Anthocyanins from Black Chokeberry ... - ACS Publications

Our results suggested that anthocyanins was a potential approach for maintaining thinking and memory in aging mice, possibly by regulating the balance...
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Anthocyanins from Black Chokeberry (Aroniamelanocarpa Elliot) Delayed Aging-Related Degenerative Changes of Brain Jie Wei,*,† Guokun Zhang,‡ Xiao Zhang,† Dexin Xu,† Jun Gao,§ Jungang Fan,§ and Zhiquan Zhou§ †

School of Life Science, Liaoning University, Shenyang, Liaoning 110036, China Chinese Academy of Agricultural Sciences, Institute of Special Wild Economic Animal and Plant Science, Changchun, Jilin 130112, China § Liaoning Academy of Forestry Science, Shenyang, Liaoning 110032, China ‡

ABSTRACT: Aging is the greatest risk factor for most neurodegenerative diseases, which is associated with decreasing cognitive function and significantly affecting life quality in the elderly. Computational analysis suggested that 4 anthocyanins from chokeberry fruit increased Klotho (aging-suppressor) structural stability, so we hypothesized that chokeberry anthocyanins could antiaging. To explore the effects of anthocyanins treatment on brain aging, mice treated with 15 or 30 mg/kg anthocyanins by gavage and injected D-galactose accelerated aging per day. After 8 weeks, cognitive and noncognitive components of behavior were determined. Our studies showed that anthocyanins blocked age-associated cognitive decline and response capacity in senescence accelerated mice. Furthermore, mice treated with anthocyanins-supplemented showed better balance of redox systems (SOD, GSH-PX, and MDA) in all age tests. Three major monoamines were norepinephrine, dopamine, and 5-hydroxytryptamine, and their levels were significantly increased; the levels of inflammatory cytokines (COX2, TGF-β1, and IL-1) transcription and DNA damage were decreased significantly in brains of anthocyanins treated mice compared to aged models. The DNA damage signaling pathway was also regulated with anthocyanins. Our results suggested that anthocyanins was a potential approach for maintaining thinking and memory in aging mice, possibly by regulating the balance of redox system and reducing inflammation accumulation, and the most important factor was inhibiting DNA damage. KEYWORDS: chokeberry, anthocyanins, brain aging, DNA damage, molecular docking

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INTRODUCTION Aging is a common risk factor to many neurodegenerative diseases, including Parkinson’s disease and Alzheimer’s disease (AD). In addition, the occurrence of these neurodegenerative diseases may also increase in relation to population aging.1 The common feature of most degenerative diseases, except those that may be involved in aging, is the result of neuronal death. Excessive accumulation of DNA damage may play a key role in progressive neuronal death. The common feature of most degenerative diseases, except those that may be involved in aging, is the result of neuronal death. Excessive accumulation of DNA damage may play a key role in progressive neuronal death. DNA damage as playing a role in aging.2,3 Without proper function of DNA damage response and repair pathways, proliferation stresses (such as injury or transplant) could lead to increase levels of unrepaired DNA damage and loss of brain function. DNA damage reactions are organized by multiple signal transduction processes, of which the ATM-Chk2 and ATR-Chk1 pathways are important, respectively, by DNA double-strand breaks (DSBs) and single-stranded DNA activation.2 The activation of these pathways is for the checkpoint, and proper coordination of the DNA repair process is critical; however, they can also modulate other biological outcomes such as apoptosis or cell senescence.2,4 In recent years, DNA damage has become the core of antiaging. At the same time, the body antioxidant capacity and inflammation level is also an important part of the aging mechanism.5,6 © 2017 American Chemical Society

Black chokeberry (Aronia melanocarpa Elliot) is a member of the Rosaceae family. It has gained popularity because of their high content of anthocyanins with antioxidant activity. In fact, they have the highest antioxidant activity in the studied chokeberry and other fruits on the basis of the determination of oxygen radical scavenging capacity (ORAC) assay.7 Anthocyanins present many health benefits such as anticarcinogenic, anti-inflammatory, or antidiabetic effects.8−11 Anthocyanins also have beneficial neurological protection.12−14 Some of them have the ability to penetrate the blood-brain barrier and spread through the central nervous system.15−17 Some epidemiological studies have shown that diets rich in anthocyanins can affect neurodegenerative diseases.18 Anthocyanins have neuroprotective effects in reducing age-related oxidative stress and improving cognitive brain function.12−14,19 A neuroprotective mechanism of action has been proposed to show that anthocyanins play their activities by reducing the DNA damage. DNA accumulation may play a critical role in brain senescence and regulate the activity of intracellular signal transduction molecules. Because of the aging population, there is a need to find more efficient ways to prevent or retard the eventual progression from aging. On the basis of the antiaging of anthocyanins reported previously, the current study was designed to identify and clarify Received: Revised: Accepted: Published: 5973

May 7, 2017 June 28, 2017 June 28, 2017 June 28, 2017 DOI: 10.1021/acs.jafc.7b02136 J. Agric. Food Chem. 2017, 65, 5973−5984

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Journal of Agricultural and Food Chemistry effects from chokeberry through molecular dynamic simulations (MD) and animal text to investigate the potential mechanism associated with the antiaging activity of these constituents.

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Table 1. Composition of the Mice Diet in the Experiment composition of experimental diets Macronutrient Composition protein, % of energy fat, % of energy carbohydrate, % of energy energy/MJ kg−1 Ingredient soybean/g kg−1 corn starch/g kg−1 flour/g kg−1 bran/g kg−1 fish meal/g kg−1 fiber (cellulose)/g kg−1 calcium/g kg−1 phosphorus/g kg−1

MATERIALS AND METHODS

Materials. Black chokeberry (Aronia melanocarpa Elliot) fruit was provided by Liaoning Academy of Forestry (Shenyang, China). D-galactose and epigallocatechin gallate (EGCG) was purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Anthocyanin extraction and separation steps are as follows: Chokeberryfruit were extracted by impregnating with MeOH (0.5% trifluoroacetic acid, TFA v/v) at room temperature for 24 h and evaporating most of the solvent in vacuo. The ethyl acetate (4 × 0.5 L) separated the concentrated water-enriched extract (0.5 L) and the organic phase was discarded. The water-phase was concentrated, then purified by using water on a bed of Amberlite XAD-7HP (5 cm × 50 cm column, Sigma-Aldrich, St. Louis, MO) until the eluent was pH 6 and then eluted with 1 L of MeOH (0.5% TFA). The anthocyanin-rich extract was passed through a Sephadex LH-20 column (5 cm × 100 cm, GE Healthcare, Uppsala, Sweden) by gradient elution using 15% (3 L) and 30% (4 L) MeOH (0.1% TFA v/v). The 200 mL fractions were collected and analyzed by HPLC. Purity acceptance was defined as >96% of the total area of the analyte peak area detected at 520 and 280 nm. MeOH and TFA were purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). The anthocyanins were isolated as pure compounds: cyanidin 3-arabinoside (4.7 mg/g), cyanidin 3-galactoside (4.7 mg/g), cyanidin 3-xyloside (0.47 mg/g), and cyanidin 3-glucoside (0.1 mg/g). Molecular Dynamic Simulations (MD) and Docking Studies. MD simulations were carried out using the AutoDockTools-1.5.6 software package. The crystal structure of human Cytosolic Neutral β-Glycosylceramidase (Klotho-related Prote:KLrP) complex (PDB entry 2E9L) was downloaded from the Protein Database (RCSB). The model of 4 anthocyanins monomer were constructed using the Chem3D 16.0 software package. Docking studies on the interaction between 4 anthocyanins and KLrP were carried out using AutoDock 4.2.5.1 (Molecular Graphics Laboratory, The Scripps Research Institute). The 3D structure of 4 anthocyanins was downloaded from Pubchem-Open Chemistry Database (htEGCGs: //pubchem.ncbi.nlm.nih.gov/substance). Both KLrP and 4 anthocyanins molecules were prepared using AutoDock Tools 1.5.6 before docking. The docking was carried out with 126 × 126 × 126 0.375 Å spacing grids covering the entire surface of KLrP. The Lamarckian genetic algorithm, which is considered one of the most appropriate docking methods available in AutoDock, was used in the docking analysis. Animals and Treatments. Adult male Kunming (6−8 weeks) mice weighing 30−35 g were provided by Changsheng Co., Ltd. (Shenyang, China). The number of the medical animal license is SCXK2015-0001. A total of 60 mice were randomly divided into the control group (Young) and 4 experience groups. The experience groups mice were abdominally and subcutaneously injected D-galactose (Sangon Biotech Co., Ltd. (Shanghai, China)) 150 mg/kg/day to advance the aging process for 8 weeks.20−22 The diet composition in the experiment was shown in Table 1. Experience groups include the negative model control group (treated with normal saline, aged), anthocyanins low dose group (15 mg/kg, anthocyanins-15, anthocyanins high dose group (30 mg/kg, anthocyanins-30), and positive control group (EGCG, EGCG-15). All drugs were administered by gavage at 8 weeks. Memory and Response Measurement. Morris Water Maze (MWM). The MWM was used to test spatial memory.23−25 All groups were evaluated for swimming before testing. None of the animals showed swimming during training, directed swim, or climbed to the tips on the platform and did not feel the movement defect, as determined by a series of neurobehavioral tasks performed before the test. Briefly, mice were given 3 trials per day for 20−30 min (min) at a temperature of 24.0 ± 1.0 °C by adding a nontoxic coating in a tank filled with water. Trained the animals to find a 12 cm × 12 cm submerged (1 cm below water surface) platform placed in a quadrant of the pool and then

levels 20 70 10 18.8 56 300 250 80 258 40 10 6

released the animals at different locations every 60 s trial. They would gently guide it if mice did not find the platform in 60 s. After keeping the platform for 20 s, remove the animals, and place them in a dry cage under a warm heating lamp. The water tank is surrounded by opaque black panels, geometric patterns about 30 cm from the edge of the pool, as distal cues. The animals were trained for 4 days. At the end of training, a 30-s probe trial was administered and the platform from the pool was removed. The platform location retention was determined by measuring the number of times that each animal crossed the previous platform location. A computer-based video tracking system (Water 2100, HVS Image, U.K) recorded the performance in all tasks. Data were compiled offline using HVS Image and compiled using Microsoft Excel before statistical analysis. Rotating Rod Test. The rotating rod performance could monitor the coordination of motor behaviors.26 The mice were placed on a rotating rod (diameter 4 cm, manufactured by Ugo Basile Biological Research Apparatus, model no. 47600) starting at 4 rpm and incrementally accelerated every 20 s to record the last 40 rpm and the latency to fall. Each experimental mouse was subjected to nine trials (3 trials/day, 1 h interval) over a 3-day period. Monoamine Neurotransmitters Measurement. After taking the whole brain, the cerebellum was removed and the brain tissue was weighed and placed in 3 mL of precooled acidic n-butanol, homogenized, then poured into a centrifuge tube, and then filled with n-butanol (1 g/30 mL). The homogenate was mixed with 1 min in a rapid mixer and then centrifuged with 3000 r/min for 10 min and the supernatant is collected. The 1.5 mL sample solution was taken for determination of norepinephrine (NE) and dopamine (DA). Under alkaline conditions, they react with iodine reagents and have very strong fluorescence. Determination of 5-hydroxytryptamine (5-HT) in another 1.0 mL of solution, in alkaline conditions, can be with the development of formaldehyde condensation, formed with fluorescent compounds. Its fluorescence intensity and concentration within a certain range by a linear relation can be quantitative by measuring the fluorescence intensity. n-Butanol, iodine, NE, DA, and 5-HT were purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Redox Related Index Measurement. Blood was centrifuged with 2300 rpm for 5 min to make serum. Fresh tissue was ground with liquid nitrogen and homogenized. The glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), and malondialdehyde (MDA) in the serum and brain tissue (hippocampus, prefrontal cortex, whole brain) were analyzed using test kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). RNA Extraction and Quantitative RT-PCR. The whole brain tissues (20 mg, stored at −80 °C) were ground with liquid nitrogen in the mortar containing RNase. Total RNA was extracted from three individual mice per group to determined the quality and quantity of RNA. Quantitative real-time PCR analysis was performed using the ABI Prism 7900-HT sequence detection system (96 wells, ABI).22 Designed for mouse cytokine analysis and synthesis of the 5974

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Figure 1. continued

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Figure 1. Use of Autodock software to analyze 50 docking models of four anthocyanins and proteins. Panoramic view showing the binding mode between cyanidin 3-arabinoside (A), cyanidin 3-galactoside (B), cyanidin 3-glucoside (C), cyanidin 3-xyloside (D), and KLrP. The most conformations of the binding energy are −4.95, −5.54, −7.43, and −5.55. and a 590 nm shielded filter. The Tail Length of each comet (the head of the comet center of gravity), the Tail DNA %, the Tail Moment, and the Olive Tail Moment (OTM, Tail Length × Tail DNA content) of each comet were calculated using the CASP analysis software. Western Blot Analysis. The expression of ataxia telangiectasia mutated (ATM), ATM and Rad3-related protein (ATR), H2AX, γ-H2AX, checkpoint kinase 1 (Chk1), p-Chk1, checkpoint kinase 2 (Chk2), p-Chk2, p53, p-p53, cell division control protein 25 (CDC25), and CDC2 proteins were determined by Western blotting. In each group, the protein was extracted from the same portion of the mouse liver. Protein samples (100 g) were isolated by 10% SDS-PAGE. Polyclonal rabbit anti-ATM, anti-ATR, anti-Chk1, anti-p-Chk1, anti-Chk2, anti-p-Chk2, anti-H2AX, anti-γ-H2AX, anti-p53, anti-p-p53, anti-CDC25, anti-CDC2 antibodies, and anti-α-tubulin antibodies (Proteintech Group, Inc., China) were used.

following primers: cyclooxygenase-2(COX-2), forward 5′-TGAGCAACTATTCCAAACCAGC-3′ and reverse 5′-GCACGTAGTCTTCGATCACTATC-3′; interleukin (IL-1), forward 5′-GCAACTGTTCCTGAACTCAACT-3′ and reverse 5′-ATCTTTTGGGGTCCGTCAACT-3′; transforming growth factor-β1 (TGF-β1), forward 5′-ACCTGCAAGACCATCGACAT-3′ and reverse 5′-GGTTTTCTCATAGATGGCGT-3′. Note that β-actin was used as an internal control. Comet Assay. The comet assay was performed according to the literature and our previous studies.27 Briefly, 1 × 105 cells in each sample suspended in 0.5% low melting agarose were spread on the normal melted agarose (1%) slides. After lysis, the slides were electrophoresed at 25 V (300 mA) for 25 min at 4 °C and stained with 10 mg/mL ethidium bromide. A total of 100 randomly selected cells (comets) were scored using a fluorescence microscope equipped with a 515−560 nm excitation filter 5976

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Journal of Agricultural and Food Chemistry Statistical Analysis. All of the results were expressed as means ± SDs of three determinations at each concentration for each sample. Analysis of variance (ANOVA) was performed to do the mean separation by LSD (p ≤ 0.05) using the SPSS 16.0 program for Windows (SPSS Inc., Chicago, IL).

shiny hair and sensitive reflectivity ability and food ration. The dynamic changes in body weight were measured during 8 weeks of feeding. As shown in Table 2, there was no significant difference in body weight. As shown in Figure 2, the heart index and kideny index also had no obvious difference in all groups mice. However, brain index and liver index were all significant lower in aged model mice than the young control, and anthocynins 30 mg/kg treated could increase the brain index and liver index for aging mice. The results showed that anthocynins could maintain the weight of the brain and liver in aging mice and prevent atrophy. Anthocyanins Treatment Maintains Spatial Memory and Response in Aging Mice. The spatial memory ability was determined with morris water maze (MWM). The results of the latent period and crossing times were shown in Table 3. They had the opposite results, that the latent period of aged model group was longer than that of the young control group (p < 0.01) and the crossing times were just the opposite. Both EGCG and anthocyanins treatment could alleviate the symptomsto a certain extent, and the dose-dependency of the anthocyanin-treated group was significantly reduced (30 mg/kg was better than 15 mg/kg). The response ability was determined with the rotating rod test. The results of latency to fall from retard was shown in Figure 3. The latency of the aged model group was significantly lower than the young control group (aged vs young, 94.00 vs 218.33 s; p < 0.01), the latency of the anthocyanins-treated groups was significantly decreased in a dose-dependent manner (30 mg/kg was better than 15 mg/kg), and the latency of anthocyanins

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RESULTS Computational Analysis of the Binding between Anthocyanin and Klotho. We carried out docking simulations to investigate the possible 4 anthocyanins-binding site on KLrP. The binding energy of 50 models in docking is shown in Figure 1. Compared with the binding patterns, anthocyanins probably bound to KLrP and was located in a region between the α-domain and the β-domain, as depicted in Figure 1. Consequently, the stabilizing effect contributed by anthocyanins on the appendant structure of KLrP may prevent the occurrence of domain swapping, so as to redirect KLrP away from the fibril-forming pathway and into forming nontoxic, unstructured, and off-pathway aggregates.28 In this study, we speculated that 4 anthocyanins can inhibit the fibrillation of human Cytosolic Neutral beta-Glycosylceramidase (Klotho-related Prote:KLrP) complex.28,29 This observation is particular significant as these four anthocyanins could protect the stability of Klotho that have an antiaging effect after interacting with the protein. This observation is particular significant as these four anthocyanins combined to avoid the occurrence of redox reaction, so as to achieve the role of the antiaging effect after interacting with the protein. Effects of Anthocynins Treatment on the Bodyweight and Organ Index. All anthocynins treatment group mice had

Table 2. Effects of Anthocyanins on Body Weight in Mice during Aginga time (week) group

0

1

2

3

4

5

6

7

8

young aged EGCG-15 anthocyanins-15 anthocyanins-30

32.23 ± 1.46 32.72 ± 1.39 32.73 ± 2.04 31.82 ± 2.31 32.4 ± 1.47

35.38 ± 2.76 36.78 ± 2.04 36.50 ± 1.94 35.37 ± 3.77 33.79 ± 2.99

39.9 ± 2.24 39.18 ± 2.68 38.3 ± 2.38 32.2 ± 2.64 35.05 ± 2.84

42.13 ± 2.55 40.90 ± 3.51 41.95 ± 4.00 39.40 ± 3.00 37.80 ± 4.21

40.92 ± 3.96 41.43 ± 3.18 43.76 ± 3.41 40.33 ± 2.91 39.86 ± 4.84

43.80 ± 3.73 42.95 ± 3.92 43.04 ± 4.59 39.86 ± 2.81 40.00 ± 4.59

43.81 ± 3.59 40.38 ± 13.06 44.62 ± 3.6 43.04 ± 3.62 42.11 ± 4.14

43.42 ± 4.57 43.92 ± 4.28 40.57 ± 5.07 41.67 ± 3.43 41.23 ± 3.76

47.65 ± 2.75 46.25 ± 4.45 48.26 ± 2.54 46.64 ± 4.31 45.17 ± 3.52

The weight of mice weighed every week during the whole text, but they did not show significant differences; values are mean ± SEM. The body weight unit of mice are grams. a

Figure 2. Effects of anthocyanins on the organ index in experimental mice. Values are means ± SEM. An asterisk indicate significant differences in expression of these proteins with respect to Young control only at the (*) p ≤ 0.05 and (**) p ≤ 0.01 levels, respectively. Hashes indicate significant differences with respect to the aged model only at the (#) p ≤ 0.05 and (##) p ≤ 0.01 levels, respectively. 5977

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Anthocyanins Treatment Enhances the Levels of Monoamines (NE, DA, and 5-HT) in Brain of Aging Mice. Monoamine neurotransmitters are involved in the regulation of cognitive processes such as emotion, arousal, and certain types of memory.30,31 As shown in Figure 4, the levels of NE, DA, and 5-HT were all decreased in the aging mice. However, the anthocyanins and EGCG treatment had no significant changes on NE and 5-HT except DA. The level of DA with anthocyaninstreated groups increased in a dose-dependent manner (30 mg/kg was better than 15 mg/kg), and the level of anthocyanins 30 mg/kg group was significantly higher than the aged model (p < 0.001), and it had a better effect than EGCG 15 mg/kg. Anthocyanins Treatment Regulates Redox Balance in Blood and Brain Tissue of Aging Mice. Redox system is an important factor on maintaining the enzymes activities such as GSH-PX and SOD and resisting excessive accumulation of ROS in the body. The stability of the redox system is an important

Table 3. Effect of Anthocyanins on Latent Period and Crossing Times in Morris Water Maze (MWM) Text for the Micea after 8 weeks group

treatment (mg kg−1)

latent period (s)

crossing times

young aged EGCG anthocyanins

normal saline normal saline 15 15 30

12.29 ± 3.17 34.33 ± 7.02b 20.31 ± 2.62b,c 20.34 ± 2.11b,c 16.51 ± 1.25b,c

6.54 ± 0.54 2.47 ± 0.39b 4.55 ± 0.16b,c 4.38 ± 0.47b,c 5.36 ± 0.71b,c

a

Detection of latent period and crossing times by morris water maze (MWM) text. There were significant differences between the groups; values are mean ± SEM. bSignificant differences with respect to young mice at the p < 0.01 level. cSignificant differences with respect to aged mice at the p < 0.01 level.

30 mg/kg group was significantly higher than the aged model (187.67 s, p < 0.05).

Figure 3. Effect of anthocyanins on latency to fall from retard in rotating rod test. Each experimental mouse performed nine trials over a span of 3 days (3 trials/day with an interval of 1 h).Values are means ± SEM. The asterisks indicate significant differences in expression of these proteins with respect to Young control only at the (**) p ≤ 0.01 levels. Hashes indicate significant differences with respect to the aged model only at the (#) p ≤ 0.05 levels.

Figure 4. Effect of anthocyanins on monoamine neurotransmitters content in brain.The levels of the three were detected by chemical fluorimetry that used brain homogenate. Values are means ± SEM. The hashes indicate significant differences in expression of these proteins with respect to the aged model only at the (#) p ≤ 0.05, (##) p ≤ 0.01, and (###) p ≤ 0.001 levels, respectively. 5978

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Figure 5. Effect of anthocyanins on the GSH-PX, T-SOD, and MDA content in serum and brain tissue. The levels of the three were detected by ELISA and the chemical method that used brain homogenate and serum. Values are means ± SEM. The asterisks indicate significant differences in expression of these proteins with respect to the Young control only at the (*) p ≤ 0.05, (**) p ≤ 0.01, and (***) p ≤ 0.001 levels, respectively. The hashes indicate significant differences with respect to the aged model only at the (#) p ≤ 0.05, (##)p ≤ 0.01, and (###) p ≤ 0.001 levels, respectively.

index to judge aging and health.32−34 As shown in Figure 5, the level of GSH-PX and T-SOD was significantly lower than the young control group compared to the aged model group both in serum and brain tissue (p < 0.05). Anthocyanins significantly raise the decrease of GSH-PX and T-SOD levels in a dose-

dependent manner (30 mg/kg was better than 15 mg/kg) in serum and brain tissue (hippocampus, prefrontal cortex, brain). However, the level of MDA had no significant changes except in the hippocampus in which the anthocyanins 30 mg/kg group was significant lower than the aged model group (p < 0.05). 5979

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in the mammalian brain.3Unrepaired DNA damage contributes to genomic instability and the aging process.2 To further investigate the mechanism of anthocyanins treatment on aging, the data of the comet assay (Figure 7) showed statistically significant differences in the aged model group on Tail Length, Tail DNA %, Tail Moment, and Olive Tail Moment (OTM) compared to the young control groups (P < 0.001). The dosedependency of the anthocyanin-treated group was significantly reduced (30 mg/kg was better than 15 mg/kg), and the level of anthocyanins 30 mg/kg group was significantly reduced than the aged model (p < 0.001), and EGCG also had a good effect that was better than anthocyanins15 mg/kg group. Anthocyanins Treatment Regulates the Proteins Expression in DNA Damage Signaling Pathway of Aging Mice Brain. Ataxia telangiectasia mutated (ATM) and ATM and Rad3-related protein (ATR) were sensors of the DNA damage signaling pathway.2 The expression levels of ATM and ATR were shown in Figure 8A, and the expression of ATM and ATR were obviously increased in the aged model group

Anthocyanins Treatment Inhibits Excessive Accumulation of Inflammatory Cytokines (COX2, IL-1, and TGFβ1) in Brain of Aging Mice. Inflammatory cytokines contribute to inflammatory diseases that have been linked to different diseases, such as atherosclerosis and cancer.35 The dysregulation of inflammatory cytokines have also been linked to depression and other neurological diseases such as brain aging.36 To further probe the effects of anthocyanins treatment on aging, the levels of inflammatory markers COX2, IL-1, and TGF-β1 were measured in brain. As the results show in Figure 6, the levels of inflammatory markers of the young control group mice were lower than the aged model group mice, and the level of IL-1 has significance (p < 0.01). Anthocyanins significantly decreased the levels of IL-1, COX2, and TGF-β1 levels especially and could significantly reduce the levels of IL-1 (p < 0.01). Also, the EGCG also had the effect on reducing inflammation, but the effect was weaker than anthocyanins. Anthocyanins Treatment Inhibits DNA Damage in Brain Cell of Aging Mice. DNA damage accumulates with age

Figure 6. Changes in the expression levels of marker genes involved in inflammation (COX-2, IL-1 and TGF-β1) in brain induced by the anthocyanins. RNA was extracted from brain tissue for real-time PCR analysis.Values are means ± SEM.Asterisk indicate significant differences in mRNA levels of IL-1, COX-2, or TGF-β1 with respect to young control only at the (*) p ≤ 0.05 and (**) p ≤ 0.01 levels, respectively. Hashes indicate significant differences with respect to the aged model only at the (##) p ≤ 0.01 levels.

Figure 7. Effect of anthocyanins on DNA damage levels in the brain with the comet assay. Cell suspension made of brain tissue for the comet assay, and each comet was calculated using CASP analyzed software. Values are means ± SEM. Asterisks indicate significant differences in expression of these proteins with respect to the young control only at the (*) p ≤ 0.05, (**) p ≤ 0.01, and (***) p ≤ 0.001 levels, respectively. Hashes indicate significant differences with respect to the aged model only at the (#) p ≤ 0.05, (##)p ≤ 0.01, and (###) p ≤ 0.001 levels, respectively. 5980

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Figure 8. Regulating effect of anthocyanins on DNA damage signaling pathway in the brain: (A) expression of ATM and ATR, (B) expression of H2AX and γ-H2AX, (C) expression of Chk1 and p-Chk1, (D) expression of Chk2 and p-Chk2, (E) expression of p53 and p-p53, and (F) expression of CDC25 and CDC2. β-Actin was used as a loading control. Statistical data are shown by the relative density ratio. Values are means ± SEM. Asterisks indicate significant differences in expression of these proteins with respect to the young control only at the (*) p ≤ 0.05, (**) p ≤ 0.01, and (***) p ≤ 0.001 levels, respectively. Hashes indicate significant differences with respect to the aged model only at the (#) p ≤ 0.05, (##)p ≤ 0.01, and (###) p ≤ 0.001 levels, respectively.

compared with the young control group. Anthocyanins treatment significantly reduced the expression of ATM in a dose-dependent

manner (30 mg/kg was better than 15 mg/kg), and the effect of anthocyanins 30 mg/kg was better than the EGCG group. 5981

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models of the group confirmed that D-galactose injection could cause significant aging of mice. Our results showed that anthocyanins could maintain the weight of the brain and liver of aging mice and prevent atrophy; the memory and response could be improved through treatment with anthocyanins in the aged model group; anthocyanins could increase the level of NE, DA, and 5-HT in brain tissue; anthocyanins could increase GSH-PX and T-SOD content, decrease the MDA content, indicating that anthocyanins had the effect of antiaging on aging induced by D-galactose in vivo. Inflammatory mediators such as COX2, IL-1, and TGF-β1 were over transcription, and anthocyanins could significantly decrease the inflammatory accumulation. DNA damage responses are caused by multiple signal transduction processes, and these signaling pathways are dominated by a number of key proteins.41 The activation of these pathways is essential for proper coordination of the checkpoint, DNA repair process, and aging.42 The anthocyanins from chokeberry could decrease the level ATM, ATR, H2AX, Chk1, Chk2, and p53. Meanwhile, the phosphorylated forms of H2AX, Chk1, Chk2, and p53 were also decreased. Also the expression of CDC2 and CDC25 were increased. These proteins control the pathways of ATM-Chk2 and ATR-Chk1, which are respectively activated by DNA double-strand breaks (DSBs) and single-stranded DNA.2 The DNA damage has been shown that was closely related to aging.3 Therefore, anthocyanins regulate cell cycle and senescence by regulating the expression of DNA damage signaling pathway related proteins. In summary, our results demonstrated that anthocyanins from chokeberry could antiaging by inhibiting DNA damage add up (preserving genome integrity) and reduce inflammation. Moreover, DNA damage signaling pathway was also involved in the antiaging mechanism of the tested compounds. Therefore, the current tested compounds from chokeberry may prove to be good potential agents for the therapy of aging. However, it should be noted that aging treatment should take into account the versatility of its pathogenesis and should act upon all pathways involved. As the highest content of anthocyanins worldwide, the current findings will no doubt have a positive effect on the research for protection against aging; however, considering the contents analysis results in the current study, it may be difficult to acquire the necessary amount of the anthocyanins for antiaging unless the sufficient intake of chokeberry can be ensured. Therefore, it may be necessary to produce chokeberry anthocyanins and use them as a food supplement. Further study of the chokeberry anthocyanins on suppressing the development of aging at the clinical level and the specific action of each anthocyanin monomer will be necessary.

The expression of ATR in the EGCG group was lower than the anthocyanins-treated group, and 30 mg/kg anthocyanins had a significant comparison to the aged model group (P < 0.05). H2AX and γ-H2AX were mediators of the DNA damage signaling pathway. The expression levels of H2AX and γ-H2AX were shown in Figure 8B, compared with the young control group, the expression of H2AX and γ-H2AX were significantly increased in the aged model group (P < 0.001). Anthocyanins (15, 30 mg/kg) significantly decreased the expression of H2AX and γ-H2AX in a dose-dependent manner. Checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2) were effectors of DNA damage signaling pathway.2 The expression levels of Chk1and Chk2 were shown in Figure 8C,D, compared with the young control group, the expression of Chk1,p- Chk1, Chk2, p-Chk2 was all significantly increased in the aged model group. Anthocyanins treatment significantly reduced the expression of them in a dose-dependent manner except anthocyanins 15 mg/kg for p-Chk1 that it was higher than the young control group. Both p53 and p-p53 were important factor for cell growth and reproduction.37 The expression levels of p53 and p-p53 were shown in Figure 8E, compared with the young control group, the expression of p53 and p-p53 were significantly increased in the aged model group (P < 0.01). Anthocyanins (15, 30 mg/kg) and EGCG significantly decreased the expression of p53 and p-p53 compared aged model group. Anthocyanins treatment significantly decreased the expression of them in a dose-dependent manner, and EGCG had the better effect for reduction than the anthocyanins. CDC25 and CDC2 were bispecific phosphatases that are considered to be subclasses of protein tyrosine phosphatases.2,38 The CDC25 proteins controls entry into and progresses at various stages of the cell cycle, including mitosis and S (“Synthesis”) stages.38 The expression levels of CDC25 and CDC2 were shown in Figure 8F, compared with the young control group, and the expression of CDC25 and CDC2 were obviously decreased in the aged model group. Anthocyanins treatment significantly increased the expression of them in a dose-dependent manner, and the effect of anthocyanins 30 mg/kg was better than the EGCG group.

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DISCUSSION The method of calculating the protein structure and ligand− protein interaction has been successfully applied for decades in biochemical research.39,40 In our study, using molecular docking to simulate the interaction between anthocyanins of black chokeberry (cyanidin 3-arabinoside, cyanidin 3-galactoside, cyanidin 3-xyloside, and cyanidin 3-glucoside) and klotho. The klotho gene was identified as an “aging-suppressor” gene in mice that accelerated aging when disrupted and extended the life span when overexpressed.28,29The results showed that these four anthocyanins could protect the stability of klotho that have antiaging effect after interacting with protein. This observation is particular significant as these four anthocyanins combined to avoid the occurrence of redox reaction, so as to achieve the role of antiaging effect after interacting with protein. Because of the strong inhibitory effect of klotho on aging, we hypothesized that anthocyanins have an antiaging effect. To this end, we conducted a series of animal experiments to verify and study. D-galactose injection is the most extensively studied model of aging in rats and mice.20,21 The best feature of these models is the rapid accumulation of free radicals leading to accelerated aging of tissues and organs.22 The behavioral and biochemical elderly

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86-24-62202232. ORCID

Jie Wei: 0000-0002-2943-7252 Funding

This work was supported by grants from Science and Technology Government Department Fund in Liaoning province (Grant No. LYB201611), the Foundation of Ocean and Fishery Hall in Liaoning Province (Grant No. 201407), the Social Science Fund Plan in Liaoning Province (Grant No. L15BJY029), and China National Natural Science Foundation of China. 5982

DOI: 10.1021/acs.jafc.7b02136 J. Agric. Food Chem. 2017, 65, 5973−5984

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Journal of Agricultural and Food Chemistry Notes

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The authors declare no competing financial interest.

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ABBREVIATIONS USED ATM, ataxia telangiectasia mutated; ATR, ATM and Rad3related protein; CDC2, cell division control protein 2; CDC25, cell division control protein 25; Chk1, checkpoint kinase 1; Chk2, checkpoint kinase 2; COX-2, cyclooxygenase-2; DA, dopamine; DSBs, DNA double-strand breaks; EGCG, epigallocatechin gallate; GSH-PX, glutathione peroxidase; 5-HT, 5-hydroxytryptamine; IL-1, interleukin-1; MD, molecular dynamic simulations; MDA, malondialdehyde; MWM, morris water maze; NE, norepinephrine; OTM, Olive Tail Moment; SOD, superoxide dismutase; TGF-β1, transforming growth factor-β1

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