Lycopene Supplementation Attenuates Oxidative Stress

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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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ACS Paragon Plus Environment


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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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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

7

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

306

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

313

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

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

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ABBREVIATIONS

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

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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,

321

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

322

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

326

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

327

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

328

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

329

central universities (2452017141) supported this research

330

AUTHOR CONTRIBUTIONS

331

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.

336 337

17



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Page 25 of 37


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

25



Page 26 of 37

477

FIGURE LEGENDS

478

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

479

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

480

memory

481

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

482

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

Lycopene Supplementation Attenuates Oxidative Stress

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