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Bioactive Constituents, Metabolites, and Functions
Alterations of the brain proteome and gut microbiota in Dgalactose-induced brain aging mice with krill oil supplementation Qinqin Jiang, Chenyang Lu, Tingting Sun, Jun Zhou, Ye Li, Tinghong Ming, Linquan Bai, Zaijie Jim Wang, and XiuRong Su J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03827 • Publication Date (Web): 14 Aug 2019 Downloaded from pubs.acs.org on August 14, 2019
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Journal of Agricultural and Food Chemistry
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Alterations
of
the
brain
proteome
and
gut
microbiota
in
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D-galactose-induced brain aging mice with krill oil supplementation
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Qinqin Jiang,†,‡,‖ Chenyang Lu,†,‡,‖,* Tingting Sun,†,‡ Jun Zhou,†,‡ Ye Li,†,‡
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Tinghong Ming,†,‡ Linquan Bai,⊥ Zaijie Jim Wang,§ Xiurong Su,†,‡,*
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†
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University, Ningbo, China
7
‡
School of Marine Science, Ningbo University, Ningbo, China
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§
Department of Biopharmaceutical Sciences, University of Illinois, Chicago,
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USA
State Key Laboratory for Quality and Safety of Agro-products, Ningbo
State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University,
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⊥
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Shanghai, China
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‖
13
*Corresponding
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Dr. Chenyang Lu
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Email address:
[email protected] (C.Y.L.)
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Postal address: Ningbo University, 169 Qixing South Road, Ningbo, China
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Dr. Xiurong Su
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E-mail address:
[email protected] (X.R.S.)
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Postal address: Ningbo University, 169 Qixing South Road, Ningbo, China
These two authors contributed equally to this work author
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ABSTRACT
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Brain aging is commonly associated with neurodegenerative disorders,
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but the ameliorative effect of krill oil and the underlying mechanism remain
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unclear. In this study, the components of krill oil were measured, and the
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antiaging effects of krill oil were investigated in mice with D-galactose
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(D-gal)-induced brain aging via proteomics and gut microbiota analysis. Krill oil
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treatment
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cAMP-regulated phosphoproteins and proteins involved in the calcium
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signaling pathway. In addition, the concentrations of dopamine were increased
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in the serum (p0.05) due to the enhanced expression of
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tyrosine-3-monooxygenase and aromatic L-amino acid decarboxylase.
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Moreover, krill oil alleviated gut microbiota dysbiosis, decreased the
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abundance of bacteria that consume the precursor tyrosine and increased the
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abundance of Lactobacillus spp. and short-chain fatty acid producers. This
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study revealed the beneficial effect of krill oil against D-gal-induced brain aging
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and clarified the underlying mechanism through proteomics and gut microbiota
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analysis.
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Keywords: Krill oil, Brain aging, Gut microbiota, Proteomics, Dopamine
decreased
the
expression
of
truncated
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INTRODUCTION
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Worldwide, the population of individuals older than 60 years is growing
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rapidly and is expected to reach more than 2 billion by 2050.1 Unless an
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effective therapy is found to attenuate or cure age-related neuronal
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degradation, healthcare costs will continue to increase. Previous studies have
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advanced numerous theories to explain the mechanisms of brain aging,
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among which the free radical theory is accepted by researchers and has
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strong support.2 Reactive oxygen species (ROS), the moderately toxic species
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and free radicals that are formed when oxygen is reduced, are highly reactive
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and attack various classes of biomolecules during the development of
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age-related disorders. In particular, the reaction of ROS with lipids causes a
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chain reaction called lipid peroxidation, and its terminal products, such as
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malondialdehyde (MDA), which are more stable than free radicals, can trigger
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further damage to proteins or nucleic acids in the brain.3 On the other hand,
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several antioxidant defense mechanisms have developed in living organisms
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to protect cells from ROS attack.2 These mechanisms include scavenging
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ROS precursors, antagonistic binding of metal ions involved in ROS formation
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catalysis, and increased endogenous antioxidant defenses. Antioxidant
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defense molecules are classified into two major groups: low-molecular weight
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antioxidant compounds (e.g., vitamins C and E, ubiquinone) and antioxidant
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enzymes (e.g., glutathione peroxidase (GSH-Px), superoxide dismutase
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(SOD)). Considering the important effect of oxidation on aging progression,
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various antioxidant supplements have been used to antagonize behavioral
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deficits during brain aging to promote healthy aging.4
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Previous studies have indicated that the absorption of fatty acids attached
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to phospholipids is better than that of fatty acids attached to triglycerides in
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animals.5 Krill oil is composed of phospholipids and triglycerides, whereas fish
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oil is composed of only triglycerides. In addition, the primary phospholipid in
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krill oil is phosphatidylcholine, and 40% of the eicosapentaenoic acid (EPA)
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and docosahexaenoic acid (DHA) in krill oil have been found to be attached to
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phosphatidylcholine.6 The unique composition of krill oil endows it with more
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profound health effects than fish oil. Previous studies have indicated that krill
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oil significantly improves cognitive function in elderly individuals, decreasing
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P300 latency (a parameter that reflects cognitive function),7 increasing
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oxyhemoglobin concentrations (markers of neural activity in the cerebral cortex)
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and antioxidant enzyme activity in the serum and brain.8 Furthermore, Zhang
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et al. analyzed the proteomics of the mouse brain and identified differentially
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expressed proteins (DEPs) in krill oil-treated groups compared to the control
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group. The results showed that the protein encoded by the Ppp1r1b gene
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mediated the beneficial effects of krill oil via the dopaminergic synapse
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pathway.9 However, we propose that the DEPs only identified with
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comparisons between krill oil-treated and control groups might miss some
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important information and that DEPs with restored abundance after krill oil
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treatment should be further taken into consideration.
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Previous studies indicate that diets play vital roles in brain aging via the
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regulation of gene transcription and protein expression.10-14 However, recent
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studies showed that the modulation of the gut microbiota (GM) induced by
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diets also affected brain-aging pathogenesis and alleviation.15 The GM
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comprises one hundred billion microorganisms with exponential population
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growth from the proximal end of the gastrointestinal tract to the distal end.
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Various studies have indicated that the composition and metabolism of the GM
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are associated with the development of neuropsychiatric disorders, including
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autism, anxiety, depression, dementia, and anorexia nervosa.16 The GM and
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central nervous system (CNS) engage in crosstalk via neurons, circulating
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hormones and other neuromodulatory molecules through the brain-gut axis.17
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On the one hand, previous study suggested that exposure to social stressors
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for only 2 h significantly changes the relative proportions of the
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Parabacteroides and Lactobacillus spp.18 On the other hand, treatments with
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the probiotics Lactobacillus helveticus and Bifidobacteria longum were
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confirmed to modulate GM composition and subsequently relieve anxiety in
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rats and humans.15 Moreover, the effects of krill oil on GM composition
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modulation have been previously reported in mice with diet-induced
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hyperlipidemia and obesity.19-20 However, the underlying mechanisms of the
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antiaging activity of krill oil in the context of the GM remain unclear.
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D-galactose (D-gal) can be converted to galactitol after long-term
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administration at a high dose, which induces enhanced ROS production,
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osmotic stress and inflammation in the brain and subsequently causes
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accelerated aging in the brain.21 In this study, brain aging was induced in mice
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by D-gal injection, and different doses of krill oil were administered. Key
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indicators related to aging were analyzed, the proteome of the mouse brain
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was measured via isobaric tags for relative and absolute quantitation (iTRAQ),
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and 16S rRNA gene sequencing of the GM in fecal samples was performed.
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This study provides proteome- and GM-related insights into the underlying
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antiaging mechanism of krill oil.
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MATERIALS AND METHODS
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Reagents
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Krill oil was purchased from Sino-Ocean Co., Ltd. (Dalian, Liaoning,
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China). D-gal was purchased from Sigma-Aldrich (ST. Louis, MO, USA).
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Donepezil (a drug for mild cognitive impairment) was purchased from Target
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Molecule Corp. (Washington, MA, USA). A mouse dopamine (DA) ELISA kit
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was purchased from Chundu Biotechnology Co., Ltd. (Wuhan, Hubei, China).
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An iTRAQ Reagent 8 Plex One Assay Kit was purchased from AB Sciex Pte.
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Ltd. (Boston, MA, USA). All other chemicals were of standard analytical grade.
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Measurement of krill oil components
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Fatty acid methyl ester was prepared as previously described and 22.
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subsequently measured via GC-MS
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mixed with 4 mL hexane, 1 mL nonadecanoic acid (19:0) (internal standard)
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and 2 mL BF3 (14% in methanol) in a 50 mL centrifuge tube. The tube was
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flushed with nitrogen and heated in a hot plate at 100 °C for 120 min under
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continuous stirring. After cooling to room temperature, hexane (1 mL) and
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distilled water (2 mL) were added to the mixture. After shaking vigorously for 1
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min and centrifugation at 650 x g for 3 min, the upper (hexane) phase
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containing the fatty acid methyl ester was directly analyzed by GC-MS with an
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Agilent 7890/M7-80EI system with a VOCOL column (60 m × 0.32 mm).
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Helium was used as the carrier gas for GC-MS. The GC oven temperature was
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increased from 60 °C to 260 °C at a rate of 5 °C/min and was maintained at
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260 °C for 40 min. The gas flow rate was 50 mL/min, and the injector
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temperature was 260 °C. The mass detection range was from 30 to 425 m/z.
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A total of 200-300 mg krill oil was
Astaxanthin in the krill oil was measured with an SS Exil ODS column (250
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× 4.6 mm, 5 μm). The mobile phase, a mixture of water, methanol,
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dichloromethane, and acetonitrile at a 4.5:28:22:45.5 ratio (v/v/v/v), was
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filtered through a 0.45 μm membrane and degassed before use. HPLC was
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carried out at a flow rate of 1.0 mL/min at room temperature. Chromatograms
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were recorded at 476 nm. Quantification was performed using a calibration
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curve of standard astaxanthin (Sigma-Aldrich, ST. Louis, MO, USA).
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Animal experimental design
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All animal procedures were approved by the Zhejiang Laboratory Animal
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Common Service Platform and the Ningbo University Laboratory Animal
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Center with permit no. SCXK (ZHE 2008-0116).
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Seventy-two six-week-old male ICR mice (20±1 g) were obtained from the
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Animal Breeding Center of Zhejiang Province (SCXK 2014-0001, No.
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1605200003). The mice were housed at 22±2 °C with an alternating 12 h
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light/dark cycle and a humidity of 50±10%. The mice were fed a granular
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irradiated diet (MD 17121) purchased from Medicience Co., Ltd. (Yangzhou,
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Jiangsu, China). The diet contained protein (180 g/kg), fat (40 g/kg), nonfiber
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carbohydrates (526 g/kg), fiber (50 g/kg), calcium (18 g/kg), phosphorus (12
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g/kg), lysine (8.2 g/kg), methionine and cystine (a total of 5.3 g/kg), and the
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gross energy was 13.8 MJ/kg. Following a one-week acclimatization to their
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home cages, the mice were randomly divided into 6 groups (12 mice per group
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and 3 mice per cage): i) the control group, in which the mice were
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subcutaneously injected with normal saline and were intragastrically
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administered distilled water; ii) the model group, in which the mice were
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subcutaneously injected with D-gal at 100 mg·kg-1·d-1 and were intragastrically
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administered distilled water; iii-v) the KO100, KO200 and KO600 groups, in
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which the mice were subcutaneously injected with D-gal at 100 mg·kg-1·d-1 and
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were given krill oil at the dosages of 100 mg·kg-1·d-1, 200 mg·kg-1·d-1, and 600
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mg·kg-1·d-1, respectively, by intragastric gavage; and vi) the donepezil group,
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in which the mice were subcutaneously injected with D-gal at 100 mg·kg-1·d-1
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and were intragastrically administered donepezil at a dosage of 1.2 mg·kg-1·d-1
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(Figure S1). Previous clinical studies reported that the dosages of krill oil used
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in human experiments ranged from 1.0 to 3.0 g·d-1 (16.7-50.1 mg·kg-1·d-1), 23-24
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which equivalent to 205.6-616.7 mg·kg-1·d-1 in mice based on body surface
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area.25 Therefore, in this study, the dosages of krill oil for the low-, moderate-
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and high-dosage groups were set to 100, 200 and 600 mg·kg-1·d-1, which were
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equivalent to 8.1, 16.2 and 48.7 mg·kg-1·d-1 in humans, respectively. In
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addition, the dosages of D-gal and donepezil used in the experiment were
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based on previous research.26
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The body weights and food consumption of the mice were measured
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weekly. After 7 weeks of administration, 6 mice in each group were randomly
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selected to perform the Morris Water Maze (MWM) test. The MWM test was
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performed as previously described.27 The detailed processes of behavioral
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analysis via the MWM are described in Supporting Information Method 1.
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After 8 weeks of administration, fecal samples were collected, and all mice
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were anesthetized with 1 min of exposure to 5 vol% isoflurane (Abcam Trading
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Co., Ltd., Shanghai, China) and exsanguinated from the orbital plexus.
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Euthanasia was subsequently performed by cervical dislocation. Organs from
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each mouse, including the brain, liver, heart, spleen and lungs, were
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immediately collected, weighed and stored at -80 °C. Whole brain tissues were
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homogenized in precooled PBS. Brain homogenates and blood were
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centrifuged at 3,500 × g for 10 min at 4 °C to obtain supernatants for
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biochemical analysis. The levels of GSH-Px, SOD, MDA and DA in the brain
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homogenates and serum were measured with kits (Solarbio, Peking, China)
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following the manufacturers’ instructions.
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Analysis of the proteome in the brain
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The proteome of the whole brain was analyzed by Ji Yun Biotechnology
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Co. Ltd. (Shanghai, China) with various methods, including protein digestion,
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iTRAQ labeling, strong cation exchange fractionation, LC-MS/MS analysis,
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protein identification, and protein quantitation. The whole brain was
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homogenized using a hand homogenizer with lysis buffer (4% SDS, 100 mM
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HEPES, 0.1% PMSF, 1× cocktail). Samples were then centrifuged at 10,000 ×
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g for 15 min at 4 °C, and the supernatants were collected. The obtained total
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protein solutions were assessed for quality with SDS-PAGE and quantified
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with the BCA method. All samples were normalized to the same concentration
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(1 mg/mL) for iTRAQ analysis. The detailed procedures for proteome analysis
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with iTRAQ are described in Supporting Information Method 2. DEP
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identification in this study was performed with previously described cutoffs:28 a
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fold change between at least one krill oil-treated group and the model group of
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≥1.2 or ≤ 0.8 and p
