Journal of Ethnopharmacology 171 (2015) 196–204

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Mori Fructus improves cognitive and neuronal dysfunction induced by beta-amyloid toxicity through the GSK-3β pathway in vitro and in vivo Hyo Geun Kim a,b, Gunhyuk Park b, Soonmin Lim b, Hanbyeol Park b, Jin Gyu Choi a, Hyun Uk Jeong a, Min Seo Kang a,1, Mi Kyeong Lee c, Myung Sook Oh a,b,n a College of Pharmacy and Kyung Hee East–West Pharmaceutical Research Institute, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701, Republic of Korea b Department of Life and Nanopharmaceutical Science, Graduate School, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701, Republic of Korea c College of Pharmacy, Chungbuk National University, Cheongju 361-763, Republic of Korea

art ic l e i nf o

a b s t r a c t

Article history: Received 4 February 2015 Received in revised form 30 May 2015 Accepted 31 May 2015 Available online 9 June 2015

Ethnopharmacological relevance: A growing body of literature supports the concept that antiaging herbs may be potential candidates for use in treating age-related neurodegeneration, including Alzheimer's disease (AD). Mori Fructus is a well-known traditional herbal medicine, food, and dietary supplement. This study employed models of amyloid beta (Aβ)-induced AD to investigate the protective effects of Mori Fructus ethanol extract (ME) against age-related disease and cognitive deficits. Materials and methods: To examine the protective effect of ME, we measured cell viability, cytotoxicity, and survival in rat primary hippocampal cultures. We performed behavioral tests and histological analysis in mouse models of AD induced by Aβ25–35 toxicity. To investigate the mechanism underlying the protective effect, we performed western blotting using antibodies against apoptotic markers as well as the nonphosphorylated and phosphorylated forms of Akt, glycogen synthase kinase-3β (GSK-3β), and tau. We also measured apoptotic marker fluorescence intensity. Results: ME significantly attenuated Aβ-induced cell damage, enhanced Akt and GSK-3β phosphorylation, and reduced tau phosphorylation. ME reduced apoptotic markers that were activated by GSK-3β, and reduced reactive oxygen species production. Further, ME decreased the B-cell lymphoma 2 (Bcl-2)/Bcl-2associated X expression ratio, mitochondria depolarization, cytochrome c release from mitochondria, and caspase-3 activation. We confirmed that ME treatment improved cognitive impairment and neuronal cell death induced by Aβ25–35 toxicity in the mouse hippocampus via its antiapoptotic activity. Conclusions: These results indicate that ME protects cognition and neurons in AD-like models induced by Aβ via reduction of tau phosphorylation and apoptosis through GSK-3β inactivation. & 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Mori Fructus Amyloid beta Glycogen synthase kinase-3β Apoptosis Neuroprotection Learning and memory

1. Introduction Aging has a profound effect on cognition (Miller and Shukitt-Hale, 2012). Many older people develop a cognitive deficit called dementia, the primary cause of which is Alzheimer's disease (AD) (Kim and Oh,

2012). AD is the most common neurodegenerative disease in the elderly, and its prevalence exponentially increases with age (Feng et al., 2013), reaching almost 50% by 85 years of age (Kim and Oh, 2012). Therefore, this neurodegenerative disease causes quality-of-life issues and is a public health concern (Kim and Oh, 2012).

Abbreviations: AD, Alzheimer's disease; Aβ, amyloid-beta; ABC, avidin–biotin peroxidase complex; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X; DAB, 3,3diaminobenzidine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; GSK-3β, glycogen synthase kinase-3β; LDH, lactate dehydrogenase; LiCl, lithium chloride; ΔψM, mitochondrial membrane potential; ME, Mori Fructus ethanol extract; NM, neurobasal media; NFTs, neurofibrillary tangles; NeuN, neuronal nuclei; NORT, novel objective test (); PFA, paraformaldehyde; PB, Phosphate buffer; PBS, phosphate buffered saline; PLL, poly-L-lysine; ROS, reactive oxygen specie n Corresponding author at: College of Pharmacy and Kyung Hee East–West Pharmaceutical Research Institute, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701, Republic of Korea. Tel./fax: þ 82 2 961 9436. E-mail address: [email protected] (M.S. Oh). 1 Present address: Concordia International School, Shanghai 999, Mingyue Road, Jinqiao, Pudong, Shanghai 201206, China. http://dx.doi.org/10.1016/j.jep.2015.05.054 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.

H.G. Kim et al. / Journal of Ethnopharmacology 171 (2015) 196–204

Since Alois Alzheimer first described AD in 1907, neuroscientists have attempted to elucidate its pathogenesis. However, its cause remains unclear. Nevertheless, amyloid-beta (Aβ) and tau proteins play well-established roles in AD by forming insoluble fibrils (amyloid plaques) and neurofibrillary tangles (NFTs), the main pathological hallmarks of AD (Shipton et al., 2011). It has been suggested that Aβ accumulation directly or indirectly causes synaptic loss and neuronal death through oxidative stress, calcium dysregulation, neuroinflammation, mitochondrial dysfunction, and apoptosis, resulting in memory impairment (Kim et al., 2010a). In addition, Aβ-induced tau phosphorylation plays a crucial role in cognitive decline (Shipton et al., 2011). Therefore, many therapeutic strategies focus on reducing Aβ-induced toxicity in experimental models of AD (Kim and Oh, 2012). Mori Fructus (Morus alba L. fruit, mulberry), a member of the Moraceae family, has been naturalized and is widely cultivated in Asia, Europe, America, Africa, and India (Khan et al., 2013). Mori Fructus is commonly consumed as a food, dietary supplement, and remedy; it is also a traditional oriental medicine used as an antiaging agent to enhance health and promote longevity (Kim et al., 2010b). Because anti-aging agents is believed to be control not only physical but also mental energy the traditional theory, this approach in anti-aging herbs seems to be reasonable in prevention and treatment of aging-associated neurological disorders (Ho et al., 2010). A growing body of studies revealed multibioactive functions of mulberry, including anti-inflammatory, antioxidant, anticarcinogenic, and antiapoptotic properties (Kim et al., 2010b, 2012; Kim and Oh, 2013; Liu and Lin, 2012). In addition, it was recently reported that mulberry exerts neuroprotective effects in several models (e.g., Parkinson's disease, AD, cerebral ischemia, vascular dementia, and senescence-accelerated cognitive deficiency) (Kaewkaen et al., 2012; Kang et al., 2006; Kim et al., 2010b; Shih et al., 2010; Song et al., 2014), as well as neuromodulatory effects by inducing nerve growth factor (Kim and Oh, 2013). Based on these actions of Mori Fructus, we hypothesized that it might exert beneficial effects in an Aβ toxin-treated AD-like model, considering that the effects of Mori Fructus have not yet been investigated. Therefore, in the present study, we evaluated the protective effects of a Mori Fructus ethanol extract (ME) on neuronal damage and memory deficits induced by Aβ25–35 toxin which is the neurotoxic core of the full-length Aβ (Bergin et al., 2015). Subsequently, we explored its possible mechanisms with respect to glycogen synthase kinase-3β (GSK-3β)-related proteins involved in tau phosphorylation and apoptosis.

2. Materials and methods 2.1. Materials Neurobasal media (NM), B27 and penicillin–streptomycin were purchased from Gibco Industries Inc. (Auckland, NZ). 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), L-glutamine, dimethyl sulfoxide, paraformaldehyde (PFA), poly-Llysine (PLL), 3,3-diaminobenzidine (DAB), sodium chloride, LY294002, lithium chloride (LiCl), phosphatase inhibitor cocktail, phosphate buffered saline (PBS), glycine, trizma base, Aβ25–35, 2,7dichlorodihydrofluorescein diacetate (H2DCF-DA), and JC-1 were purchased from Sigma-Aldrich (St. Louis, MO, USA). TEMED, protein assay, tween 20, ammonium persulfate, acrylamide, ECL reagent, and skim milk were purchased from Bio-Rad Laboratories (Hercules, CA, USA). A caspase-3 assay kit, lactate dehydrogenase (LDH) -cytotoxicity assay kit, and Mitochondria/Cytosol Fractionation kit were purchased from BioVision, Inc. (Mountain View, CA, USA). A rabbit anti-microtubule-associated protein 2 (MAP-2),

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rabbit anti-cleaved caspase-3, and mouse anti- neuronal nuclei (NeuN) antibodies were obtained from Millipore Corp. (Billerica, MA, USA). Rabbit anti-B-cell lymphoma 2 (Bcl-2), rabbit anti-Bcl2-associated X (Bax), rabbit mouse anti-cytochrome c, rabbit antitau (phospho-ser396), goat anti-tau (tau), rabbit anti-GSK-3β (phospho-ser9), rabbit anti-GSK-3β, rabbit anti-Akt (phosphoser473), rabbit anti-Akt, and mouse anti-β-actin antibodies were obtained from Santa Cruz Biotechnology, Inc., (Delaware Avenue, CA, USA). Anti-mouse, anti-rabbit, and anti-goat-HRP secondary antibodies were purchased from Assay Designs Inc. (Ann Arbor, MI, USA). Biotinylated anti-rabbit antibody, normal goat serum, and an avidin-biotin peroxidase complex (ABC) were purchased from Vector Laboratories (Burlingame, CA, USA). Aβ35–25 was purchased from Abcam (Cambridge, UK). 2.2. Sample preparation The extract of dried mulberry fruit (ME) was the same as that used in previous study (Kim et al., 2010b; Kim and Oh, 2013) in which chemical profiling and standardization of ME had been performed (Supplementary data 1). Briefly, dried mulberry fruit was purchased from Jung Do Herbal Drug Company (Seoul, Korea), and was extracted with 70% ethanol for 24 h at room temperature. Then, the extract was filtered, evaporated on a rotary vacuum evaporator and finally lyophilized. The powder (yield, 20.53%) was kept at 4 1C. 2.3. Aβ aggregation Aβ25–35 and Aβ35–25 were reconstituted in sterile water at a concentration of 500 μM or 1 mg/mL for in vitro and in vivo assays, respectively. Aliquots were incubated at 37 1C for 72 h or 96 h to form aggregated amyloid (Kim et al., 2010a; Zussy et al., 2011). For in vitro assays, 500 μM of Aβ25–35 and Aβ35–25 were diluted to 8 μM in PBS. 2.4. Cultures of rat hippocampal neuronal cells Cell Cultures were prepared from the hippocampus of 18-day embryos of timed pregnant Sprague–Dawley rats (Daehanbiolink Co. Ltd., Choongju, Korea). Hippocampus was dissected, collected, dissociated, and plated in PLL pre-coated 96-well plates, 24-well plates with cover slips, and 60-mm dishes at densities of 1.3  104 cells/well, 2  105 cells/mL, and 1  106 cells/dish, respectively. Cultures were maintained in a humidified incubator of 5% CO2 at 37 1C in a NM with 2 mM glutamine, 2% B27, and 1% penicillin/ streptomycin. After 3 days incubation, the medium was replaced with a new medium. On day in vitro 7 (DIV 7), cells were treated with ME at 0.1, 1, 10, and 100 μg/mL, LiCl at 10 mM, and LY294002 at 10 μM for 24 h. The treated cells were stressed with 8 μM Aβ25–35 or 8 μM Aβ35–25 for the last 4 h and 23 h to examine reactive oxygen specie (ROS) and the others, respectively. The control, Aβ25–35 and 8 μM Aβ35–25 groups were treated with the same volume of PBS. 2.5. Measurement of cell viability and cytotoxicity Treated cells with ME at 0.1, 1, 10, and 100 μg/mL and 8 μM Aβ25–35 or 8 μM Aβ35–25 were incubated with 1 mg/mL of MTT as described (Park and Oh, 2012) using a spectrophotometer (Versamax microplate reader; Molecular Device) at a wavelength of 570 nm. And the supernatants of the treated cells were transferred to a separate plate and release of LDH was by using a LDH kit according to the manufacturer's protocol as previously described (Kim et al., 2010a). Then the cell viability and cytotoxicity were expressed as a percentage of the control value.

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2.6. Measurement of reactive oxygen species level The intracellular ROS level was measured using a fluorescent probe, H2DCF-DA as previously described (Park and Oh, 2012). The cells were treated with ME at 0.1, 1, 10 μg/mL and/or Aβ25–35 then they were incubated with 10 mM H2DCF-DA at 37 1C for 30 min, the fluorescence intensity of DCF was measured at an excitation wavelength of 480 nm and an emission wavelength of 530 nm using a fluorescence microplate reader. The fluorescence intensity was expressed as a percentage of the control value. 2.7. Assessment of mitochondrial membrane potential (ΔψM) The ΔψM was measured using a fluorescence dye, JC-1 reagent as previously described (Kim et al., 2010a) after the cells were treated with ME at 0.1, 1, 10 μg/mL and/or Aβ25–35. The red and green fluorescence were measured using a fluorescence microplate reader (SpectraMax Gemini EM; Molecular Devices), with excitation at 585 and 510 and emission at 590 and 527, respectively. Then the results were expressed as the ratio of red to green fluorescence. 2.8. Measurement of caspase-3 activation The caspase-3 activity assay was performed according to the manufacturer's protocol as previously described (Kim et al., 2010a) after the cells were treated with ME at 0.1, 1, 10 μg/mL and/or Aβ25–35. The fluorescence was detected using a fluorescence microplate reader, with 360 nm excitation and 450 nm emission filters. The fluorescence intensity was expressed as a percentage of the control value. 2.9. Animals, surgery procedure, and treatment 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, South Korea. The experimental protocols were approved by the Institutional Animal

Care and Use Committee of Kyung Hee University. Male ICR mice (6 weeks, 25–28 g), were purchased from the Daehan Biolink Co. Ltd. (Eumseong, Korea). Animals were housed 3–4 per cage, had free access to water and food, and maintained under a constant temperature (23 71 1C), humidity (60 710%), and a 12 h light/dark cycle. For the surgery, mice were anesthetized and tied up in a stereotaxic apparatus (My-neuro Lab, St. Louis, USA). 3 μL of Aβ25–35 at 1 mg/mL or saline was injected over 6 min using a Hamilton micro syringe (fitted with a 26-gauge needle) into intracerebroventricular (i.c.v.) which coordinates 0.5 mm posterior and 1.0 mm lateral from bregma and 2.0 mm ventral from the skull surface (Paxinos and Franklin, 2001). The needle remained in position for an additional 5 min after injection. The incision was closed with black silk (Ailee, Busan, Korea) and the animal was allowed to recover under a heat lamp. Then, the mice were randomly divided into 6 groups (n ¼8 in each group). The mice were orally administered with ME (20 mg/kg, 100 mg/kg, and 500 mg/kg) or donepezil (2 mg/kg) for 14 consecutive days and the stereotaxic injection of Aβ25–35 was performed on the seventh day of the treatment. ME was dissolved in normal saline and the control group and Aβ25–35 group were treated with the same volume of saline. The gavage doses of ME were derived from the previous study (Kim et al., 2010b).

2.10. Novel object recognition test and Y-maze test Novel objective test (NORT) was carried out in a black open field box (45 cm  45 cm  50 cm) as previously described (Huh et al., 2014). Results were expressed as percentage of novel object recognition time: Novel object recognition index¼ [(time of exploring novel object)/(time of exploring novel objectþtime of the familiar object)]  100. Y-maze test was carried out in a black three-arms with equal angles between all arms (40 cm  3 cm  12 cm) as previously described (Huh et al., 2014). 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.

Fig. 1. Protective effects of ME against Aβ25–35 neurotoxicity. The cell viabilities by MTT assay (A) and the level of general cell death which detected the release of LDH in culture medium (B) were determined. The numbers of MAP-2-positive neurons were also counted (C) and representative images of experiments were shown (D). Scale bar¼ 100 μm. The results are expressed as a percentage of the controls (cells treated with vehicle for 24 h).Values are indicated as the mean 7SEM (n¼ 4 or 5). nnnpo 0.001 compared with the control group, #po 0.05 compared with the Aβ25–35-only treated group.

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2.11. Brain tissue preparation For immunohistochemistry, the mice (n ¼4 in each group) were anesthetized and rapidly perfused transcardially with PBS, followed by 4% PFA in 0.1 M phosphate buffer (PB). Then, brains were rapidly taken out, post-fixed in 4% PFA solution, and processed for

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cryoprotection in 30% sucrose at 4 1C. Frozen brains were cut into 30 μm coronal sections using a cryostat microtome. Then, the tissues were stored in storing solution containing glycerin, ethylene glycol, and PB at 4 1C for immunohistochemistry. For western blotting, the mice (n ¼4 in each group) were decapitated and each hippocampus of the brains were isolated and stored at 80 1C. 2.12. Western blot analysis The treated cells or hippocampal tissues were lysed with triple detergent lysis buffer (50 mM Tris–Cl, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.02% sodium azide, 0.5% sodium deoxycholate, 100 μg/ ml phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin) for lysis whole protein to detection Akt, GSK-3β, tau, and caspase-3. For the detection of Bax, Bcl-2, and cytochrome C, cells or tissues were fractionated into mitochondria and cytosol using the Mitochondria/Cytosol Fractionation kit according to the manufacturer's instructions. The lysates were loaded on 12 or 15% SDS-polyacrylamide gel electrophoresis, and then separated proteins were electrophoretically transferred to a membrane. The membranes were incubated with 5% skim milk in TBST (25 mM tris–Cl, 150 mM NaCl, 0.005% tween-20) for 1 h. Then they were incubated with anti-ptau (1:1000 dilution), anti-tau (1:1000 dilution), anti-GSK-3β (1:500 dilution), anti-pGSK-3β (1:500 dilution), anti-Akt (1:500 dilution), anti-pAkt (1:500 dilution), anti-cleaved caspase-3 (1:1000 dilution), anti-Bax (1:1000 dilution), anti-Bcl-2 (1:2000 dilution), anti-cytochrome C (1:500 dilution), and anti-βactin (1:2000 dilution) antibodies overnight at 4 1C, followed by incubated with HRP-conjugated secondary antibodies for 1 h. Antibody detection was carried out using ECL detection kit and visualized by LAS-4000 mini system (Fujifilm Corporation, Tokyo, Japan). The intensities of the bands were normalized to the β-actin band intensity using Multi Gauge software (Fujifilm Corporation, Tokyo, Japan). 2.13. Immunocytochemistry and immunohistochemistry Rat hippocampal cells on cover slips or free-floating sections were rinsed in PBS at room temperature before immunostaining. They were pre-treated with 1% hydrogen peroxide in PBS for

Fig. 2. Neuroprotective effects of ME via regulation of PI3K cascade including Akt and GSK-3β. The cell viabilities by MTT assay (A) were determined. The graphs display densitometric analyses of the expression ratios of the phosphorylated Akt/Akt (B), phosphorylated GSK-3β/GSK-3β (C). Values are indicated as the mean 7 SEM (n¼ 4–6). nnnpo 0.001, *po 0.05 compared with the control group, ### p o 0.001, #p o 0.05 compared with the Aβ25–35-only treated group, ψ p o 0.05 compared between the MEþ Aβ25–35 and MEþ LY294002 þAβ25–35 treated groups.

Fig. 3. Effect of ME on tau hyperphosphorylation induced by Aβ25–35. The graph displays densitometric analyses of the expression ratio of the phosphorylated tau (ser396)/β-actin. Values are indicated as the mean7SEM (n¼ 4). nnnpo0.001 compared with the control group, #po0.05 compared with the Aβ25–35-only treated group, ψ po0.05 compared between the MEþ Aβ25–35 and MEþLY294002þAβ25–35 treated groups.

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15 min to remove endogenous peroxidase activity. Then, the rat hippocampal cells or the brain sections were incubated overnight at room temperature with a rabbit anti-MAP-2 antibody (1:1000 dilution) or a mouse anti-NeuN antibody (1:100 dilution) for neuronal cell detection. They were then incubated with a biotinylated anti-rabbit or mouse IgG for 90 min, followed by incubation in ABC solution for 1 h at room temperature. The peroxidase activity was visualized with DAB for 3 min. After every incubation step, the cells or tissues were washed three times with PBS. Finally, the cortical neurons on cover slips were mounted on gelatincoated glass slides, air dried, and photographed with a research microscope (BX51T-32F01; Olympus Corporation, Tokyo, Japan). For quantification of the effect of ME in the rat hippocampal cells, MAP-2-immunopositive cells were counted on at least four cover slips from independent experiments for each condition. Data presented are as percentages of control group values. The freefloating brain tissues were mounted on gelatin-coated glass slides, dehydrated, cleared with xylene, cover slipped using histomount medium, and photographed with a research microscope. 2.14. Statistical analysis All quantitative data were analyzed. The results are expressed as means 7standard error or the mean (SEM) for all assays. Statistical significance was determined by one way analysis of variance followed by the Tukey's post hoc test using GraphPad Prism 5.0 software (GraphPad Software Inc.; San Diego, CA, USA). P values less than 0.05 were deemed to be statistically significant.

3. Results 3.1. Protective effect of ME on Aβ25–35-induced neurotoxicity in rat hippocampal neurons Prod. Type: To determine the protective effects of ME in rat hippocampal neurons, cell viability and cell cytotoxicity were measured using MTT and LDH assays, respectively. Treatment with several ME concentrations for 24 h did not influence cell proliferation and did not cause cell toxicity. Also, ME treatment with reversal sequence of Aβ35– 25 did not affect cell viability. However, decreased cell viability induced by 8 μM Aβ25–35 was prevented by ME treatment at 1, 10, and 100 μg/ mL (75.5773.03–83.1771.96%) compared with the control (Fig. 1(A)). In addition, 0.1, 1, 10 and 100 μg/mL ME treatment reduced Aβ25–35triggered LDH release by 138.1471.76%, 130.32711.50%, 119.327 9.78%, and 116.7577.44%, respectively, compared with the control

(Fig. 1(B)). Then, we counted cell body number using MAP-2-positive immunoreactivity. Aβ25–35 neurotoxicity was defined as a 56.2874.71% reduction in the survival rate compared with the control. ME treatment (0.1, 1, and 10 μg/mL) increased the survival of hippocampal neurons to 60.3575.38%, 69.5471.58%, and 78.3773.90%, respectively, compared with the control (Fig. 1(C and D)). These results suggested that ME prevented Aβ25–35-induced neuronal damage. 3.2. Regulatory effect of ME on Aβ25–35-induced GSK-3β activity in rat hippocampal neurons To identify the role of the PI3K cascade in the neuroprotective effects of ME, a PI3K inhibitor, LY294002, was co-administered with ME. ME and LY294002 co-treatment decreased cell viability to 70.73 71.37%, whereas ME treatment alone resulted in 78.8371.43% viability following 8 μM Aβ25–35 toxin treatment (Fig. 2(A)). This result suggested that the PI3K pathway mediated the protective effect of ME. GSK-3β inactivation can be mediated by PI3k-dependent Akt kinase activity (Noh et al., 2009). Therefore, we determined whether the effect of ME involved GSK-3β regulation via the PI3K/Akt pathway. As shown in Fig. 2(B and C), ME treatment with Aβ25–35 resulted in phosphorylation of non-phosphorylated Akt (ser473) and GSK-3β (ser9) (108.237 7.96% and 56.99 75.38% of the control), whereas Aβ25–35 treatment resulted in Akt (ser473) and GSK-3β (ser9) phosphorylation that was 64.87 75.13% and 30.8271.69% of the control, respectively. However, ME-induced effects on the Akt and GSK-3β signaling pathways were blocked in the presence of LY294002. Collectively, ME enhanced GSK-3β inhibition via the PI3k/Akt pathway, resulting neuroprotection against Aβ25–35 toxicity in hippocampal neurons. 3.3. Effect of ME on Aβ25–35-induced tau hyperphosphorylation in rat hippocampal neurons Aβ-induced GSK-3β activation contributes to increased tau phosphorylation (Mines et al., 2011). Therefore, we performed western blotting with a tau (ser396) antibody to investigate the effect of ME on tau phosphorylation. The ptau/tau ratio was increased to 246.33 718.43% of the control in the Aβ25–35 toxicity group, whereas ME significantly decreased the ratio to 178.86 7 14.75% of the control. However, ME and LY294002 co-treatment decreased this effect to 205.72 78.59% of the control (Fig. 3). This result indicated that ME could reduce tau hyperphosphorylation mediated by GSK-3β inactivation.

Fig. 4. Effect of ME on ROS production and Bax translocation induced by Aβ25-35. ROS production was measured using H2DCF-DA (A). The graph displays densitometric analyses of the expression ratio of the mitochondrial Bax/Bcl-2 (B) Values are indicated as the mean7 SEM (n¼ 4). nnnp o 0.001, nnp o 0.01 compared with the control group, ### p o 0.001, #po 0.05 compared with the Aβ25–35-only treated group.

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3.4. Effect of ME on Aβ25–35-induced mitochondria-mediated apoptosis in rat hippocampal neurons Several reports suggested that GSK-3β inhibition might be neuroprotective in AD by reducing oxidative stress and apoptosis (Mines et al., 2011). To investigate the protective effect of ME against oxidative stress and apoptosis, we performed a variety of fluorescence assays and western blotting using ROS- and apoptosis-related markers. Exposure to Aβ25–35 resulted in significantly elevated ROS (198.39 72.40%) compared with the control, whereas ME treatment reduced ROS generation, dosedenendently (Fig. 4(A)). In addition, ME treatment increased the Bcl-2/Bax expression ratio by decreasing Bax and increasing Bcl-2 in the mitochondria (Fig. 4(B)). We then examined the impact of ME on ΔψM using JC-1, which accumulates in mitochondria based on membrane potential. The ratio between green fluorescence (low ΔψM) and red fluorescence (high ΔψM) indicates depolarized ΔψM. In the present study, Aβ25–35 toxicity decreased ΔψM to 50.8375.62% of the control. However, 10 μg/mL ME treatment significantly reduced depolarization of the mitochondrial membrane to 84.1778.26% of the control (Fig. 5(A)). Furthermore, we found that ME reduced cytochrome C release into the cytosol and caspase-3 activation whereas Aβ25–35 toxicity increased these parameters (Fig. 5(B and C, respectively)).

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cytochrome C expression level was increased to 244.43720.30% in the Aβ25–35 group compared to the control, whereas 100 and 500 mg/ kg/day ME treatment ameliorated it to 186.43725.08% and 119.80720.06%, respectively (Fig. 7(B)). Moreover, the cleaved caspase-3 expression level in the hippocampus of Aβ25–35-injected mice increased to 229.06715.76% compared to the control mice; the increase was inhibited to 171.75723.10% and 110.36718.48% by 100 and 500 mg/kg/day ME treatment, respectively (Fig. 7(C)). These results suggested that the neuroprotection observed in this model involved the antiapoptotic activity of ME.

3.5. Protective effect of ME against Aβ25–35-induced memory impairment neuronal cell death in mice A NORT and a Y-maze test were employed to investigate whether ME improved cognition, working, and spatial memory in mice after Aβ25–35-induced toxicity, as several studies indicated that GSK3 plays a pivotal role in learning and memory (Ma, 2014). During the training session on the first day, no significant differences were observed in exploratory preferences in any of the groups. During the test session on the following day, control mice spent more time exploring the novel object than the familiar object (68.6072.11%). However, the Aβ25–35-injected mice spent similar amounts of time exploring the two objects (43.8076.03%). Treatment with 20, 100, and 500 mg/kg/ day ME improved Aβ25–35-induced cognitive deficits by 57.3171.93%, 62.2773.07% and 64.6173.12%, respectively (Fig. 6(A)). In addition, the Aβ25–35-injected mice showed spontaneous memory impairment (53.5372.26%) compared to the control (75.3973.09%). However, 20, 100, and 500 mg/kg/day ME treatment protected against this effect (57.6972.85%, 66.8872.57%, and 69.1472.84%, respectively; Fig. 6(B)). Aβ injection into mouse brain caused AD-type neurodegeneration, which resulted in memory deficits (Liu et al., 2012). Therefore, to determine the protective effects of ME against neuronal damage, we performed staining in mouse hippocampus using a neuron-specific nuclear marker, NeuN. Compared to the control group, a partial loss of neurons was observed in the CA1 and CA3 hippocampal region in the Aβ25–35-injected group. However, neuronal loss was prevented by 14-day ME administration (Fig. 6 (C)). 3.6. Effect of ME on Aβ25–35-induced mitochondria-mediated apoptosis in mice To determine the mechanisms underlying the protective effect of ME in our mouse model, we examined the changes in Bcl-2, Bax, cytochrome C, and cleaved caspase-3 protein levels by western blotting. In this study, the mitochondrial Bcl-2/Bax expression level in the hippocampus of Aβ25–35-injected mice was decreased to 58.1075.02% compared to the control. However, 100 and 500 mg/ kg/day ME treatment ameliorated the decrease (67.4170.85% and 80.7073.70%, respectively; Fig. 7(A)). In addition, the cytosolic

Fig. 5. Effect of ME on apoptosis induced by Aβ25–35. Depolarized ΔψM production was measured using the JC-1 reagent (A). The graph displays densitometric analyses of the expression ratio of the cytosolic cytochrome C/β-actin (B). Caspase-3-like activity and cleaved caspase-3 form were determined (C). Values are indicated as the mean7 SEM (n¼ 4). nnnp o 0.001, nnp o0.01 compared with the control group, ##p o 0.01, #p o 0.05 compared with the Aβ25–35-only treated group.

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Fig. 6. Protective effect of ME on memory impairment and neuronal damage of the mouse hippocampus induced by Aβ25–35 toxicity. The time of exploring the novel object (A) and spontaneous alternation index (B) were recorded. Then, neurons in the mouse hippocampus were visualized with NeuN immunostaining (C). Values are indicated as the mean7 SEM (n¼ 8). Scale bar¼ 200 μm. nnnPo 0.001; mean values were significantly different from the control group. ##Po 0.01; mean values were significantly different from the Aβ25–35-only treated group.

4. Discussion The purpose of this study was to investigate the neuroprotective effects of ME against Aβ-induced toxicity in rat primary hippocampal cells and mice. We specifically examined GSK-3β pathway-mediated reduction of tau phosphorylation and apoptosis. ME protected against Aβ25–35-induced cell damage by increasing cell viability and cell survival, as well as by decreasing cell cytotoxicity. However, LY294002, a PI3K pathway inhibitor, abrogated the protective effects of ME. In addition, Akt and GSK-3β phosphorylation mediated the neuroprotective effects of ME. These results suggested that the neuroprotective effect of ME was related to GSK-3β activity via the PI3K/Akt pathway. GSK-3β is a serine-threonine kinase that mediates various processes, including cellular signaling pathways, metabolic control, embryogenesis, cell death, and oncogenesis (Liang et al., 2012). In particular, it appears to be linked to all of the major pathological mechanisms that have been identified in AD (Mines et al., 2011). Experimental studies suggested that Aβ toxin activates GSK-3β through the PI3K/Akt pathway, which normally maintains the inhibitory serine-phosphorylation of GSK-3β (Mines et al., 2011). GSK-3β promotes hyperphosphorylation of the tau protein, resulting in NFT formation (Liang et al., 2012). Hyperphosphorylated tau dissociates from microtubules and destabilizes them, resulting in neuronal dysfunction, neurodegeneration, and ultimately, functional deficits (Xu et al., 2012). In this study, Aβ25–35 toxin treatment increased tau phosphorylation, whereas ME and a positive control, LiCl, attenuated it. However, co-treatment with ME and LY294002 failed to result in a significant inhibition of phosphorylation. In addition, GSK-3β promotes ROS generation and intrinsic apoptotic signaling, which is often induced by intracellular damage that leads to mitochondrial release of cytochrome c and the activation of intracellular cysteine proteases known as caspases, eventually resulting in apoptosis (Huang et al., 2012; Mines et al., 2011). In this study, ME treatment reduced oxidative stress, which generally contributes to apoptosis (Zhang et al., 2005). We also showed that ME reduced Aβ25–35-induced translocation of Bax to mitochondria. ME decreased Bcl-2 levels and depolarization of the mitochondrial membrane potential, resulting in reduced cytochrome c release (Fig. 5(B)), followed by caspase-3 activation. In addition, the

antiapoptotic effect of ME against Aβ25–35-toxicity was confirmed by reducing the above apoptotic cascade in our mouse model. Taken together, these results indicated that ME improved tau phosphorylation and mitochondria-mediated apoptosis caused by the Aβ25–35 toxin via inactivating GSK-3β by autophosphorylation. Moreover, ME prevented Aβ-induced AD-type memory impairment and neuronal damage. Neuronal degeneration and loss are observed in the brains of AD patients, and Aβ injection into rodent brain ventricles resulted in cell death (Zussy et al., 2011). Aggregated Aβ containing senile plaques are an important pathological marker that induce neuronal cell death, neurite atrophy, synaptic loss, and memory impairment; therefore, it is considered a good neurotoxin for use in generating AD-type in vitro and in vivo models (Kim et al., 2010a). We found that ME-treated mice had better spatial and object recognition memory in the NORT and Ymaze test, resulting in improved memory impairment and reduced Aβ25–35-induced neuronal damage in the hippocampus. Taken together, we assume that the effect of ME on the AD-type memory impairment in the present study was due to reduction of Aβinduced apoptosis. As mentioned previously, the pathological mechanism underlying Aβ-induced neurotoxicity remains unclear; however, many studies suggested that an imbalance of biological systems (e.g., oxidative stress, chronic inflammatory response, and apoptotic cascades) contributes to neurodegeneration (Kim and Oh, 2012; Miller and Shukitt-Hale, 2012). Therefore, several clinical and experimental studies focused on the beneficial role of herbal medicines or functional foods in cellular signaling in AD (Khodagholi and Ashabi, 2012; Kim and Oh, 2012; Miller and Shukitt-Hale, 2012). Mori Fructus is an antiaging agent that contains active phytochemicals. As we previously described, ME showed a total phenolic content of 1.78% (Kim et al., 2010b) and those compounds may exert antioxidant and anti-inflammatory activities that enhance health, particularly in the brain (Kim and Oh, 2012, 2013). Especially, anthocyanins which are well known to be rich in ME have been shown to modulate the progression of AD (Qin et al., 2013; Shih et al., 2010; Song et al., 2014). For example, one of the major anthocyanin in ME, cyanidin 3-O-glucoside, has improved learning and memory against Aβ toxin via modulation

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cognitive dysfunction and neuronal damage in several experimental models by inhibiting apoptosis, oxidative stress, autophagy, and inflammation, underlying pathways of AD-like pathological condition (Joseph et al., 2010; Sabogal-Guáqueta et al., 2015; Tsai et al., 2015). We hypothesize that the multiple bioactive effects of ME on neuroassociated models and its various phytochemicals contributed to the neuroprotective effect observed in the present study. However, further investigation is necessary to fully understand not only toxicity of ME, but also functional phytochemicals. In conclusion, the present study provides evidence that Mori Fructus, an antiaging agent, may be beneficial for cognitive function by protecting neurons, an effect mediated by reduction of tau phosphorylation and apoptosis through GSK-3β regulation following Aβ-induced toxicity.

Conflict of interest statement The authors declare that there are no conflicts of interest.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2015.05.054. References

Fig. 7. Effect of ME on apoptosis of the mouse hippocampus induced by Aβ25–35 toxicity. The graphs display densitometric analyses of the expression ratios of the mitochondrial Bax/Bcl-2 (A), cytosolic cytochrome C/β-actin (B), and cleavedcaspase-3/β-actin. Values are indicated as the mean 7SEM (n¼ 4). nnPo 0.01 and n P o0.05; mean values were significantly different from the control group. ## Po 0.01 and #Po 0.05; mean values were significantly different from the Aβ25–35-only treated group.

of GSK-3β and tau (Qin et al., 2013). In addition, Mori Fructus contains numerous flavonoids such as caffeic acid, quercetin, chlorogenic acid, and ferulic acid (Natić et al., 2015). Caffeic acid attenuated tau phosphorylation by the reduction of GSK-3β activation against Aβ-induced neurotoxicity (Sul et al., 2009). Quercetin, chlorogenic acid, and ferulic acid have been shown to protect

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Mori Fructus improves cognitive and neuronal dysfunction induced by beta-amyloid toxicity through the GSK-3β pathway in vitro and in vivo.

A growing body of literature supports the concept that antiaging herbs may be potential candidates for use in treating age-related neurodegeneration, ...
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