Anthocyanins Extracted from Aronia melanocarpa Protect SH-SY5Y

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Bioactive Constituents, Metabolites, and Functions

Anthocyanins extracted from Aronia melanocarpa protect SHSY5Y cells against amyloid-beta (1–42)-induced apoptosis by regulating Ca2+ homeostasis and inhibiting mitochondrial dysfunction Lingshuai Meng, Guang Xin, Bin Li, Dongnan Li, Xiyun Sun, Tingcai Yan, Li Li, Lin Shi, Sen Cao, and Xianjun Meng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05404 • Publication Date (Web): 11 Nov 2018 Downloaded from http://pubs.acs.org on November 12, 2018

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Journal of Agricultural and Food Chemistry

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Anthocyanins extracted from Aronia melanocarpa protect SH-SY5Y

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cells against amyloid-beta (1–42)-induced apoptosis by regulating

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Ca2+ homeostasis and inhibiting mitochondrial dysfunction

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Lingshuai Meng †, Guang Xin †, Bin Li †, Dongnan Li †, Xiyun Sun †, Tingcai Yan †, Li

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Li †, Lin Shi † Sen Cao ‡, Xianjun Meng *, †

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†

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110866, P. R. China

College of Food Science, Shenyang Agricultural University, Shenyang, Liaoning

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‡

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Guizhou 550000, P. R. China

School of Food and Pharmaceutical Engineering, Guiyang College, Guiyang,

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* Corresponding

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E-mail: [email protected]; Tel.: 13390117107

Author: Xianjun Meng

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Abstract

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We investigated the cytoprotective effects of anthocyanins in Aronia melanocarpa

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against apoptosis induced by Aβ1-42, a key mediator of AD pathophysiology. We

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measured intracellular calcium with a colorimetric kit, cellular apoptosis with DAPI,

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intracellular ROS with the fluorescent marker 2,3-dimethoxy-1, 4-naphthoquinone,

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mitochondrial membrane potential with JC-1, and ATP with a colorimetric kit. Gene

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transcription and protein expression levels of calmodulin, cytochrome c, caspase-9,

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cleaved caspase-3, Bcl-2, and Bax were analyzed by RT-PCR and western blotting.

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The results showed that pretreatment with anthocyanins significantly inhibited

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Aβ1-42-induced apoptosis, decreased intracellular calcium and ROS, and increased

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ATP and mitochondrial membrane potential. RT-PCR and western blotting revealed

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that anthocyanins upregulated the gene transcription and protein expression of

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calmodulin and Bcl-2 and downregulated those of cytochrome c, caspase-9, cleaved

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caspase-3, and Bax. A. melanocarpa anthocyanins protected SH-SY5Y cells against

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Aβ1-42-induced apoptosis by regulating Ca2+ homeostasis and apoptosis-related genes

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and inhibiting mitochondrial dysfunction.

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Keywords: Aronia melanocarpa; Anthocyanins; Aβ1-42; SH-SY5Y cell; Apoptosis;

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Ca2+ homeostasis; Mitochondrial dysfunction

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INTRODUCTION

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Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that results

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in memory loss, cognitive dysfunction, behavioral disorders, and social barriers; it is

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also the most common cause of dementia1. An estimated 24 million people worldwide

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have dementia, the majority of whom are thought to have AD2. This disorder is

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generally considered an age-related disease most prevalent in people 65 years or

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older3, 4. Consequently, AD remains one of the principal health issues for the aging

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population, with its numbers likely to increase significantly in the coming decades. At

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present, there are several hypotheses to explain the pathogenesis of AD, including the

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involvement of beta-amyloid (Aβ) peptide and tau protein hyperphosphorylation as

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major contributors to its pathogenesis5, 6. In addition, a growing body of evidence

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points to the critical role of Ca2+ in AD via its regulation of apoptosis7, 8.

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The mitochondrial dysfunction associated with the loss of Ca2+ homeostasis has

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long been recognized to play a major role in apoptosis9, 10. Recent studies have also

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identified the roles of both death receptor and mitochondrial pathways in programmed

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cell death. In the mitochondrial apoptotic pathway, mitochondria-mediated calcium

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flow is an important indicator of apoptosis. When the concentration of intracellular

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calcium increases, mitochondria absorb Ca2+ such that its concentration is maintained

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within the physiological range. However, when calcium intake exceeds the limits of

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the mitochondria, the outer membrane of the mitochondria will rupture and the

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interstitium will overflow, resulting in decreased mitochondrial membrane potential

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and increased permeability. Calcium is known to activate several intracellular 3 ACS Paragon Plus Environment


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enzymes such as phospholipase A2, nitric oxide synthase, xanthine dehydrogenase,

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calcineurin, and endonucleases, many of which can induce the generation of

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endogenous ROS. Moreover, when taken up by the mitochondria, calcium can

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physiologically increase ATP generation by activating matrix dehydrogenases11. An

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increase in mitochondrial calcium can also promote the generation of ROS directly12.

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Additionally, an increase in mitochondrial membrane permeability will result in the

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release of cytochrome c. Within the cytosol, cytochrome c initiates the formation of

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the apoptosome—a multimeric complex—after binding to the apoptotic protease

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activating factor-1 (Apaf-1) in the presence of deoxyATP. This complex is

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responsible for activating caspase-9, which as an initiator, induces the apoptotic

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cascade by activating caspase-3. Thus, mitochondrial dysfunction and the activation

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of caspases eventually lead to cell death13.

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The Bcl-2 family members, including Bax, Bad, and Bcl-2, are key players in

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mitochondrial apoptotic pathways14, 15. The proapoptotic protein Bax was previously

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reported to mediate the release of cytochrome c by opening the permeability transition

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pore (PTP) in a process regulated by cyclosporin A. However, Bax was also

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demonstrated to trigger cytochrome c release from isolated mitochondria independent

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of the Ca2+-inducible, cyclosporin A-dependent PTP. Moreover, Bax was shown to be

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translocated from the cytosol to the mitochondria, forming homooligomers

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responsible for cytochrome c release16, 17. Bcl-2, known as ced-9 in C. elegans, was

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one of the first antiapoptotic proteins recognized to prevent the release of

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mitochondrial cytochrome c18. Moreover, Bcl-2 has been shown to inhibit the 4 ACS Paragon Plus Environment

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intracellular formation of free radicals, shifting the redox potential of cells toward

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reduction19 and increasing the capacity of mitochondria to accumulate Ca2+.

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Among the various antioxidants, natural substances isolated from medicinal herbs

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have distinct advantages over synthetic chemicals due to the potential for severe

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adverse effects posed by the strong radical scavenging abilities of many synthetics.

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Previous studies have shown that anthocyanins protect cells against apoptosis through

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various pathways20, 21 and that A. melanocarpa has been shown to be a rich source of

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anthocyanins22,

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including cyanidin-3-O-(galactoside, glucoside, arabinoside, xyloside) from A.

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melanocarpa in order to investigate the ability of the extracted anthocyanins to protect

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against

23.

For the present study, we obtained high-purity anthocyanins

amyloid-beta

(1–42)-induced

apoptosis

by

inhibiting

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dysfunction and regulating Ca2+ homeostasis in SH-SY5Y cells.

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MATERIALS AND METHODS

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Chemicals and reagents

mitochondrial

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Cyanidin-3-O-(galactoside, glucoside, arabinoside, xyloside) standards were

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purchased from Tokiwa Phytochemical Co., Ltd. (Chiba, Chiba Prefecture, Japan).

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Amyloid beta peptide (1-42) (Aβ1-42) was purchased from Bioss (Beijing, China).

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DMEM, fetal bovine serum (FBS), and Gluta-Max were purchased from Gibco

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(Gibco, Grand Island, NY). DMSO was purchased from Solarbio (Shanghai, China).

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MTT kits were purchased from Beyotime (Shanghai, China). Calcium colorimetric

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assay kits, 2,3-dimethoxynaphthalene-1,4-dione, ATP colorimetric/fluorometric assay

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kits, DAPI (4', 6-diamidino-2-phenylindole), and mitochondrial membrane potential 5 ACS Paragon Plus Environment


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detection kits (JC-1) were purchased from Sigma-Aldrich (St. Louis, MS).

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Calmodulin antibody, cytochrome c antibody, caspase-9 antibody, cleaved caspase-3

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antibody, Bcl-2 antibody, Bax antibody, GAPDH antibody, goat anti-rabbit IgG H&L

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(HRP), and goat anti-mouse IgG H&L (HRP) were purchased from Abcam

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(Cambridge, England). BCA protein quantification assay kits were purchased from

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Wanleibio (Shenyang, China).

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A. melanocarpa anthocyanins

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A. melanocarpa, named “Fu Kangyuan NO.1,” was harvested and transported at 4

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°C from Liaoning Fu Kangyuan Black Chokeberry Technology Co., LTD.

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(40°47′41″N, 122°40′42″E) located in Haicheng City, Liaoning Province, China, on

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September 9, 2016, then stored at the north 120 laboratory of the College of Food

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Science at Shenyang Agricultural University at -80 °C. Anthocyanins were extracted

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with ethanol and purified by absorbent resin coupled with semi-preparative reverse

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high-performance liquid chromatography (RPLC). Then, the anthocyanins were

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identified and quantified by high-performance liquid chromatography (HPLC) using

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anthocyanin standards.

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SH-SY5Y cell culture and sample treatment

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The SH-SY5Y neuroblastoma cell line was obtained from the Chinese Academy of

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Sciences Shanghai Cell Bank (Shanghai, China) and cultured in an incubator (CB60,

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Eppendorf, Hamburg, Germany) with 5% CO2 at 37 °C. A total of 200 mg

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anthocyanin was dissolved in 10 mL DMSO to prepare an anthocyanin stock solution

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of 20 mg/mL which was stored at 4 °C until use. A total of 2 mg Aβ1-42 was dissolved 6 ACS Paragon Plus Environment

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in 1.8 mL DMSO to prepare an Aβ1-42 stock solution at 257 mM which was stored at 4

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°C until use.

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MTT assay and test groups

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SH-SY5Y cells in the late logarithmic growth period were seeded in 96-well

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culture plates after adjusting cell suspension concentration; each well was filled with

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90 µL SH-SY5Y cells and the edge wells were filled with PBS without cells. Then,

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the 96-well culture plate was placed in an incubator with 5% CO2 at 37 °C until a

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single layer of cells covered the bottom of the wells. Then, the SH-SY5Y cells were

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treated with the test compounds. To assess the cellular protection and toxicity of

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anthocyanins, SH-SY5Y cells were treated with anthocyanins at 10 μg/mL, 50 μg/mL,

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100 μg/mL, 200 μg/mL, 300 μg/mL, 500 μg/mL, and 800 μg/mL. To assess the

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damaging effect of Aβ1-42 on SH-SY5Y cells, SH-SY5Y cells were treated with Aβ1-42

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at 10 nM, 50 nM, 100 nM, 200 nM, 300 nM, 500 nM, 800 nM, and 1 μM. To assess

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the protective effect of anthocyanins on SH-SY5Y cell injury induced by Aβ1-42,

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SH-SY5Y cells were first treated with anthocyanins and placed in an incubator with

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5% CO2 for 24 h at 37 °C and then treated with Aβ1-42. Finally, all SH-SY5Y cells

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were placed in an incubator with 5% CO2 for 24 h at 37 °C and cell viability was

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assessed using the MTT colorimetric assay. Briefly, 20 μL MTT (5 mg/mL) was

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added to the cells, followed by incubation for 4 h at 37 °C. Then, the culture

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supernatant was removed and the MTT formazan crystals were dissolved in 150 μL

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DMSO. Absorbance was measured at 490 nm in an ELISA microplate reader

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(iMark™; Bio-Rad, Hercules, California). 7 ACS Paragon Plus Environment


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Based on the MTT test results, the following experimental groups were established:

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Control group—SH-SY5Y cells were placed in an incubator containing 5% CO2 at 37

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°C; Aβ1-42 group—Aβ1-42 was incubated for 24 h at 37 °C and added to the cultured

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SH-SY5Y cells at a final concentration of 1 μM, then the cells were placed in an

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incubator containing 5% CO2 at 37 °C; Anthocyanins (20 µg/mL) + Aβ1-42

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group—cells were pretreated with 20 µg/mL anthocyanins for 24 h in an incubator

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with 5% CO2 at 37 °C, followed by a 24-h treatment with 1 μM Aβ1-42 that had been

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incubated for 24 h at 37 °C; Anthocyanins (40 µg/mL) + Aβ1-42 group—cells were

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pretreated with 40 µg/mL anthocyanins for 24 h in an incubator with 5% CO2 at 37 °C,

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followed by a 24-h treatment with 1 μM Aβ1-42 that had been incubated for 24 h at 37

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°C; and Anthocyanins (60 µg/mL) + Aβ1-42 group—cells were pretreated with 60

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µg/mL anthocyanins for 24 h in an incubator with 5% CO2 at 37 °C, followed by a

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24-h treatment with 1 μM Aβ1-42 that had been incubated for 24 h at 37 °C.

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Measurement of intracellular calcium ([Ca2+]i)

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The intracellular calcium ion concentration was determined using a calcium ion

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colorimetric kit. Briefly, 1 × 106 cells were collected at each concentration, diluted

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with 100 μL of PBS containing 1% protease inhibitor, and centrifuged at 12 000 rpm

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for 10 min, after which the supernatant was collected. The 500 mM standard was

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diluted to 5 mM and 0, 2, 4, 6, 8, and 10 μL aliquots of the diluted standards were

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added to 96-well plates, with each well filled to 50 µL with water (Q.S. to 50 µL).

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Each sample well contained 25 µL sample and was filled to 50 µL with water. Ninety

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microliters of chromogenic reagent was added to each well and mixed, and 60 μL 8 ACS Paragon Plus Environment

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calcium assay buffer was added to each well and mixed. Finally, the reaction system

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was incubated at room temperature (25 °C) for 5–10 min in the dark and analyzed

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with a microplate reader (iMark™; Bio-Rad) at 575 nm. A standard curve was

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generated from the concentrations of the standards and the concentration of each

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sample well was calculated according to the standard curve. Sample well

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concentration (μg/μL) = concentration per sample well / sample volume per well;

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sample concentration (μg/μL) = sample well concentration × 40 μg/μmol.

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DAPI (4',6-diamidino-2-phenylindole) staining

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SH-SY5Y cells were treated according to the experimental groups established

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above. The cell culture medium in each well was removed and cells were washed

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thrice with PBS, fixed in 4% paraformaldehyde for 30 min, and again washed thrice

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with PBS. Then, nuclei were stained with DAPI staining solution for 10 min at room

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temperature in the dark. Finally, cells were coated with 50% glycerol and observed

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under a fluorescence microscope (BX43F; Olympus Corporation, Shanghai, China) at

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400× magnification; images were captured for further analysis.

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Evaluation of intracellular ROS

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SH-SY5Y cells were stained with 2,3-dimethoxy-1,4-naphthoquinone to observe

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intracellular ROS levels using fluorescence microscopy (BX43F; Olympus

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Corporation). Briefly, the culture medium was removed from SH-SY5Y cells cultured

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in a 96-well plate, cell surfaces were washed with PBS, the PBS was discarded, and

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the cells were then digested with trypsin at 37 °C in a cell culture incubator. After the

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digestion was complete, trypsin was used to halt digestion, the bottom of the culture 9 ACS Paragon Plus Environment


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bottle was blown, and the cells were transferred to a centrifuge tube and centrifuged at

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1000 rpm for 5 min. Then, the supernatant was discarded and the cells were

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repeatedly washed three times with 1 mL complete medium and mixed. Then, 3 × 105

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cells were collected from each well, the cells were treated according to the

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experimental groups established above and placed in an incubator with 5% CO2 for 24

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h at 37 °C. The cells were washed twice with 2 mL PBS and stained with

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2,3-dimethoxy-1,4-naphthoquinone to observe the level of intracellular ROS using

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fluorescence microscopy.

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Measurement of mitochondrial membrane potential

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JC-1, a lipophilic cationic fluorescent dye, selectively enters mitochondria. When

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the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial

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matrix and exists in a polymerized form (red fluorescence). When the mitochondrial

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membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix and

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exists in a monomer form (green fluorescence). Therefore, the ratio of red

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fluorescence to green fluorescence is commonly used to examine changes in

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mitochondrial membrane potential. For this test, SH-SY5Y cells (1 × 106/well) were

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seeded in 6-well culture plates and incubated overnight, then treated according to the

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established experimental groups and placed in an incubator with 5% CO2 for 24 h at

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37 °C. After 24 h, the cells were digested with trypsin, collected, washed with PBS,

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and centrifuged at 1000 × g for 5 min. The cells were resuspended in PBS and mixed

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with 5 μg/mL JC-1, then the reaction system was incubated at 37 °C for 20 min in the

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dark. FACSCalibur™ flow cytometry was used for detection and analysis (BD C6; 10 ACS Paragon Plus Environment

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Becton Dickinson, Shanghai, China).

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Detection of cellular ATP levels

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An ATP colorimetric kit was used to determine the ATP content in mitochondria

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24-26.

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4, 6, 8, and 10 μL aliquots of ATP standard solution (1 nmol/μL) were placed in the

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standard wells of a 96-well plate and each well was filled to 50 μL with ATP assay

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buffer (Q.S. to 50 µL). Then, 1 × 106 cells were collected at each concentration,

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diluted with 100 μL ATP assay buffer containing 1% protease inhibitor, and

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centrifuged at 12 000 rpm for 10 min. The supernatant was collected to remove the

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protein and a 20 μL sample was then added to each well filled to 50 μL with ATP

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assay buffer. The reaction system was incubated at room temperature for 30 min in

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the dark and the wells were analyzed with a microplate reader (iMark™; Bio-Rad) at

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570 nm.

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Protein extraction and western blot analysis

Briefly, a 10 nmol/μL ATP standard solution was diluted to 1 nmol/μL, then 0, 2,

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SH-SY5Y cells were treated according to the experimental groups established

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above. Protein was extracted as described previously27. Protein levels were measured

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using a BCA protein quantification assay kit and proteins were separated by sodium

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dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto

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a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The membrane was soaked

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with PBST, transferred to blocking solution for 1.5 h at room temperature, then

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incubated with PBST-diluted primary antibodies against calmodulin, cytochrome c,

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caspase-9, cleaved caspase-3, Bcl-2, and Bax overnight at 4 °C (GAPDH was used for 11 ACS Paragon Plus Environment


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normalization). The membrane incubated with secondary antibodies at a dilution of

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1:2000 were prepared in the same manner described above: the membrane was

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incubated with secondary antibody at room temperature for 1.5 h then washed with

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PBST three times at room temperature for 10 min. The chemiluminescence reaction

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was performed with an ECL chromogenic kit (Sigma-Aldrich). Immunoreactive bands

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were visualized with an ECL luminescence instrument (5200, Tianon; Shanghai,

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China) and analyzed densitometrically with a gel image processing system (5200,

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Tianon

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RNA extraction and RT-PCR analysis

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SH-SY5Y cells in the late logarithmic growth period were removed from the

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culture solution, the cell surfaces were washed with PBS, the PBS was discarded, and

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the cells were then digested with trypsin at 37 °C in a cell culture incubator. After the

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digestion was complete, trypsin was used to halt digestion, the bottom of the culture

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bottle was blown, and the cells were transferred to a centrifuge tube and centrifuged at

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1000 rpm for 5 min. The supernatant was discarded and the cells were repeatedly

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washed three times with 1 mL complete medium and mixed, then cultured at 37 °C in

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a cell culture incubator overnight. The cells were treated overnight according to the

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experimental groups established above and placed in an incubator with 5% CO2 for 24

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h at 37 °C, then collected. Total RNA was extracted from cultured SH-SY5Y cells

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using TRIzol (Gibco) and reverse transcription was carried out using a TaKaRa kit

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(Gibco). RT-PCR analysis was performed using an Exicycler™ 96 real-time

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quantitative fluorescence detector (Bioneer; Daejeon, Korea). Forward and reverse 12 ACS Paragon Plus Environment

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primers (10 μM; Table 1), SYBR green solution at working concentration, and a PCR

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master mix were added and thermocycled as follows: 95 °C for 10 min followed by

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40 cycles at 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s. GAPDH, a

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housekeeping gene, was used as an internal standard to control for variability in

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amplification due to differences in starting concentrations of mRNA. The copy

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number of each transcript was calculated as the relative copy number normalized with

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the GAPDH copy number. One microgram of total RNA was subjected to RT-PCR

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and the products were quantified using a quantitative fluorescence PCR instrument.

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The results were evaluated using the 2−ΔΔCT method.

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Statistical analysis

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Each experiment was done in triplicate and mean values with standard deviations

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were analyzed using Excel software (Version 2003, Microsoft Corp., Redmond, WA).

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The statistical product and service solutions (SPSS) statistics software (Version 17.0,

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SPSS Inc./IBM Corp.; Armonk, NY) was used for statistical analysis of the data. The

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significant difference between any two mean values was determined using one-way

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ANOVA at a 95% confidence level (p ≤ 0.05) and significant differences among

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groups were determined using a Least Significant Difference test (LSD) and Duncan’s

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multiple range test (p < 0.05). Graphs were obtained using Excel software.

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RESULTS

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A. melanocarpa anthocyanins

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Anthocyanins extracts were purified with NKA-9 resin and further purified by

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RPLC (Supplementary Fig. 1) and the anthocyanins were identified and quantified by 13 ACS Paragon Plus Environment


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HPLC. The identification of anthocyanins by HPLC (Supplementary Fig. 2) indicated

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that anthocyanins consisted of cyanidin-3-O-galactoside, cyanidin-3-O-glucoside,

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cyanidin-3-O-arabinoside, and cyanidin-3-O-xyloside. The anthocyanin content was

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930.30 mg/g dry weight (DW), including cyanidin-3-O-galactoside (497.87 mg/g

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DW), cyanidin-3-O-glucoside (33.32 mg/g DW), cyanidin-3-O-arabinoside (320.47

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mg/g DW), and cyanidin-3-O-xyloside (78.64 mg/g DW), based on the

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cyanidin-3-xyloside calibration curves (Supplementary Table 1).

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Cell viability by MTT assay

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To assess the toxicity of anthocyanins on SH-SY5Y cells, cells were treated with

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different concentrations of anthocyanins for 24 h, then cell viability was measured by

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the MTT method. The results are shown in Fig. 1. When the anthocyanin

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concentration was 10–100 µg/mL, anthocyanins promoted the proliferation of

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SH-SY5Y cells; the proliferation effect was the most significant (p < 0.05) at an

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anthocyanin concentration of 50 µg/mL. At a concentration of 200 µg/mL,

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anthocyanins had a significant toxic effect on SH-SY5Y cells (p < 0.05). With further

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increases in anthocyanin concentration, the toxic effect became increasingly stronger.

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Therefore, anthocyanin concentrations of 10–100 µg/mL were selected for subsequent

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experiments.

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To assess the toxicity of Aβ1-42 on SH-SY5Y cells, cells were treated with different

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concentrations of Aβ1-42 for 24 h, then cell viability was determined by MTT assay as

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shown in Fig. 2. SH-SY5Y cell viability decreased significantly as the concentration

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of Aβ1-42 increased (p < 0.05). When the concentration of Aβ1-42 was 1 µM, cell 14 ACS Paragon Plus Environment

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viability reached its lowest (74.08%), with SH-SY5Y cell viability decreased by

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25.92% compared with that of the control group. These results indicated that Aβ1-42

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damaged cells and inhibited cell growth. Therefore, 1 µM Aβ1-42 was used for

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subsequent modeling experiments.

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To assess the protective effects of anthocyanins on SH-SY5Y cell injury induced

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by Aβ1-42, SH-SY5Y cells were treated with anthocyanins at 10 µg/mL, 20 µg/mL, 40

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µg/mL, 50 µg/mL, 60 µg/mL, 80 µg/mL, and 100 µg/mL for 24 h then treated with 1

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µM Aβ1-42 for 24 h. The cell viability results determined by the MTT assay are shown

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in Fig. 3. Compared with the control group, the cell viability of the Aβ1-42 treatment

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group decreased significantly (p < 0.05) by 35.2%. Compared with the Aβ1-42

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treatment group, when the concentration of anthocyanin was 10 µg/mL and 20 µg/mL,

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there was no significant change in cell viability (p > 0.05), which indicated that

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anthocyanin had no significant protective effect on the cells (p > 0.05) at these

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concentrations. When the concentration of anthocyanin was 30 µg/mL, cell viability

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began to increase significantly as the anthocyanin concentration increased (p < 0.05),

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which indicated that anthocyanin had a protective effect on the cells at these higher

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concentrations. Anthocyanin had the best protective effect on the cells at a

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concentration of 60 µg/mL, with cell viability increasing 19.33% compared with that

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of the Aβ1-42 treatment group. As the concentration of anthocyanins continued to

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increase, cell viability began to decrease.

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Therefore, based on the MTT results, five groups were established for the

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follow-up trials: Control group; Model group (Aβ1-42 group, 1 µM); Low-dose drug 15 ACS Paragon Plus Environment


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protection group [anthocyanin (20 µg/mL) + Aβ1-42 group]; Medium-dose drug

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protection group [anthocyanin (40 µg/mL) + Aβ1-42 group]; and High-dose drug

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protection group [anthocyanin (60 µg/mL) + Aβ1-42 group].

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Measurement of intracellular calcium ([Ca2+]i)

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The intracellular calcium ion concentration was measured using a calcium ion

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colorimetric assay kit; the results are shown in Fig. 4. The [Ca2+]i levels in cells

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treated with Aβ1-42 increased by 155.14.1% relative to the levels in control cells.

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Compared with the control group, when the increase in anthocyanin concentration in

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the drug protection group reached 20 µg/mL, the calcium ion concentration began to

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decrease, but not significantly (p > 0.05). When the anthocyanin concentration

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continued to increase, the intracellular calcium concentration decreased significantly

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(p < 0.05). When the anthocyanin concentration was 60 µg/mL, the calcium ion

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concentration reached a minimum of 1.42 µg/µL and the [Ca2+]i levels decreased by

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48.84% relative to the levels in the model group.

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DAPI staining

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The cells were stained with DAPI and observed under a fluorescence microscope,

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as shown in Fig. 5(A). The cell morphology in the control group was relatively intact;

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however, the cell morphology in the model group changed conspicuously, including

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chromatin border sets, nuclear pyknosis, nuclear fragmentation, and even the

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formation of apoptotic bodies, indicating that cell apoptosis had occurred. Compared

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with the model group, cell apoptosis was alleviated in the groups treated with

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anthocyanins. Moreover, the numbers of total cells and apoptotic cells were counted 16 ACS Paragon Plus Environment

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under a fluorescence microscope and the apoptosis rate was calculated; the results are

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shown in Fig. 5(B). Compared with the control group, the apoptotic rate in the model

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group reached 20.65% and increased significantly (p < 0.05), indicating that Aβ1-42

356

induced apoptosis in SH-SY5Y cells. Compared with the model group, the apoptotic

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rate significantly decreased as the anthocyanin concentration increased in the drug

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protection groups (p < 0.05); the apoptotic rate was 11.35% when the anthocyanin

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concentration was 60 µg/mL, which was still significantly higher than that in the

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control group (p < 0.05). These results indicated that anthocyanins attenuated the

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apoptosis induced by Aβ1-42 in SH-SY5Y cells and protected SH-SY5Y cells against

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apoptosis.

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Evaluation of intracellular ROS

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As shown in Fig. 6, the oxygen free radicals in the model group (Aβ1-42) increased

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significantly compared with the level in the control group, indicating that Aβ1-42

366

induced oxidative stress in SH-SY5Y cells. In addition, compared with the model

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group, oxygen free radicals in the drug protection groups were significantly reduced

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and as the anthocyanin concentration increased, the oxygen free radicals in the cells

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also continuously decreased. Among the drug protection groups, the high-dose drug

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protection group (Aβ1-42 + anthocyanin (60 µg/mL) had the lowest level of oxygen

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free radicals. These results suggest that anthocyanins attenuated the oxidative stress

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induced by Aβ1-42 in SH-SY5Y cells and played a protective role.

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Measurement of mitochondrial membrane potential

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The mitochondrial membrane potential results are shown in Fig. 7. The ratio of red 17 ACS Paragon Plus Environment


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fluorescence to green fluorescence in the model group was 92.86%, which was

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significantly lower than that in the control group (303.49%), indicating that a decrease

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in mitochondrial membrane potential in SH-SY5Y cells was induced by Aβ1-42. For

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the drug protection groups, the ratio of red fluorescence to green fluorescence

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gradually increased as the anthocyanin concentration increased, indicating that the

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mitochondrial membrane potential also increased gradually. When the anthocyanin

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concentration was 60 µg/mL, the ratio of red fluorescence to green fluorescence

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(510.83%) was significantly higher than these ratios in the model group and the

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control group. These results reveal that anthocyanins protected SH-SY5Y cells from

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the decrease in mitochondrial membrane potential induced by Aβ1-42.

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Detection of cellular ATP levels

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An ATP colorimetric assay kit was used to detect the concentration of

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mitochondrial ATP in cells; the results are shown in Fig. 8. Compared with that in the

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control group (9.55 nmol/μL), the ATP concentration in the model group decreased

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significantly (p < 0.05) to 7.09 nmol/μL, indicating that ATP was decreased by Aβ1-42.

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In the drug protection groups, when the anthocyanin concentration was 20 µg/mL, the

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ATP concentration did not change significantly (p > 0.05) compared with that in the

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model group. As the anthocyanin concentration increased, the ATP concentration

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began to decrease significantly (p < 0.05) compared with that in the model group; at

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60 µg/mL anthocyanins, the ATP concentration was 8.29 nmol/μL, which was still

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significantly lower than that in the control group. Thus, anthocyanins attenuated the

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decrease in ATP concentration induced by Aβ1-42 in SH-SY5Y cells. 18 ACS Paragon Plus Environment

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Western blot analysis

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The expression of proteins associated with apoptosis was analyzed by western

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blotting; the results are shown in Fig. 9. Calmodulin and Bcl-2 protein expression in

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the Aβ1-42 group was downregulated compared with that in the control group, and

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cytochrome c, caspase-9, cleaved caspase-3, and Bax protein expression in the Aβ1-42

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group was upregulated compared with that in the control group. In the drug protection

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groups, as the anthocyanin concentration increased, calmodulin and Bcl-2 protein

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expression was gradually upregulated and cytochrome c, caspase-9, cleaved caspase-3,

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and Bax protein expression was gradually downregulated compared with those in the

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model group.

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RT-PCR analysis

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Apoptosis-related gene transcription was monitored by RT-PCR; the results are

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shown in Fig. 10. Calmodulin and Bcl-2 gene transcription in the Aβ1-42 group was

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significantly downregulated compared with that in the control group (p < 0.05) and

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cytochrome c, caspase-9, cleaved caspase-3, and Bax gene transcription in the Aβ1-42

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group was significantly upregulated compared with that in the control group (p

Anthocyanins Extracted from Aronia melanocarpa Protect SH-SY5Y

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