Fucoxanthin Inhibits β-Amyloid Assembly and Attenuates β-Amyloid

Key Laboratory of Industrial Fermentation Microbiology of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300...
0 downloads 16 Views 5MB Size
Download PDF
Article pubs.acs.org/JAFC

Fucoxanthin Inhibits β‑Amyloid Assembly and Attenuates β‑Amyloid Oligomer-Induced Cognitive Impairments Siying Xiang,† Fufeng Liu,§ Jiajia Lin,† Huixin Chen,† Chunhui Huang,‡ Liping Chen,† Yiying Zhou,† Luying Ye,† Ke Zhang,† Jiukai Jin,† Jiacheng Zhen,† Chuang Wang,† Shan He,‡ Qinwen Wang,† Wei Cui,*,† and Jinrong Zhang*,‡ †

Ningbo Key Laboratory of Behavioural Neuroscience, Zhejiang Provincial Key Laboratory of Pathophysiology, School of Medicine, and ‡School of Marine Sciences, Laboratory of Marine Natural Products, Ningbo University, Ningbo 315211, China § Key Laboratory of Industrial Fermentation Microbiology of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China ABSTRACT: β-Amyloid (Aβ) can form aggregates through self-assembly and produce neurotoxicity in the early stage of Alzheimer’s disease (AD). Therefore, the inhibition of Aβ assembly is considered as the primary target for AD therapy. In this study, we reported that fucoxanthin, a marine carotenoid, potently reduced the formation of Aβ fibrils and oligomers. Moreover, the fucoxanthin-triggered modification significantly reduced the neurotoxicity of Aβ oligomers in vitro. Molecular dynamics simulation analysis further revealed a hydrophobic interaction between fucoxanthin and Aβ peptide, which might prevent the conformational transition and self-assembly of Aβ. Most importantly, fucoxanthin could attenuate cognitive impairments in Aβ oligomer-injected mice. In addition, fucoxanthin significantly inhibited oxidative stress, enhanced the expression of brain-derived neurotrophic factor, and increased ChAT-positive regions in the hippocampus of mice. On the basis of these novel findings, we anticipated that fucoxanthin might ameliorate AD via inhibiting Aβ assembly and attenuating Aβ neurotoxicity. KEYWORDS: fucoxanthin, β-amyloid, Alzheimer’s disease, marine carotenoid, neuroprotection

■

INTRODUCTION Alzheimer’s disease (AD) is one of the most prevalent neurodegenerative disorders affecting elderly worldwide.1 With the rapid increase in the aging population, AD becomes a huge burden in both developing and developed countries. Unfortunately, no effective treatment is available for this disease. AD is characterized by the reduction of learning and memory and the loss of functional neurons in the brain.2 Although the exact pathogenesis of AD remains unknown, recent studies have suggested that β-amyloid (Aβ) aggregates, including Aβ fibrils and Aβ oligomers in particular, are the main neurotoxins in AD.3 In the brain of AD patients, extracellular Aβ can assemble into oligomers and fibrils.4 Aβ fibrils further form plaques, the pathological markers of AD.5 Aβ oligomers can induce neuronal loss and cognitive impairments via multiple pathways, including increasing oxidative stress, decreasing expression of neurotrophic factors, and inhibiting cholinergic transmission, in the early stage of AD.6 Therefore, it is widely accepted that the reduction of Aβ neurotoxicity via inhibiting Aβ assembly should be the primary target for AD prevention. Many studies have shown that Aβ monomers can form soluble oligomers and fibrils in vitro through self-assembly.7,8 Chemicals could manipulate the Aβ assembly, leading to the reduction of soluble Aβ oligomers and Aβ fibrils. These chemicals can inhibit Aβ neurotoxicity in vitro and reverse cognitive impairments in vivo.9−11 For example, curcumin is found to block the formation of Aβ oligomers and fibrils, and enhance learning and memory in AD animals.9 O4, an ocreinrelated molecule, can inhibit the formation of soluble Aβ © 2017 American Chemical Society

oligomers from Aβ monomers and reverse Aβ oligomerinduced neurotoxicity in hippocampal slices.10 We have reported that bis(heptyl)-cognitin, a dimer of tacrine, reduces the Aβ assembly and reverses Aβ-induced cognitive impairments in mice that received hippocampal injection of Aβ oligomers.11 These results strongly suggested that chemicals with the ability to inhibit Aβ assembly may reverse Aβ-induced cognitive impairments, and therefore be used for AD prevention. Many carotenoids, produced by plants and microorganisms but not animals, are reported to inhibit Aβ assembly As a carotenoid extracted from ocean microorganisms,12 fucoxanthin is safe for animals and humans. Previous studies have suggested that fucoxanthin can reduce oxidative stress, decrease inflammation, and exert antiobesity effects.13,14 Therefore, fucoxanthin has been used for antiobesity therapy.15 Recently, there are studies showing that fucoxanthin can inhibit Aβrelated oxidative stress in microglia cells and prevent Aβinduced neuronal death in cortical neurons.16,17 We have also demonstrated that fucoxanthin can ameliorate scopolamineinduced amnesia in mice.18 These studies suggested that fucoxanthin may decrease neurotoxicity and improve cognitive performance, and therefore be used in the AD treatment. Many in vitro assays could evaluate Aβ aggregates. For example, thioflavin T (ThT) could bind to Aβ fibrils and emit Received: Revised: Accepted: Published: 4092

February 28, 2017 May 7, 2017 May 7, 2017 May 7, 2017 DOI: 10.1021/acs.jafc.7b00805 J. Agric. Food Chem. 2017, 65, 4092−4102

Article

Journal of Agricultural and Food Chemistry fluorescence. Oligomer-specific antibody A11 could recognize oligomeric forms of Aβ. Moreover, the morphology of Aβ aggregates could be detected by transmission electron microscopy (TEM). In the present study, we aimed to explore the direct effects of fucoxanthin on Aβ assembly. We also investigated the interaction between fucoxanthin and Aβ by using molecular dynamics (MD) simulation and evaluated the effects of fucoxanthin on cognitive performance in Aβ oligomer-treated mice.

■

structure of Aβ1−42 was obtained from the Protein Data Bank (PDB code: 1Z0Q).22 The 3D structure of fucoxanthin was generated by the program Sybyl 6.92. The GROMOS96 53a6 force field parameters of fucoxanthin were supplied by Automated Topology Builder and Repository 2.0 Web server (https://atb.uq.edu.au/). The atomic charges and charge groups of fucoxanthin were corrected to agree well with the GROMOS96 53A6 force field parameter set. MD Simulation. Aβ1−42 was first placed into a 6 nm cubic box. Next, 10 fucoxanthin molecules were randomly located around the monomer. Subsequently, water molecules were added into the box, and two water molecules were replaced by the same number of positive ions (Na+) to neutralize the negative charge of the simulated system. The simple point charge (SPC) model was used to describe water. An energy minimization of 1000 steps was performed to relax the simulation system. The relaxed system then was successively equilibrated for 1 ns under an isochoric−isothermal ensemble and isothermal−isobaric ensemble using the Berendsen weak-coupling method. Finally, three MD simulations of 100 ns under different initial conditions were carried out by assigning different initial velocities on each atom of the simulation system. All of the MD simulations were performed at physiological temperature (i.e., 300 K) and a pressure of 1 bar. All atomic MD simulations were performed using the GROMACS 5.1.1 package together with the GROMOS96 53A6 force field. Newton’s classical equations of motion were integrated using the Verlet Leapfrog algorithm with a 2 fs time step. All short-range nonbonded interactions were cut off at 1.4 nm, with dispersion correction applied to energy and pressure to determine the truncation of van der Waals interactions. Long-range electrostatic interactions were calculated with the smooth particle mesh Ewald method using cubic-spline interpolation and a Fourier grid spacing of approximately 0.12 nm. The neighbor list was updated every 10 simulation steps. All bond lengths were constrained with the LINCS algorithm with a relative geometric tolerance of 10−4. Initial velocities were assigned according to a Maxwell distribution. For all simulations, the atomic coordinates were saved every 50 ps for the following analysis. MD Simulation Analysis. The auxiliary programs provided by GROMACS 5.1.1 package were used to analyze the simulation trajectories. The program gmx energy was used to calculate the Lennard-Jones and Coulomb potential energies between fucoxanthin molecules and Aβ1−42. The number of contacts between fucoxanthin and residues within 0.5 nm was calculated by the program gmx mindist. The snapshots were made by visual MD software version 1.9.2. Drug Treatment for Animal Study. Use and care of animals followed the guidelines of the Ningbo University Animal Research Advisory Committee. Male Institute of Cancer Research (ICR) mice weighing 25−30 g were obtained from Zhejiang Academy of Medical Sciences (Hangzhou, Zhejiang, China). The animals were maintained with a 12-h light/dark cycle under controlled temperature (22 ± 2 °C) and humidity (50 ± 10%), and standard diet and water were provided. Animals were allowed to acclimatize for 3 days prior to experiments. Mice were anesthetized by intraperitoneal (i.p.) administration of sodium pentobarbital (50 mg/kg) before being placed in a stereotaxic apparatus (RWD life science, Shenzhen, China). Hippocampal injection of Aβ1−42 oligomers was performed according to a previous publication.23 Briefly, 1 μL of Aβ1−42 oligomers was injected into bilateral hippocampal regions of the mice using the following coordinates: AP, 1.7 mm from bregma; ML, 1.8 mm from the midline; and DV, 2.0 mm from pia mater. Control mice were administered with an equal volume of saline. Fucoxanthin and huperzine A were dissolved in sterile saline. Mice were randomly assigned into six groups with eight animals in each group as follows: sham operation, Aβ1−42 oligomers, Aβ1−42 oligomers plus low (50 mg/kg), medium (100 mg/kg), and high (200 mg/kg) concentrations of fucoxanthin, and Aβ1−42 oligomers plus huperzine A (0.2 mg/kg). Fucoxanthin was given by oral gavage. Huperzine A was given by i.p. injection. Huperzine A or fucoxanthin was given 30 min prior to each trial once a day for 17 consecutive days. Mice were sacrificed for biochemical study on the 17th day. All animals received the last injection of drugs 30 min prior to sacrifice.

MATERIALS AND METHODS

Chemicals and Reagents. Fucoxanthin was extracted from Sargussum horneri as previously described.19 Briefly, fucoxanthin-rich fraction was isolated by ethanol (the ratio of ethanol and sample was 1:4) at 30 °C for 2 h. This solution was further concentrated at 25 °C. Waste, lipid, and chlorophylls were precipitated when the ethanol content reached 63% in concentrated solution. Fucoxanthin was then purified through precipitation when ethanol reached approximately 40% in the solution. The purity of fucoxanthin was greater than 90% as determined by HPLC. Huperzine A was purchased from Sigma (St. Louis, MO). Synthetic Aβ1−42 peptide was obtained from GL Biochem (Shanghai, China). Preparation of Aβ1−42 Fibrils and ThT Assay. Aβ1−42 fibrils were prepared as previously described.9 Briefly, Aβ1−42 lyophilized powder was dissolved in hexafluoroisopropanol (HFIP, Sigma) to form monomers. HFIP was completely evaporated, and Aβ1−42 monomers were further dissolved in 20 mM NaOH to obtain 1 mM Aβ1−42 solution. Moreover, 2 μL of Aβ1−42 was added to 200 μL of PBS containing 5 μM ThT (Sigma, U.S.), and the system was incubated at 37 °C for 6 days in the dark without shaking. Fluorescence intensity was measured by a microplate reader at the excitation and emission wavelengths of 440 and 485 nm, respectively. Preparation of Aβ1−42 Oligomers. Soluble Aβ1−42 oligomers were prepared as previously described.11 Briefly, Aβ1−42 lyophilized powder was dissolved in HFIP to form Aβ1−42 monomers. Aβ1−42 monomers were further spin-vacuumed in 10% HFIP Milli-Q water solution. Subsequently, HFIP was evaporated to obtain 50 μM Aβ1−42. Moreover, 10 μL of Aβ1−42 was added to 40 μL of fucoxanthin. The mixture was incubated at 25 °C for 2 days under stirring and then centrifuged at 14 000g for 15 min at 4 °C. The supernatant consisting mainly of soluble Aβ1−42 oligomers was collected and quantified by bicinchoninic acid assay (Thermo Fisher Scientific, Waltham, MA). Dot Blotting Analysis. Dot blotting analysis was performed as previously described.11 Briefly, 2 μL of Aβ1−42 oligomer sample was spotted onto the nitrocellulose membrane and then air-dried. The membrane was blocked in TBST solution containing 10% milk overnight and then incubated with antioligomer antibody A11 (Thermo Fisher Scientific, 1:1000) or anti-Aβ1−17 antibody 6E10 (Sigma, 1:1000) for 1 h with gentle shaking. After three washes with TBST, the membrane was incubated with secondary antibodies for 1 h and developed with an enhanced chemiluminescence plus kit. Transmission Electron Microscopy. TEM analysis was performed as previously described.11 Briefly, TEM samples were prepared by placing 2 μL of the preincubated solution onto a carbon-coated grid. The samples were stained with 1% uranylacetate and then placed onto a clean paper to remove excess staining solution. The grids were thoroughly examined using a TEM (JEOL, Tokyo, Japan). Culture of SH-SY5Y Cells. SH-SY5Y cells were maintained in high glucose modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/mL)/streptomycin (100 μg/mL) at 37 °C with 5% CO2. The medium was refreshed every 2 days. Before experiments, SH-SY5Y cells were seeded in DMEM supplemented with 1% FBS for 24 h. Cell Viability Measurement. 3-(4,5-Dimethylthiazol-2-yl)-2.5diphenyltetrazolium bromide (MTT) assay based on our previous protocol was used to measure cell viability.20,21 Simulation System. Aβ1−42 monomers were used to explore the molecular interactions between fucoxanthin and Aβ1−42. The initial 4093

DOI: 10.1021/acs.jafc.7b00805 J. Agric. Food Chem. 2017, 65, 4092−4102

Article

Journal of Agricultural and Food Chemistry Open Field Test. Open field test was performed according to a previous publication.11 To analyze the exploratory behavior and locomotor activity, animals were placed in the left rear quadrant of an open field (50 × 50 × 39 cm) with white plywood walls and a brown floor divided into four equal squares of equal dimensions (25 × 25 cm). The animals were placed one by one at the center of the box and allowed to explore it for 5 min. Hand-operated counters and stopwatches were used to score the number of line crossing with four paws and the number of rearing (number of times the animals stood on its hind legs), which were used as indicators of exploratory behavior and locomotor activity, respectively. The drug status of the subjects was blindly monitored by an independent investigator. To avoid perturbation of the animals due to urine and feces, the open field was cleaned with 10% ethanol solution and dry cloth between two tests. Novel Objective Recognition (NOR) Tests. The NOR tests were conducted according to a previous publication.24 Briefly, an open-field arena (30 × 30 × 30 cm) was constructed with polyvinyl chloride, plywood, and acrylic. The task included acclimation, training, and retention over three consecutive days. On day 1, the animals were acclimated to the experimental area for 5 min without exposure to any behavioral stimulus. On day 2 of training session, the animals explored two identical objects (black plastic cubes, 5 × 5 × 5 cm) for 5 min. On day 3 of recognition session, one of the objects was replaced by a gray plastic square pyramid with a new shape and color (5 × 5 × 7 cm), and the animals were again acclimated to the area for 5 min. The field was decontaminated with 10% ethanol solution and dry cloth between the tests. The animals explored the test area by sniffing or touching the objects with their nose and/or forepaws at a distance of less than 2 cm. Sitting or turning around the objects was not considered as an exploratory behavior. The exploratory behavior was manually evaluated using a video camera by an observer blinded to the test conditions. Total exploration time referred to the amount of time devoted to location of the two objects. The cognitive function was measured using a recognition index, which was the exploration time involving either of the two objects (training session) or the novel object (retention session) as compared to the total exploration time. Morris Water Maze Tests. Spatial memory was tested by the Morris water maze tests as previously described.25 The water maze consisted of a circular pool (110 cm in diameter) filled with water at 23 ± 2 °C and a platform. The platform was always positioned in the middle of the northwest quadrant except for on the last day. Swimming was recorded by a video camera linked to a computer-based image system. Learning was evaluated in four consecutive days. Each mouse was trained to locate the platform during four trials daily. The time required to enter the hidden platform was measured. On day 5, a probe trial was conducted by removing the platform and training the mice to swim for 90 s to locate it. Swimming time in the four quadrants of the pool was calculated. Preference for a previous quadrant occupied by the platform indicated spatial memory. Measurements of Antioxidative Enzyme Activity and MDA Level. The activities of specific oxidative stress markers, including antioxidative superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH), and malondialdehyde (MDA) levels, were determined using specific reagents and kits according to the manufacturer’s instructions (Nanjing Jiancheng Biotechnology Institute, Nanjing, Jiangsu, China). Immunohistochemical Staining. Immunohistochemical staining was performed according to a previously reported protocol with minor modifications.26 Briefly, after the behavioral tests, brains were dissected and incubated with 4% paraformaldehyde for 1 day. The brain specimens were dehydrated, embedded in paraffin, and cut into 4-μmthick sections. The hippocampal sections were dewaxed and rehydrated. Treatment with 3% H2O2 for 10 min was carried out to inhibit the cellular peroxidase activity. The sections were incubated with primary antibodies against brain-derived neurotrophic factor (BDNF, Santa Cruz Biotechnology, Santa Cruz, CA) and choline acetyltransferase (ChAT, Santa Cruz Biotechnology) at 4 °C overnight. The specimens were rinsed and incubated with secondary antibodies at 37 °C for 30 min. The sections were labeled with DAB

and colorimetrically analyzed. Image Pro 6.0 (Media Cybernetics Inc., MD) was used to analyze the immunohistochemical index, defined as the average of integral optical density. Data Analysis and Statistics. Data were expressed as means ± SD. Statistical significance was determined by one-way ANOVA and Tukey’s test for post hoc multiple comparison, with the exception of mean escape latency, which was analyzed using two-way repeatedmeasures ANOVA followed by LSD post hoc test. A p < 0.05 was considered as statistically significant.

■

RESULTS Fucoxanthin Inhibits the Formation of Aβ1−42 Fibrils. Previous studies have demonstrated that Aβ aggregates, such as Aβ fibrils and Aβ oligomers, are the main neurotoxins in the brain of AD patients. Therefore, we first evaluated the effects of fucoxanthin on the formation of Aβ aggregates. By using the ThT assay, we found that Aβ1−42 monomers in NaOH solution could form fibrils after 6 days of incubation at 37 °C (Figure 1).

Figure 1. Fucoxanthin significantly prevents the formation of Aβ1−42 fibrils in a concentration-dependent manner. Aβ1−42 monomers (10 μM) were incubated with or without fucoxanthin or curcumin in NaOH solution at the indicated concentrations at 37 °C for 6 days. The amount of Aβ1−42 fibrils was analyzed by ThT assay. Data, expressed as percentage of control, were the mean ± SD of three separate experiments; **p < 0.01 versus the control group (one way ANOVA and Tukey’s test).

Coincubation of fucoxanthin (0.1−30 μM) and Aβ 1−42 monomers largely suppressed the formation of Aβ1−42 fibrils (Figure 1). Moreover, fucoxanthin exhibited a greater potency of inhibiting the formation of Aβ1−42 fibrils as compared to curcumin, a compound previously identified as an inhibitor of Aβ fibrils. Fucoxanthin Inhibits the Formation of Aβ 1−42 Oligomers. We further evaluated the effects of fucoxanthin on the formation of Aβ1−42 oligomers. By using the dot blotting assay, we found that Aβ1−42 monomers could form oligomers after 2 days of incubation under shaking at 25 °C (Figure 2A). TEM analysis demonstrated that globular Aβ1−42 oligomers with a diameter around 10−50 nm were mostly presented in the Aβ 1−42 alone sample (Figure 2B). However, the coincubation of fucoxanthin (0.1−1 μM) substantially reduced the formation of Aβ1−42 oligomers as evidenced by the dot blotting assay (Figure 2A). Moreover, small amounts of irregular aggregates were observed in the sample after coincubation of fucoxanthin and Aβ1−42, suggesting that fucoxanthin largely altered the shape of Aβ1−42 assemblies (Figure 2B). 4094

DOI: 10.1021/acs.jafc.7b00805 J. Agric. Food Chem. 2017, 65, 4092−4102

Article

Journal of Agricultural and Food Chemistry

monomers to form fucoxanthin-modified Aβ1−42 monomers. Cell viability in cells treated with fucoxanthin (0.3−1 μM)modified Aβ1−42 monomers was significantly increased as compared to cells treated with Aβ1−42 oligomers, suggesting that fucoxanthin-modified Aβ1−42 oligomers were less toxic to SH-SY5Y cells as compared to Aβ1−42 oligomers. Fucoxanthin Directly Binds to Aβ1−42 Peptide via Hydrophobic Interactions. To explore the inhibitory mechanism of fucoxanthin on Aβ aggregation, we analyzed direct interactions between fucoxanthin and Aβ1−42 peptide based on the trajectories of all-atom MD simulations. Figure 4A reveals that nine fucoxanthin molecules clustered each other, bound to Aβ1−42 peptide, and formed stable aggregates, which could inhibit the conformational transition of Aβ1−42 and the subsequent aggregation. We further analyzed the atomic contacts between fucoxanthin molecules and Aβ1−42 monomers. Figure 4B shows that the number of atomic contacts between fucoxanthin and Aβ1−42 peptide was sharply increased within the first 10 ns from the initial 180 to around 1500. Moreover, the number of the atomic contacts between Aβ1−42 peptide and fucoxanthin fluctuated around 1300. We also calculated the intermolecular Lennard-Jones and electrostatic energies between Aβ1−42 peptide and fucoxanthin. Figure 4C exhibits that the Lennard-Jones energy contributed the overwhelming majority to the interactions between fucoxanthin and Aβ1−42 peptide. For example, the last 50 ns electrostatic energy between fucoxanthin and Aβ1−42 peptide was −79.3 kJ/mol. However, the last 50 ns Lennard-Jones energy between fucoxanthin and Aβ1−42 peptide was −540.8 kJ/mol, which was more than 6 times stronger as compared to the electrostatic interactions between fucoxanthin and Aβ1−42 peptide. These results indicated that fucoxanthin directly bound to Aβ1−42 peptide mainly via hydrophobic interactions. Fucoxanthin Does Not Change Locomotor Activity in Mice. We further investigated whether fucoxanthin could prevent Aβ-related neurotoxicity and cognitive impairments in vivo. Previous studies have shown that hippocampal injection of Aβ1−42 oligomers can induce the loss of cholinergic neurons, the reduction of BDNF expression, and the impairments of cognition in rodents, which might mimic the pathological events in the early stage of AD patients. Therefore, we tested the effects of fucoxanthin by using this model. Figure 5 displays that mice were anesthetized and injected with 1 μL of Aβ1−42 oligomers in bilateral hippocampal regions to induce neurotoxicity and cognitive impairments. Fucoxanthin or huperzine A was daily given 30 min prior to behavioral tests. We evaluated the locomotor activity by using the open field tests on the seventh day. Treatments of Aβ1−42 oligomers, fucoxanthin or huperzine A, did not change the number of line crossing [oneway ANOVA, F(5, 42) = 1.260, p > 0.05, Figure 6] or rearing [one-way ANOVA, F(5, 42) = 1.836, p > 0.05, Figure 6] in the tests, suggesting that fucoxanthin or huperzine A could not alter the locomotor activity of animals. Fucoxanthin Significantly Improves Recognition Performance in Aβ1−42 Oligomer-Treated Mice. We also evaluated recognition performance in mice at days 8−10 after injection of Aβ1−42 oligomers. In the training session, the recognition index in various groups was not significantly altered [one-way ANOVA, F(5, 42) = 0.575, p > 0.05, Figure 7A]. In the recognition session, the recognition index was significantly different among various groups [one-way ANOVA, F(5, 42) = 4.186, p < 0.01, Figure 7B]. The recognition index in the Aβ1−42 oligomer-treated group was significantly lower as compared to

Figure 2. Fucoxanthin prevents the formation of Aβ1−42 oligomers in a concentration-dependent manner. Aβ1−42 monomers (10 μM) were incubated with or without fucoxanthin in Milli-Q solution at the indicated concentrations under stirring at 25 °C for 2 days. The solution was further centrifuged at 14 000g for 10 min. (A) The supernatants of samples were examined by the dot blotting analysis with antioligomer antibody (A11) and general anti-Aβ antibody (6E10). (B) The supernatants of Aβ1−42 oligomers treated without or without 1 μM fucoxanthin were examined by TEM.

Fucoxanthin-Modified Aβ1−42 Oligomers Are Less Toxic to SH-SY5Y Cells As Compared to Aβ 1−42 Oligomers. We further evaluated the neurotoxicity of fucoxanthin-modified Aβ1−42 oligomers in SH-SY5Y cells using the MTT assay. We found that the cell viability of SHSY5Y cells was decreased to around 50% by treatment of 1 μM Aβ1−42 oligomers for 24 h (Figure 3). Therefore, we coincubated fucoxanthin (0.1−1 μM) with 1 μM Aβ1−42

Figure 3. Fucoxanthin-modified Aβ1−42 oligomers are less toxic than Aβ1−42 oligomers to induce neuronal death in SH-SY5Y cells. (B) Aβ1−42 monomers (1 μM) were incubated with or without fucoxanthin at the indicated concentrations under stirring at 25 °C for 2 days. The solution was further centrifuged at 14 000g for 10 min. The supernatants of samples were added to SH-SY5Y cells. After 24 h, the MTT assay was used to analyze cell viability. Data, expressed as percentage of control, were the mean ± SD of three separate experiments; ##p < 0.01 versus the control group, **p < 0.01 versus the Aβ1−42 oligomer group (ANOVA and Tukey’s test). 4095

DOI: 10.1021/acs.jafc.7b00805 J. Agric. Food Chem. 2017, 65, 4092−4102

Article

Journal of Agricultural and Food Chemistry

Figure 4. Fucoxanthin directly binds to Aβ1−42 peptide via hydrophobic interactions. (A) A representative structure of fucoxanthin molecules binding to Aβ1−42 monomers. For clarity, water molecules are not shown. The main chain of Aβ1−42 peptide is represented by a red NewCartoon model. Fucoxanthin molecules are shown in Licorice model. The atoms of fucoxanthin are colored green for carbon, red for oxygen, and white for hydrogen. (B) Time dependence of atom contacts between Aβ1−42 monomers and fucoxanthin molecules. The cutoff of 0.5 nm was used to calculate the atomic contacts between Aβ1−42 peptide and fucoxanthin. (C) Analysis of Lennard-Jones and Coulomb energies between Aβ1−42 peptide and fucoxanthin. The results were all averaged for three trajectories. The corresponding dotted lines indicated the average level of potential energies in the last 50 ns.

oligomer-treated group spent significantly longer time to find the hidden platform as compared to the control group, suggesting that Aβ1−42 oligomers could induce the impairments of spatial learning in mice (one-way ANOVA, Tukey’s test, p < 0.05, Figure 8A). Moreover, the mice treated with fucoxanthin (100−200 mg/kg) or huperzine A (0.2 mg/kg) spent a significantly shorter time finding the hidden platform as compared to the Aβ1−42 oligomer-treated group, indicating that fucoxanthin and huperzine A could attenuate Aβ1−42 oligomer-induced impairments of spatial learning (one-way ANOVA, Tukey’s test, p < 0.05, Figure 8A). In the probe trial, the platform was removed, and mice were allowed to swim freely. The duration of time mice spent in the target quadrant was significantly different among various groups [one-way ANOVA, F(5, 42) = 5.215, p < 0.01, Figure 8B]. The mice in the Aβ1−42 oligomer-treated group spent more time in the target quadrant as compared to the control group, suggesting that Aβ1−42 oligomers also caused impairments of spatial memory (one-way ANOVA, Tukey’s test, p < 0.01, Figure 8B). Interestingly, both fucoxanthin (50−200 mg/kg) and huperzine A (0.2 mg/kg) significantly reversed Aβ1−42 oligomer-induced impairments of spatial memory in mice, as evidenced by the longer duration in the target quadrant in the fucoxanthin and huperzine A groups as compared to the Aβ1−42 oligomer group (one-way ANOVA, Tukey’s test, p < 0.05, Figure 8B). Fucoxanthin Significantly Reverses the Reduction of SOD, CAT, and GSH Activities, and the Increase of MDA Level in Aβ1−42 Oligomer-Treated Mice. Mice were sacrificed for biochemical study on the 17th day after hippocampal injection of Aβ1−42 oligomers. The activities of SOD, CAT, and GSH, and the MDA level were measured in the hippocampal regions of brain samples. The activities of SOD, CAT, and GSH were significantly higher in the hippocampus of Aβ1−42 oligomer-treated mice as compared to

Figure 5. Chronological sequence of design of animal experiments. At day 1, mice were anesthetized and injected with 1 μL of Aβ1−42 oligomers in bilateral hippocampal regions. Fucoxanthin, huperzine A, or vehicle was daily given for 17 consecutive days. Mice were allowed to recover for 7 days. The locomotor activity of mice was analyzed by open field tests on the 7th day. The cognitive performance of mice was analyzed by novel objective recognition tests and Morris water maze tests at days 8−10 and 11−16, respectively. Mice were sacrificed for biochemical study on the 17th day.

the control group, suggesting that hippocampal injection of Aβ1−42 oligomers could induce recognition impairments in mice (one-way ANOVA, Tukey’s test, p < 0.01, Figure 7B). Moreover, both fucoxanthin (100−200 mg/kg) and huperzine A (0.2 mg/kg) reversed the Aβ1−42 oligomer-induced impairments of recognition in mice, as evidenced by the higher recognition index in the fucoxanthin and huperzine A groups as compared to the Aβ1−42 oligomer group (one-way ANOVA, Tukey’s test, p < 0.05, Figure 7B). Fucoxanthin Significantly Improves Spatial Learning and Memory in Aβ1−42 Oligomer-Treated Mice. Morris water maze tests were performed to evaluate spatial learning and memory in mice at days 11−16 after injection of Aβ1−42 oligomers. At the last day of training, the escape latency was significantly different among various groups [one-way ANOVA, F(5, 42) = 3.931, p < 0.01, Figure 8A]. The mice in the Aβ1−42 4096

DOI: 10.1021/acs.jafc.7b00805 J. Agric. Food Chem. 2017, 65, 4092−4102

Article

Journal of Agricultural and Food Chemistry

Figure 6. Fucoxanthin and huperzine A do not change locomotor activity in Aβ1−42 oligomer-treated mice. The numbers of line crossing (A) and rearing (B) were evaluated by the open field tests at day 7 after hippocampal injection of Aβ1−42 oligomers. Fucoxanthin and huperzine A neither changed the number of line crossing, nor altered the number of rearing in Aβ1−42 oligomer-treated mice. Data represent the mean ± SD (n = 8).

Figure 7. Fucoxanthin and huperzine A reverse Aβ1−42 oligomerinduced impairments of recognition in mice. The NOR tests were used to evaluate recognitive performance on days 8−10 after hippocampal injection of Aβ1−42 oligomers. (A) In the training session of NOR, the recognition index was not changed among various groups. (B) In the recognition session of NOR, fucoxanthin and huperzine A significantly reversed the decrease of recognition index induced by Aβ1−42 oligomers. Data represent the mean ± SD (n = 8); ##p < 0.01 versus the control group, *p < 0.05 and **p < 0.01 versus the Aβ1−42 oligomer group (one-way ANOVA and Tukey’s test).

the control mice (one-way ANOVA, Tukey’s test, p < 0.05, Table 1). Moreover, the MDA level was significantly lower in the brain of Aβ1−42 oligomer-treated mice as compared to the control mice (one-way ANOVA, Tukey’s test, p < 0.01, Table 1). These results suggested that Aβ1−42 oligomers could increase oxidative stress in the hippocampus. Moreover, huperzine A (0.2 mg/kg) and fucoxanthin (100−200 mg/kg) could attenuate Aβ1−42 oligomer-induced changes of SOD, CAT, and GSH activities and MDA level, leading to ameliorated oxidative stress in the hippocampus (one-way ANOVA, Tukey’s test, p < 0.05, Table 1). Fucoxanthin Significantly Attenuates Aβ 1 − 4 2 Oligomer-Induced Down-Regulation of BDNF and ChAT in Mice. In the present study, we evaluated the expression of BDNF and ChAT, two important proteins for cognition in the hippocampus, using immunohistochemical staining. BDNF-positive area in the hippocampus of Aβ1−42 oligomer-treated mice was significantly smaller as compared to the control mice, demonstrating that Aβ1−42 oligomers decreased the expression of BDNF (one-way ANOVA, Tukey’s test, p < 0.01, Figure 9). Both fucoxanthin (100−200 mg/kg) and huperzine A (0.2 mg/kg) significantly elevated the BDNFpositive area as compared to the Aβ1−42 oligomer-treated group, suggesting that fucoxanthin and huperzine A could increase the expression of BDNF (one-way ANOVA, Tukey’s test, p < 0.05, Figure 9). ChAT-positive area in the hippocampus of mice

treated with Aβ1−42 oligomers was also significantly smaller as compared to the control mice, suggesting that Aβ1−42 oligomers triggered the reduction of ChAT expression (one-way ANOVA, Tukey’s test, p < 0.01, Figure 10). Furthermore, fucoxanthin (100−200 mg/kg) and huperzine A (0.2 mg/kg) significantly increased the ChAT-positive area as compared to the Aβ1−42 oligomer-treated group, demonstrating that fucoxanthin and huperzine A could attenuate Aβ1−42 oligomer-induced downregulation of ChAT (one-way ANOVA, Tukey’s test, p < 0.01, Figure 10).

■

DISCUSSION In this study, we, for the first time, found that fucoxanthin, a marine carotenoid, could directly inhibit Aβ assembly. We further revealed a hydrophobic interaction between fucoxanthin and Aβ peptide, which might contribute to the inhibition of conformational transition and self-assembly of Aβ. Finally, we demonstrated that fucoxanthin effectively reversed cognitive impairments possibly via inhibiting oxidative stress and upregulating expression of BDNF and ChAT in Aβ1−42 oligomertreated mice, suggesting that fucoxanthin reduced Aβ neurotoxicity in vivo, which might be useful in AD prevention. 4097

DOI: 10.1021/acs.jafc.7b00805 J. Agric. Food Chem. 2017, 65, 4092−4102

Article

Journal of Agricultural and Food Chemistry

formation of Aβ fibrils. Our findings showed that fucoxanthin reduced the formation of Aβ fibrils from monomers, suggesting that fucoxanthin could directly act on Aβ peptide and inhibit Aβ assembly. Moreover, the half maximal inhibition concentration of fucoxanthin (around 1 μM) to prevent the formation of Aβ fibrils was much lower than that reported for lutein (around 10 μM), crocin (around 15 μM), and zeaxanthin (around 20 μM), indicating that fucoxanthin was potent to inhibit Aβ assembly.27,29 Recent studies have suggested that soluble Aβ oligomers are more toxic than other Aβ aggregates, and they are the main neurotoxins to induce neurotoxicity in the early stage of AD.1,30 Therefore, we further examined the effects of fucoxanthin on the formation of Aβ oligomers. According to our dot blotting analysis, fucoxanthin (0.3−1 μM) substantially reduced the amount of Aβ oligomers, suggesting its effective inhibitory function on the formation of Aβ oligomers. Moreover, as evidenced by the TEM study, the morphology of fucoxanthinmodified Aβ oligomers was different from that of normal Aβ oligomers, indicating that fucoxanthin could alter Aβ assembly procedures. Furthermore, we examined if fucoxanthin-modified Aβ oligomers were still toxic to neurons. Our data showed that the neuronal death of SH-SY5Y cells in fucoxanthin-treated group was less as compared to the Aβ oligomer-treated group, suggesting that the fucoxanthin-triggered modification of Aβ oligomers could substantially decrease the neurotoxicity. Therefore, fucoxanthin might function via reducing the amount of Aβ oligomers and decreasing the Aβ neurotoxicity in the early stage of AD. How could fucoxanthin act on Aβ peptide and inhibit Aβ assembly? Fucoxanthin has a hydrophobic chain consisting of eight conjugated double bonds. Moreover, there are hydroxyl groups on each side of the fucoxanthin. Aβ1−42 peptide, a hydrophobic molecule, contains many hydrophobic residues, including Phe, Val, Leu, Val, Ala, Ile, and Met. The assembly of Aβ involves the formation of highly ordered β-sheet structures from its hydrophobic peptides.4 A previous study has suggested that the inhibition of Aβ by carotenoids may be mainly caused by the interaction between the hydroxyl groups of carotenoids and the acceptor groups of Aβ peptide.29 However, our results obtained from MD analysis suggested that fucoxanthin acted on the Aβ1−42 peptide mainly via a hydrophobic interaction, possibly due to the binding between the hydrophobic chain of fucoxanthin and hydrophobic peptides of Aβ. The interaction between fucoxanthin and Aβ peptide further prevented the conformational transition and self-assembly of Aβ. Therefore, fucoxanthin could effectively inhibit the formation of Aβ fibrils and oligomers. Moreover, the interaction between fucoxanthin

Figure 8. Fucoxanthin and huperzine A reverse Aβ1−42 oligomerinduced impairments of spatial cognition in mice. The Morris water maze tests were used to evaluate spatial cognitive performance on days 11−16 after hippocampal injection of Aβ1−42 oligomers. (A) On the 4th day of training session of the Morris water maze tests, fucoxanthin and huperzine A significantly decreased the prolonged escape latency of mice to find the hidden platform induced by Aβ1−42 oligomers. (B) In the probe trial of the Morris water maze tests, fucoxanthin and huperzine A significantly reversed the decrease of the duration in the target quadrant induced by Aβ1−42 oligomers. Data represent the mean ± SD (n = 8); #p < 0.05 and ##p < 0.01 versus the control group, *p < 0.05 and **p < 0.01 versus the Aβ1−42 oligomers group (one-way ANOVA and Tukey’s test).

Many carotenoids, such as lutein, crocin, and zeaxanthin, can inhibit the formation of Aβ fibrils.27−29 Therefore, we evaluated whether fucoxanthin, a marine carotenoid, could affect the

Table 1. Fucoxanthin Reverses Aβ1−42 Oligomer-Induced Decrease of SOD, CAT, and GSH Activities and Increase of MDA Level in the Hippocampus of Micea Aβ oligomer fucoxanthin (mg/kg) control SOD (U/mg) CAT (U/mg) GSH (U/mg) MDA (nmol/mg)

11.7 166.2 58.2 0.92

± ± ± ±

0.7 13.9 2.1 0.07

vehicle 4.2 70.9 45.0 1.72

± ± ± ±

0.9## 11.2## 2.0# 0.07##

50 7.2 79.9 39.6 1.27

± ± ± ±

0.1** 11.5 1.1 0.04*

HupA (mg/kg)

100 7.9 104.5 53.8 0.94

± ± ± ±

0.6** 8.9** 1.4* 0.04**

200 9.9 149.4 59.7 0.71

± ± ± ±

1.1** 12.3 1.5* 0.04**

0.2 8.4 90.4 58.9 1.33

± ± ± ±

0.5** 8.5* 1.5* 0.05*

a At day 17 after hippocampal injection of Aβ1−42 oligomers, animals were sacrificed for biochemical study. The activities of SOD, CAT, GSH, and MDA level in hippocampus were evaluated by using specific kits. Data represent the mean ± SD (n = 4); #p < 0.05 and ##p < 0.01 versus the control group. *p < 0.05 and **p < 0.01 versus the Aβ1−42 oligomer group (one-way ANOVA and Tukey’s test).

4098

DOI: 10.1021/acs.jafc.7b00805 J. Agric. Food Chem. 2017, 65, 4092−4102

Article

Journal of Agricultural and Food Chemistry

Figure 9. Fucoxanthin and huperzine A reverse Aβ1−42 oligomer-induced decrease of BDNF-positive region in the hippocampus of mice. (A) Representative pictures of BDNF staining in various groups as indicated. (B) Quantitative results showed that fucoxanthin and huperzine A reversed Aβ1−42 oligomer-induced decrease of BDNF-positive region in the hippocampus of mice. Data represent mean ± SD (n = 4); ##p < 0.01 versus the control group, *p < 0.05 and **p < 0.01 versus the Aβ1−42 oligomer group (one-way ANOVA and Tukey’s test).

and Aβ peptide might lead to the formation of fucoxanthinmodified Aβ oligomers with irregular shapes, which were different from those of normal Aβ oligomers. Could fucoxanthin reach the brain to exert its neuroprotective effects? Fucoxanthin is safe in terms of toxicity and mutagenicity for mice. A previous study reported that no morality, no abnormalities in gross appearance, and no abnormal changes in liver, kidney, spleen, and gonadal tissues were observed in ICR mice treated with single dose of 2000 mg/kg fucoxanthin or repeated dose of 1000 mg/kg fucoxanthin for 30 days.31 Moreover, the incidence of micronucleus was not changed between control mice and mice treated with 2000 mg/kg fucoxanthin.32 Our preliminary study suggested that 200 mg/kg fucoxanthin could penetrate the blood−brain barrier, and be retained in the brain of mice (data not shown). Previous studies have shown that fucoxanthin can delay the onset of stroke in stroke-prone spontaneously hypertensive rats.33 We have also found that repeated oral administration of fucoxanthin prevents scopolamine-induced cognitive impairments in mice.18 All of these results suggested that fucoxanthin could exert its neuroprotective effects in the brain. Animal studies are essential to study the pharmacological activities of chemicals on diseases. Although fucoxanthin is found to inhibit Aβ oligomers formation in vitro, it is important to evaluate the effects of fucoxanthin on Aβ oligomer-induced neurotoxicity in vivo. Hippocampal injection of Aβ oligomers can induce the impairments of learning and memory in rodents.

In these models, preformed Aβ oligomers could induce neuronal impairments via increasing oxidative stress. Moreover, Aβ inhibitors might reverse Aβ oligomers-induced cognitive impairments in vivo. We have previously found that bis(heptyl)-cogntin, a chemical that could inhibit the formation of Aβ oligomer in vitro, reversed Aβ oligomer-induced cognitive impairments in mice.11 Therefore, we used this model to evaluate the effects of fucoxanthin on Aβ oligomer-induced cognitive impairments in vivo. Huperzine A, a naturally occurring acetylcholinesterase inhibitor, is used for the treatment of AD in China. Moreover, huperzine A was reported to inhibit Aβ oligomers-induced ATP reduction and mitochondrial swelling in isolated cortical mitochondria.34 Huperzine A could also improve learning and memory in triple transgenic AD mice.35 Therefore, we used huperzine A as a positive control in our study. We found that hippocampal injection of Aβ oligomers could induce impairments of recognition and spatial cognition, as evidenced by NOR and Morris water maze tests, respectively. Moreover, our findings indicated that fucoxanthin effectively attenuated Aβ oligomerinduced cognitive impairments with effectiveness similar to that of huperzine A, strongly suggesting that fucoxanthin could produce anti-Aβ oligomer cognitive enhancing effects in vivo. How could fucoxanthin improve cognitive performance in Aβ oligomer-treated mice? Previous studies have suggested that Aβ oligomers can increase the oxidative stress.4,36 As a metalloenzyme, SOD mainly scavenges oxygen radicals. CAT specifically catalyzes H2O2 into H2O and O2. GSH is a major 4099

DOI: 10.1021/acs.jafc.7b00805 J. Agric. Food Chem. 2017, 65, 4092−4102

Article

Journal of Agricultural and Food Chemistry

Figure 10. Fucoxanthin and huperzine A reverse Aβ1−42 oligomer-induced decrease of ChAT-positive region in the hippocampus of mice. (A) Representative pictures of ChAT staining in various groups as indicated. (B) Quantitative results showed that fucoxanthin and huperzine A reversed Aβ1−42 oligomers-induced decrease of ChAT-positive region in the hippocampus of mice. Data represent mean ± SD (n = 4); ##p < 0.01 versus the control group, **p < 0.01 versus the Aβ1−42 oligomer group (one-way ANOVA and Tukey’s test).

induced loss of cholinergic neurons. Previous studies have suggested that fucoxanthin can directly inhibit acetylcholinesterase and increase cholinergic transmission in mice.18,40 Therefore, we speculated that fucoxanthin exerted its effects on the enhancement of cholinergic system possibly via concurrently suppressing Aβ oligomer-induced loss of cholinergic neurons and inhibiting acetylcholinesterase. However, additional experiments are required to investigate the underlying mechanisms of fucoxanthin on the cholinergic system. Besides antioxidative system, neurotrophic factors, and cholinergic system, fucoxanthin might act on other targets to reverse Aβ oligomer-induced cognitive impairments. For example, fucoxanthin was reported to regulate signaling pathways, such as mitogen-activated protein kinase pathway and protein kinase A cascade, to exert neuroprotective effects.13 Moreover, fucoxanthin can reduce the production of proinflammatory factors, including inducible nitric oxide synthase, tumor necrosis factor-α, and cyclooxygenase-2, in PC12 cells.41 However, the involvements of these targets in fucoxanthininduced elevation of cognition in our study remained to be exclusive. Many studies have suggested that dietary may influence risk, raising the possibility of AD.42 Therefore, the prevention strategies through diet may be effective in AD. For example, high vegetable intakes were associated with reduced cognitive decline.43 Higher intakes of vitamin E from food sources were associated with reduced AD incidence.44 Dietary intake of fucoxanthin was reported to prevent or treat obesity. For

intracellular thiol, and it scavenges reactive oxygen species. Aβ oligomers could decrease the activities of SOD, CAT, and GSH, resulting in the accumulation of intracellular free radicals, the degradation of polyunsaturated lipids to form MDA, and eventually neurodegeneration. We found that fucoxanthin effectively enhanced the activity of antioxidative system and reduced the MDA level, suggesting that fucoxanthin exerted its neuroprotective effects at least partially via inhibiting oxidative stress in the brain. These results were in accordance with previous studies that fucoxanthin has a strong antioxidative stress property.13,14 Neurotrophic factors are affected by Aβ oligomers. BDNF, a neurotrophic factor facilitating synaptic plasticity, plays an important role in learning and memory.37 In our study, fucoxanthin attenuated Aβ oligomer-induced down-regulation of BDNF in the hippocampal regions, suggesting that fucoxanthin improved learning and memory via acting on neurotrophic factors. These results were also in accordance with a previous study that fucoxanthin can promote the BDNF expression in microglial cells.38 The cholinergic system is essential in regulating learning and memory. Aβ oligomers can specifically impair cholinergic transmission in the hippocampus via inducing the loss of cholinergic neurons.39 ChAT is an enzyme responsible for the acetylcholine production. Therefore, ChAT is generally considered as a marker of cholinergic neurons. In our study, fucoxanthin reversed Aβ oligomerinduced ChAT reduction in the hippocampal regions, suggesting that fucoxanthin could attenuate Aβ oligomer4100

DOI: 10.1021/acs.jafc.7b00805 J. Agric. Food Chem. 2017, 65, 4092−4102

Article

Journal of Agricultural and Food Chemistry

(9) Yang, F. S.; Lim, G. P.; Begum, A. N.; Ubeda, O. J.; Simmons, M. R.; Ambegaokar, S. S.; Chen, P. P.; Kayed, R.; Glabe, C. G.; Frautschy, S. A.; Cole, G. M. Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo. J. Biol. Chem. 2005, 280, 5892−5901. (10) Bieschke, J.; Herbst, M.; Wiglenda, T.; Friedrich, R. P.; Boeddrich, A.; Schiele, F.; Kleckers, D.; del Amo, J. M. L.; Gruning, B. A.; Wang, Q. W.; Schmidt, M. R.; Lurz, R.; Anwyl, R.; Schnoegl, S.; Fandrich, M.; Frank, R. F.; Reif, B.; Gunther, S.; Walsh, D. M.; Wanker, E. E. Small-molecule conversion of toxic oligomers to nontoxic beta-sheet-rich amyloid fibrils. Nat. Chem. Biol. 2012, 8, 93− 101. (11) Chang, L.; Cui, W.; Yang, Y.; Xu, S. J.; Zhou, W. H.; Fu, H. J.; Hu, S. Q.; Mak, S. H.; Hu, J. W.; Wang, Q.; Ma, V. P. Y.; Choi, T. C. L.; Ma, E. D. L.; Tao, L.; Pang, Y. P.; Rowan, M. J.; Anwyl, R.; Han, Y. F.; Wang, Q. W. Protection against beta-amyloid-induced synaptic and memory impairments via altering beta-amyloid assembly by bis(heptyl)-cognitin. Sci. Rep. 2015, 5, 1 DOI: 10.1038/srep10256. (12) Sugawara, T.; Baskaran, V.; Tsuzuki, W.; Nagao, A. Brown algae fucoxanthin is hydrolyzed to fucoxanthinol during absorption by Caco2 human intestinal cells and mice. J. Nutr. 2002, 132, 946−51. (13) Liu, C. L.; Chiu, Y. T.; Hu, M. L. Fucoxanthin Enhances HO-1 and NQO1 Expression in Murine Hepatic BNL CL.2 Cells through Activation of the Nrf2/ARE System Partially by Its Pro-oxidant Activity. J. Agric. Food Chem. 2011, 59, 11344−11351. (14) Gammone, M. A.; D’Orazio, N. Anti-Obesity Activity of the Marine Carotenoid Fucoxanthin. Mar. Drugs 2015, 13, 2196−2214. (15) Maeda, H.; Hosokawa, M.; Sashima, T.; Miyashita, K. Dietary combination of fucoxanthin and fish oil attenuates the weight gain of white adipose tissue and decreases blood glucose in obese/diabetic KK-A(y) mice. J. Agric. Food Chem. 2007, 55, 7701−7706. (16) Pangestuti, R.; Vo, T. S.; Ngo, D. H.; Kim, S. K. Fucoxanthin ameliorates inflammation and oxidative reponses in microglia. J. Agric. Food Chem. 2013, 61, 3876−83. (17) Xin, Z.; Shiping, Z.; Chunna, A.; Hongning, Z.; Yi, S.; Yanmei, L.; Xiaoping, P. Neuroprotective effect of fucoxanthin on β-amyloidinduced cell death. J. Chin. Pharm. Sci. 2015, 24, 467−474. (18) Lin, J. J.; Huang, L.; Yu, J.; Xiang, S. Y.; Wang, J. L.; Zhang, J. R.; Yan, X. J.; Cui, W.; He, S.; Wang, Q. W. Fucoxanthin, a Marine Carotenoid, Reverses Scopolamine-Induced Cognitive Impairments in Mice and Inhibits Acetylcholinesterase in Vitro. Mar. Drugs 2016, 14, 67. (19) Lin, J.; Huang, L.; Yu, J.; Xiang, S.; Wang, J.; Zhang, J.; Yan, X.; Cui, W.; He, S.; Wang, Q. Fucoxanthin, a Marine Carotenoid, Reverses Scopolamine-Induced Cognitive Impairments in Mice and Inhibits Acetylcholinesterase in Vitro. Mar. Drugs 2016, 14, 67. (20) Cui, W.; Zhang, Z.; Li, W.; Hu, S.; Mak, S.; Zhang, H.; Han, R.; Yuan, S.; Li, S.; Sa, F.; Xu, D.; Lin, Z.; Zuo, Z.; Rong, J.; Ma, E. D.; Choi, T. C.; Lee, S. M.; Han, Y. The anti-cancer agent SU4312 unexpectedly protects against MPP(+) -induced neurotoxicity via selective and direct inhibition of neuronal NOS. Br. J. Pharmacol. 2013, 168, 1201−14. (21) Cui, W.; Zhang, Z. J.; Hu, S. Q.; Mak, S. H.; Xu, D. P.; Choi, C. L.; Wang, Y. Q.; Tsim, W. K.; Lee, M. Y.; Rong, J. H.; Han, Y. F. Sunitinib produces neuroprotective effect via inhibiting nitric oxide overproduction. CNS Neurosci. Ther. 2014, 20, 244−52. (22) Tomaselli, S.; Esposito, V.; Vangone, P.; van Nuland, N. A.; Bonvin, A. M.; Guerrini, R.; Tancredi, T.; Temussi, P. A.; Picone, D. The alpha-to-beta conformational transition of Alzheimer’s Abeta-(1− 42) peptide in aqueous media is reversible: a step by step conformational analysis suggests the location of beta conformation seeding. ChemBioChem 2006, 7, 257−67. (23) Jiang, L. T.; Huang, M.; Xu, S. J.; Wang, Y.; An, P. Y.; Feng, C. X.; Chen, X. W.; Wei, X. F.; Han, Y. F.; Wang, Q. W. Bis(propyl)cognitin Prevents beta-amyloid-induced Memory Deficits as Well as Synaptic Formation and Plasticity Impairments via the Activation of PI3-K Pathway. Mol. Neurobiol. 2016, 53, 3832−3841.

example, dietary fucoxanthin (0.2%) significantly increased oxygen consumption and reduced white adipose tissue mass in mice.45 Moreover, KK-Ay mice fed with a diet containing 0.2% fucoxanthin for 4 weeks significantly increased HDL-cholesterol and non-HDL-cholesterol levels.46 Our findings that fucoxanthin could reverse Aβ oligomer-induced cognitive abnormities in mice suggest that fucoxanthin might be taken through diet to prevent AD. Taken together, we, for the first time, reported that fucoxanthin effectively inhibited Aβ assembly possibly via a hydrophobic interaction. We also demonstrated that fucoxanthin potently reversed Aβ oligomer-induced impairments of learning and memory via inhibiting oxidative stress, increasing BDNF expression, and elevating cholinergic system, concurrently. Our findings suggested that fucoxanthin could be developed as a potential intervention for AD prevention.

■

AUTHOR INFORMATION

Corresponding Authors

*Tel.: (86) 574 8760 9589. E-mail: [email protected]. *Tel.: (86) 574 8760 0458. E-mail: [email protected]. ORCID

Wei Cui: 0000-0002-8645-4278 Funding

This work was supported by the Natural Science Foundation of Zhejiang Province (LY15H310007), the Applied Research Project on Nonprofit Technology of Zhejiang Province (2016C37110), the National Natural Science Foundation of China (21576199, U1503223, 41406163, 81673407), the Ningbo International Science and Technology Cooperation Project (2014D10019), Ningbo municipal innovation team of life science and health (2015C110026), 863 Program of China (2013AA092902), National 111 Project of China, the Scientific Research Foundation for the Returned Overseas Chinese Scholars, LiDakSum Marine Biopharmaceutical Development Fund, and the K. C. Wong Magna Fund in Ningbo University. Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Scheltens, P.; Blennow, K.; Breteler, M. M. B.; de Strooper, B.; Frisoni, G. B.; Salloway, S.; Van der Flier, W. M. Alzheimer’s disease. Lancet 2016, 388, 505−517. (2) Garber, K. NEURODEGENERATION Potential Alzheimer’s Drug Spurs Protein Recycling. Science 2014, 344, 351−351. (3) Kulshreshtha, A.; Piplani, P. Current pharmacotherapy and putative disease-modifying therapy for Alzheimer’s disease. Neurol Sci. 2016, 37, 1403−1435. (4) Breydo, L.; Uversky, V. N. Structural, morphological, and functional diversity of amyloid oligomers. FEBS Lett. 2015, 589, 2640−2648. (5) Gu, L.; Guo, Z. F. Alzheimer’s A42 and A40 peptides form interlaced amyloid fibrils. J. Neurochem. 2013, 126, 305−311. (6) Kayed, R.; Head, E.; Thompson, J. L.; McIntire, T. M.; Milton, S. C.; Cotman, C. W.; Glabe, C. G. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 2003, 300, 486−489. (7) Potapov, A.; Yau, W. M.; Ghirlando, R.; Thurber, K. R.; Tycko, R. Successive Stages of Amyloid-beta Self-Assembly Characterized by Solid-State Nuclear Magnetic Resonance with Dynamic Nuclear Polarization. J. Am. Chem. Soc. 2015, 137, 8294−8307. (8) Zhang, Y. L.; Hashemi, M.; Lv, Z. J.; Lyubchenko, Y. L. Selfassembly of the full-length amyloid A beta 42 protein in dimers. Nanoscale 2016, 8, 18928−18937. 4101

DOI: 10.1021/acs.jafc.7b00805 J. Agric. Food Chem. 2017, 65, 4092−4102

Article

Journal of Agricultural and Food Chemistry

forms to incident Alzheimer disease and to cognitive change. Am. J. Clin. Nutr. 2005, 81, 508−14. (44) Devore, E. E.; Grodstein, F.; van Rooij, F. J.; Hofman, A.; Stampfer, M. J.; Witteman, J. C.; Breteler, M. M. Dietary antioxidants and long-term risk of dementia. Arch. Neurol. 2010, 67, 819−25. (45) Wu, M. T.; Chou, H. N.; Huang, C. J. Dietary fucoxanthin increases metabolic rate and upregulated mRNA expressions of the PGC-1alpha network, mitochondrial biogenesis and fusion genes in white adipose tissues of mice. Mar. Drugs 2014, 12, 964−82. (46) Beppu, F.; Hosokawa, M.; Niwano, Y.; Miyashita, K. Effects of dietary fucoxanthin on cholesterol metabolism in diabetic/obese KKA(y) mice. Lipids Health Dis. 2012, 11, 112.

(24) Bevins, R. A.; Besheer, J. Object recognition in rats and mice: a one-trial non-matching-to-sample learning task to study ’recognition memory’. Nat. Protoc. 2006, 1, 1306−1311. (25) Morris, R. Spatial localization does not require the presence of local cues. Learning and Motivation 1981, 2, 239−260. (26) Koga, T.; Bellier, J. P.; Kimura, H.; Tooyama, I. Immunoreactivity for Choline Acetyltransferase of Peripheral-Type (pChAT) in the Trigeminal Ganglion Neurons of the Non-Human Primate Macaca fascicularis. Acta Histochem. Cytochem. 2013, 46, 59−64. (27) Ghahghaei, A.; Bathaie, S. Z.; Kheirkhah, H.; Bahraminejad, E. The protective effect of crocin on the amyloid fibril formation of a beta 42 peptide in vitro. Cell Mol. Biol. Lett. 2013, 18, 328−339. (28) Asadi, F.; Jamshidi, A. H.; Khodagholi, F.; Yans, A.; Azimi, L.; Faizi, M.; Vali, L.; Abdollahi, M.; Ghahremani, M. H.; Sharifzadeh, M. Reversal effects of crocin on amyloid beta-induced memory deficit: Modification of autophagy or apoptosis markers. Pharmacol., Biochem. Behav. 2015, 139, 47−58. (29) Katayama, S.; Ogawa, H.; Nakamura, S. Apricot Carotenoids Possess Potent Anti-amyloidogenic Activity in Vitro. J. Agric. Food Chem. 2011, 59, 12691−12696. (30) Jarosz-Griffiths, H. H.; Noble, E.; Rushworth, J. V.; Hooper, N. M. Amyloid- Receptors: The Good, the Bad, and the Prion Protein. J. Biol. Chem. 2016, 291, 3174−3183. (31) Beppu, F.; Niwano, Y.; Tsukui, T.; Hosokawa, M.; Miyashita, K. Single and repeated oral dose toxicity study of fucoxanthin (FX), a marine carotenoid, in mice. J. Toxicol. Sci. 2009, 34, 501−10. (32) Beppu, F.; Niwano, Y.; Sato, E.; Kohno, M.; Tsukui, T.; Hosokawa, M.; Miyashita, K. In vitro and in vivo evaluation of mutagenicity of fucoxanthin (FX) and its metabolite fucoxanthinol (FXOH). J. Toxicol. Sci. 2009, 34, 693−8. (33) Ikeda, K.; Kitamura, A.; Machida, H.; Watanabe, M.; Negishi, H.; Hiraoka, J.; Nakano, T. Effect of Undaria pinnatifida (Wakame) on the development of cerebrovascular diseases in stroke-prone spontaneously hypertensive rats. Clin. Exp. Pharmacol. Physiol. 2003, 30, 44−48. (34) Yang, L.; Ye, C. Y.; Huang, X. T.; Tang, X. C.; Zhang, H. Y. Decreased accumulation of subcellular amyloid-beta with improved mitochondrial function mediates the neuroprotective effect of huperzine A. J. Alzheimer’s Dis. 2012, 31, 131−42. (35) Ratia, M.; Gimenez-Llort, L.; Camps, P.; Munoz-Torrero, D.; Perez, B.; Clos, M. V.; Badia, A. Huprine X and huperzine A improve cognition and regulate some neurochemical processes related with Alzheimer’s disease in triple transgenic mice (3xTg-AD). Neurodegener. Dis. 2013, 11, 129−40. (36) Blennow, K.; de Leon, M. J.; Zetterberg, H. Alzheimer’s disease. Lancet 2006, 368, 387−403. (37) Schjetnan, A. G. P.; EscobAr-Rodriguez, M. L. Memory coding and retention: Brain-derived neurotrophic factor (BDNF) in synaptic plasticity. Rev. Neurologia 2007, 45, 409−417. (38) Pangestuti, R.; Vo, T. S.; Ngo, D. H.; Kim, S. K. Fucoxanthin Ameliorates Inflammation and Oxidative Reponses in Microglia. J. Agric. Food Chem. 2013, 61, 3876−3883. (39) Ferreira-Vieira, T. H.; Guimaraes, I. M.; Silva, F. R.; Ribeiro, F. M. Alzheimer’s Disease: Targeting the Cholinergic System. Curr. Neuropharmacol. 2016, 14, 101−115. (40) Kawee-ai, A.; Kuntiya, A.; Kim, S. M. Anticholinesterase and Antioxidant Activities of Fucoxanthin Purified from the Microalga Phaeodactylum tricornutum. Nat. Prod. Commun. 2013, 8, 1381−1386. (41) Tan, C. P.; Hou, Y. H. First Evidence for the Anti-inflammatory Activity of Fucoxanthin in High-Fat-Diet-Induced Obesity in Mice and the Antioxidant Functions in PC12 Cells. Inflammation 2014, 37, 443−450. (42) Barnard, N. D.; Bush, A. I.; Ceccarelli, A.; Cooper, J.; de Jager, C. A.; Erickson, K. I.; Fraser, G.; Kesler, S.; Levin, S. M.; Lucey, B.; Morris, M. C.; Squitti, R. Dietary and lifestyle guidelines for the prevention of Alzheimer’s disease. Neurobiol. Aging 2014, 35 (Suppl 2), S74−8. (43) Morris, M. C.; Evans, D. A.; Tangney, C. C.; Bienias, J. L.; Wilson, R. S.; Aggarwal, N. T.; Scherr, P. A. Relation of the tocopherol 4102

DOI: 10.1021/acs.jafc.7b00805 J. Agric. Food Chem. 2017, 65, 4092−4102