Ginsenoside Rg3 prevents cognitive impairment by improving

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Bioactive Constituents, Metabolites, and Functions

Ginsenoside Rg3 prevents cognitive impairment by improving mitochondrial dysfunction in the rat model of Alzheimer’s disease Yan Zhang, Xiaomei Yang, Shuang Wang, and Shuang Song J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03793 • Publication Date (Web): 18 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019

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Journal of Agricultural and Food Chemistry

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Ginsenoside Rg3 prevents cognitive impairment by

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improving mitochondrial dysfunction in the rat model

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of Alzheimer’s disease

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Yan Zhang†, *, Xiaomei Yang$, Shuang Wang‡, Shuang Song‡

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†School

of Chemical and Pharmaceutical Engineering, Jilin Institute of Chemical

Technology, Jilin 132022, PR China $Nutritional

Department, Jilin Medical University Affiliated Hospital, Jilin 132013, PR

China ‡Graduate

School, Jilin Institute of Chemical Technology, Jilin 132022, PR China

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*Correspondence to: Yan Zhang, School of Chemical and Pharmaceutical Engineering,

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Jilin Institute of Chemical Technology, Jilin 132022, PR China.

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E-mail: [email protected]

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Tel: +86-432-62185230

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ORCID: 0000-0002-8938-4906

ACS Paragon Plus Environment


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ABSTRACT

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Ginseng, the roots and rhizomes of Panax ginseng C. A Meyer, is not only used as

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herbal medicine but also used as functional food to support body functions. Ginsenoside

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Rg3 (GRg3) is a major bioactive component in ginseng. In this study, the beneficial

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effects of GRg3 on Alzheimer’s disease (AD) rats were evaluated via the behavioral

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experiment and anti-oxidant capacity. Moreover, metabolomic analysis based on

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UPLC-QTOF-MS/MS and apoptosis analysis were used to obtain the change between

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AD and GRg3-administrated rats to assess the underlying mechanisms on improving

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mitochondrial dysfunction. Results showed that GRg3 could prevent cognitive

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impairment of AD rats by improving the mitochondrial dysfunction. The potential

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mechanisms were related to regulate abnormality of energy metabolism, electron

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transport chain, amino acid metabolism, purine metabolism, and anti-apoptosis. These

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findings support the exploitation of GRg3 as an effective complementary and functional

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food to prevent and delay AD.

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KEYWORDS: ginsenoside Rg3, Alzheimer’s disease, mitochondrial dysfunction,

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metabolomics, apoptosis


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INTRODUCTION

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Alzheimer’s disease (AD) appears in more than 50% of dementia cases, which is a

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prevalent illness among the elderly population above 65 years of age1. AD is first

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reported in 1906 and affects an enlarging number of patients year by year, with the

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number of patients up to more than 35 million worldwide2. Individuals of AD with

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cognitive impairment usually undergo changes in cognitive ability and ultimately lack

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of ability to manage life independently3. Cholinesterase inhibitors have been used to

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treat AD since 1993; however, they still have several limitations and even show adverse

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effects4. Thus, the demand to focus on effective complementary and functional food to

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prevent and delay AD is increasing.

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Panax ginseng C. A Meyer is a naturally widely distributed herbal plant in Asian

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countries, such as China, Korea, and Japan. Ginseng, the roots and rhizomes of P.

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ginseng C. A Meyer, is not only used in medicine but also used as functional food to

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support body functions. Several kinds of ginseng functional food, including red ginseng

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pill, ginseng chicken soup, red ginseng candy, ginseng tea, and ginseng chips, are

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popular5-9. Ginsenoside Rg3 (GRg3) is a major bioactive component of ginseng.

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Previously published works have reported the beneficial effects of GRg3 on AD due to

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the effects of inhibition beta-amyloid peptide (Aβ) deposition and anti-inflammatory

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activity. Su Kil Jang et al.10 reported that GRg3 could prevent AD pathogenesis by

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enhancing Aβ42 uptake, endocytosis, and degradation in AD mice. Lingling Yang et

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al.11 revealed that GRg3 could promote Aβ42 and Aβ40 degradation by enhancing

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neprilysin expression in SK-N-SH cells. Min Suk Kang et al.12 illustrated that GRg3



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could reduce Aβ42 production by inhibiting γ-secretase activity in CHO cells and AD

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mice. Bombi Lee et al.13 stated that GRg3 could alleviate learning and memory

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impairments of AD rats by suppressing the mRNA expression of inflammatory factors.

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Although the beneficial effects of GRg3 on AD have been reported, the underlying

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mechanisms are still unclear.

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Mitochondria, with the main function of producing ATP by coupling of oxidative

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phosphorylation with respiration, are the most complex and metabolically active

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organelles in the cell14. They are the ultimate site for the metabolism of carbohydrate,

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lipid, and protein and are usually described as the “energy house of the cell.”

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Furthermore, mitochondria are an important apoptotic hub. Mitochondrial dysfunction

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of AD shows as turbulent the electron transport chain (ETC), abnormal energy

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metabolism, and obviously neuronal apoptosis15. Current evidence indicates that

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mitochondrial dysfunction is an event in progress of AD and is a vital intracellular

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mechanism in the deterioration of AD16-19. Thus, improving the mitochondrial function

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becomes a new strategy for preventing and delaying AD20. In this study, the beneficial

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effects of GRg3 on AD were evaluated on the D-galactose (D-gal)-induced AD rat

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model via the Morris water maze (MWM) test and anti-oxidant capacity. Moreover,

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metabolomic analysis cooperated with UPLC-QTOF-MS/MS and apoptosis analysis

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were used for assessing the underlying mechanisms on improving mitochondrial

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dysfunction.

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MATERIALS AND METHODS

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Chemicals and primary detection kits


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GRg3 (98% purity) was obtained from Shanghai Yuanye Biotechnology (Shanghai,

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China). Detection kits including malondialdehyde (MDA), catalase (CAT), superoxide

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dismutase (SOD) and glutathione peroxidase (GSH-Px) were all obtained from Nanjing

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Jiancheng Bioengineering Institute (Nanjing, Jiangsu Province, China). DeadEnd

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Fluorometric TUNEL Detection Kit, POD was obtained from Roche (Roche Diagnostic,

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Mannheim, Gemany). The rabbit monoclonal antibodies of anti-glyceraldehyde-3-

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phosphate dehydrogenase (GAPDH), anti-Bax, anti-Bcl-2, anti-apoptosis-inducing

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factor (AIF), anti-cytochrome C (Cyt C), anti-caspase-3, anti-caspase-9, and goat anti-

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rabbit second antibody were all obtained from Bioss (Beijing, China). RIPA lysis buffer,

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BCA protein assay kit, and Beyo-emitter coupled logic (ECL) substrate were all

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acquired from Beyotime Institute of Biotechnology (Shanghai, China). D-gal was

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acquired from Sigma (St. Louis, MO, USA). HPLC-grade of formic acid and

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acetonitrile were both obtained from Fisher Scientific (Nepean, Ontario, Canada).

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AD model modeling and administration

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Male Wistar rats, weighing about 180–220 g used in the experiments were purchased

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from the experimental animal center of Jilin University. Experiments were conducted

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according to the Care and Use Guide of Laboratory Animals (Document of Jilin

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institute of chemical technology No.45) and were approved by Animal Ethics

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Committee of Jilin Province. Rats were randomly by weighing divided into three groups,

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namely, the control group, the D-gal group, and the D-gal+GRg3 group, with each

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group having 10 rats. The control group was administrated with the water. Except for

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the control group, the other two groups were administrated with D-gal (60 mg per kg



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per day for 60 days, intraperitoneally) for modeling AD. Simultaneously, the D-

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gal+GRg3 group was administrated with GRg3 (20 mg per kg per day for 60 days,

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intragastrically), while the two other groups received the water.

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MWM test

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After modeling for 60 days, the MWM test was operated by a water maze system

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(Techman, Chengdu City, Sichuan Province, China) to evaluate the cognitive capacity

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of rats. Following previous methods21, place navigation and space probe were the two

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session of MWM test. In the place navigation session, rats were studied to find out the

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platform in two miuites, which was hidden in 2 cm under the water surface for five

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successive days. On the sixth day, rats tried to search the platform that had already been

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taken away in the space probe session. Swimming thermal infrared trajectories of rats

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were tracked and escape latency time (ELT) was obtained by a video tracking system

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(Techman, Chengdu City, Sichuan Province, China).

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Detection of anti-oxidant capacity

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Serum samples were obtained after the rats were sacrificed. Levels of MDA, SOD,

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CAT and GSH-Px were detected using commercially available kits.

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Sample preparation for metabolomic analysis

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24 h urine samples were collected after MWM test. After centrifuging at 10000 rpm

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for 10 min, the supernatants were diluted with purified water at a proportion of 1:10.

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The serum samples were added acetonitrile at a pr oportion of 1:4 to remove the protein

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for 10 min, then supernatants were obtained by centrifuging at 12000 rpm for 15 min.

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The Brain tissues were collected after the rats were sacrificed and homogenized with


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0.5% formic acid water at a proportion of 1:10 and the supernatants were obtained by

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centrifuging at 12000 rpm for 20 min. At last, all the supernatants were filtered through

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0.22 μm filter membrane.

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Metabolomic analysis

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Metabolomic analysis of brain homogenate, serum and urine were carried out by

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means of liquid chromatography-mass spectrometry (LC-MS/MS). The instrument for

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detection was an ACQUITY UPLC H-class (Waters, Milford, MA) connected to a

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SYNAPT G2 HDMS Q-TOF mass spectrometer (Waters, UK). UPLC BEH C18

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column (2.1 mm×50 mm, 1.7 μm, Waters) was used at the flow rate of 0.40 mL/min

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and 35°C with 5 μL injection volume. Mobile phase A was 0.1% (v/v) formic acid in

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water and mobile phase B was acetonitrile. The elution conditions of the brain

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homogenate were as follows: started at 5% B and increased to 20%B within 3 min, then

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increased to 40%B within 3 min, went on increasing to 100%B within 2 min and held

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2 min, went back to 5% B within 0.1 min and held 4 min. The elution conditions of the

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serum were as follows: started at 5% B and increased to 60% B within 2 min, then

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increased to 70%B within 2 min, went on increasing to 100%B within 3 min and held

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1 min, went back to 5% B within 0.1 min and held 4 min. The elution conditions of

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urine were as follows: started at 5% B and increased to 20% B within 4 min, then

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increased to 40%B within 1 min, went on increasing to 100%B within 1 min, went back

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to 5% B within 0.1 min and held 4 min. The eluant was imported to the Q-TOF-MS

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with an electrospray ionization (ESI) source in both positive and negative modes.

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Sodium formate was used to establish the quality axis standard curve, and leucine



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enkephalin was corrected in real time. The parameters were set as follows: cone voltage

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30 kV; extraction cone voltage, 5 V; capillary voltage, 2.50 kV; desolvation gas flow,

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nitrogen, 600 L/h; cone gas flow, nitrogen, 50 L/h; source temperature, 120 °C;

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desolvation temperature, 400 °C; data acquisition, centroid mode from m/z 100 to

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1000m/z. MS/MS analysis utilized MSE mode. Argon was collision gas. The optimal

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collision energy was set at 5 eV for a low collision energy and 25–35 eV a high collision

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energy.

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Hematoxylin and eosin (H–E) staining

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Collected brain samples were preserved in 10% formalin solution. After embedding

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in paraffin, 4 μm sections were sliced, then dewaxed and rehydrated, subsequently

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stained with H–E and examined under a standard light microscopy for general

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histopathology examination.

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TUNEL staining and analysis

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TUNEL staining of brain paraffin sections was used to assess neural apoptosis using

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commercially available kits. Apoptotic cells (TUNEL positive cells) were stained dark

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brown. Five non-overlapping fields per paraffin section were taken to record the total

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number of cells and apoptotic number of cells, respectively. Apoptotic cells rate was

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calculated as follows: Apoptotic cells%= Apoptotic number of cells/Total number of

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cells×100%.

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Immunohistochemical staining and analysis

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Immunohistochemical staining was operated on brain paraffin sections to detect

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protein expressions of Bax, Bcl-2, Cyt C, AIF, caspase-3 and caspase-9. Positive


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expressions were stained in brown-yellow. The positive expressions of each group were

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photographed and processed by NIS-Elemnt’s BR image systerm (Nikon, Tokyo,

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Japan). Integrated optical density (IOD) was used for statistics.

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Western blot analysis

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The brain tissue samples were lysed and protein concentrations were measured

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following by the manufacturer’s instructions of assay kits. Equal amounts of proteins

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(10 μg) were resolved by SDS–PAGE (BioRad, CA, USA) and then electrophoretically

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transferred to 0.45 μm PVDF membranes (Millipore, USA). Subsequently, the

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membranes were blocked in 5% BSA for 2 h to reduce non-specific binding and

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incubated with primary antibodies including anti-GAPDH, anti-caspase-3, anti-

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caspase-9, anti-Cyt C, anti-AIF, anti-Bax, anti-Bcl-2 overnight at 4 °C. After

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thoroughly washing with TBST and reacting with HRP-conjugated secondary antibody,

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Beyo-emitter coupled logic (ECL) substrate were was applied to quantify the relative

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expression.

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Statistical analysis

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Data of different groups were statistical analyzed by GraphPad Prism 6.0 using one-

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way analysis of variance (ANOVA) and Dunnett’s test. All results were presented as

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mean ± standard deviation (SD). The criterion for statistical significance was set at

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Ginsenoside Rg3 prevents cognitive impairment by improving

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