Anthocyanins from Lycium ruthenicum Murr. Ameliorated d-Galactose

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Bioactive Constituents, Metabolites, and Functions

Anthocyanins from Lycium ruthenicum Murr. ameliorated D-galactose-induced memory impairment, oxidative stress, and neuroinflammation in adult rats Shasha Chen, Haonan Zhou, Gong Zhang, Jing Meng, Kai Deng, Wu Zhou, Honglun Wang, Zhenhua Wang, Na Hu, and Yourui Suo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06402 • Publication Date (Web): 28 Feb 2019 Downloaded from http://pubs.acs.org on March 1, 2019

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Journal of Agricultural and Food Chemistry

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Anthocyanins from Lycium ruthenicum Murr. ameliorated D-

2

galactose-induced memory

3

neuroinflammation in adult rats

4

Shasha Chen†,¶, Haonan Zhou†,¶, Gong Zhang†,¶, Jing Meng†,¶, Kai Deng†,¶,

5

Wu Zhou‡, Honglun Wang†,‡,§, Zhenhua Wang†, Na Hu*†,‡,§, Yourui

6

Suo*†,‡,§

7 8 9 10 11 12 13

†

impairment,

oxidative

stress,

and

Key Laboratory of Tibetan Medicine Research, Northwest Institute of

Plateau Biology, Chinese Academy of Sciences, Xining, 810008, China; ‡

State Key Laboratory of Plateau Ecology and Agriculture (Qinghai

University), Xining 810016, China; §

Qinghai Provincial Key Laboratory of Tibetan Medicine Research,

Xining, 810001, P.R. China; ¶

University of Chinese Academy of Sciences, Beijing 100049, China.

14 15

Corresponding authors

16

* Na Hu.: E-mail: [email protected]. Tel: +8618797320636.

17

Fax: +869716143857.

18

* Yourui Suo.: E-mail: [email protected]. Tel: +8613709763482.

19

Fax: +869716143857.

20 21

ACS Paragon Plus Environment


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ABSTRACT: Lycium ruthenicum Murr. (LR) is a perennial shrub

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commonly used as a nutritional food and medicine. Herein, we identified

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12 anthocyanins from LR, with petunidin derivatives constituting

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approximately 97% of the total anthocyanin content. Furthermore, the

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potential mechanism of anthocyanins exerting neuroprotective effects in

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D-galactose (D-gal)-treated rats was explored. Behavioral results showed

28

that anthocyanins relieved D-gal-induced memory disorder. Additionally,

29

anthocyanins reduced receptor for advanced glycation end products

30

(RAGE) and suppressed oxidative stress caused by D-gal. Anthocyanins

31

suppressed microgliosis and astrocytosis, reduced the overexpression of

32

nuclear

33

cyclooxygenase-2 (COX-2), and tumor necrosis factor-alpha (TNF-α).

34

Moreover, anthocyanins lowered C-jun N-terminal kinase (p-JNK),

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caspase-3 levels, and the B-cell lymphoma 2-associated X protein/ B-cell

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lymphoma 2 (Bax/Bcl-2) ratio. Thus, anthocyanins from LR attenuated

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memory disfunction, neuroinflammation, and neurodegeneration caused

38

by D-gal, possibly through the RAGE/NF-κB/JNK pathway, representing

39

a

40

neurodegenerative diseases.

41

KEYWORDS: Lycium ruthenicum Murr., anthocyanins, oxidative stress,

42

memory impairment, neuroinflammation, neurodegeneration

factor

promising,

kappa

safe

B

(NF-κB),

candidate

for

interleukin-1-beta

prevention

43


and

(IL-1β),

therapy

of

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INTRODUCTION

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Aging is a complex physiological process playing a crucial role in

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neurodegenerative diseases.1 Increasing evidence has emerged, indicating

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that oxidative stress is associated with aging.2,3 Oxidative stress can

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damage proteins, lipids, DNA, and other cellular components, as well as

49

disrupt normal mechanisms of cellular signaling.4,5 Indeed, oxidative stress

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is injurious to DNA in the neocortex and hippocampus, eventually

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contributing to neuronal cell death.6 Therefore, the inhibition of oxidative

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stress-induced nerve injury could function as the underlying strategy in the

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treatment or prevention of aging and neurodegenerative diseases.

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The D-galactose (D-gal)-treated model involves brain senescence and

55

can develop Alzheimer's disease (AD) like symptoms in rodents.

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Additionally, there is growing evidence that chronic treatment with D-gal

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for 6–10 weeks causes oxidative stress, neuroinflammation, apoptosis, and

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memory dysfunction.7,8 D-gal can be combined with the intracorporal free

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amines and produce advanced glycation end products (AGEs). The

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combination of AGEs and their receptors (RAGE) can induce increased

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levels of ROS and inflammatory factors, which gradually lead to impaired

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cognitive function.9,10 Therefore, D-gal-treated animal models have been

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extensively used for anti-ageing studies.7,8,10-12

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Lycium ruthenicum Murr. (LR), a well-known perennial shrub

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belonging to the Solanaceae family and widespread throughout the



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Qinghai Tibet Plateau, has been commonly used as a traditional herb to

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treat heart disease, menstrual disorder, eyesight correction, and as an

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antihypertensive drug for hundreds of years.13 The bioactivity of LR has

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recently been shown to include antioxidative, antihyperlipidemic, and anti-

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inflammatory properties.14,15 Further, phytochemical analyses revealed LR

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to contain a considerable amount of anthocyanins.16,17 Anthocyanins,

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which are flavonoids present in flowers, vegetables, fruits, berries, and

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other plants, have attracted increasing attention in recent years due to their

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multifunctional pharmacological activities, including antioxidative, anti-

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inflammatory, anti-ischemic, anticancer, and cardiovascular protection

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effects.18,19 In particular, anthocyanins have been reported to protect

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against amyloid beta (Aβ) peptide-induced toxicity and to reduce neuronal

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apoptosis and necrosis.20 Indeed, various studies have shown that long-

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term consumption of anthocyanins has a beneficial effect on cognitive

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promotion and neuroprotection against neurodegenerative disorders.21-25

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However, the neuroprotective effects of anthocyanins from LR, as well as

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the underlying mechanisms, remain unknown.

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Herein, we assessed the neuroprotective effects of anthocyanins from

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LR on D-gal-induced memory dysfunction in adult rats. The potential

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underlying mechanisms were also discussed in terms of the RAGE/NF-

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κB/JNK signaling pathway.

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MATERIALS AND METHODS


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Materials and chemicals. Dried LR fruit was purchased from Yuan

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Xintang Biotechnology Co. Ltd. (Golmud, Qinghai, China). D-gal was

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purchased from Sigma-Aldrich, Co. (St. Louis, MO, USA).

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Preparation of anthocyanins. Anthocyanins from dried LR fruit were

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extracted with 40-fold 80% ethanol by ultrasound-assisted extraction for 1

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h, repeated three times. The extracts were concentrated by rotary

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evaporation at 45°C under vacuum and subsequently loaded onto a column

95

(5 × 100 cm) filled with AB-8 macroporous resin. Firstly, twice the bed

96

volume of ultrapure water was used to remove the strong polar constituents.

97

Then, the anthocyanins were eluted with 95% ethanol, and passed through

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a medium 0.22 µm filter paper. The filtrate was concentrated at 45°C under

99

vacuum, then dried with a vacuum drying chamber (DZF-6030A, Shanghai,

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China). The resulting anthocyanin powder sample was stored at –20°C.

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Determination of the total anthocyanin content in the sample was

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performed using a pH-differential method, as previously reported.17

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Absorbances against distilled water were recorded at wavelength of 520

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nm and 700 nm at pH 1.0 and pH 4.5, respectively. Data was presented as

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mg of cyanidin 3-glucoside (C3G) equivalents per g of sample, was 875.4

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± 7.727 C3G mg/g of sample.

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HPLC-DAD analysis. For HPLC analysis, 13 mg of anthocyanin was

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dissolved in 1 mL of 40% acetonitrile, then treated in an ultrasonic cleaner

109

for 10 min and filtered with a 0.22 µm filter. The samples were analyzed



110

using an Agilent 1260 Series HPLC system, equipped with an ultraviolet–

111

visible (UV/VIS) detector and a C18 column (100 mm × 4.6 mm, Agilent,

112

USA). A 5 μL aliquot of the anthocyanin/acetonitrile solution was injected.

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The Chromatogram was recorded at wavelength of 520 nm. The eluents

114

were 1% methanoic acid in water (A) and 1% methanoic acid in acetonitrile

115

(B). The gradient elution was performed as 0–3 min, 93%–86% A; 3–7 min,

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86%–80% A; 7–10 min, 80%–75% A; 10–13 min, 75%–69% A; 13–15

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min, 69%–63% A; 15–19 min, 63%–55% A; 19–23 min, 55%–25% A; 23–

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28 min, 25%–93% A; and 28–30 min, 93% A isocratic. The flow rate was

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1.0 mL/min and temperature was 30°C.

120

UPLC-Triple-TOF/MS. An Acquity™ UPLC detecting system (Waters

121

Company, USA) coupled to a triple time-of-flight (TOF) 5600+ mass

122

spectrometer was employed for UPLC-Triple-TOF/MS analysis. An

123

electrospray ionization source (AB SCIEX company, USA) and a

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ZORBAXSB C18 analytical column (100 mm × 4.6 mm i.d., 1.8 μm,

125

Agilent, USA) were used. The gradient elution conditions described above

126

were employed to separation different peaks. The mass spectrometry (MS)

127

conditions were a positive ion scanning mode, as described previously.17

128

Animals and treatment. Sprague–Dawley (SD) female rats (12 weeks

129

old, 200–240 g average body weight) were purchased from Hunan Slake

130

Jingda Experimental Animals Co. Ltd. (Changsha, Hunan, China). Rats

131

were habituated for 1 month in the animal house under a 12 h/12 h


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light/dark cycle. The temperature was 23°C and humidity was 60% ± 10%.

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Rats were randomly divided into five groups and treated with either

134

normal saline (Control), 100 mg/kg of D-gal (D-gal group), 100 mg/kg of

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D-gal plus 200 mg/kg of anthocyanins (high-dose; An-H), 100 mg/kg of

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D-gal plus 100 mg/kg of anthocyanins (middle-dose; An-M) or 100 mg/kg

137

of D-gal plus 50 mg/kg of anthocyanins (low-dose; An-L). D-gal was

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administered by intraperitoneal injection and anthocyanins by intragastric

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treatment once-daily for 7 weeks. Whereafter, behavioral analysis was

140

performed. Finally, all rats were sacrificed for further analysis. All the

141

experimental procedures were carefully performed comply with the local

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animal ethics committee of the Northwest Institute of Plateau Biology,

143

Chinese Academy of Sciences, China.

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Morris water maze (MWM) test. Following intraperitoneal injection

145

and intragastric administration, behavioral experiments (n = 11 per group)

146

were performed. Spatial learning and memory ability were evaluated using

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the MWM test, as previously described, with minor modifications.11 The

148

main component of water maze setup was a round pool (125 cm in diameter

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and 51 cm in height), which was filled with water (23 ± 1°C). Milk powder

150

was added to make the water opaque. A transparent platform (10 cm in

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diameter, 28 cm in height) was placed 1 cm under the water at the center

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of one quadrant. A 5-day training for each rat was performed, in which the

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escape latency (Latency) and the rats’ moving trajectory for each trial were



154

recorded. On day 6, the platform was removed and the probe test was

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carried out, in which rat was allowed to swim freely for 1 min. Latency to

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the removed platform, time spent in the target quadrant, and number of

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platform crossings were measured.

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Step-down-type passive avoidance test. The same rats (n = 11 per

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group) were subjected to the step-down-type passive avoidance test. The

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main apparatus was made up of a black Perspex chamber (30 × 30 × 40 cm

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high) and an electrified latticed floor (36 V). A wooden platform (7.5 × 15

162

× 4 cm) was fixed at one corner of the chamber. A 15 W lamp was used for

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illumination during the test. For the training experiment, each rat was

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placed on the wooden platform. It received an electric shock (9 s, 0.5 mA)

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when it stepped down to the floor. The number of electric shocks required

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within 120 s was recorded as error numbers for each rat (the time taken for

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the rat to escape from the floor onto the platform was not recorded in the

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total time). The retention test was performed 24 h after the training

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experiment, with no electric shock administered. The step-down latency

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for each rat was recorded, with a blocking time of 120 s.

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Protein extraction. The rats were sacrificed after passive avoidance test.

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The brains were immediately removed, and hippocampus was dissected on

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ice, frozen with liquid nitrogen and stored at −80°C. The hippocampus

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tissue was ground in liquid nitrogen and homogenized in 0.2 M PBS

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containing phosphatase and protease inhibitor. Finally, the homogenate


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was centrifuged at 13,000 rpm at 4°C for 25 min, and supernatant was

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stored at −80°C.

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Determination of ROS and lipid peroxidation (LPO). Determination

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of ROS and LPO in the hippocampus was essential for the assessment of

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oxidative stress. ROS was detected as previously described, with minor

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modifications.8 Specifically, the hippocampus tissue homogenate was

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diluted 10 times with ice-cold Lock’s buffer (pH 7.4). Subsequently, 180

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µL of Lock’s buffer, 10 µL of homogenate, and 10 µL of DCFH-DA (200

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µM) were added into a 96-well plate and incubated at 37°C for 20 min to

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form DCF. A fluorescence microplate reader (PerkinElmer Enspire, US)

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was employed for the determination of DCF. The excitation and emission

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were at 485 nm and 530 nm, respectively. Quantification of ROS was

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expressed as pmol DCF/mg of protein. The LPO level was determined by

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measuring its marker free malondialdehyde (MDA) using an MDA Assay

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Kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). The

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specific procedure used followed the kit manufacturer’s instructions.

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RNA extraction and Q-PCR assay. Total RNA was extracted from the

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hippocampus tissue using the Quick Tissue/Culture Cells Genomic DNA

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Extraction Kit (Dongsheng Biotech, Guangzhou, Guangdong, China). The

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extracted RNA was immediately reversed-transcribed into cDNA using the

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iScript™ gDNA Clear cDNA Synthesis Kit (Bio-rad, Hercules, California,

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USA). cDNA targets were quantified using the QuantiTect SYBR® Green



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PCR Kit (Qiagen, Hilden, Germany). The Q-PCR reaction was performed

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in triplicate in 96-well plates under the following conditions: initial

200

activation at 95°C for 10 min, followed by 45 cycles of three-step cycling

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consisting of denaturation at 94°C for 30 s, then 30 s of annealing at 60°C,

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and finally extension at 72°C for 30 s. Primers were designed as follows:

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CAGCGGATGCCAGTGATAGAG

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TCCTGAGTGGGATGACGAGC (anti-sense) for cyclooxygenase-2

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(COX-2);

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GCCTCGGGCCAGTGTATG

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GATGTCTGCAATACCCAGGCA

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CTCCAAGGGGCTTCTTCCTA (anti-sense) for p-JNK.

(sense)

CCCAGAAAAGCAAGCAACCA (anti-sense)

for (sense)

and

(sense) TNF-α;

and and and

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Western blot analysis. The total protein concentration of each sample

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was measured using the BCA Assay Kit (Solarbio, Beijing, China).

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Samples were mixed with 2× loading buffer, followed by denaturing for 10

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min at 98°C in a metal bath. Equal amounts of total proteins (25–30 μg)

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were electrophoresed using SDS-PAGE, then transferred to a PVDF

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membrane (Merck Millipore, Immobilon-P, USA) using a transblot device.

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The PVDF membrane was blocked in 5% skimmed milk in TBS-T buffer,

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and incubated with corresponding primary antibodies. After reaction with

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IgG secondary antibody, the target bands were detected by a Gel Doc XR+

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system (Bio-Rad) using ECL detection reagents (Millipore, Billerica,

219

USA). The density values were quantified using Image J software (Image


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220 221


J 1.37C, USA). Immunohistochemical analysis. For tissue collection, rats (n = 4 rats

222

per

group)

were

perfused

transcardially

with

4%

ice-cold

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paraformaldehyde. Rat brain tissue was fixed with 4% paraformaldehyde

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for 72 h to make paraffin-embedded blocks. The tissue blocks were

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sectioned at 5 µm using a RM2235 Rotary Microtome (Leica, Wetzlar,

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Hesse, Germany). The sections were mounted on positively charged slides

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(CITOGLAS, Haimen, Jiangsu, China), deparaffinized, rehydrated, treated

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for 2 min at 121°C in 0.01Mcitric buffer (pH 6), cooled at room

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temperature for 20 min, rinsed in osmosed water (1×5 min), and washed

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(2×5 min) in TBS. Immunohistochemical studies were conducted as

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described previously.8 The positive cells in the cortex and hippocampus

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were counted using the Image J program.

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Antibodies. Anti-RAGE, anti-BACE-1, anti-Aβ1-42, anti-GFAP, anti-

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Iba-1, and anti-β-actin were purchased from Abcam (Massachusetts, USA).

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Anti-NF-κB p65, anti-p-JNK, and anti-tubulin were obtained from CST

236

(Boston, USA). Anti-OGG1 was purchased from Novus (Colorado, USA).

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Anti-IL-1β, anti-caspase-3, anti-Bax, and anti-Bcl-2 were obtained from

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Bioss (Beijing, China).

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Statistical analysis. The GraphPad Prism 7.0 software (GraphPad

240

Software, Inc., USA) was employed for the statistical analyses. Significant

241

differences were determined by Student’s t test and one-way ANOVA. That



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p value less than 0.05 was considered to be significant. Experimental data

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were expressed as mean ± SEM.

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RESULTS

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Anthocyanin identification. UPLC with simultaneous online MS was

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used to characterize the presence of anthocyanins. The chromatogram at

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520 nm was shown in Fig. 1 and MS data was summarized in Table 1.

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Peaks 2 and 8 accounted for 97.03% of the total peak area, with peak 8

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accounting for 81.4%. Although 10 peaks are visible, MS revealed the

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presence of 12 anthocyanins; indeed, peaks 5 and 8 corresponded to two

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structurally different but similar polarity eluents each. MS/MS data

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revealed that three aglycones of anthocyanins were present, and they were

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petunidin (m/z 317), delphinidin (m/z 303) and malvidin (m/z 331). The

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most abundant aglycone, petunidin, constitute approximately 97% of the

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total anthocyanin content.

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Anthocyanins reversed D-gal-induced memory impairment in rats.

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MWM tests were performed to assess spatial learning and hippocampus-

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dependent memory of rats (Fig. 2a–f). The mean latency decreased

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progressively during the training period for all groups (Fig. 2a).

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Nevertheless, rats in D-gal group showed longer latencies than rats in

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control group (p < 0.05), indicating that spatial learning and memory were

262

impaired. Additionally, rats in anthocyanin groups performed significantly

263

(p < 0.05) shorter latencies compared to rats in D-gal group, which reveals


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that anthocyanins could improve learning and memory. Furthermore, rats

265

in the high- and mid-dose groups showed shorter latencies than rats in the

266

low-dose group.

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The swimming path for each rat was recorded on day 5; representative

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paths for the different groups are exhibited in Fig. 2b. Rats in the control

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group found the platform in the best search mode, yet rats in the D-gal

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group searched for the platform without direction. Rats in the three

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anthocyanin treatment groups performed better in the search mode than rats

272

in the D-gal group. In addition, rats in high-dose group performing

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similarly to those in the control group.

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The probe test was performed on day 6 (Fig. 2c–e). Rats in the D-gal

275

group exhibited significantly increased latency to platform compared to the

276

control group (p < 0.001; Fig. 2c). High- and mid-dose anthocyanin

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treatment significantly reduced latency to platform in D-gal rats (p < 0.001

278

and p < 0.01, respectively), yet low-dose anthocyanin treatment failed to

279

show such a reduction (p > 0.05). The number of platform crossings and

280

time spent in the target quadrant were significantly decreased in D-gal

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group as compared to the control group (p < 0.01 and p < 0.001,

282

respectively; Fig. 2d, e). Conversely, anthocyanin treatment (all doses)

283

significantly increased the number of crossings and time spent in the target

284

quadrant compared to the D-gal group (p < 0.05), indicating that

285

anthocyanins could attenuate D-gal-induced memory impairment in rats.



286

Furthermore, rats in the high-dose anthocyanin group performed better

287

than those in the mid-dose group, which in turn had a better performance

288

than those in the low-dose group.

289

Results of step-down-type passive avoidance tests were shown in Fig.

290

2f–g. Rats in the D-gal group made a significantly increased number of

291

errors when compared to rats in the control group (p < 0.001), yet this

292

increase was reversed in the high- and mid-dose anthocyanin-treated

293

groups (An-H, p < 0.01; An-M, p < 0.05). Moreover, D-gal significantly

294

reduced the step-down latency (p < 0.001), while high-dose and mid-dose

295

anthocyanin intake noticeably (p < 0.01) prolonged the step-down latency

296

in the D-gal-treated rats. However, the enhancement effect of low-dose

297

anthocyanin intake in passive avoidance tests was not significant (p > 0.05).

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These results indicated that anthocyanins enhanced memory ability and

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passive avoidance reaction in D-gal-treated rats. Additionally, high-dose

300

anthocyanin treatment led to a greater improvement in memory than mid-

301

dose treatment, while low-dose treatment showed no significant effect

302

compared to the D-gal group.

303

Anthocyanins reduced D-gal-induced upregulated expression of

304

RAGE. Previous findings have indicated that chronic D-gal administration

305

in rodents causes increased formation of RAGE, which contributes to

306

memory impairment by reducing memory-related protein.26 The western

307

blot result indicated a markedly increased expression of RAGE in the


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hippocampus of rats treated by D-gal in comparison with saline-treated

309

controls (p < 0.001). Conversely, anthocyanin administration (all doses)

310

significantly downregulated RAGE levels in D-gal-treated rats (p < 0.01;

311

Fig. 3a), with the expression of RAGE being lower in the high-dose group

312

than in the mid-dose group, and being lower in the mid-dose group than in

313

the low-dose group.

314

Anthocyanins reduced D-gal-induced overexpression of beta-site

315

amyloid precursor protein-cleaving enzyme 1 (BACE-1) and Aβ42

316

protein in rats. Aβ is a pathogenetic peptide in AD cleaved from the

317

amyloid precursor protein; specifically, the 42-amino acid Aβ isoform

318

(Aβ42) is more prone to aggregation than the remaining isoforms.27 BACE-

319

1 is essential for the generation of Aβ in AD. Our western blot data revealed

320

overexpression of BACE-1 and Aβ42 in the hippocampus of rats in the D-

321

gal group compared to rats in the control group (p < 0.01 and p < 0.001,

322

respectively). They were significantly down-regulated by high- and mid-

323

dose anthocyanins (p < 0.05), yet low-dose anthocyanin intake failed to

324

reduce the Aβ42 levels (p > 0.05). Moreover, the BACE-1 and Aβ42 levels

325

in the high-dose group were lower than in the mid-dose group (Fig. 3a).

326

Anthocyanins inhibited D-gal-induced activation of astrocytes and

327

microglia cells in rats. Glial fibrillary acidic protein (GFAP) and ionized

328

calcium-binding adaptor molecule 1 (Iba-1) are markers for activated

329

astrocytes and microglia, respectively.28 The western blot results



330

demonstrated elevated GFAP and Iba-1 levels D-gal group, as compared to

331

the control group (p < 0.001 and p < 0.01, respectively). Importantly,

332

anthocyanin treatment (all doses) notably inhibited the increase in GFAP

333

and Iba-1 in D-gal-treated rats (p < 0.05; Fig. 3b). Moreover, they were

334

lower in the high-dose anthocyanin group than in the mid-dose group,

335

which in turn were lower than in the low-dose group.

336

Anthocyanins reversed D-gal-induced oxidative stress in rats.

337

Oxidative stress is a crucial factor of many neurodegenerative diseases.2 A

338

ROS assay was performed to assess the level of oxidative stress. Our results

339

suggested that D-gal significantly (p < 0.05) increased ROS levels

340

compared to the control group, while anthocyanins significantly (p < 0.01

341

for all doses) reduced ROS levels in the hippocampus of rats treated with

342

D-gal (Fig. 4a). In addition, the result of MDA implied that LPO levels

343

increased significantly in the D-gal group (p < 0.001), while anthocyanin

344

intake significantly reduced LPO levels compared to rats in the D-gal

345

group (p < 0.001 for all doses; Fig. 4b).

346

8-Oxoguanine, a marker of DNA oxidative damage, has been found

347

to increase with age in the brain and muscle of rodents and human.29 In

348

particular, 8-oxoguanine glycosylase (OGG1), known as a DNA

349

glycosylase enzyme, plays a central role in the specific recognition and

350

excision repair of 8-oxoguanine and has been shown to be obviously

351

decreased in the brain of AD patients and in AD mouse models.30,31 Our


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352

western blot data revealed reduced OGG1 levels in the hippocampus of rats

353

in D-gal group in comparison to those in the control group (p < 0.001).

354

However, co-treatment of anthocyanins significantly improved the

355

expression of OGG1 compared to rats only treated with D-gal (p < 0.001

356

for all doses; Fig. 4c). Furthermore, IHC-P results also evidenced that D-

357

gal treatment reduced OGG1 level, while anthocyanins facilitated OGG1

358

expression in the cortex and CA1, CA3, and DG regions of hippocampus

359

of rats (Fig. 4d).

360

Anthocyanins suppressed D-gal-induced activation of NF-κB and

361

other inflammatory factors in rats. It was demonstrated in earlier studies

362

that D-gal activated NF-κB through interaction with RAGE, contributing

363

to neuroinflammation in the brain.11 The western blot results showed a

364

significantly increase in the expression level of NF-κB in the D-gal group

365

as compared to the control group (p < 0.001, Fig. 5a). Moreover, the IHC-

366

P images also illustrated the extensive expression of NF-κB in the

367

hippocampus of rats in D-gal group, with increased NF-κB positive cells

368

in CA1, CA3, and DG regions (Fig. 5b).

369

We also examined the effects of various inflammatory mediators,

370

including IL-1β, COX-2, and TNF-α, through western blotting or Q-PCR.

371

The IL-1β (Fig. 5c), COX-2 (Fig. 5d) and TNF-α (Fig. 5e) levels were

372

obviously elevated by D-gal in comparison with the control group (p
0.05; Fig. 5a-e).

379

Anthocyanins inhibited D-gal-induced neuronal apoptosis in rats. It

380

is well known that p-JNK regulates cell growth, differentiation, survival

381

and apoptosis, and other key cellular functions. Previous studies reported

382

that D-gal can upregulate the p-JNK level in rats.7,8,24 Herein, we analyzed

383

p-JNK by western blot and observed an activation of p-JNK in D-gal group

384

compared to that in control group (p < 0.01). However, anthocyanins

385

significantly suppressed D-gal-induced activation of p-JNK (An-H, p
0.05) was observed in the reduction of

395

the Bax/ Bcl-2 ratio by low-dose anthocyanin.


Page 18 of 41

Page 19 of 41

396


DISCUSSION

397

Herein, 12 anthocyanins from LR were characterized by UPLC-

398

Triple-TOF/MS. The MS/MS data revealed the presence of only three

399

aglycones, namely, petunidin, delphinidin and malvidin, with petunidin

400

being the most abundant. Furthermore, we studied the neuroprotective

401

effects of anthocyanins through an alleviation of memory disfunction,

402

oxidative stress, neuroinflammation, and neurodegeneration caused by D-

403

gal in adult rats. It was confirmed that long-term injection of D-gal causes

404

ROS formation.32 Moreover, the metabolic process of D-gal that produces

405

advanced glycation end products (AGEs), and in turn AGEs activate

406

RAGE, leading to oxidative stress and inflammation, ultimately causes

407

neuronal damage and memory impairment.3,8-10,24,33 Anthocyanins were

408

shown to reverse D-gal-induced memory impairment as observed through

409

a reduction in latency to the platform, an increase in the number of

410

crossings and time spent in the target quadrant in the MWM test, and a

411

reduction in the number of errors and increased step-down latency in the

412

passive avoidance tasks.

413

Excessive ROS threaten organismal health and survival by damaging

414

the structures of biological macromolecules, including DNA, lipids, and

415

proteins, further accelerating aging.3 In our work, elevated ROS and LPO

416

levels and decreased OGG1 levels were observed in rats following long-

417

term D-gal injection over 7 weeks. Conversely, anthocyanin administration



418

reduced ROS and LPO levels, and increased OGG1 level in the

419

hippocampus of rats treated with D-gal. Thus, anthocyanins reduced nerve

420

damage likely by scavenging ROS and suppressing oxidative stress. These

421

observations are consistent with those in previous studies.21,25 In addition,

422

a previous study revealed that the main anthocyanin from LR showed

423

protective effects against ROS damage in neuron-like cells (rat adrenal

424

pheochromocytoma cell line),18 which is in agreement with our results.

425

BACE-1is conducive to the Aβ production, while oxidative stress was

426

reported to induce the accumulation of BACE-1.34-36 Various studies have

427

indicated overexpression of BACE-1 and Aβ in AD animal models and AD

428

patients.24,37,38 Herein, we showed that they were lowered by anthocyanins

429

administration. Additionally, elevated ROS and RAGE levels mediate the

430

activation of NF-κB, which upregulates various inflammatory cytokines,

431

leading to exaggerated inflammatory responses, damaging neurons and

432

finally impairing learning and memory.11,39-41

433

Overexpression of NF-κB and RAGE can activate microglia and

434

astrocytes. Subsequently, activation of microglia can release ASC specks,

435

which contribute to Aβ aggregation and spreading, gradually causing

436

neuroinflammation, memory impairment, and cognitive dysfunction.42

437

Moreover, activated microglia can produce ROS as a result of increased

438

production of proinflammatory cytokines, mediating inflammatory tissue

439

damage.43 Activation of the expression of NF-κB p65 protein was found in


Page 20 of 41

Page 21 of 41


440

hippocampus in aging and AD models.8,11,44 Herein, we also detected

441

elevated NF-κB p65, COX-2, TNF-α, and IL-1β levels in rats treated with

442

D-gal. Nevertheless, supplementation of D-gal with anthocyanins

443

significantly suppressed the activation of these inflammation factors in rats.

444

Additionally, the western blot results showed overexpression of GFAP and

445

Iba-1 in rats treated with D-gal, respectively indicating astrocytosis and

446

microgliosis. Anthocyanins considerably inhibited the D-gal-induced

447

proliferation

448

neuroinflammation and memory impairment in rats.

of

astrocytes

and

microglia,

further

preventing

449

Inflammatory cytokines, ROS, and other stimuli, including oxidative

450

stress, can activate p-JNK. Conversely, p-JNK promotes oxidative stress-

451

induced cell death.45 As previously reported, D-gal increased ROS

452

production, which activated p-JNK kinases, gradually leading to neuron

453

apoptosis and neurodegenerative disorders.7 Herein, chronic D-gal

454

administration for 6 weeks elevated caspase-3 levels and the Bax/Bcl-2

455

ratio, accelerating the apoptosis and neurodegeneration process.

456

Nevertheless, anthocyanins suppressed the activation of p-JNK, reduced

457

caspase-3 levels, and reduced the Bax/Bcl-2 ratio, thus showing that the

458

speed of apoptosis of a nerve cell can be retarded. A previous study

459

reported that anthocyanins suppressed neuroinflammation and apoptotic

460

through inhibition of the p-JNK/NF-κB pathway in Aβ1–42-treated AD

461

models, which is consistent with our results,46 which is consistent with our



462

Page 22 of 41

results.

463

In conclusion, our results revealed that anthocyanins from LR could

464

ameliorate memory disfunction, oxidative stress, neuroinflammation, and

465

neurodegeneration induced by D-gal in adult rats, with high-dose

466

anthocyanin showed better neuroprotective effects than mid-dose, and

467

mid-dose showed a better effect than low-dose. In addition, the potential

468

mechanism in terms of the RAGE/NF-κB/JNK signaling pathway was

469

summarized in Fig. 7. Further study on the preparation of monomeric

470

compound from anthocyanins in LR, as well as their neuroprotective

471

effects and corresponding mechanisms, are in progress.

472

ABBREVIATIONS USED

473

Aβ, Amyloid beta; BACE-1, Beta-site APP cleaving enzyme-1; RAGE,

474

receptor for advanced glycation end products; NF-κB, Nuclear

475

kappa B; TNF-α, Tumor necrosis factor-alpha; p-JNK, C-jun N-terminal

476

kinase; ROS, Reactive oxygen species; Q-PCR, real-time quantitative PCR.

477

FUNDING SOURCES

478

This study was supported by the National Natural Science Foundation of

479

China (31800297), Qinghai Provincial Science Foundation (2017-SF-A8),

480

Innovation Platform for the Development and Construction of Special

481

Project of Key Laboratory of Tibetan Medicine Research of Qinghai

482

Province (2017-ZJ-Y11), and Qinghai Province International Cooperation

483

Project (2018-HZ-812).


factor

Page 23 of 41


484

Author Contributions

485

S. Chen, Y. Suo, and N. Hu conceived and designed the experiments. S.

486

Chen performed the research with the help of H. Zhou, G. Zhang, J. Meng,

487

K. Deng, and W. Zhou. S. Chen analyzed the data and wrote the manuscript.

488

Y. Suo, N. Hu, H. Wang and Z. Wang critically revised the draft. All authors

489

discussed the results and revised the manuscript.

490

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491

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2533-2544.

635



636

Figure captions

637

Figure 1. The HPLC chromatogram at 520 nm of anthocyanins from

638

Lycium ruthenicum Murr..

639 640

Figure 2. Anthocyanins improved memory impairment in D-gal-treated

641

rats (n = 11 per group). (a) Mean latency (sec) to reach platform during

642

training session of Morris water maze (MWM). (b) The representative

643

swimming paths of rats during training session of MWM. (c) Latency to

644

previous platform during the probe test of MWM. (d) The number of

645

platform crossing during the probe test of MWM. (e) Time spent in the

646

target quadrant (where the platform was located during training session)

647

during the probe test of MWM. (f) Number of each rat stepped down onto

648

the floor during training session of passive avoidance task. (g) Latency of

649

rat first stepped down onto the floor during testing session of passive

650

avoidance task. #Significantly different from the control group.

651

*Significantly different from the D-gal group. Significance = * or #, p

Anthocyanins from Lycium ruthenicum Murr. Ameliorated d-Galactose

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