The number of merged cells with GFAP and S100- in the DG increased in the AO-injected group compared with the vehicleinjected group, while the extent of GFAP and S100- colocalization was evidently declined in the BEE administration group (20 and 100 mg/kg/day).
DISCUSSION It is well established that AO, rather than amyloid plaques, are the major players underlying AD pathogenesis. Although several lines of evidence indicate that BE is an effective therapeutic agent for a variety of diseases, there is a lack of evidence demonstrating that BE directly influences AO-induced brain dysfunction. The current data showed that systemically administered BEE prevented memory impairments, neuronal loss, gliosis, and cytokine (S100-) production in an AO-injection mouse model of AD. Additionally, the direct inhibitory effects of BE on neurodegeneration in PC12 cells and NO generation in BV-2 cells (Supplementary Figure 1) were confirmed. It has been suggested that AD is a result of altered activity in multiple pathological pathways that are responsible for various functions [12, 13]. Thus, new
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Fig. 7. Inhibitory effect of BEE on AO-induced S100- production from astrocytes in the DG region. Mice were treated with vehicle or BEE for 14 days and with stereotaxic injection of AO (10 M) on the fifth day. The of S100- cytokine expression in activated astrocytes was examined using double-staining with GFAP of S100-. Representative photomicrographs are shown for the immunoreactivity of GFAP (A, D, G, J), S100- (B, E, H, K) from each group. The number of GFAP and S100--immunoreactive cells in the DG were elevated by AO injection, while BEE treatment inhibited the increase in number of GFAP and S100--stained cells in the DG. White arrow indicate the clearly merged cells in the DG. Scale bar = 50 m. (A–C) sham group; (D–F) AO and saline-treated group; (G–I) AO + BEE (20 mg/kg/day) group; (J–L) AO + BEE (100 mg/kg/day) group.
multi-functional pharmacological therapeutic strategies are needed to act on the multiple neural and biochemical targets underlying the pathogenesis of AD. Taking this into consideration, natural compounds or medicinal herbal extracts composed of multiple factors represent promising drug candidates because they possess multi-functional effects. The current findings demonstrate that BE is not neurotoxic (Fig. 4D). BE is known to contain a number of biogenic components or physiologically active substances, including chlorophyll, sodium copper chlorophyllin, pectin,
phytol, 1-deoxynojirimycin, megastigmane sesquiterpenes, flavonoids, and mulberry leaves [20–26]. These components have been reported to exert antioxidant effects in the brain and have multiple activities, including antiviral, anti-cancer, and blood-glucose-lowering properties [20–26]. Therefore, it is proposed here that BE is an excellent pharmacological candidate for the treatment of neurodegenerative disorders due to its effects on multiple targets. Previous studies have confirmed the central role of AO and its fibrils in the pathogenesis of AD [45].
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Recent findings have demonstrated that soluble AO, which is stable and can lead to synaptic dysfunction, are the most toxic of the A species to neurons and can ultimately result in memory deficits [46, 47]. AO also induce several markers of a neuroinflammatory condition, such as glial cell-driven pathological materials, and AO-induced toxicity plays a role in this cascade and ultimately accelerates neuronal death via various actions in the brain [48, 49]. To date, no studies have examined the effects of BE on the brain or neuronal function. Therefore, to determine the effective doses of BE for the treatment of AD-like pathologies, a scopolamine-treatment model was used to examine the effects of BE on cognitive function (Fig. 2). Scopolamine, an anticholinergic agent, inhibits central cholinergic neuron activity, and impairs learning and memory acutely [50]. Thus, scopolamine-treatment models are useful for the investigation of cognitive function and cognitionenhancing agents. It has been shown that unilateral intra-hippocampal injections of A induce cognitive impairments in rodents [51, 52] and that AO impairs long-term potentiation-mediated memory formation [53]. The present study demonstrated that BE attenuated memory impairments in both the AO-injection mouse model of AD and the scopolamine-treatment cognitive impairment model. These data suggest that BE can directly regulate or enhance cognitive functions following AD-like damage. Significant neuronal degeneration has been observed in the brains of AD patients [54]. Likewise, the intra-hippocampal administration of AO in rats induces apoptosis-mediated cell death [55]. Nissl staining data from the current study revealed that BEE treatment significantly ameliorated AO-induced cell loss and atrophy in a mouse model of AD. To examine the direct protective effects of BEE against AO-induced toxicity, cell viability was examined using the MTT assay in PC12 cells. Pre-treatment and post-treatment with BEE significantly protected PC12 cells against AO-induced stress and resulted in no toxicity to the cells. These data suggest that BE can act as a neuroprotectant against AO toxicity. Moreover, because neuronal death has been shown to result in the release of microglial activators such as laminin, MMP3, and ␣-synuclein (which all cause memory deficits) [41, 56], it is possible that the memory-enhancing and anti-inflammatory effects of BE might be, at least in part, mediated by the prevention of AO-induced neuronal death. In the present study, BEE treatment resulted in a significant reduction of AO-induced glial hyperac-
tivation. BE significantly inhibited the AO-induced release of NO in a dose-dependent manner as well as the production of S100- in a mouse model of AD. One of the characteristic pathological features of AD is a strong inflammatory response that is associated with the extracellular deposition of A proteins [57]. The activation of glial cells, which is the result of acute neuronal inflammation, is abundantly observed following the accumulation of A peptides in the AD brain [58, 59]. An examination of the postmortem brains of AD patients reveals the heavy presence of proinflammatory cytokines and chemokines such as interlukin-1 (IL1), tumor necrosis factor-␣, and oxygen radicals [48, 60]. Inflammatory processes are also evident in transgenic AD mice that exhibit the accumulation of human A peptides that develop into AO [61, 62]. Moreover, previous studies have identified the presence of astrogliosis and microgliosis in an AD mouse model that utilized intra-hippocampal injections of AO [55]. It has been assumed that the inflammatory response is detrimental and contributes to neuronal degeneration [63] and, because activated glial cells produce several toxic molecules such as superoxide, NO, and cytokines that all directly result in cognitive impairments [40, 64, 65], it is possible that the cognition-enhancing effects of BE are, in part, mediated via anti-inflammatory action. It is known that the astrocytic overexpression of S100- is an important pathogenic factor in the generation of neuritic plaques in AD [66]. S100- that is released from activated astrocytes causes further microglial activation, which culminates in an aggravation of inflammatory responses and the stimulation of neuropathological processes thought to underlie AD [67]. Additionally, NO has been reported to mediate the regulation of A action and A clearance, which indicates that elevated levels of NO may contribute to the maintenance, self-perpetuation, and progression of AD [68, 69]. During the early stages of AD progression, the presence of A, S100-, and NO could result in a vicious perpetuating cycle of neuronal death and A generation between glial cells and neurons, which, in turn, leads to progressive and sustained inflammation [41, 44]. The current data demonstrate that BEE can inhibit the AO-stimulated release of NO and S100- from glial cells, which suggests that BE can regulate A plaque deposits and neuronal death via the modulation of these compounds. The current data show that BE is a multi-functional compound that exerts a variety of activities, including neuroprotective, anti-inflammatory, and cognitionenhancing effects. Based on the present findings
M. Moon et al. / Therapeutic Effects of BE in Model of AD
and previous reports, it may be speculated that BE (1) directly enhances cognitive function, (2) attenuates memory loss via the inhibition of AO-induced cell death and inflammation, (3) inhibits memory impairments and neuroinflammation by means of its neuroprotective effects against AO toxicity and (4) reduces cognitive deficits and neuronal death through its inhibition of the release of inflammatory mediators such as NO and S100- (Supplementary Figure 1). Further detailed examination of BE is required to elucidate the molecular mechanisms underlying its influence on the deactivation of glial cells, cognition-enhancing effects, protection versus neuronal death, and the suppression of inflammatory mediators. In summary, a water extract of BE may directly protect against the cognitive deficits induced by AO. This action is likely mediated via the inhibition of neuronal loss and inflammation. BE significantly reduced the release and/or production of proinflammatory factors, such as NO and S100-, from microglial cells and astrocytes, suggesting it to be a potential candidate agent for AD treatment.
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ACKNOWLEDGMENTS [14]
This study was supported by a Grant of the Oriental Medicine R&D Project, Ministry for Health & Welfare & Family Affairs, Republic of Korea (B090039). The authors thank Soohyun Kwak (Newton South High School, MA, USA) for drawing the illustration. Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=2149).
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SUPPLEMENTARY MATERIAL
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The supplementary figure is available in the electronic version of this article: http://dx.doi.org/ 10.3233/JAD-140270.
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