Journal of Ethnopharmacology 154 (2014) 206–217

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Protective effects of bajijiasu in a rat model of Aβ25-35-induced neurotoxicity Di-Ling Chen a, Peng Zhang b, Li Lin b,n, He-Ming Zhang a, Shao-Dong Deng b, Ze-Qing Wu b, Shuai ou b, Song-Hao Liu a, Jin-Yu Wang b a b

Southern Institute of Pharmaceutical Research, South China Normal University, Guangzhou, Guangdong 510631, People's Republic of China College of Chinese Materia Medical, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong 510006, People’s Republic of China

art ic l e i nf o

a b s t r a c t

Article history: Received 2 December 2013 Received in revised form 5 March 2014 Accepted 2 April 2014 Available online 14 April 2014

Ethnopharmacological relevence: Neurodegenerative diseases (NDs) caused by neurons and/or myelin loss lead to devastating effects on patients' lives. Although the causes of such complex diseases have not yet been fully elucidated, oxidative stress, mitochondrial and energy metabolism dysfunction, excitotoxicity, inflammation, and apoptosis have been recognized as influential factors. Current therapies that were designed to address only a single target are unable to mitigate or prevent disease progression, and disease-modifying drugs are desperately needed, and Chinese herbs will be a good choice for screening the potential drugs. Previous studies have shown that bajijiasu, a dimeric fructose isolated from Morinda officinalis radix which was used frequently as a tonifying and replenishing natural herb medicine in traditional Chinese medicine clinic practice, can prevent ischemia-induced neuronal damage or death. Materials and methods: In order to investigate whether bajijiasu protects against beta-amyloid (Aβ25-35)induced neurotoxicity in rats and explore the underlying mechanisms of bajijiasu in vivo, we prepared an Alzheimer's disease (AD) model by injecting Aβ25-35 into the bilateral CA1 region of rat hippocampus and treated a subset with oral bajijiasu. We observed the effects on learning and memory, antioxidant levels, energy metabolism, neurotransmitter levels, and neuronal apoptosis. Results: Bajijiasu ameliorated Aβ-induced learning and memory dysfunction, enhanced antioxidative activity and energy metabolism, and attenuated cholinergic system damage. Our findings suggest that bajijiasu can enhance antioxidant capacity and prevent free radical damage. It can also enhance energy metabolism and monoamine neurotransmitter levels and inhibit neuronal apoptosis. Conclusion: The results provide a scientific foundation for the use of Morinda officinalis and its constituents in the treatment of various AD. Future studies will assess the multi-target activity of the drug for the treatment of AD. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Bajijiasu Alzheimer's disease Neurotoxicity Mechanism Apoptosis Chemical compounds studied in this article: Beta-amyloid (PubChem CID: 3407255) Norepinephrine bitartrate (PubChem CID: 297812) Dopamine hydrochloride (PubChem CID: 65340) Serotonin (PubChem CID: 5202) Acetylcholine (PubChem CID: 187) Malondialdehyde (PubChem CID: 10964)

1. Introduction Neurodegenerative diseases (NDs), including Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD) are a major problem worldwide (Hardy and Gwinn-Hardy, 1998; Muqit and Feany, 2002; Palop et al., 2006). According to the World Health Organization, the morbidity due to NDs will soon exceed that of cancer, becoming the second highest cause of death in 2040. As such, it has become a serious social problem and has attracted considerable attention in the medical profession. NDs can develop for various reasons, including oxidative stress,

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Corresponding author. Tel.: þ 86 2039358270. E-mail address: [email protected] (L. Lin).

http://dx.doi.org/10.1016/j.jep.2014.04.004 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

mitochondrial dysfunction, excitotoxicity, inflammation, and apoptosis. Over the past several years, a new paradigm has emerged that simultaneously targets multiple disease pathways. Such “multi-target-designed drugs” have shown great promise as useful neuroprotective agents in preclinical studies and are able to afford symptomatic relief to decrease the day-to-day burden of these illnesses. Currently, there is little that can be done to slow the progression of NDs. Their multifaceted profiles necessitate a change in the paradigm toward designing effective therapies. As more multi-target-designed drugs are tested in clinical trials and clinical applications, support from society is growing, and the feasibility of this approach is now recognized by many. Alzheimer’s disease (AD) is the most common form of dementia in the elderly and is clinically characterized by the deterioration of learning, memory, and other higher cognitive functions

D.-L. Chen et al. / Journal of Ethnopharmacology 154 (2014) 206–217

Fig. 1. Chemical structure of Bajijiasu.

(Glenner and Wong, 1984; Grundke-Iqbal et al., 1986). AD is an irreversible and progressive neurodegenerative disease. Senile plaques, neurofibrillary tangles, and extensive neuronal loss are the main histological hallmarks observed in AD brains (Do Carmo and Cuello, 2013). Beta-amyloid peptide (Aβ), a major protein component of senile plaques, is considered a critical cause of the pathogenesis of Alzheimer’s disease (AD). Modulation of Aβ-induced neurotoxicity has emerged as a possible therapeutic approach to ameliorate AD onset and progression (Selkoe, 2000; Sambamurti et al., 2002). Morinda officinalis is one of the best-known herbs in China, Korea, and Japan and is especially popular in Southern China. It is a component herb that contains many herbal formulae, such as hexasaccharide and heptasaccharide which have been shown to ameliorate symptoms in an animal model of depression (Li et al., 2001, 2004, 2008; Deng et al., 2012). Previous studies have demonstrated that bajijiasu (BJ), a dimeric fructose isolated from Morinda officinalis (chemical structure is shown in Fig. 1, previous name: Bajisu) is able to reinforce population spikes (PSs) and longterm potentiation (LTP) (Chen et al., 1999), ameliorate cognitive deficits induced by D-galactose in mice, and protect against ischemia-induced neuronal damage or death (Tan et al., 2000a, 2000b). Cell culture experiments have shown that bajijiasu protects against Aβ25-35-induced neurotoxicity in PC12 cells (Chen et al., 2013). The protective effect of bajijiasu might be exerted through enhancing antioxidant abilities, stabilizing mitochondrial membrane potential and intracellular Ca2 þ concentration, and reducing neuronal apoptosis (Chen et al., 2000). Collectively, these results provide evidence that bajijiasu protects against ischemiainduced neuronal damage or death (Lin et al., 2008). Although the effects of multi-target bajijiasu have been described in different models of neurotoxicity, there is no direct evidence regarding the protective function of bajijiasu in the case of Aβ exposure. Thus, the aim of the present study was to investigate whether bajijiasu is protective against Aβ-induced neurotoxicity in rats and to explore the underlying mechanisms in vivo.

2. Materials and methods 2.1. Subjects Adult male Sprague Dawley rats (180–220 g, obtained from Center of Laboratory Animal of Guangzhou University of Chinese Medicine, SCXK [Yue] 2008-0020, SYXK [Yue] 2008-0085) were pair-housed in plastic cages in a temperature-controlled (25 1C) colony room on a 12/12-h light/dark cycle. Food and water were available freely. All experiment protocols were approved by the Center of Laboratory Animals of Guangzhou University of Chinese Medicine. All efforts were made to minimize the number of animals used. 2.2. Bajijiasu extraction and acute toxicity Each 1000 g Morinda officinalis root powder was mixed in 3000 mL of distilled water and soaked for 6 h. Then heated reflux extraction 1.0 h in a round-bottom flask twice, merged the filtrate,

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concentrated to 1000 mL under reduced pressure in a rotary evaporator (90 1C). Adjusted the pH value of the concentrated solution to 6.0 with diluted hydrochloric acid at 45 1C, traversed the anion exchange resin (D-900, free amine type, purchased from Hebei Cangzhou Baoen adsorption materials technology Co., Ltd.) at the speed of 1.0 Bv/h, repeated 3 times, and then concentrated to 500 mL, when cooled to the room temperature added alcohol to the concentration of 90% of alcohol, refrigerated at 4 1C for 48 h. The crystallization was extracted by 65% of acetonitrile in aqueous solution at 80 1C, and the undissolved substance extracted by 65% of acetonitrile in aqueous solution 3 times, the residue crystallization was the bajijiasu. The purity of bajijiasu was analysed by a high performance liquid chromatography (HPLC) system consisted of a pump (Waters, Smartline system 600, including degasser and autosampler system), coupled to an evaporative light scattering detector (ELSD detector Alltech 2000). Briefly, with the hydrophilic chromatography column of Rezex RCM (Phenomenex, 300 mm  7.8 mm) and column temperature 35 1C, 100% of deionized water as mobile phase, flow rate at 0.6 mL/min, drift tube temperature and the nitrogen gas flow rate of ELSD were at 100 1C and 2.0 L/min, respectively. Adult KM mice (18–22 g, half of male, obtained from Center of Laboratory Animal of Guangdong province, SCXK [Yue] 0058740) were pair-housed in plastic cages in a temperature-controlled (25 1C) colony room on a 12/12-h light/dark cycle. Food and water were available freely. All experiment protocols were approved by the Center of Laboratory Animals of Guangzhou University of Chinese Medicine. All efforts were made to minimize the number of animals used, and the experimental operation in accordance with the technical guidelines for acute toxicity studies of Chinese and natural medicines. 20 male and female mice for each group, the test set by the maximum concentration (500 mg/mL) and maximum volume (0.4 mL/10 g body weight) administered at a dose of 20 g/kg body weight last two weeks. And the solvent control group was given the same amount of pure water. The animal weight, diet, appearance, behavior, secretions, excretions and animal deaths and so on were observed, especially the reactions of mice after administration 4 h every day, record all of the situations, and all the appear, volume, color, texture and other changes of the tissues and organs. 2.3. Drugs and treatment procedures Aβ25-35 (A4559-1 mg), purchased from Sigma-Aldrich (St Louis, MO, USA), was dissolved in stroke-physiological saline solution to the concentration of 2 g/L and stored at  20 1C. Before being used, it was incubated at 37 1C for 4 days. Other materials including norepinephrine bitartrate (NE, 100169-199402), dopamine hydrochloride (DA, 100070- 200405), 5-hydroxy-indole-3-acetic acid (5-HIAA, 140737-200501) and serotonin hydrochloride (5-HT, 111656-200401) were purchased from the China Drugs and Biological Products Inspection Institute. Acetonitrile (1489730-925) was purchased from Merck & Co. Inc.; Sodium 1-octanesulfonate (B8, AR, 20081229) was obtained from the Tianjin Damao chemical reagent factory; and phosphoric acid (AR, 20080427), perchloric acid (HPLC, 20070901-1), and sodium dihydrogen phosphate (AR, 20080801-1) were purchased from the Guangzhou chemical reagent factory. Pentobarbital sodium was purchased from MYM Biological (USA). Normal saline and medicinal alcohol were purchased from WJ Biotechnology (China). Assay kits for malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GSH-Px), acetylcholine (ACh), acetylcholinesterase (AChE), and Na þ /K þ -ATPase were purchased from Nanjing JianCheng Bioengineering Institute. (China). All other reagents and chemicals used in the study were of analytical grade.

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2.4. Model preparation The procedures were similar to those described previously (Yamaguchi and Kawashima, 2001). Rats were intraperitoneally anesthetized with 30 g/L pentobarbital sodium (40 mg/kg, i.p.; Sigma-Aldrich) and placed in a stereotaxic frame (RWD Life Science Co., Ltd., Shenzhen, China). The hair was shaved, the scalp was opened, and holes were drilled with an electric dental drill (brushless motor, 30,000 rpm) according to a mouse brain atlas (AP-3.6 mm, ML 72.5 mm, DV3.0 mm). Then, 5 μL (10 μg) Aβ25-35, fibrillar state for one hole (Chen et al., 2013), was slowly injected into the CA1 region of the hippocampus over 5 min, and the needle was kept in for 5 min. Then, the wound was sutured, and penicillin (30 U/kg) was injected intramuscularly to protect against infection. Then the rats were isolated in a warm box until they recovered consciousness. After 15 days, the rats were screened with water maze tests to identify animals that were appropriate models, and they were randomly divided into seven groups as follows: control group (received oral distilled water), sham-operation group (animals underwent surgery but did not received Aβ25-35, oral distilled water was given), model group (Aβ25-35 and oral distilled water were given), positive control group (oral donepezil HCl, 0.125 mg/ [kg  d]), low-dose group (oral BJ, 8 mg/[kg  d]), medium-dose group (oral BJ, 24 mg/[kg  d]), high-dose group(oral BJ, 48 mg/ [kg  d]). Every group had 14 animals, and the experiments lasted 25 days. 2.5. Water maze tests Rat spatial learning and memory abilities were tested in the Morris water maze (MWM, DMS-2, Chinese Academy of Medical Sciences Institute of Medicine) using procedures similar to those described previously (Novarino et al., 2004; Hamid et al., 2009; Dong, Z.F. et al., 2013). The MWM consisted of a circular fiberglass pool (200-cm diameter) filled with water (25 71 1C) that was made opaque by non-toxic black paint. The pool was surrounded by light blue curtains, and three distal visual cues were fixed to the curtains. Four floor light sources of equal power provided uniform illumination to the pool and testing room. A CCD camera suspended above the center of the pool recorded animal swim paths, and video output was digitized by an EthoVision tracking system (Noldus, Leesburg, VA). The water maze tests included three periods: initial spatial training, spatial reversal training, and the probe test. Initial spatial training: Twenty-four hours before spatial training, animals were allowed to adapt to the maze during a 120-s free swim. Rats were then required to swim to find a hidden platform (15 cm  15 cm, located at NW) submerged 2 cm below the water surface. Rats were placed into the water facing the pool wall at four starting positions (N, S, E, and W). Animals were then trained in the initial spatial learning task for 4 consecutive days. There were four trials in every experimental day and between every two trials were 10-min inter-trial intervals. During each trial, rats were allowed to swim until they found the hidden platform, where they remained for 20 s before they were removed to a holding cage. Rats that failed to find the hidden platform in 120 s were guided to the platform, where they also remained for 20 s. Spatial reversal training: After the initial spatial learning task, a reversal learning protocol was conducted with a subset of rats. During the reversal learning task, the hidden platform was moved to the opposite quadrant (e.g., from NW to SE). The reversal learning task entailed 3 additional days with 4 trials per day, similar to the initial training period. Rats exposed to acute stress were trained with a modified reversal protocol that could be completed in a single session. This protocol was used to avoid

confounding the results by exposing the rats to acute stress more than once. Probe test: Twenty-four hours after the final reversal training trial, rats were placed in the pool from a new drop point with the hidden platform absent for 120 s, and their swim path was recorded.

2.6. Animal parameters The animals' fur color and appearance and behavior were observed and recorded every day. Animal weight was measured every 3 days during the drug administration period.

2.7. Enzymatic assays After the water maze tests, the animals were sacrificed, and their hearts were washed twice in 4% paraformaldehyde solution. The entire brain was dissected, and the left brain tissues of half of the number of animals in each group were homogenized with 9 times the volume of physiological saline in an ice bath and centrifuged for 10 min at 3500g. The supernatant fluid was stored at 20 1C until being used. The activities of malondialdehyde (MDA), total superoxide dismutase (T-SOD), catalase (CAT), glutathione reductase (GSH-Px), and the levels of acetylcholine (ACh), acetylcholinesterase (AChE), and Na þ /K þ -ATPase were measured with detection kits. Protein concentrations were determined using the Coomassie Brilliant Blue G250 assay. The kits were all purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, PR China). The procedures were performed according to the manufacturer’s instructions and following previously described methods (Wei et al., 2013; Dong, D. et al., 2013). Levels were normalized to the protein concentration of each sample and expressed as percentage of non-treated controls.

2.8. Monoamine neurotransmitter assay The right brain tissue was dissected from eight rats in each group, following previously described methods (Zhou et al., 2011). Briefly, the tissue was homogenized with HClO4 and filtered in ultrafiltration centrifuge tubes (3K, volume 1.5 mL, Vivaspin 500) to remove the high-molecular weight proteins. Then, the supernatants were directly injected into the chromatographic system (HPLC-ECD) that contained a chromatographic column (Phenomenex Luna C18 [150  4.6 mm2, 5 μm]) and a mobile-phase system that contained two solvents. Solvent A was prepared by dissolving 31.2 g sodium dihydrogen phosphate and 1.0 g sodium octane sulfonate into 1 L deionized water and adjusting the pH value to 4.0. Solvent B was 13% acetonitrile (V/V). During the detection, the solvent speed was 1.0 mL/min, the column temperature was 35 1C, and the detection voltage was set at 350 mV or 700 mV.

2.9. HE staining After water maze testing, the whole brains were dissected, and six brains from each group were fixed in 4% paraformaldehyde solution and prepared as paraffin sections that were stained with hematoxylin–eosin (HE) and observed under light microscopy. The hippocampal histopathological abnormalities were investigated under a light microscope. The number of cells in the hippocampal CA1 region of each section was examined by 2 pathologists in a blinded manner, and the average number was taken as the final result.

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2.10. Hippocampal neuron apoptosis assay

3.3. Effect of bajijiasu on animal appearance and weight

Hippocampal neuron apoptosis was measured by flow cytometry (FCM) using propidium iodide (PI) staining. The experimental procedure was as follows: the bilateral hippocampi were added to 2 mL precooled saline, homogenized in an ice bath, filtered with cell strainers, and centrifuged for 10 min at 1200 rpm. The supernatant was removed, and 1.5 mL phosphate-buffered saline (PBS) solution was added and dispersed, the sample was filtered again, centrifuged 5 min at 1200 rpm, and the supernatant was discarded. Finally, we added 2 mL 70% ethanol for storage at 4 1C for 24 h. Then, the sample was centrifuged, the supernatant was discarded, 1 mL PBS was added, the sample was centrifuged for 5 min at 1500 r/min, the supernatant was removed, 200 μL PBS and 200 μL PI dye was added, incubated in the dark for 20 min at 4 1C, and then measured in a flow cytometer (Cytomics TM FC 500, Beckman Coulter, USA) at Ex 488 nm and Em 630 nm.

One animal in the model group died during the treatment; it did not defecate, and its belly was swollen and angular, suggesting that this rat was severely constipated. The fur of the treated animals was obviously much smoother than that of the model group. Weight records showed that there was no significant difference (p 40.05) in average weight between the treated and model groups. They weighed approximately 250 g at the beginning and 340 g at the end of the experiment, which implied that neither the surgery nor the drugs had a serious impact on animal weight.

2.11. Statistical analysis Behavioral assessment data were analyzed using two-way analysis of variance (ANOVA) followed by least significant difference (LSD) test. Dunnett's test was used for each test and control group. Significance level was set at po 0.05. Statistical Package for the Social Science software (SPSS 17.0, SPSS Inc.) was used for all statistical analyses.

3. Results 3.1. Bajijiasu analysis The HPLC-ELSD test result was show in Fig. 2, there were no other peaks except the bajijiasu.

3.4. Effect of bajijiasu on behavior The initial spatial training results are shown in Fig. 3. On the first day, the latency of the control group (74.25 737.01 s) was significantly lower than that of model group (103.81 716.11 s), and this difference was significant (p o0.01). However, the shamoperation group (87.90 7 20.02 s) showed no significant difference (p 40.05) compared to the control group. On the fourth day, the incubation period of the control group was (29.31 72.66 s), while for the model group it was (56.18 719.96 s) (compared with the control group, po 0.01), and the sham-operation group was (30.40 71.29 s) (p4 0.05 compared with the control group and po 0.01 compared with the model group). These results demonstrate that injecting Aβ25-35 into CA1 could cause memory loss and that the surgery did not cause any side effect. Compared with the model group, the incubation period for each BJ-treated group was significantly shorter. The low-dose BJ group incubation period was (80.30 7 21.16 s), while for the medium-dose group it was (71.46 733.54 s), and for the highdose group it was (53.47 713.93 s), and for the positive control group it was (69.44 726.43 s) on the first day. Compared with the

3.2. Acute toxicity of bajijiasu In the 14 days of testing, the activity ability, fur and secretions were all normal, and there were no death. The weight of each measurement time points were no significant difference compared to the solvent control group (p 40.05), as shown in Table 1. And the volume, color, texture of the tissues and organs were no changes.

Fig. 2. The HPLC–ELSD fingerprint of bajijiasu extracted from Morinda officinalis.

Fig. 3. Effect of bajijiasu on escape latency of Aβ25-35-treated rats in the MWM. The graph Control, control group; Sham, sham-operation group; Model, model group; Positive, positive group (Aβ25-35 20 μgþ donepezil HCl, 0.125 mg/[kg  d]); BJ-8 mg, low-dose BJ group (Aβ25-35 20 μg þBJ 8 mg/[kg  d]); BJ-24 mg, medium-dose BJ group (Aβ25-35 20 μgþ BJ 24 mg/[kg  d]); BJ-48 mg, high-dose BJ group (Aβ25-35 20 μgþ BJ 48 mg/[kg  d]). Values given are the mean 7 SD n¼ 14).

Table 1 Results of the acute toxicity of bajijiasu (mean 7 SD, g). Groups

Solvent control Bajijiasu

n

40 40

Dose/(g/kg)

– 20

Measurement time points

Death rate/%

D0

D1

D3

D7

D14

21.4 71.7 21.6 71.8

23.4 7 2.0 24.37 2.1

23.4 7 2.0 25.2 7 1.7

29.9 7 1.8 30.17 1.9

34.0 7 2.4 36.2 7 3.1

0 0

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model group, the differences were significant (po0.01). On the fourth day, the low-dose BJ group incubation period was (31.30 74.69 s), for the medium-dose group it was (27.64 7 3.35 s), for the high-dose group it was (32.08 77.20 s), and for the positive group it was (26.52 71.57 s), and the differences were significant compared to the model group (po 0.01). These results showed that bajijiasu can ameliorate Aβ25–35-induced learning and memory dysfunction in rats. The variation in total swimming distance in each group was similar to the variation observed for the latency period. The total distances of the control group were 8119 7274, 4947 7245, 3947 7336, and 3205 7 329 cm from the first to the fourth day, respectively. The corresponding values for the model group were 11,3527 304, 6936 7 263, 5187 7347, and 6144 7356 cm, respectively. The differences were significant between the model and control groups (p o0.01). The swimming distances of the positive group were 7594 7 269, 3946 7 236, 3432 7 331, and 2900 7 327 cm, respectively. Compared to the model group, the differences were significant (p o0.01). The swimming distances of the low-dose BJ group were 8782 7280, 5075 7246, 41357338, and 3423 7331 cm; those of the medium-dose group were 7815 7271, 4157 7238, 2969 7327, and 3023 7328 cm; and those of the high-dose group were 5847 7253, 5305 7249, 3690 7334, and 35087 332 cm. Compared to the model group, the differences of all BJ-treated groups were significant (p o0.01). However, the differences were not significant compared to the control group (p 40.05), and the average swimming speed was similar among all groups (p 40.05). Probe test results showed that there was no significant difference (p4 0.05) among the groups with regard to total swimming distance or speed. The swimming time of the control group in the NW quadrant (27.36 73.38 s) was longer than in the other three quadrants (23.217 5.03 s, 25.00 74.11 s, 25.437 5.27 s), and the differences among them were significant (p o0.01). The swimming time of the model group was 20.7775.63 s, which was significantly shorter than the control group (p o0.01), suggesting that the rats remembered the location where the platform was placed. The swimming time of the low-, medium- and high-dose BJ groups were 26.00 76.95 s, 28.907 3.96 s, and 31.93 73.39 s, which were significantly shorter than the control group. Compared to the control group, the differences were significant (p o0.01), as shown in Figs. 4 and 5, the swimming trajectory of the BJ-high dose group was obviously denser in the NW quadrant than the others. These results suggest that bajijiasu could ameliorate Aβ25-35induced learning and memory dysfunction in the rat model.

Fig. 4. The swimming time in the platform quadrant during the spatial probe test. Values given are the mean 7 SD n¼ 14, ♯p o 0.01 vs. control group, np o 0.05 vs. model group, nnpo 0.01 vs. model group.

3.5. Enzymatic assays As shown in Fig. 6, values were expressed as percentage of control group, control group levels of SOD, MDA, CAT and GSH-PX were 195.91 712.71 NU/mg protein, 3.68 70.0.44 nmol/pg protein, 8.53 70.35 U/mg protein, and 2.96 70.20 U/mg protein, respectively. The same measurement values in the model group were 177.23 7 12.17, 4.58 70.41, 4.58 70.12, and 2.667 0.36, respectively. The differences for all of these were significant (p o0.05), which suggested that Aβ25-35 influenced antioxidant levels in rat brain tissue, and the same results as in the PC12 cells experiment (Chen et al., 2013). As shown in Fig. 6A, the SOD levels in the low-, medium-, and high-dose BJ groups were 192.2279.21, 195.0476.43, and 190.757 9.94 NU/mg protein, respectively, compared to 190.3779.27 and 177.23712.17, in the positive and model groups, respectively. The differences of low-, medium-, high-dose BJ groups, and the positive control group were significant compared to the model group (po0.05), suggesting that bajijiasu encouraged SOD production. As shown in Fig. 6B, MDA levels significantly decreased after bajijiasu treatment. The MDA levels of the low-, medium-, and high-dose BJ groups were 3.81 70.26, 3.65 7 0.15 and 3.46 7 0.25 nmol/pg protein, respectively, and that of the positive group was 3.68 70.40, all of which were lower than the model group (4.58 70.12). The differences of low-, medium-, high-dose BJ group, and positive control group were significantly different compared to the model group (p o0.05), and the MDA level of the medium-dose group dropped to the same as the control group, which suggesting that bajijiasu inhibits MDA production. As shown in Fig. 6C, bajijiasu significantly increased CAT levels. That in the low-, medium and high-dose BJ groups were 7.89 70.21, 8.20 70.17 and 8.32 70.21 U/mg protein, respectively, and that of the positive group was 8.677 0.35, all of which were higher than the model group (4.58 70.12) and increased in a concentration-dependent manner. The differences of low-, medium-, high-dose BJ group, and positive control group were significant compared to the model group (p o0.05). As shown in Fig. 6D, bajijiasu treatment significantly increased GSH-Px levels. The GSH-Px level of the low-, medium- and highdose groups were 2.53 70.27, 2.77 70.2 and 3.037 0.36 U/mg protein, and the positive group was 3.28 70.48, all of which were higher than the model group (4.58 70.12) and were again concentration-dependent. The differences of low-, medium-, highdose BJ groups, and the positive control group were significant compared to the model group (po0.05). To evaluate the protective efficacy of bajijiasu on energy metabolism in Aβ25-35-treated rats, we evaluated Na þ /K þ -ATPase levels in brain tissue. As shown in Fig. 7, Na þ /K þ -ATPase was significantly lower in the model group (0.96 70.52 μmol/pg protein) compared to the control group (1.31 70.21) (p o0.05). The levels of all BJ treated groups were significantly increased, the values of the low-, medium-, and high- dose groups and positive group were 1.02 70.16, 1.48 70.45, 1.62 70.52 and 1.777 0.43, respectively, all of which were higher than the model group (0.96 70.52). The differences of low-, medium-, high-dose BJ group, and positive control group were significantly different compared to the model group (po 0.05), and they were also concentration-dependent activity, while the specific mechanism needs further researches. Cholinergic system damage and abnormal ACh levels are observed in AD patients. The results are as shown in Fig. 8, the control group levels of ACh and AChE were 119.84718.82 μmol/mg protein and 4.8670.58 μmol/pg protein, respectively, compared to 107.68710.29 and 7.7470.15 in the model group, respectively. Both of these differences were significant (po0.05). After treatment with different concentrations (8, 24, 48 mg/[kg  d]) of bajijiasu, the

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Fig. 5. The swimming trajectory of each group during the probe test in Aβ25-35-induced model rats. CP, control group; SP, sham-operation group; MP, model group; PP, positive group (Aβ25-35 20 μg þ donepezil HCl, 0.125 mg/[kg  d]); BLP, low-dose BJ group (Aβ25-35 20 μgþ BJ 8 mg/[kg  d]); BMP, medium-dose BJ group (Aβ25-35 20 μgþ BJ 24 mg/[kg  d]); BHP, high-dose BJ group (Aβ25-35 20 μg þBJ 48 mg/[kg  d]).

Fig. 6. Effect of bajijiasu on SOD, MDA, CAT, and GSH-Px levels in Aβ25-35-induced model rats. The graphs show levels of (A) SOD, (B) MDA, (C) CAT, (D) GSH-Px. Values given are the mean 7SD n ¼8 and expressed as percentage of control group, ♯p o 0.01 vs. control group, np o 0.05 vs. model group, nnp o 0.01 vs. model group.

ACh levels of model rats increased to 118.91719.25, 119.82717.70 and 121.07719.32, respectively. Compared with the model group (107.68710.29), the differences were significant (po0.05).

The AchE decreased to 6.7570.19, 6.2470.52 and 5.2870.18, respectively. These differences were significant (po0.05) compared with the model group (7.7470.15).

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Fig. 7. Effect of bajijiasu on Na þ /K þ -ATPase in Aβ25-35-induced model rats. Values given are the mean 7SD n¼ 8, ♯p o 0.01 vs. control group, nnp o 0.01 vs. model group.

Fig. 9. The results of neurotransmitter assays in the brain tissue of Aβ25-35-induced model rats. The graphs show levels of (A) NE and (B) DA. Values given are the mean7 SD n¼ 8 and expressed as percentage of control group, np o0.01 vs. control group, np o 0.05 vs. model group, nnp o0.01 vs. model group.

Fig. 8. Effect of bajijiasu on AchE and Ach in Aβ25-35-induced model rats. Values given are the mean 7SD n ¼8, ♯p o0.01 vs. control group, np o 0.05 vs. model group, nnpo 0.01 vs. model group.

3.6. Monoamine neurotransmitters levels As shown in Figs. 9 and 10, levels of NE, DA, 5-HT and 5-HIAA in the control group were 3.80 70.48, 1.82 7 0.16, 2.18 70.69, and 6.10 70.54 μg/g, respectively. In the model group, they were

3.7170.33, 1.8070.08, 1.8770.19, and 5.5170.41 μg/g, respectively. All of these differences were significant (po0.05) except the DA level, which suggested that Aβ25-35 influences the levels of monoamine neurotransmitters in rat brain tissue. As shown in Fig. 9A, the levels of NE in the low-, medium-, and high-dose BJ groups were 3.72 7 0.46, 4.03 70.55 and 4.41 70.61, and 3.91 7 0.42 in the positive group, except the low- all of which were higher than the model group (3.80 70.48). The differences of medium-, high-dose BJ group, and positive control group were significant compared with the control group (po 0.05). This result showed that bajijiasu promoted NE secretion in a concentrationdependent manner. As shown in Fig. 9B, DA levels in the low-, medium- and highdose BJ groups were 1.83 7 0.23, 1.97 70.13, and 1.98 70.23, and that in the positive group was 1.80 70.06, all of which were higher than that in the model group (1.80 70.08), and the differences of medium- and high-dose BJ group were significant compared with the control group (p o0.05), which showed that bajijiasu stimulated DA secretion in a concentration-dependent fashion. As shown in Fig. 10A, the levels of 5-HT in the low-, medium-, and high-dose BJ groups were 2.1870.19, 2.0770.38, and 2.8470.84, respectively, and that in the positive control group was 2.2070.36, all of which were higher than the model group (1.8770.19), and the differences of low-, medium-dose BJ group, and positive control group were significantly different compared with the control group

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Fig. 10. The results of neurotransmitter assays in the brain tissue of Aβ25-35induced model rats. The graphs show levels of (A) 5-HT, and (B) 5-HIAA. Values given are the mean 7SD n¼ 8 and expressed as percentage of control group, ♯ p o 0.01 vs. control group, np o0.05 vs. model group, nnp o 0.01 vs. model group.

(po0.05), demonstrating that bajijiasu stimulated NE production and in a concentration-dependent manner. Fig. 10B illustrates that the levels of 5-HIAA in the low-, medium-, and high-dose groups were 5.81 70.90, 6.26 70.56, and 6.35 71.12, respectively, and that of the positive group was 6.007 0.65, the medium- and high-dose groups were higher than the model group (6.007 0.65), and the differences of high-dose BJ group, and positive control group were significant compared with the control group (p o0.05),which suggested that bajijiasu stimulated concentration-dependent 5-HIAA production.

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untreated and bajijiasu treated groups, but were overall worse than those in the control group. Fig. 12A graphs the neuronal counts in each group. The number of CA1 pyramidal cells in the control group was 30 72 (n ¼ 6). However, the number in the model group significantly decreased to 15 73 (p o0.01). Compared with the model group, the number of neurons in the bajijiasu-treated (high-, medium- and low-dose) groups increased to 44 74, 31 73, and 25 73, respectively. These values were positively correlated to dose, and the differences between all the groups were significant (p o0.01). The pyramidal cell number in the positive group was 277 3, which was significantly higher than that in the model group (p o0.01) but not with the medium and low-dose BJ groups (p4 0.05). As shown in Fig. 12B, the neuronal number in the CA1 region of the hippocampus in the control group was 477752, compared to 3787 60 in the model group (p o0.01). The neuronal numbers in the bajijiasu-treated groups were 491 752 (high-dose), 5577 46 (medium-dose), and 503 7 57 (low-dose), all of which were significantly different from the model group (p o0.01). The number in the positive group was 491 752, which was also significantly different compared with the model group (p o0.01). The results in Fig. 13A demonstrate that the number of neurons in the cerebral cortex was 474 753 in the control group and 481 757 in the sham-operation group, compared to 378 765 in the model group, which was significantly lower (p o0.01). This finding shows that Aβ25-35 had neurotoxic effects. Compared with the model group, the bajijiasu-treated group numbers increased to 489 732 (high-dose), 464 741 (medium-dose), and 4187 31 (low-dose), and these values were significantly different compared to the model group (p o0.01). The number in the positive group was 495743, and this difference was also significant compared with the model group (po 0.01). The results as in Fig. 13B, which illustrate that the numbers of forebrain basal ganglia neurons were 643799 in the control group and 648 791 in the sham-operation group, compared to 464 740 in the model group, and the difference between them was significant (p o0.01). Compared with the model group, the bajijiasu-treated groups increased to 639 7 93 (high-dose), 568 768 (medium-dose), and 511 768 (low-dose), and the differences were significant and dose dependent (p o0.01). The number of neurons in the positive group was 635756, which was also significantly different compared to the model group (p o0.01). 3.8. Hippocampal neuron apoptosis assay As shown in Fig. 14, the apoptotic rate significantly increased after rats were treated with Aβ25-35. The apoptotic rate was 27.31 70.98%. However, after they were treated by bajijiasu concentrations of 8, 24, 48 mg/(kg d), the apoptotic rate decreased to 13.277 1.29%, 6.35 70.89%, and 3.277 1.10%, respectively (p o0.05 compared with control group).

3.7. Histopathologic morphology HE staining revealed no remarkable neuronal abnormalities in the hippocampus of rats in the control group. The pyramidal cells in the CA1 region were arranged neatly and tightly, and no cell loss was found. Additionally, for the control group, cells were round and intact with nuclei stained clear, dark blue (Fig. 11). However, obvious hippocampal histopathological damage was observed in the model and bajijiasu treated groups. The pyramidal layered structure was disintegrated, and neuronal loss was found in the CA1 region. Neurons with pyknotic nuclei and with shrunken or irregular shape were also observed. These abnormalities were attenuated by bajijiasu treatment. The cells in bajijiasu group had better cell morphology and were more numerous than those in the

4. Discussion Alzheimer’s disease (AD) has been recognized as a senile neurodegenerative disease affecting the life quality of patients. It has been characterized as progressive memory capacity damage and cognitive dysfunction in clinic. Pathological evidences support the fact that a large number of senile plaques (SP) deposit in hippocampus and cerebral cortical neurons of AD patients. A great many evidences have been found to confirm that the accumulation of intracellular β-amyloid (Aβ) may be an early event in the development of AD (Pathan et al., 2006; Hampel, 2013; Li et al., 2013).

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Fig. 11. Pathological sections of hippocampus and cerebral cortex from Aβ25-35-induced model rats after stained with hematoxylin–Eosin (HE) (4  200). Rats in the control group did not show histopathological abnormalities. In the model and high-dose BJ groups, the number of cells in the hippocampal CA1 region appeared decreased. Furthermore, the remnants of the pyramidal cells were arranged irregularly and some exhibited shrunken and irregular shape. The cells in the BJ group had better cell morphology and were more numerous than those in the model and high-dose BJ groups.

Learning and memory abilities are considered an important aspect of cognition, and also reflect the advanced integrative functions of the brain. Thus, it is useful to assess learning and memory abilities to diagnose and evaluate the therapeutic effects of treatments for AD, which is clinically characterized by intellectual decline, memory loss, and related behavioral dysfunction. The water maze test results showed that the latency induced by Aβ25-35 were significantly longer than that of the control group, but these changes were ameliorated by bajijiasu, and the time in the quadrant target also increased markedly. These results demonstrated that the model was successfully established and that bajijiasu improved learning and memory abilities in the dementia model rats. Aβ is a peptide of 36–43 amino acids that is formed by a large transmembrane glycoprotein expressed on the cell, such as amyloid precursor protein (APP). Aβ may activate inflammatory and neurotoxic process, including the excessive generation of free radical and oxidative damage among intracellular proteins and other macromolecules. Oxidative stress is defined as a disturbance of the balance between the production of reactive oxygen species (ROS) and antioxidant defense systems. Excessive ROS production is known to cause oxidative damage to major macromolecules in cells, including DNA, lipids, and proteins, and disrupts cellular functions and integrity (Gardner et al., 1997; Fiers et al., 1999) and can result in cell death and tissue damage. ROS are implicated in several diseases, including cancer, diabetes, cardiovascular diseases, and aging. Previous studies (Markesbery, 1997; GilgunSherki et al., 2003; Polidori, 2004) have indicated that oxidative damage is an early factor in NDs. Therefore, it may be possible to delay AD progression with antioxidants. The results of this study showed that SOD, CAT, and GSH-Px levels were lower in the model group compared to the control group, but MDA levels were higher than the control group. After bajijiasu treatment, the levels of SOD,

CAT and GSH-Px increased, but MDA decreased markedly, which demonstrated that bajijiasu alleviated the oxidative damage, and hence delayed the onset of dementia, but further researches will be encouraged. Cholinergic deficiencies, particularly in the hippocampus, cortex, and septal nuclei of basal forebrain, is another pathological phenomenon observed in AD. And studies have showed that the impaired memory function may also be associated with the cholinergic system synthase such as ChAT and hydrolytic enzyme such as AChE. The levels of acetylcholine transferase enzyme (ChAT) and AChE are decreased in AD patients, and acetylcholine (Ach) concentration is increased. These changes have been postulated to cause delirium and cognitive decline (Volpicelli and Levey, 2004; Marcantonio et al., 2006; Hshieh Tammy et al., 2008; Cabeza et al., 2012). Currently, AChE inhibitors (AChEIs) are primarily used to maintain cognitive functions of AD patients in clinical practice (Cracon et al., 1998; Borroni et al., 2001; De la Fuente et al., 2003; Wolff et al., 2005; Jay, 2005). The results of this study showed that acetylcholinesterase levels decreased, and those of acetylcholine increased in the bajijiasu-treated groups. Modern research has shown that learning and memory abilities are closely related to central nervous system function, and monoamine neurotransmission plays an important role in the regulation of learning and memory, and NE, DA, and 5-HT levels change with age (Coyle et al., 1983; Rehman and Masson, 2001; Bohnen and Albin, 2011). The results described here demonstrate that the levels of NE, DA, 5-HT, and 5-HIAA recovered to normal levels following bajijiasu-treatment. Previous studies have confirmed that mitochondrial dysfunction and energy metabolism abnormalities are early and common pathological phenomena (Raichle and Gusnard, 2002; BouzierSore et al., 2003; Wang et al., 2008; Zhao et al., 2010). Thus, ameliorating energy metabolism abnormalities may be effective

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Fig. 12. Effect of bajijiasu on pyramidal cells and neurons in hippocampus of Aβ25–35induced model rats, cerebral cortex and forebrain basal ganglia. The graph (A) pyramidal cells number in CA1 region of hippocampus, (B) neurons number in CA1 region of hippocampus. Values given are the mean7SD n¼ 6, ♯po0.01 vs. control group, nnpo0.01 vs. model group.

for treating AD. We found that Na þ /K þ -ATP levels of the model group were lower than those of the control group, but treatment with bajijiasu attenuated this decrease, suggesting that bajijiasu can ameliorate energy metabolism abnormalities associated with dementia. In recent years, apoptosis was found in some specific nerve tissues of ND models (Chuang et al., 2002; Iqbal et al., 2002; Vassar, 2002; Wang et al., 2006), especially in the hippocampus and cortex of mutant APP-overexpressing transgenic mice. The evidence indicates that apoptosis is involved in AD pathogenesis, and current research seeks to identify methods or drugs to inhibit apoptosis. We determined that the numbers of neurons in the bajijiasu-treated group, including the CA1 region of the hippocampus, cortex and septal nuclei of basal forebrain, were higher than in the control group, and the apoptotic rate of hippocampal neurons was lower. It is possible that bajijiasu inhibited neuronal apoptosis, but this mechanism should be confirmed by further study. One important property of carbohydrate drugs is that most of them interact with cell-surface molecules, such as cell recognition, cell differentiation, and cell interaction molecules. These kinds of interactions have important effects on human physiology and pathological processes, such as cell growth and differentiation, immune response, bacterial infection, and tumor metastasis (Wang, 2001). Because they interact on the cell surface and do

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Fig. 13. Effect of bajijiasu on neurons in hippocampus of Aβ25-35-induced model rats, cerebral cortex and forebrain basal ganglia. The graph (A) neurons number in cerebral cortex and (B) neurons number in forebrain basal ganglia. Values given are the mean7SD n¼ 6, ♯po0.01 vs. control group, npo0.05 vs. model group, nnpo0.01 vs. model group.

Fig. 14. Effect of bajijiasu on hippocampal neuron apoptosis in Aβ25-35-induced model rats. Values given are the mean7 SD n ¼6, ♯p o0.01 vs. control group, nn p o 0.01 vs. model group.

not enter the intracellular space, they are associated with fewer side effects. Therefore, carbohydrates and their related compounds are used to both treat disease and promote health; they are even being developed as functional foods. These novel treatments have

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great potential as anti-dementia drugs. Bajijiasu, a dimeric fructose, may be one choice for AD, similar to the marine sulfated oligosaccharide HSH-971. In the present study, our results demonstrate that bajijiasu protected against Aβ25-35-induced neurotoxicity in rats. The protective effect was mediated through inhibition of oxidative stress and neuronal apoptosis, restoration of normal energy metabolism, and amelioration of cholinergic system defects. Also in our previous vitro study have demonstrated that treatment with bajijiasu significantly increased the cell viability and mitochondrial membrane potential, while decreased oxidative stress, and alteration of P21, CDK4, E2F1, Bax, NF-κB p65, Caspase-3 protein and mRNA was involved in neuroprotective action of bajijiasu against Aβ-induced cellular toxicity (Chen et al., 2013). These observations laid the scientific foundation for the use of Morinda officinalis in the treatment of various AD. In addition bajijiasu is considered a promising natural chemical constituent for treating AD, and the multi-target activity of this drug will be further studied for treating AD.

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Protective effects of bajijiasu in a rat model of Aβ₂₅₋₃₅-induced neurotoxicity.

Neurodegenerative diseases (NDs) caused by neurons and/or myelin loss lead to devastating effects on patients׳ lives. Although the causes of such comp...
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