Effects of Astaxanthin and Docosahexaenoic-Acid-Acylated

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Functional Structure/Activity Relationships

The effects of astaxanthin and docosahexaenoic acid-acylated astaxanthin on Alzheimer's disease in APP/PS1 double transgenic mice Hongxia Che, Qian Li, Tiantian Zhang, Dandan Wang, Lu Yang, Jie Xu, Teruyoshi Yanagita, Changhu Xue, Yaoguang Chang, and YuMing Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00988 • Publication Date (Web): 25 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018

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Journal of Agricultural and Food Chemistry

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The effects of astaxanthin and docosahexaenoic acid-acylated astaxanthin on Alzheimer's disease

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in APP/PS1 double transgenic mice

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Hongxia Che, † Qian Li,† Tiantian Zhang,† Dandan Wang,† Lu Yang,† Jie Xu,† Teruyoshi Yanagita,‡ Changhu Xue,†,

4

⊥

⊥,*

Yaoguang Chang,†, * Yuming Wang†,

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6

†

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Shandong, China

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‡

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Saga University, Saga, 840-8502, Japan

10 11

College of Food Science and Engineering, Ocean University of China, Qingdao, 266003,

Laboratory of Nutrition Biochemistry, Department of Applied Biochemistry and Food Science,

⊥

Qingdao National Laboratory for Marine Science and Technology, Laboratory of Marine Drugs

& Biological products, Qingdao 266237, Shandong, China

ACS Paragon Plus Environment


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ABSTRACT

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Alzheimer's disease (AD) is a progressive neurodegenerative disorder with the characteristics of

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senile plaques, neuro-inflammation, neurofibrillary tangles and destruction of synapse structure

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stability. Previous studies have verified the protective effects of astaxanthin (AST). However,

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whether synthesized docosahexaenoic acid-acylated AST diesters (AST-DHA) could delay AD

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pathogenesis remains unclear. In the present study, APP/PSEN1 (APP/PS1) double transgenic

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mice were administrated with AST and AST-DHA for 2 months. The results of radial 8-arm maze

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and Morris water maze tests showed that AST-DHA exerted more significant effects than AST in

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enhancing learning and memory levels of APP/PS1 mice. Further mechanical studies suggested

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that AST-DHA was superior to AST in regulating the parameters of oxidative stress, reducing Tau

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hyper-phosphorylation, suppressing neuro-inflammation and regulating inflammasome expression

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and activation in APP/PS1 mice. The findings suggested AST-DHA attenuated cognitive disorders

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by reducing pathological features in APP/PS1 mice, suggesting AST-DHA might be a potential

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therapeutic agent for Alzheimer's disease.

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KEYWORDS: Alzheimer's disease, astaxanthin, cognitive disorder, DHA-acylated AST esters,

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neuro-inflammation

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INTRODUCTION

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The main pathological features of Alzheimer's disease (AD) are senile plaques,

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neuro-inflammation, and destruction of synapse structure stability 1. There are very few AD drugs

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on the present market, and these drugs only provide minimal symptomatic relief rather than

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changes in disorder progression. Therefore, there is a great need for the therapeutic agents to

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modify AD.

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Astaxanthin (AST, Fig.1A), one of natural carotenoids, is widely present in marine organisms

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such as shrimp, crab, krill, salmon, and microalgae 2. Importantly, the hydroxyl and keto moieties

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on each ionone ring in AST imply its unique properties, especially, the ability to be esterified with

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fatty acids to increase stability. Usually, AST occurs in esterified forms in nature, which is

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chemically bound to various types of fatty acids such as oleic, eicosanoic, palmitic and stearic acid.

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The AST in red crab langostilla (Pleuroncodes planipes) is comprised of about 70% diesters, 12%

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monoesterified and 10% unesterified AST 3. Interestingly, it has been reported that the main

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existing form of AST in Atlantic salmon is unesterified AST 4.

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The bioactivity of AST is usually related to the reduced markers of oxidative damage.

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Notably, recent evidence has emerged to indicate AST has a broad range of bioactivities including

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anti-inflammatory, anti-apoptotic properties5. Moreover, AST plays a vital role in reducing

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neurotoxicity in cell models of AD. In addition, AST could protect PC12 cells from Aβ-induced

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cytotoxicity by up-regulating heme oxygenase-1 expression via ERK1/2 pathway5-6. Lobos et al.

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provided further evidence to indicate that AST protected neurons from the noxious effects of Aβ

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on mitochondrial ROS production and calcium dysregulation 7. Moreover, Cheong et al. have

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verified that krill oil could enhance cognitive capability and modulate proteomic alterations in

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brain of D-galactose induced aging mice 8.

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Docosahexaenoic acid (DHA) is well known for various bioactivities 9. However, few study

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focused on the protective effects of DHA-acylated AST ester. The APP/PSEN1 (APP/PS1)

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transgenic mice, co-expressing the mutated Swedish APP gene and the exon-9-deleted variant of

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the presenilin-1 (PS1) gene, are a successfully established transgenic animal models for AD


10

.


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This model displays age-related plaque pathology, inflammatory response, oxidative damage,

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age-related memory deficits11-12. In the present study, DHA-acylated AST diesters (AST-DHA)

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was prepared, and APP/PS1 mice were used to compare the neuroprotective actions of AST and

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AST-DHA and illustrate the possible underlying mechanisms.

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MATERIALS AND METHODS

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Chemicals and Reagents. Astaxanthin was obtained from Xinxiang Kesen Food Additives

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Corporation (Tongxiang, Zhejiang, China). Aβ42 and Aβ40 ELISA kits were purchased from

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Wuhan Uscn Life Science, Inc. (Wuhan, Hubei, China). The superoxide dismutase (SOD) assay

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kit and nitric oxide (NO) assay kit were obtained from Nanjing Jiancheng Bioengineering Institute

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(Nanjing, Jiangsu, China). The Nitric Oxide Synthase ELISA kit was from R&D System

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(Minneapolis, MN, USA). p-Tau (Ser396) antibody, Tau antibody, p- GSK-3β (Y216+Y279)

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antibody, GSK-3β antibody, CD11b antibody, GFAP antibody, IL-1β antibody, TNF-α antibody,

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NLRP3 antibody, Caspase-1 antibody, Bax antibody, Bcl-2 antibody, caspase-9 antibody and

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β-actin antibody were from Abcam (Cambridge, UK). Caspase-3 antibody and cleaved caspase-3

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antibody were purchased from Cell Signaling Technology (Boston, USA).

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Preparation of AST-DHA. AST-DHA (Fig.1A) was synthesized by 4-dimethylaminopyridine

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catalyzed reaction of AST and DHA in the presence of 1-(3-Dimethylaminopropyl)-

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3-ethylcarbodiimide hydrochloride system with nitrogen protection and light isolation according

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to our previous study

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25 ℃. The organic phase is recovered following the subsequent cleaning by 1 M hydrochloric acid,

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saturated sodium bicarbonate and saturated sodium chloride. The organic mixture was evaporated

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to dryness to obtain the AST-DHA (the purity > 90%), which was confirmed by HPLC-DAD

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according to our previously published study.

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Animals and Treatments. The animal study protocol was proved by the Animal Ethics

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Committee of College of Food Science and Engineering of Ocean University of China. All the

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animals were housed at the Laboratory Animal Facility at the Ocean University of China. The

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research was conducted in accordance with the Guide for the Care and Use of Laboratory Animals

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. Dichloromethane was added to the system after the reaction for 3 h at


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(8 th edition, Institute of Laboratory Animal Resources on Life Sciences, National Research

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Council, National Academy of Sciences, Washington DC). APP/PS1 transgenic mice (half male

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and half female, weight of 20-25 g, aged 3 months) and eight of their wild-type littermates as

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normal control group (Control) were obtained from Vital River Laboratories (Beijing, China). All

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the mice were acclimatized for 1 week under a 12 h/12 h light/dark cycle at 23 °C with 60 ± 10%

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humidity and provided with food and water ad libitum. The APP/PS1 transgenic mice were

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randomly divided into Model group, AST group and AST-DHA group. All the mice were

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supplemented with AIN-93G diet. The mice in AST and AST-DHA group were supplemented

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with 0.2% AST and AST-DHA for 60 days, respectively. Then the water maze and radial 8-arm

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maze tests were used to determine learning and memory levels. After that, the mice were

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sacrificed by rapid decapitation. The cerebral cortex and hippocampus were separated from the

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whole brain and weighed, then frozen with liquid nitrogen and stored at -80 °C until use.

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Radial 8-Arm Maze Test. Spatial learning ability was determined by the radial 8-arm maze test

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according to a previous method

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radiating arms was placed at 1 m above the floor. A food cup was located at the end of each

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radiating arm. Before training, the mice were deprived of half of the diet during the test. The baits

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were restricted to the food cups. On the first two training days, the food pellet (45 mg) were

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located at the food cups (eight trails) and central octagonal plate, the mice of the same group were

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allowed to explore for food for 10 minutes together. On the third and fourth days, every mouse

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was allowed to explore for food for 10 minutes. On the last day, four identical arms were baited

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with a single 45 mg food pellet. Each trial continued until all four baits had been consumed or

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until 10 minutes had elapsed. The numbers of reference memory errors and working memory

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errors were conducted.

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Water Maze Test. A circular stainless-steel pool with water (21-23 °C) was divided into four

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quadrants. A circular black escape platform was located in the center of one quadrant. The mice

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were trained to find the platform for five consecutive days. The time of finding the platform was

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recorded as latency. The swim paths, distances, and latencies taken to the platform were

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monitored with a video camera. Probe test without platform on the sixth day was used to

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. The apparatus composed of a central octagonal plate and 8



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determine spatial memory retention. The mice were placed in a position opposite the platform

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location and allowed to swim for 60 sec. The number crossing over the previous position of the

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platform and the time spent in the target quadrant were recorded as measures for spatial memory.

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Immunohistochemistry. For paraffin sections, mice were killed and systemically perfused with

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phosphate buffered saline (PBS) and 4% buffered paraformaldehyde through the left ventricle to

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wash out blood cells. Then brain samples were collected, ehydrated and embedded in paraffin

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using standard techniques. Sections (5 µm) were cut and deparaffinized. After incubation in

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methanol containing 3% H2O2 for 15 minutes to block endogenous peroxidase activity,

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nonspecific binding was done with universal blocking reagent for 30 minutes at room temperature.

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The sections were incubated with the primary antibody against anti-Aβ (1:100; Servicebio),

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anti-Iba1 (1:500; Servicebio) and anti-GFAP (1:500; Servicebio) at 4 °C for overnight followed by

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several washing steps in PBS. Following incubation with biotinylated goat anti-rabbit IgG (1/200)

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for 50 minutes, staining was done through incubation with peroxidase streptoavidin and

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diaminobenzidine (DAB) at room temperature. Specific primary antibody was omitted in negative

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control of the reactions. After counter staining nuclei with Mayer’s haematoxylin, the

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immunopositive amyloid plaques in cortex and hippocampus were observed and counted. The

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images were taken using a 5 MP Canon A95 camera integrated to the microscope and were

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evaluated using image-J analysis.

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Determination of Aβ Concentration by ELISA. Soluble and insoluble Aβ were extracted by the

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previous method 15. Briefly, the right hippocampus was homogenized in TBS (pH8.0) containing

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protease inhibitors. Samples were sonicated and centrifuged. The supernatant was used to

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determine soluble Aβ40 and Aβ42 concentrations, whereas the TBS-insoluble pellet was firstly

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sonicated in 2% SDS. To analyze the insoluble Aβ content, the SDS-insoluble pellet was

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dissolved and sonicated in 70% formic acid. The extract was neutralized with 0.5 M Tris before

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loading on the ELISA plate. The soluble/insoluble Aβ40 and Aβ42 levels were determined by

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ELISA kits according to the manufacture’s instruction.

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The Measurement of Oxidative Stress Parameters. The brain was prepared as a tissue

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homogenate in 0.9% saline solution for the determination of protein concentration using a BCA


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protein assay kit. The concentrations of nitric oxide (NO), inducible nitric oxide synthase (NOS)

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and the activity of superoxide dismutase (SOD) were measured by the manufacture’s instruction.

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Western Blot Analysis. The total protein and RNA of hippocampus were extracted by the

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total-DNA-RNA-Protein kit. Equal amounts of protein were separated on 5-12% SDS-PAGE gels

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and transferred to poly membranes. The membranes were incubated with antibodies against p-Tau

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antibody (1:5000), Tau antibody (1:2000), p-GSK3β antibody (1:1000), GSK-3β antibody (1:500),

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CD11b antibody (1:500), GFAP antibody (1:5000), IL-1β antibody (1:5000), TNF-α antibody

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(1:200), NLRP3 (1:2000), Caspase 1 (1:1000) at 4 °C for overnight. After this, membranes were

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incubated with specific horse radish peroxidase (HRP)-conjugated secondary antibodies (1:3000)

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at room temperature 2 h and the blots were evaluated with chemiluminescent horseradish

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peroxidase substrate. Then the blots were visualized by enhanced chemiluminescence (ECL)

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substrate with UVP Auto Chemi Image system. Protein loading was evaluated by anti-β-actin

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antibody (1:2000)..

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Statistical Analysis. Data were expressed as mean ± standard deviation (SD). Statistical analyses

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were evaluated by Student’s t test and Tukey’s test using SPSS 18.0. P < 0.05 was considered

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statistically significant. Different letters indicated significant differences between each group.

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RESULTS

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AST and AST-DHA Improved Cognitive Disorder in APP/PS1 Mice. The spatial learning was

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measures by radial 8-arm maze and Morris water maze tests. The radial 8-arm maze results

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showed that APP/PS1 transgenic mice remarkably enhanced the number of reference memory

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errors and working memory errors in comparison with non-APP/PS1 transgenic mice (Fig 1B and

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C). Interestingly, AST and AST-DHA obviously reduced the number of reference memory errors

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and working memory errors in APP/PS1 transgenic mice (Fig 1B and C), and no remarkable

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difference was observed between these two groups.

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The results of escape latency showed that the mice of control group had better performance

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than APP/PS1 transgenic mice from day 1 to day 5, suggesting APP/PS1 transgenic mice

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exhibited significant deficiency in spatial learning ability (Fig. 1D). The treatment with AST and



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AST-DHA significantly improved the behaviors of APP/PS1 transgenic mice. Interestingly,

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AST-DHA had a more significant effect than AST in alleviating spatial deficits of APP/PS1

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transgenic mice.

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The spatial memory results showed that APP/PS1 transgenic mice in model group spent less

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time in the target quadrant and less crossing number than the control group (Fig. 1E and F). The

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mice treated with AST and AST-DHA exerted similar effects with no statistical difference in

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improving the time spent in the target quadrant. Interestingly, AST-DHA was superior to AST in

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improving the number of crossing platform.

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AST and AST-DHA Regulated Aβ Levels in APP/PS1 Mice. Brain sections were

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immunostained with anti-Aβ antibody to show the Aβ deposition in the cortex and hippocampus

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(Fig.2A), which was analyzed the number and area of plaques (Fig.2B and C). Compared with

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control group, the amyloid plaques number in the cortex and hippocampus of APP/PS1 transgenic

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mice were remarkably increased. Interestingly, AST and AST-DHA treatment could reduce

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amyloid plaques number in cortex and hippocampus of APP/PS1 transgenic mice. Importantly,

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AST-DHA was superior to AST in suppressing the number of amyloid plaques in cortex. The

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results of quantitative analysis of Aβ showed that AST and AST-DHA suppressed the Aβ load.

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AST-DHA exhibited more statistical effects than AST in reducing the Aβ load in hippocampus.

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The soluble/insoluble Aβ40 and Aβ42 in the hippocampus were determined by ELISA kits,

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and the results were shown in Fig. 3 A-D. The APP/PS1 transgenic mice exhibited higher soluble

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and insoluble Aβ40 and Aβ42 levels than control group. Supplementation of AST and AST-DHA

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exhibited a remarkable decrease of soluble and insoluble Aβ40 and Aβ42 levels in a certain degree.

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Notably, AST-DHA exhibited more statistical effects than AST in reducing soluble Aβ40/Aβ42

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levels. Unexpectedly, AST showed a better effect in reducing insoluble Aβ40 level than

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AST-DHA group. No significant difference between AST and AST-DHA was observed in

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inhibiting insoluble Aβ42 generation.

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Following the soluble/insoluble Aβ40 and Aβ42 measurement, ADAM10, BACE1 and

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Nicastrin were detected by western blotting. Compared with control group, the ADAM10 level of


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model group was significantly decreased, and BACE1 and Nicastrin expression were obviously

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increased. Notably, AST and AST-DHA treatment exerted similar effects in decreasing Nicastrin

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level. However, there were no significant improvement for AST and AST-DHA in protein

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expressions of ADAM10 and BACE1.

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Effects of AST and AST-DHA on Oxidative Stress. Oxidative stress plays a central role in the

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physiopathology of AD. Thus, we further investigated the effects of AST and AST-DHA on

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oxidative stress to confirm the neuroprotective effects of AST and AST-DHA against AD. The

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indexes of oxidative stress including SOD, NO, NOS were detected (Fig.4). Compared with

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control group, the SOD activity in model group was significantly decreased, meanwhile, NOS

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activity and NO level were obviously increased. Importantly, AST and AST-DHA treatment

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significantly recovered the activity of SOD as well as declining NO and NOS levels, in which

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AST-DHA was superior to AST in up-regulating SOD activity and down-regulating NO and NOS

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levels.

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The Effects of AST and AST-DHA on the Expression of p-GSK-3β and p-Tau. The effects of

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AST and AST-DHA treatment on GSK-3β activity and p-Tau protein expression were evaluated

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by Western blotting (Fig. 5A). Compared with the control group, the expression of p-GSK-3β

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level in model group was obviously increased (p < 0.05, Fig. 5B). AST-DHA significantly

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suppressed the expression of GSK-3β phosphorylation. Notably, AST treatment had no effects in

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regulating the p-GSK-3β expression.

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Following GSK3β activity, we also investigated the effects of dietary supplements of AST

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and AST-DHA on p-Tau protein expression. The expression of p-Tau in model group was

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remarkably increased compared with control group (Fig. 5C). Notably, dietary supplementation of

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AST and AST-DHA could obviously reverse these changes, and AST-DHA exerted more

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significant effects than AST in reducing the expression of p-Tau.

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Inhibitory Effects of AST and AST-DHA on Neuro-inflammation in APP/PS1 Mice. The

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neuro-inflammation induced by activated microglia and astrocytes is related to AD development.

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To test the effects of AST and AST-DHA on neuro-inflammatory processes in APP/PS1



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transgenic mice, immunohistochemical analysis and western blotting assay of astrogliosis and

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microgliosis were performed using the astroglial marker (GFAP) and the microglial marker (Iba1

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and CD11b, Fig. 6 A-D). The results showed that GFAP, Iba1 and CD11b were markedly

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increased in the APP/PS1 transgenic mice but were significantly reduced in the AST and

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AST-DHA treated mice both in immunohistochemical analysis and western blotting assay.

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Importantly, AST-DHA was superior to AST in regulating the activation of microglia and

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astrocytes.

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Cytokines secreted by activated microglia and astrocytes are crucial in the inflammatory

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processes of AD. The levels of IL-1β and TNF-α in AD mice were determined to investigate the

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effects of AST and AST-DHA on cytokine production (Fig. 6 B, E and F). Compared with

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no-transgenic mice, the expression of TNF-α in the APP/PS1 transgenic mice was markedly

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increased. Notably, AST and AST-DHA supplementation could significantly reduce the

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expression of TNF-α, and AST-DHA was superior to AST. Only AST-DHA significantly

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suppressed the expression of IL-1β, and no statistical difference was found between AST group

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and model group.

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Inhibitory Effects of AST and AST-DHA on Inflammasome Activation in APP/PS1 Mice.

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The NLRP3 inflammasome activation initiates an inflammatory response through caspase-1

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activation, resulting in inflammatory cytokine IL-1β℃maturation and secretion. Therefore,

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western blotting assay was performed to detect the NLRP3 inflammasome activation-related

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proteins (Fig. 7). Unexpectedly, no statistical difference was observed in the expression of NLRP3

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in these four groups. Compared with the control group, the expression of ASC in APP/PS1

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transgenic mice was significantly reduced. However, AST and AST-DHA supplementation further

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decreased the expression of ASC protein. Compared with the non-transgenic mice, the expression

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of Caspase 1 and Pro-IL-1β in APP/PS1 transgenic mice was remarkably increased. Interestingly,

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AST and AST-DHA supplementation obviously reversed the increase of Caspase 1 and Pro-IL-1β,

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and AST-DHA exerted more significant effects than AST.

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AST and AST-DHA Suppressed Apoptosis in APP/PS1 Mice. Western blotting assay was

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performed to detect the apoptosis-related proteins in APP/PS1 mice (Fig. 8). Unexpectedly, AST


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and AST-DHA supplementation had no corresponding improvement in regulating Bcl-2 and Bax

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protein expression. The relative densities of Caspase-9 and Caspase-3 in APP/PS1 transgenic mice

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were remarkably increased compared with the mice in the control group. Following AST and

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AST-DHA administration, the protein levels of Caspase-9 and Caspase-3 were obviously reduced.

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Notably, AST-DHA was superior to AST in reducing the protein expressions of Caspase-9/-3. Both

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AST and AST-DHA could obviously suppress cleaved Caspase-3 expression with a similar degree.

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DISCUSSION

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The pathological characteristics of AD brains mainly include extracellular Aβ plaques,

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intracellular

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neuro-inflammation 1. APP/PS1 transgenic mouse is one of well-established AD animal model16-17.

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To illustrate the possibility of false positivity, we determined the efficacy of AST and AST-DHA in

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8-Arm maze and Morris water maze test in the present study. Both AST and AST-DHA treatment

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could improve spatial learning ability in APP/PS1 mice by 8-arm maze apparatus, which were

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further corroborated by the Morris water maze test. The results showed that both AST and

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AST-DHA could improve the learning and memory skills, in which the AST-DHA was superior to

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AST in Morris water maze test. The different result of these two behavior tests may be attributed

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to that 8-Arm maze test is appetitive reinforcement, and Morris water maze test is aversive

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reinforcement. Furthermore, the learned behavior in these two tests is also different among

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learning tasks. The Morris water maze test usually learns a spatial task, in which rats have to learn

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complex behavioral strategies to recognize the platform to escape from water.

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neurofibrillary

tangles,

massive

neuronal

cell

and

synapse

loss,

and

The accumulation and deposition of Aβ are considered to play an important role in the 18

269

pathogenesis of AD, which is the dominant theory of AD in the past few decades

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produced by the proteolytic processing of amyloid precursor protein (APP). An important way to

271

process APP is the nonamyloidogenic pathway, in which α-secretase cleaves the Aβ domain in

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APP, thereby precluding the formation of intact Aβ. However, under normal circumstances, a

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small amount of APP is processed via the amyloidogenic pathway, in which Aβ is released from

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APP by β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and γ-secretases. ADAM10

275

and Nicastrin is one of the components of α-secretase and γ-secretases, respectively. Aβ as a


. Aβ is


276

monomer readily aggregates to form multimeric complexes 19. These complexes are composed of

277

soluble Aβ ranging from oligomers to protofibrils and insoluble Aβ such as amyloid plaques. The

278

immunohistochemistry results showed that AST-DHA treatment showed better effects than AST in

279

decreasing the number of senile plaques and Aβ plaques load in the cortex and hippocampus of

280

APP/PS1 mice, which was consistent with the results of ELISA kits. Interestingly, AST and

281

AST-DHA only showed notable effects in regulating the expression of Nicastrin instead of

282

BACE1 and ADAM10. The Aβ decrease might partly depend on the clearance mechanism, as it

283

has been reported that n-3 LCPUFAs significantly promoted interstitial Aβ clearance from the

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brain to resist Aβ injury 20.

285

AST treatment is usually related to the reduced markers of oxidative damage. AST may

286

increase the levels of endogenous antioxidant enzymes including superoxide dismutase and

287

catalase

288

indispensable role of DHA in AST-DHA.

21

. AST-DHA exhibited more effective antioxidant capacity than AST, indicating the

289

Increasing evidence has indicated Aβ induced hyper-phosphorylation of Tau protein by

290

GSK3β activation 22-23. The results showed that AST and AST-DHA significantly decreased p-Tau

291

and p-GSK3β, and AST-DHA was superior to AST. The cognitive improvement of AST-DHA on

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APP/PS1 transgenic mice are mainly attributed to the neuroprotective effects on GSK3β activation

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and tau hyper-phosphorylation.

294

Uncontrolled microglia and astrocytes activation, and sustained inflammatory responses may

295

contribute independently to neurodegeneration 24-25. It can not only be a consequence but also be a

296

trigger of pathology

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APP/PS1 transgenic mice, which was accompanied by a strong increase of TNF-α rather than

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IL-1β compared with the non-transgenic mice. Following AST and AST-DHA treatment,

299

pro-inflammatory cytokine levels were greatly reduced, especially in the AST-DHA group,

300

indicating that the anti-inflammatory effects of AST and AST-DHA may account for the reduced

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Aβ level and cognitive improvement in APP/PS1 transgenic mice.

26

. The activation of microglia and astrocytes were found in the brain of


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Neuro-inflammatory cascades depend on the activation of NLRP3 inflammasome, which was 27

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crucial in neurodegenerative diseases

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NLRP3 inflammasome, process IL-1β and IL-18, and finally induce AD pathology and tissue

305

damage 28. Moreover, in AD transgenic mouse model, the inhibition of NLRP3 can largely protect

306

memory loss and decrease Aβ deposition. A chronic administration of AST and AST-DHA in

307

APP/PS1 transgenic mice led to a remarkable alteration in NLRP3 inflammasome, in which

308

AST-DHA was superior to AST. The effects of AST and AST-DHA on glial activation and NLRP3

309

inflammasome confirmed the importance of AST-DHA in regulating inflammatory responses.

. It has been proved that the toxicity of Aβ can activate

310

Numerous studies on mitochondria dysfunction revealed that the mitochondria were the

311

central of oxidative stress induced apoptosis29-30. Bcl-2 is an anti-apoptotic protein, while Bax has

312

the opposite function. Caspase-9/-3 are the initiator and executioner, respectively, which typically

313

predominate in neurodegenerative diseases

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decrease the expression Caspase-9/-3 and cleaved Caspase-3, and the effect of AST-DHA was

315

superior to AST. However, no expected effects of AST and AST-DHA on reducing Bax and

316

improving Bcl-2 were observed in APP/PS1 transgenic mice. Apoptosis is a complex and precisely

317

controlled process, which is regulated by multiple pathways. We speculated that the decreased

318

Caspase-9 and Caspase-3 were not only regulated by the Bcl-2/Bax, other pathways might be

319

related to regulating Caspase family.

31

. Both AST and AST-DHA could significantly

320

In summary, both AST and AST-DHA could improve AD in different degrees. AST-DHA

321

exhibited better actions than AST in improving learning and memory abilities by the behavior

322

experiments. The further mechanical research indicated AST-DHA exerted more remarkable

323

functions than AST on inhibiting Aβ generation, regulating oxidative stress, suppressing the

324

hyperphosphorylation of Tau and GSK-3β, and reducing neuroglial activation and

325

neuro-inflammation (Fig. 9). Therefore, AST and AST-DHA may be applied as food supplements

326

and/or functional ingredients to relieve neurodegenerative disease.

327

Author Information

328

* E-mail: [email protected]; phone: +86 0532 82032597; fax: +86 0532 82032468



329

*E-mail: [email protected]; phone: +86 0532 82032597; fax: +86 0532 82032468

330

Author contributions

331

Hongxia Che and Qian Li designed and conducted the research. Qian Li, Dan-dan Wang and Lu

332

Yang analyzed the data; Hongxia Che wrote the manuscript; Tian-tian Zhang, Jie Xu and

333

Teruyoshi Yanagita revised the manuscript. Yuming Wang, Changhu Xue and Yaoguang Chang

334

had primary responsibility for the final content. All authors read and approved the final

335

manuscript.

336

Funding

337

This work was supported by grants from State Key Program of National Natural Science of China

338

(No. 31330060) and National Natural Science Foundation of China-Shandong Joint Fund for

339

Marine Science Research Centers (U1606403), and the Fundamental Research Funds for the

340

Central Universities (No. 201762028).

341

Conflicts of interest

342

All the authors declare that there is no conflict of interest for any of them.

343

List of Abbreviations

344

AD, Alzheimer’s disease; AST, Astaxanthin; AST-DHA, Docosahexaenoic acid-acylated AST

345

diesters; APP, amyloid precursor protein; PS1, presenilin; Aβ, β-amyloid; SOD, superoxide

346

dismutase; NOS, inducible nitric oxide synthase; NO, nitric oxide; GFAP, glial fibrillary acidic

347

protein; TNF-α, tumor necrosis factor α; IL-1β, interleukin 1beta; Bcl-2, B-cell lymphoma 2; Bax,

348

Bcl-2-associated X Protein; NLRP3, nucleotide-binding oligomerization domain (NOD)-like

349

receptor containing pyrin domain 3; ASC, apoptosis-associated speck-like protein containing a

350

CARD


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Figure Captions

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Fig.1 Structure and effects of AST and AST-DHA on spatial learning and memory deficiency.

437

Structure of AST and AST-DHA (A). The number of reference memory errors in radial 8-Arm

438

maze (B), number of working memory errors in radial 8-Arm maze (C). Time needed to reach the

439

hidden platform in the Morris maze (D). The time spent in the target quadrant. (E) and the number

440

of crossing platform (F) were measured for analysis of spatial memory function. Data were

441

presented as mean ± SD (n = 8), *P< 0.05 was considered statistically significant. Different letters

442

indicated significant difference between each group.

443

Fig.2 Represent photo and quantitative analysis of amyloid plaques in cortex and hippocampus of

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APP/PS1 transgenic mice (A). Plaques number of Aβ in cortex and hippocampus (B). Relative of

445

quantitative analysis of Aβ load in cortex and hippocampus (C). Scale bar = 100 µm.

446

Fig.3 Effects of AST and AST-DHA on the regulation of Aβ concentration in APP/PS1 transgenic

447

mice. Levels of insoluble Aβ40 (A), soluble Aβ40 (B), insoluble Aβ42 (C) and soluble Aβ42 (D)

448

were measured by ELISA. Representative western blots (E) and densitometry of ADAM10 (F),

449

BACE1 (G) and Nicastrin (H). Data were presented as mean ± SD (n=8); *P< 0.05 was

450

considered statistically significant. Different letters indicated significant difference between each

451

group.

452

Fig.4 Effects of AST and AST-DHA on the SOD activity (A), NO concentration (B) and NOS

453

activity (C) in brain. Data were expressed as mean± SD (n = 8), *P< 0.05 was considered

454

statistically significant. Different letter indicated significant difference between each group.

455

Fig.5. Effects of AST and AST-DHA on tau and GSK-3β hyper-phosphorylation. (A) Western

456

blot analysis of p-GSK3β and p-Tau. Densitometry analysis of p-GSK3β (B) and p-Tau (C).

457

Values were indicated as the mean ± SD (n = 8), *P< 0.05 was considered statistically significant.

458

Different letter indicated significant difference between each group APP/PS1 transgenic mice.

459

Fig.6 Effects of AST and AST-DHA on neuroglial activation and neuro-inflammation. (A) Effects

460

of AST and AST-DHA on glial markers were analyzed by immunohistochemistry mmunostaining



461

of the microglial marker Iba1astroglial marker GFAP and the Scale bar = 10 µm. (B) Western blot

462

analysis of CD11b, GFAP, IL-1β and TNF-α. Densitometry analysis of CD11b (C), GFAP (D),

463

IL-1β (E) and TNF-α (F). Values were indicated as the mean ± SD (n = 8), *p< 0.05 was

464

considered statistically significant. Different letter indicated significant difference between each

465

group.

466

Fig.7 Effects of AST and AST-DHA on inflammasome expression and activation. (A) Western

467

blot analysis of NLPR3, ASC, Caspase 1 and Pro-IL-1β. Densitometry analysis of NLRP3 (B),

468

ASC (C), Caspase 1 (D), and Pro-IL-1β (D). Values were indicated as the mean ± SD (n = 8), *p

Effects of Astaxanthin and Docosahexaenoic-Acid-Acylated

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