Lycopene Supplementation Attenuates Oxidative Stress

Mar 6, 2018 - (10−12) Thus, early management of inflammation and neurodegradation and the prevention of oxidative stress could ameliorate the effect...
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Bioactive Constituents, Metabolites, and Functions

Lycopene Supplementation Attenuates Oxidative Stress, Neuroinflammation, and Cognitive Impairment in Aged CD-1 Mice Beita Zhao, Hua Liu, Jiamin Wang, Pujie Liu, Xintong Tan, Bo Ren, Zhigang Liu, and Xuebo Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05770 • Publication Date (Web): 06 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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Journal of Agricultural and Food Chemistry

Lycopene Supplementation Attenuates Oxidative Stress, Neuroinflammation, and Cognitive Impairment in Aged CD-1 Mice

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Beita Zhao, Hua Liu, Jiamin Wang, Pujie Liu, Xintong Tan, Bo Ren, Zhigang Liu*, Xuebo Liu*

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Laboratory of Functional Chemistry and Nutrition of Food, College of Food Science and Engineering,

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Northwest A&F University, Yangling, China

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* Corresponding author:

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Prof. Xuebo Liu, College of Food Science and Engineering, Northwest A&F University, Xinong Rd. 22, Yangling 712100,

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China. Tel: +862987092492; E-mail: [email protected]

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Dr. Zhigang Liu, College of Food Science and Engineering, Northwest A&F University, Xinong Rd. 22, Yangling 712100,

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China. Tel: +862987092817; E-mail: [email protected]

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ABSTRACT

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Carotenoid pigment lycopene (LYC) possesses several types of properties such as antioxidative,

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anti-inflammatory, and neuroprotective. This study examined the effects of dietary supplementation with

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LYC on age-induced cognitive impairment, and the potential underlying mechanisms. Behavioral tests

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revealed that chronic LYC supplementation alleviated age-associated memory loss and cognitive defects.

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Histological and immunofluorescence staining results indicated that LYC treatment reversed

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age-associated neuronal damage and synaptic dysfunction in the brain. Additionally, LYC

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supplementation decreased age-associated oxidative stress via suppression of malondialdehyde levels,

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which increased glutathione, catalase, and superoxide dismutase activity, and the antioxidant enzymes

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mRNA levels including heme oxygenase-1 and NAD(P)H: quinone oxidoreductase-1. Furthermore,

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LYC supplementation significantly reduced age-associated neuroinflammation by inhibiting

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microgliosis (Iba-1) and downregulating related inflammatory mediators. Moreover, LYC lowered the

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accumulation of Aβ1-42 in the brains of aged CD-1 mice. Therefore, LYC has the potential for use in the

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treatment of several age-associated chronic diseases.

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KEYWORDS: Lycopene; Age-associated cognitive impairment; Oxidative stress; Neuroinflammation;

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Amyloidogenesis; Synaptic dysfunction

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Journal of Agricultural and Food Chemistry

INTRODUCTION

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Aging is a major cause of gradual declines in brain function and is implicated in cognitive disorders,

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memory loss, and dementia.1 To promote healthy aging and prevent age-induced health problems,

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effective and affordable anti-aging interventions need to be developed. Neuroinflammation,

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neurodegradation, and oxidative stress are often associated with age and age-induced chronic diseases,

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including Alzheimer’s disease (AD) and Parkinson’s disease (PD), which lead to cognitive decline and

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neuronal loss.2,

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neurodegenerative disease development.4, 5 IL-1β and TNF-α and other proinflammatory cytokines have

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been implicated in neuronal damage and subsequent neuronal loss.6,

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suggest that oxidative stress may also be a major contributor to the aging process.8, 9 Studies have

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reported that an activated oxidative stress response leads to a downstream pathological cascade, which

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involves the increased expression of inflammatory mediators.10-12 Thus, the early management of

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inflammation and neurodegradation, and the prevention of oxidative stress could ameliorate the effects

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of chronic neurodegenerative diseases.

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Recently, studies revealed that chronic inflammation is a key factor causing

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Evidence is accumulating to

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Recent studies have shown that carotenoid pigments, such as astaxanthin and lutein, can suppress

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D-galactose-induced neuronal damage in the brain, and LPS-induced oxidative stress and inflammatory

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responses.13 In addition, higher levels of serum carotenoid pigments, such as lycopene (LYC), lutein,

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and zeaxanthin, lowered the risk of neurodegenerative diseases.14 Furthermore, the antioxidant and

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anti-inflammatory effects of LYC have been illustrated both in vitro and in vivo, and LYC possesses

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blood-brain barrier permeability.15-17 LYC is a natural carotene and carotenoid pigment that is abundant

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in fruits and vegetables that are red in color; these include tomato, papaya, pink grapefruit, pink guava, 3

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and watermelon18, and its various bioactivities have attracted a large amount of attention. In a previous

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study, LYC prevented D-galactose-induced cognitive impairment.19 Our goal in present research was to

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determine the neuroprotective effects of LYC against age-associated cognitive impairment and synaptic

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dysfunction, elevated oxidative stress, and neuroinflammation. The effects of LYC on age-associated

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cognitive impairment were determined via behavioral tests. The levels of antioxidant and inflammatory

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cytokines were determined and morphological tests were carried out to evaluate what effect LYC has on

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oxidative stress, neuroinflammation, and synaptic dysfunction,.

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MATERIALS AND METHODS

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Reagents and antibodies

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LYC (≥ 95%) was obtained from Sigma Aldrich (St. Louis, MO, USA). Malondialdehyde (MDA),

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superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT) assay kits were purchased from

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Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Mouse TNF-α kit and Mouse IL-1β kit

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were acquired from Xinle Biology Technology (Shanghai, China). Iba-1 (ab178847), was purchased

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from Abcam, Inc. (Cambridge, MA, USA). PSD95 (D27E11), APP (2452) and BACE1 (D10E55606)

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was bought from Cell Signaling Technology (Beverly, MA, USA). SNAP-25 (sp14), BDNF (sc546), and

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HRP-conjugated secondary antibodies were bought from Santa Cruz Biotechnology (Dallas, TX, USA).

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Animals

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CD-1 male mice (8 months old) obtained from Beijing Vital River Laboratory Animal Technology

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(Beijing, China) were housed in rectangular cages in a controlled atmosphere with a 12-h light/dark

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cycle. Commercial basal chow (AIN-93M) was obtained from TROPHIC Animal Feed High-Tech Co.,

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Ltd. (Nantong, Jiangsu, China) and distilled water was provided ad libitum. After 2 weeks of adaptive

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feeding, the mice were divided into 3 groups (n = 10): (a) 2-month-old mice fed with AIN-93M

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commercial basal chow (Young Control); (b) 8-month-old mice fed with AIN-93M commercial basal

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chow for 7 months (Old Control); and (c) 15-month-old mice treated with AIN-93M commercial basal

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chow mixed with LYC (0.033% w/w) for the last 2 months; LYC was mixed in the commercial basal

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chow (AIN-93M) at approximately 50 mg/kg bodyweight per day LYC for each mouse (Old with LYC

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supplementation group or Old + LYC). The animal Ethics Committee of Northwest A & F Univ.

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approved the animal protocols. All the animal surgeries were performed under anesthesia. 5

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Behavioral Tests

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Y-maze test: Behavioral studies were performed using the Y-maze test (n = 10/group). Basic

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mnemonic processing (percentage of alternation) and exploratory activity (total number of arm choices)

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were assessed by placing each mouse at the center of a black Y-maze (20 cm x 4 cm x 40 cm high) and

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allowing its free exploration of the three arms for 8 min. The number of arm entries and their sequence

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were recorded, and 3 choices in a row were defined as an alternation.

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Morris water maze (MWM) test: After one day of rest from the Y-maze test, MWM test was

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conducted. The MWM apparatus contains a water tank (radius: 50 cm, height: 40 cm ). In target

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quadrant, a transparent platform (radius :5 cm, height: 20 cm ) was hidden 1 cm below the the water

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surface. The mice were provided with 4 training periods per day on 5 consecutive days and the escape

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latency was calculated for trials of each mouse. On the sixth day, the mice were given the opportunity to

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swim free for 60 s without the platform. During the swim, platform latency, time spent in the target

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quadrant and the number of times the mice attempted to reach the platform by crossing where it was

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located previously were enumerated. The data were recorded with a visual tracking system equipped

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with SuperMaze software (Shanghai Xinruan Information Technology Co., Ltd., China).

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Preparation of brain and serum samples

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After anesthetization, mice were killed with 10% chloral hydrate (dissolved in physiological saline,

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4 mL for one kg body). Serum samples were separated from orbital eye bleeding under anesthesia and

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stored at -80°C for experiments. In each group, samples from 5 mice were used for biochemical analysis,

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and samples from the other 5 mice were used for histopathology and immunohistochemistry. The brains

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were removed quickly with care and washed with cold physiological saline. The hippocampus was then 6

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immediately separated from the cerebrum on a cold plate and then stored in liquid nitrogen for later

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biochemical

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phosphate-buffered paraformaldehyde (pH7.4, in 0.1 M phosphate buffer) for later histopathological

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and immunohistochemical studies.

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Biochemical analysis

analysis

and

mRNA

expression

determination.

Brains

were

stored

in

4%

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The hippocampus of the mice were homogenized in tissue total protein lysis buffer (9 mL for 1 g

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tissue), and centrifuged at 4,000 g for 10 min, and the supernatant was retained for subsequent testing. A

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commercial BCA protein assay kit was used to measure the protein concentration, with bovine serum

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albumin (BSA) as the standard. GSH, MDA, CAT, and SOD activity levels in the hippocampus

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homogenate and serum were determined using assay kits. TNF-α and IL-1β in the serum were analyzed

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using commercial ELISA kits (Xinle Biology Technology, China).

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Hematoxylin and eosin (H&E) and immunohistochemical staining

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Histological brain sections, fixed in 4% (v/v) paraformaldehyde/PBS and embedded in paraffin,

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were stained with H&E, and immunohistochemical stains were observed with optical microscopy. The

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brain tissues were . For immunohistochemistry, thick sections were prepared, dewaxed, and rinsed thrice

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with PBS (pH 7.4). Collection of antigens was carried out with the Tris-EDTA buffer epitope retrieval

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method. Slices were then treated with 3% H2O2 for 10 min to eliminate the confounding peroxidase.

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After blocking of nonspecific staining, the sections were incubated with primary antibody, incubated

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with horsereadish peroxidase-coupled secondary antibody for 30 min at ambient temperature, and then

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washed and visualized with DAB kit (Zhongshan Golden Bridge Biotechnology Co. Ltd., China) for 10

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min; hematoxylin was used as the counterstain. Brain sections were dehydrated in ethanol, washed with 7

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xylene, and mounted in Permount. They were then observed under a optical microscope (Olympus, ,

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Japan) (× 200).

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Immunofluorescence staining and Thioflavin S staining

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Brain sections were incubated with the SNAP-25 (sp14, 1:500) and PSD95 (D27E11, 1:250)

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primary antibodies at 4°C overnight, then washed with PBS thrice, and again incubated with Alexa

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Fluor (555 or 488)-conjugated anti-rabbit (4413) or anti-mouse (4408) secondary antibody at 37°C for

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20 min. An inverted fluorescent microscope (Olympus, Japan) (×200) was used to acquire

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immunofluorescence images. For thioflavin S staining, the brain sections were incubated with 1%

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aqueous thioflavin S for 8 min, washed in distilled water and dehydrated in an ethanol series (70%, 90%,

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and 100%), and treated for 1 min with each ethanol solution. The sections were then placed on slides in

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a mounting medium (Solarbio, China), and thioflavin S staining was examined using a fluorescence

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microscope (Olympus, Japan) (×200).

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Transmission electron microscopy (TEM)

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The process of transmission electron microscopy (TEM) is divided into four steps: (1) Double

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fixation: Treated the specimen with 2.5% glutaraldehyde (pH 7.0) for 4 h, then washed thrice using

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phosphate buffer (PB) after treated the specimen with 1% OsO4 (pH 7.0) for 1 h, and washed in PB

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thrice. (2) Dehydration: Dehydration of the specimen is carried out using a graded ethanol series

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solutions (30%, 50%, 70%, 80%, 90%, 95%, and 100%) for 15–20 min each, and then the specimen is

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transferred to acetone for a 20 min incubation. (3) Infiltration: Placed the specimen in a acetone-resin

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mixture (1:1) for 1 h at 25 ℃, and then transferred the specimen to a 1:3 acetone-resin mixture overnight.

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(4) Embedding and ultrathin sectioning: Sections were placed in capsules of embedding medium and 8

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kept at 70°C for 9 h, stained by incubation with uranyl acetate and alkaline lead citrate for 15 min, and

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then observed using a TEM (Model H-7650).

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Quantitative Real-time Polymerase Chain Reaction

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The RNA was isolated from the brain with an RNA Extraction Kit (TaKaRaMiniBEST Universal

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RNA Extraction Kit, China) and reverse-transcribed (RT) into cDNA using a Prime ScriptTM RT Master

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Mix kit (TaKaRaPrimeScript RT Master Mix, China). Use SYBR green PCR kit (TaKaRaSYBR®

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Premix Ex TaqTM II, China) to quantify the mRNA expression, and real-time detection was performed

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using a CFX96TM real-time system (Bio-Rad, Hercules,) operated at 95°C for 10 min, followed by 40

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cycles of 15 s at 95°C and 1 min at 60°C. Gene-specific primers are listed in Table 1. The results were

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normalized to GAPDH expression levels. Gene expression levels were calculated using the ∆∆Ct

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method. All of the experiments were performed in triplicates, and the results are expressed as the

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percentage of the young group.

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Western blotting

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Brain tissue homogenates were dissolved in SDS buffer, separated by SDS-PAGE, and transferred

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onto PVDF membranes. Bands that immunoreacted with the antibodies were visualized with an

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enhanced chemiluminescence reagent. All the HRP-conjugated secondary antibodies and antibodies

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were used in the Materials and Methods that were mentioned above.

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Data analysis

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Data are presented as mean ± SD of at least three independent experiments. Significant differences

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between means were calculated by one-way ANOVA using GraphPad Prism software. Post hoc analysis

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was performed using Newman-Keuls correction for multiple comparison tests. Means were considered 9

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statistically significant for P < 0.05.

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RESULTS

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Effects of LYC on cognitive impairment in aged CD-1 mice

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The effects of LYC on aging-induced spatial working memory impairment and locomotor activity

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were investigated in the following way. The mice were grouped as described in Materials and Methods

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above. After 2 months of LYC supplementation in old mice (15 months old), Y-maze and locomotor

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activity tests were conducted (Fig. 1A). As demonstrated in Fig. 1B, the old control group showed a

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significant decreasing trend in spontaneous alternation compared with the young control group (2

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months old), demonstrating impairment of working memory. However, LYC supplementation reversed

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this effect. The results suggest that supplementation with LYC attenuated aging-induced spatial working

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memory.

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MWM is a method for revealing impairments of spatial memory and learning20. The effects of LYC

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on aging-induced cognitive impairment were investigated in the following way: the MWM was

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conducted using escape latency, latency to platform time, time spent in the target quadrant, and the

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crossing numbers (Fig. 2). Significant differences were observed between the groups in terms of the

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escape latency across successive days. The escape latency times of the old group were apparently longer

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than the young group for all the test days. Further, supplementation of LYC resulted in a significant

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decrease in the escape latency time compared to the old group on each test day (Fig. 2A). In the probe

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trial on the day immediately following the last day of hidden-platform training, the hidden platform was

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removed, and the old group demonstrated a significant deficit in cognition, as shown by a longer latency

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time to the platform compared with the young group. This result was reversed with the treatment of 10

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LYC (Fig. 2B). As shown in Fig. 2C & D, old mice spent less time in the target quadrant than young

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ones and had lower numbers of platform crossings. Results illustrated that LYC supplementation

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prevents cognitive impairment because the mice not given LYC exhibited a significant increasesof the

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average time spent in the target quadrant and crossing times.

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Effect of LYC on histopathological changes in aged CD-1 mice

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To observe the histopathological changes during aging, H&E staining tests were conducted. The

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photomicrographs of H&E-stained hippocampi of the mice are shown in Fig. 3A. For the young group,

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the neurons in the hippocampal cornu ammonis region 1 (CA1), cornu ammonis region 3 (CA3), and

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dentate gyrus region (DG) appeared normal: their cytoplasms were well-preserved, and their nuclei and

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nucleoli were prominent. However, neuronal degeneration and nuclear shrinkage were observed in the

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CA1, CA3, and DG hippocampal regions of the old group. Interestingly, LYC supplementation

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improved age-induced neuronal degeneration.

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BDNF refers to brain-derived neurotrophic factor, which mainly exists in the hippocampus. It is an

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abundant brain neurotrophic factor that is crucial to neuronal survival, genesis, growth, and synaptic

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plasticity in the brains of mammals, and it can also improve learning and the capacity for memory. As

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shown in Fig. 3B, immunohistochemical staining revealed that BDNF expression levels of the

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hippocampus were significantly lowered in the old group. However, LYC administration increased the

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BDNF levels compared to the control group.

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Effects of LYC on synaptic dysfunction in age-associated CD-1 mice

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Synaptic dysfunction is often observed in aging-associated diseases, such as AD and PD 21. In Fig. 4

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& 5, the immunofluorescence staining and western blotting results show that synaptosomal associated 11

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protein 25 (SNAP-25) and postsynaptic density protein 95 (PSD-95) were both decreased for aged mice;

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however, LYC treatment significantly increased the levels of SNAP-25 and PSD-95. As shown in Fig. 5,

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TEM results demonstrate that aged mice have a shorter and thinner postsynaptic density (PSD): however,

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supplementation with LYC reversed this condition.

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Effects of LYC on age-associated oxidative damage in CD-1 mouse serum and hippocampus

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Oxidative stress plays a major role in brain aging, and MDA is an important mark of lipid

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peroxidation, which is an indicator of the oxidative damage caused during oxidative stress 22. As shown

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in Fig. 6A & B, there was an obvious increase in MDA levels in the serum and hippocampus of old

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mice compared with the young ones; however, the increased MDA level was significantly lowered by

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the administration of LYC. GSH activity in the serum and hippocampus was also measured. As shown

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in Fig. 6C & D, old mice showed a significant decrease in GSH activity compared with the young ones

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(P < 0.05), while LYC treatment restored GSH activity. LYC supplementation markedly improved the

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elicited loss of antioxidant enzyme activity, including SOD and CAT (Fig. 6E-G). Fig. 6E-G also

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demonstrated that the LYC treatment alleviated age-associated oxidative stress.

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Effect of LYC on preventing neuroinflammation in age-associated CD-1 mice

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The expression of Iba-1 indicates the activation of microglia in response to inflammation.23 As

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shown in Fig. 7A, the expression of Iba-1 was higher in the hippocampal CA1, CA3, and DG brain

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regions of the aged mice compared to the young controls. However, LYC supplementation reversed the

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overexpression of Iba-1 in these regions. It is kmown that inflammation plays a vital role in oxidative

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stress. To examine the effects of LYC on inflammatory responses, the relative content of inflammatory

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mediators, including interleukins and tumor necrosis factors, were determined. As shown in Fig. 7B & 12

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C, the NF-κB expression was suppressed by LYC. Fig. 7D-G shows that TNF-α and IL-1β levels in the

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plasma and hippocampus were significantly higher for aged mice, and LYC supplementation suppressed

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the levels of these mediators. Fig. 7H shows that COX-2 mRNA expression levels in the hippocampus

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were also much higher for the aged mice, and LYC supplementation suppressed them. In Fig. 7I, IL-6

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mRNA expression levels show a similar trend as COX-2.

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Effects of LYC on Aβ accumulation in age-associated CD-1 mice

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Accumulation of Aβ is one factor that leads to AD. Therefore, we examined the accumulation of

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Aβ and amyloidogenesis in aged mice.24 Thioflavin S, a dye that can interact with the beta sheet-rich

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structures of Aβ, was employed for the detection of Aβ accumulation. As it is demonstrated in Fig. 8A,

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greater Aβ accumulation was detected in the aged mice brains, while a lower accumulation of Aβ was

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detected in the group treated with LYC. Amyloid precursor protein (APP) and beta-site APP cleaving

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enzyme 1 (BACE1) protein levels were also tested by Western blotting. As shown in Fig. 8B, LYC

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significantly inhibited age-associated increased the protein expression of APP and BACE1.

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DISCUSSION

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The neuroprotective mechanism of LYC against age-associated cognitive impairments and synaptic

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dysfunction, elevated oxidative stress, neuroinflammation, and neurodegradation was discovered. The

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behavioral experiments revealed that LYC could attenuate age-associated cognitive impairment,

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including locomotor activity, working memory, and spatial cognitive memory. The administration of

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LYC reversed systemic and CNS oxidative stress responses induced by aging. Moreover, LYC

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downregulated the expression of inflammatory mediators and recovered synaptic dysfunction in aged

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mouse brains. LYC also consistently reduced Aβ accumulation in aged CD-1 mice. 13

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LYC has been reported to possess blood-brain barrier permeability and potent neuroprotective

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properties.17,

25

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inflammatory mediators in Aβ1-42 or colchicine-induced neurodegenerative disease mouse models.26, 27

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Researchers have also demonstrated that the combination of LYC and vitamin E significantly attenuated

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oxidative stress in a tau-transgenic mouse model.28 Recent reports also reported that the fat-soluble

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extract of tomatoes, of which LYC and β-carotene are the major carotenoid constituents, suppressed

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oxidative stress in a D-galactose-treated mouse model.29 In a previous study, LYC prevented

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D-galactose-induced cognitive impairment, possibly through the Nrf2/NF-κB pathway.19 In this work,

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the effects of LYC on brain oxidative stress and inflammatory responses, and the underlying

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mechanisms were further determined.

Previous studies have found that LYC treatment suppressed oxidative stress and

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Previous research suggested that oxidative stress has a significant role in aging and

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neurodegenerative disease processes. Of all organs, brain is the most vulnerable to aging, and aging in

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the brain may cause several neurodegenerative diseases.30 Tissue and intracellular oxidative stress

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causes damage to biomacromolecules, such as cell membrane lipids, proteins, and DNA. MDA increase

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is an indicator of lipid peroxidation, suggesting of membrane oxidative damage.31, 32 Additionally, some

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antioxidants can interact with free radicals in vivo and stop the chain reactions before damage is incurred

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by vital molecules. Antioxidants can be either enzymatic or non-enzymatic (GSH), and SOD is able to

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prevent the deleterious effects of superoxide free radicals in tissues.33-35 The current study found that

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LYC supplementation alleviated age-associated oxidative stress indicators of abnormality, and decreased

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oxidative stress-induced damage to the hippocampus. This may be associated with cognitive

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improvement. 14

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Essential crosstalk between oxidative stress and inflammation exists. Intracellular H2O2 can activate

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NF-κB signaling pathways, leading to inflammatory responses.36 NF-κB plays a vital role in the

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processes of inflammation and oxidative stress by controlling the synthesis and release of

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proinflammatory substances. Numerous studies have shown that NF-κB, which is activated by

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lipopolysaccharide (LPS), is an important upstream modulator in the production of proinflammatory

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mediators, such as NO, TNF-α, and IL-1β in BV-2 cells.37, 38 It is beleived that NF-κB is a nuclear

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transcription factor, palying an important role in inflammatory response.39 LYC has also been shown to

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prevent the proinflammatory cytokine cascade in human macrophages via the inhibition of NF-κB.40 In

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the present study, LYC consistently restored Iba-1 expression levels and restored age-associated

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inflammatory factors, which prevented neuroinflammation and cognitive impairment.

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BDNF is a small dimer protein. It can not only regulate the neuronal development, maintenance,

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and survival, but also control the cognition and the formation of memories. BDNF may be reduced in

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the hippocampus of animals that age normally, possibly contributing to synaptic dysfunction, cellular

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loss, or memory impairment.41-44 A recent report revealed that the oral administration of LYC restored

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BDNF levels in β-amyloid-induced AD rat brains.45 This study is mainly focused on the mechanisms of

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LYC in alleviating brain aging. The results revealed that chronic LYC supplementation alleviated

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cognitive impairment and restored BDNF levels in the hippocampi of mice.

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The memory loss observed in AD development is associated with an increase in extraneuronal

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senile plaques and neurofibrillary tangles, which result from the aggregation of Aβ and

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microtubule-associated protein tau, respectively. The accumulation of Aβ leads to synaptic loss and

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cognitive deficiency in AD patients. In addition, the precursor of Aβ (APP) localizes to mitochondrial 15

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membranes, impairing mitochondrial function, increasing reactive oxygen species production, and

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leading to neuronal dysfunction and damage. Recent studies have proven that neurotrophic factors like

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BDNF can reduce the amyloidogenesis that occurs in AD.24 The current study demonstrated that LYC

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decreased Aβ accumulation in aged mice.

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Synaptic dysfunction has been studied in progressive age-associated chronic diseases. In the brains

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of AD patients and AD models. PSD95 and SNAP-25 levels demonstrated a decreasing trend, and both

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of them are involved in the functions of synaptic plasticity and memory. Previous studies have

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demonstrated that in various AD models of neurodegenerative disease, melatonin maintained the

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synaptic integrity and synaptic plasticity by recovering pre- and post-synaptic proteins expression

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levels.46 In the current study, SNAP-25 and PSD-95 were decreased in the aged mice while LYC

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treatment significantly recovered the levels of SNAP-25 and PSD-95, demonstrating that synaptic

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dysfunction was prevented by LYC treatment.

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This study also demonstrated that LYC treatment alleviated oxidative stress-induced cognitive

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impairment via improving neurodegradation, restoring antioxidant activity, and downregulating

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inflammatory responses in aged CD-1 mice. Overall, LYC supplementation is effective against aging

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related cognitive impairment, neuroinflammation, and neurodegradation. Therefore, LYC could be a

315

promising and safe antioxidant for the treatment of age-related neurodegenerative diseases.

316

ABBREVIATIONS

317

AD, Alzheimer's disease; Aβ, Amyloid beta peptides; BDNF, Brain derived neurotrophic factor; CAT,

318

Catalase; COX-2, Cyclooxygenase-2; GSH, Glutathione; H&E, Hematoxylin-eosin staining; HO-1,

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Heme oxygenase-1; Iba-1, Ionized calcium binding adaptor molecule-1; IHC, Immunohistochemistry; 16

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IL-1β, Interleukin-1 beta; IL-6, Interleukin 6; LYC, Lycopene; MDA, Malondialdehyde; NQO1,

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NAD(P)H: quinone oxidoreductase-1; PD, Parkinson's disease; SNAP-25, Synaptosomal-associated

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protein 25; PSD-95, postsynaptic density protein 95; SOD, Superoxide dismutase; TNF-α, Tumor

323

necrosis factor-α.

324

ACKNOWLEDGEMENTS

325

State key research and development plan “modern food processing and food storage and

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transportation technology and equipment” (No. 2017YFD0400200), Young talent fund of university

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association for science and technology in Shaanxi, China (20170201), General financial grant from the

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China postdoctoral science foundation (2016M602867), and the Fundamental research funds for the

329

central universities (2452017141) supported this research

330

AUTHOR CONTRIBUTIONS

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BZ, ZL, and XL conceived and designed the research; BZ, XT, PL, HL, and BR performed the

332

experiments; BZ, PL, and XT analyzed the data; BZ, BR, ZL, and XL interpreted the results of the

333

experiments; BZ, HL, and JW prepared the figures; and ZL and BZ drafted the manuscript.

334

CONFLICTS OF INTEREST

335

The authors declare that they do not have any conflicts interest.

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Table 1 Primer sequences for semi-quantitative RT-PCR analysis

467

Forward Primer

Reverse Primer

ho-1

ATGTGGCCCTGGAGGAGGAGA

CGCTGCATGGCTGGTGTGTAG

nqo1

GGATTGGACCGAGCTGGAA

AATTGCAGTGAAGATGAAGGCAAC

IL-1β

TGACGGACCCCAAAAGATGA

TCTCCACAGCCACAATGAGT

tnf-α

CCCTCACACTCAGATCATCTTCT

CTACGACGTGGGCTACAG

cox-2

GAAGTCTTTGGTCTGGTGCCT

GCTCCTGCTTGAGTATGTCG

IL-6

TTCCATCCAGTTGCCTTCTTG

TATCCTCTGTGAAGTCTCCTCTC

gapdh

TGGAGAAACCTGCCAAGTATGA

TGGAAGAATGGGAGTTGCTGT

psd-95

TCTGTGCGAGAGGTAGCAGA

AAGCACTCCGTGAACTCCTG

snap-25

CTGGCTGATGAGTCCCTGG

GACCGACTACTCAGGGACC

468 469 470 471 472 473 474 475 476

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FIGURE LEGENDS

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Table 1 Primer sequences used for semi-quantitative RT-PCR analysis

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Fig. 1 Timeline depicting the supplementation of lycopene and assessments of CD-1 mice working

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memory

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(A) Experimental scheme showing the effect of lycopene (LYC) on age-associated CD-1 mice. (B)

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Effect of LYC on spontaneous alternation. Data are presented as mean ± SD, n = 10. *P < 0.05, **P