MOLECULAR MEDICINE REPORTS 19: 3783-3790, 2019 Resveratrol improves cognition and decreases amyloid plaque formation in Tg6799 mice YUE CHEN1, GUANG-WEI SHI2, ZHI-MAN LIANG1, SHU-YUE SHENG2, YU-SHENG SHI3, LI PENG1, YA-PING WANG1,4, FANG WANG1 and XING-MEI ZHANG1 1Key Laboratory of Psychiatric Disorders of Guangdong Province, Department of Neurobiology, School of Basic Medical Sciences; 2The First Affiliated Hospital;3 Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515; 4Department of Rehabilitation, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong 510282, P.R. China Received May 12, 2018; Accepted September 29, 2018 DOI: 10.3892/mmr.2019.10010 Abstract. Alzheimer's disease (AD) is an irreversible, progres- Introduction sive neurodegenerative disorder of the central nervous system that causes severe cognitive impairment. One of the most Alzheimer's disease (AD) is a progressive neurodegen- significant pathological features of AD is the accumulation erative disorder characterized by cognitive and memory of β-amyloid (Aβ) peptide in the brain. Resveratrol (Res) is a impairments (1). There are >35 million individuals with AD polyphenol derived from peanuts, red grapes and other plants, worldwide, thus resulting in marked emotional and financial which has received increasing attention due to its neuropro- burdens on patients and society (2). The accumulation of tective features. Tg6799 mice are transgenic mice with five β-amyloid (Aβ) peptide in the brain is strongly implicated as familial AD (FAD) mutations that are also known as 5XFAD a main hallmark of sporadic and familial forms of AD (3,4). mice. The present study aimed to investigate the effects of Amyloid precursor protein (APP) -derived toxic peptides are Res on Tg6799 mice. The transgenic mice were randomly found at autopsy in the brains of individuals with AD (5). Aβ is divided into the Res treatment group and the vehicle control cleaved from APP by β-secretase 1 (BACE1) and subsequently group, and were treated with 0.5% Res solution (60 mg/kg) by γ-secretase. BACE1 is considered the rate-limiting enzyme or volume-matched normal saline, respectively. Treatment in the production of Aβ (6,7). In addition, accumulation of was administered by oral gavage daily for 60 consecutive BACE1 is observed in normal and dystrophic presynaptic days. Res reduced amyloid plaque formation and the levels terminals surrounding the amyloid plaques in brains of AD of Aβ42, and β-secretase 1 levels were also significantly mouse models and patients (8,9). decreased. Furthermore, Res was able to reduce the expres- The effects of medications that are currently approved by sion of amyloid precursor protein and its cleavage products. the USA Food and Drug Administration for the treatment of The administration of Res to Tg6799 mice also improved their AD manifestations are modest, transient and provide only spatial working memory, as measured by the Y-maze test, and symptomatic treatment (10). Recently, researchers have been rescued spatial memory deficits, as measured using the Morris paying more attention to herbal extracts for their potential water maze test; however, Res did not affect their motor func- therapeutic effects on AD. Resveratrol (Res) is a natural tion. In conclusion, this study suggested that Res may reduce polyphenol with strong effects, including anti-oxidative, Aβ-induced neuronal damage, thus preventing memory loss. anti‑inflammatory, cardiovascular protective, neuroprotective and cancer chemopreventive activities (11-13). It has been suggested that Res can act as a potent antioxidant in neurode- generative disorders. Previous studies have reported that Res has the ability to regulate Aβ toxicity or significantly increase the Aβ clearance rate in a mouse model of AD (14,15). Res can Correspondence to: Professor Fang Wang or Professor Xing-Mei also increase resistance against nerve inflammation and exerts Zhang, Key Laboratory of Psychiatric Disorders of Guangdong anti-oxidant effects that contribute to its neuroprotective Province, Department of Neurobiology, School of Basic Medical effects on the nervous system (16,17). Sciences, Southern Medical University, 1838 Guangzhou Avenue, Tg6799 mice are APP/presenelin 1 (PS1) double transgenic Guangzhou, Guangdong 510515, P.R. China E-mail: [email protected] mice that coexpress five familial AD (FAD) mutations, which E-mail: [email protected] are also known as 5XFAD mice. This mouse strain is a useful model of AD that recapitulates the relevant pathogenic features Key words: Alzheimer's disease, Tg6799 mice, resveratrol, amyloid of AD amyloid pathology. Intraneuronal Aβ42 accumulates in plaques, Y-maze test, Morris water maze the brain starting at 1.5 months, and the levels of brain Aβ42 and Aβ40 increase with age, whereas, the Aβ42/Aβ40 ratio decrease. Amyloid deposition starts at 2 months. Tg6799 mice 3784 CHEN et al: RESVERATROL IMPROVES COGNITION AND DECREASES AMYLOID PLAQUE FORMATION develop memory deficits by 4 months of age, as assessed using the time spent in the target quadrant and the speed were a Y-maze test compared with nontransgenic control mice (18). recorded using a video tracking system. The present study aimed to determine the protective effects and underlying mechanisms of Res in AD treatment. Thioflavin S staining. Four mice from each group were sacrificed with 0.75% pentobarbitone sodium (50 mg/kg) by Materials and methods intraperitoneal injection. The deeply anesthetized mice exhib- iting loss of reflexes and muscle relaxation were perfused with Animals and Res treatment. This study was approved 4% paraformaldehyde in PBS. Subsequently, the hippocampus by The Animal Ethics Committee of Nanfang Hospital, was extracted and frozen sections, measuring 35 µm in thick- Southern Medical University (application no. NFYY-2015-43; ness, were prepared at ‑25˚C. For thioflavin S immunostaining, Guangzhou, China). The experiments were performed using brain sections were allowed to defrost at room temperature male Tg6799 mice (Jackson Laboratory, Bar Harbor, ME, and were then washed with PBS for 5 min. The sections were USA), which express the human APP and PS1 genes and five then stained with freshly prepared 0.4% thioflavin S solution FAD mutations on a C57/B6XSJL background (18). These mice (Sigma-Aldrich; Merck KGaA) for 5 min in the dark at room were group-housed under standard environmental conditions temperature, washed twice for 3 min with 70% alcohol, washed (12-h light/dark cycle, 23±1˚C and 55±5% relative humidity), twice for 5 min with PBS and mounted. Image acquisition was with free access to food and water. The procedures were performed under a fluorescence microscope. Plaque counts performed in strict accordance with the National Institutes were quantified by counting thioflavin S‑positive plaques in of Health Guidelines for the Care and Use of Laboratory the hippocampus. The region of interest was manually selected Animals (19). At 2.5 months of age, 40 male Tg6799 mice under x4 magnification and all parameters were kept constant (weight, 18-22 g) were randomly divided into the control and during the analysis. Res groups (n=20/group). According to a previous study (20), the Res group was treated daily with 0.5% Res (Sigma-Aldrich; Aβ40 and Aβ42 ELISA. Another four mice from each group Merck KGaA, Darmstadt, Germany; 60 mg/kg/day) by oral were used to conduct Aβ ELISA analyses. Following Res gavage for 60 consecutive days, whereas the control group was treatment for 60 days, animals were anesthetized by pento- treated with volume-matched normal saline by gavage. barbitone sodium (50 mg/kg) and were decapitated. The required amount of homogenization buffer was prepared prior Behavioral testing to use. The hemisphere tissues (50 mg) were isolated and Open field test.The open field apparatus consisted of a rectan- homogenized using a Potter-Elvehjem homogenizer (DWK gular chamber (40x40x30 cm) made of gray polyvinyl chloride. Life Sciences, Millville, NJ, USA). Brain homogenates were A video camera, a loudspeaker that provided masking noise treated with PBS/protease inhibitor (Thermo Fisher Scientific, and a 25 W red light bulb (illumination density at the center Inc., Waltham, MA, USA) and were supplemented with guani- of the maze was 0.3 lx) were placed 180 cm above the center dine HCl to a final concentration of 5 mol/l. The brain tissue of the apparatus. The mice were gently placed at the center samples were then added to an Eppendorf tube containing and were allowed to explore the area for 5 min. The digitized the brain extraction buffer provided in the ELISA kits (cat. image of the path taken by each mouse was recorded and loco- nos. KHB3481, KHB3441 and KHB3442; Invitrogen; Thermo motor activity was subsequently analyzed using EthoVision Fisher Scientific, Inc.). The sample was ground thoroughly 7.0 software (Noldus Information Technology, Wageningen, with a tissue homogenizer, and then centrifuged at 16,000 x g The Netherlands). for 20 min at 4˚C. The supernatant was carefully transferred to a new tube and diluted with the buffer provided in the BioRad Y‑maze test. A black Y-maze was used with an arm length protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, of 40 cm, width of 3 cm and wall height of 12.5 cm. Each USA). Final guanidine HCl concentrations were <0.1 mol/l. mouse was placed in the center of the symmetrical Y-maze The protein content was measured using the Bio-Rad protein and allowed to explore freely through the maze until they assay kit. Duplicate samples were subsequently analyzed entered the arms 22 times; the sequence of 22 entries was using Aβ40- and Aβ42‑specific sandwich colorimetric ELISA recorded. Each mouse was placed at the end of one arm facing kits (cat. nos. KHB3481, KHB3441 and KHB3442; Invitrogen; the center and its movements were recorded for 8 min. Data Thermo Fisher Scientific, Inc.), according to themanufacturer's were analyzed to determine the number of arm entries without protocol.