Background

Neurodegenerative disorders, including Alzheimer’s disease (AD), Parkinson’s disease and Huntington’s disease are characterized by loss of neuronal function and memory and cognitive impairment [1]. AD is the most common neurodegenerative disorder [2]. Oxidative stress, including lipid peroxidation, free radical formation, protein oxidation and DNA oxidation, in the central nervous system (CNS) can lead to cell death and contributes to the pathogenesis of various neurodegenerative disorders [3, 4].

Glutamate is an excitatory neurotransmitter that plays a role in learning and memory, and contributes to excitotoxicity in neuronal cells [5]. Glutamate is one of the most important mechanisms known to trigger and neuroinflammation and neuronal cell death in CNS disorders. Glutamate-induced excitotoxicity may contribute to the neuronal injury in neurodegenerative diseases such as motor neuron disease and Alzheimer’s disease by neuronal injury as anoxia and reperfusion [6].

Excessive glutamate concentration can induce oxidative stress by increasing the production of reactive oxygen species (ROS) and intracellular calcium (Ca2+) levels. Glutamate excitotoxicity also results in mitochondrial dysfunction and depletion of antioxidant defense systems, including glutathione (GSH), glutathione peroxidase (GPx) and glutathione reductase (GR) by inhibiting cystine uptake [7–9].

Aronia melanocarpa (black chokeberry: A. melanocarpa), a member of the Rosaceae family, is a fruit with a high content of polyphenols, including anthocyanins (cyanidin glycosides), flavanols, flavonoids (quercetin glycosides), chlorogenic acids, triterpenes, and fibers and caffeic acid derivatives [10]. A. melanocarpa has shown high antioxidant activity, as well as hepatoprotective, gastroprotective and anti-inflammatory effects. Recent studies showed that A. melanocarpa prevents obesity in C57BL/6 J mice and reduces systolic and diastolic blood pressure [11, 12]. A. melanocarpa also protects against female skeleton damage due to chronic exposure to Cd [13]. Cyanidin-3-O-galactoside is a major compound in A. melanocarpa, has considerable antioxidant activity and protects against endothelial dysfunction and vascular failure induced by peroxynitrite [14, 15].

Trolox is water-soluble derivative of vitamin E and antioxidant to reduce oxidative stress. it play as positive control in this study.

The aim of present study was to investigate the neuroprotective effect of Aronia melanocarpa and the possible underlying mechanism against glutamate-induced death of HT22 cells.

Methods

Plant material and extraction

A. melanocarpa berries (Danyang, Chungcheongbuk-do, Korea) from a 14-year-old plant were collected and authenticated by Dr. Young Bae Seo, a professor of the College of Oriental Medicine, Daejeon University, Korea. A voucher specimen (CJ200M) has been deposited in the natural products laboratory, the Kangwon National University. A. melanocarpa extract was obtained from the Future Food Research Center (Cheng Ju, Korea).

A. melanocarpa berries were pulverized using a blender after freeze-drying for 3 days (PVTFA 10AT, ILSHIN BioBase, Dongducheon, Korea). Powdered A. melanocarpa berries were extracted in 70% ethanol (100 g/1 L) at room temperature by maceration and were filtered through a vacuum filter. The extract was concentrated by evaporation (EYELA N-1000, Tokyo Rikakikai Co, Tokyo, Japan) and then freeze-dried for 3 days.

Cell viability assay

Cell viability was investigated by MTT assay using a method described previously [16]. HT22 cells were seed at a density of 6.7 × 104/well in 48-well plates and incubated at 37 °C in 5% CO2. After incubation for 24 h, 10 and 100 μg/ml of extract, 1 and 10 μg/ml of cyanidin-3-O-galactoside, trolox (positive control, 50 μM) and glutamate were added. Then, cells were incubated for 3 h with dimethyl thiazolyl diphenyl tetrazolium salt (MTT) (1 mg/ml) solution, and dimethyl sulfoxide (DMSO) was added to dissolve MTT-formazan crystals. The optical density at 570 nm was measured using an ELISA reader.

ROS measurement

2`7`-Dichlorofluorescein diacetate (DCF-DA) was used for measurement of ROS levels. HT22 cells (6.7 × 104/well in 48-well plates) were treated with 10 and 100 μg/ml of extract, 1 and 10 μg/ml of cyanidin-3-O-galactoside, trolox (positive control, 50 μM) and 2 mM glutamate for 8 h. Then, cells were washed with PBS and incubated in 10 μM DCF-DA in Dulbecco’s modified Eagle’s medium (DMEM) without phenol red for 30 min. Cells were washed twice with phosphate buffer saline (PBS), 1% Triton X-100 added, and incubated for 10 min at 37 °C. Fluorescence was measured at an excitation wavelength of 490 nm and emission wavelength of 525 nm.

Calcium (Ca2+) measurement

Intracellular Ca2+ levels were measured using the Fura-2 AM. HT22 cells were plated in 48-well plates and incubated for 24 h at 37 °C and 5% CO2. After incubation, 10 and 100 μg/ml of extract, 1 and 10 μg/ml of cyanidin-3-O-galactoside, Trolox (positive control, 50 μM) and 2 mM glutamate were treated for 3 h, and then 2 μM Fura-AM was added to each well. Cells were then washed three times with HEPES buffer saline. Ca2+ levels were determined by measuring fluorescence intensity at an excitation wavelength of 340 and 380 nm and emission wavelength of 500 nm.

Mitochondrial membrane potential (ΔΨ) measurement

Mitochondrial membrane potential (ΔΨ) change was determined by monitoring the accumulation of the fluorescent dye, rhodamine 123 (Rho123). HT22 cells were treated with 2 mM glutamate, 10 and 100 μg/ml of A. melanocarpa extract and 1 and 10 μg/ml of cyanidin-3-O-galactoside. HT22 cells were then stained with Rho123 for 15 min at 37 °C and washed. The Rho123 concentration was measured by spectrofluorometry at an excitation wavelength of 488 nm and emission wavelength of 520 nm.

GSH measurement

Total GSH was measured by an enzymatic cycling method based on the reduction of 5′,5′-dithiobis 2-nitrobenzoic acid (DTNB) with GSH reductase and nicotinamide adenine dinucleotide phosphate (NADPH). Cells were treated with extract, cyanidin-3-O-galactoside and glutamate for 8 h and washed with 0.2 M phosphate buffer (pH 7.4). Cells were lysed with sulfosalicylic acid and centrifuged at 3000 g for 30 min at 4 °C to collect supernatants. Supernatants were mixed with 5 units/mL glutathione disulfide reductase, 0.3 mM NADPH and 0.5 mM DTNB. The reaction absorbance at 412 nm was measured within 15 min.

Antioxidant enzyme, glutathione reductase and glutathione peroxidase assays

HT22 cells were treated with extract, cyanidin-3-O-galactoside and glutamate for 8 h. Cells were washed with 0.2 M phosphate buffer (pH 7.4) and lysed with sulfosalicylic acid. After centrifugation (3000 g for 30 min at 4 °C), supernatants were collected. Glutathione reductase (GR) was measured by monitoring the reduction of oxidized GSH (GSSG) in the presence of NADPH. Glutathione peroxidase activity was determined by quantifying the rate of oxidation of GSH to GSSG. The decrease in absorbance at 340 nm was measured using a spectrophotometer.

Statistics

All data were expressed as means ± S.D. Significant differences were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Statistical significance was set at P < 0.05, 0.01 and 0.001.

Results

Neuroprotective effect of A. melanocarpa on glutamate-induced cell death in HT22 cells

We evaluated the neuroprotective effects of A. melanocarpa extract in HT22 cells (Fig. 1). A. melanocarpa extract exhibited significant neuroprotective effects by reducing glutamate-induced cell death to 16.81 ± 35.38% (relative protection at 100 μg/ml). Cyanidin-3-O-galactoside also significantly protected HT22 cells against glutamate-induced neurotoxicity, with relative protection of 35.38 ± 12.43% at 10 μg/ml.

Fig. 1
figure 1

Effect of A. melanocarpa extract (10 and 100 μg/ml) and cyanidin-3-O-galactoside (1 and 10 μg/ml) on glutamate-induced death of HT22 cells. Data are means ± S.D. *p < 0.05 versus the glutamate-treated group

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ROS production

Inhibition of ROS production by A. melanocarpa extract was evaluated using 2`7`-dichlorofluorescein diacetate (Fig. 2). Glutamate treatment increased the ROS level in HT22 cells compared to the control (126.7 ± 1.32%). A. melanocarpa extract at 100 μg/ml significantly decreased glutamate-induced ROS production to 87.32 ± 7.50% (p < 0.05). Cyanidin-3-O-galactoside at 10 μg/ml also decreased ROS production to 87.23 ± 5.30%.

Fig. 2
figure 2

Effect of A. melanocarpa extract (10 and 100 μg/ml) and cyanidin-3-O-galactoside (1 and 10 μg/ml) on ROS production in HT22 cells. Data are means ± S.D. *p < 0.05 versus the glutamate-treated group

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Intracellular Ca2+ production

High concentrations of glutamate lead to intercellular Ca2+ accumulation. We investigated intracellular Ca2+ levels using Fura-AM to determine the effect of A. melanocarpa extract in HT22 cells (Fig. 3). A. melanocarpa extract at 100 μg/ml significantly decreased the intracellular Ca2+ concentration in HT22 cells compared to glutamate-treated cells (100.81 ± 1.89% (p < 0.05) at 100 μg/ml). Cyanidin-3-O-galactoside at 10 μg/ml also significantly decreased intracellular Ca2+ levels to 104.37 ± 1.80%.

Fig. 3
figure 3

Effect of A. melanocarpa extract (10 and 100 μg/ml) and cyanidin-3-O-galactoside (1 and 10 μg/ml) on Ca2+ influx in HT22 cells. Data are means ± S.D. *p < 0.05 versus the glutamate-treated group

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Mitochondrial membrane potential (ΔΨ)

We investigated the effect of A. melanocarpa extract on mitochondrial membrane potential (ΔΨ) in HT22 cells using Rho123 dye (Fig. 4). Glutamate-treated HT22 cells exhibited a decreased mitochondrial membrane potential to 80.94 ± 8.70%. A. melanocarpa extract at 100 μg/ml significantly increased the mitochondrial membrane potential to 98.84 ± 11.90% of the control. In addition, Cyanidin-3-O-galactoside improved the mitochondrial membrane potential decreased by glutamate (92.25 ± 2.62% at 10 μg/ml).

Fig. 4
figure 4

Effect of A. melanocarpa extract (10 and 100 μg/ml) and cyanidin-3-O-galactoside (1 and 10 μg/ml) on glutamate-induced disruption of mitochondrial membrane potential. Data are means ± S.D. *p < 0.05 versus the glutamate-treated group

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GSH level and GR and GPx activity

We evaluated the effect of A. melanocarpa extract on GSH level, and GR and GPx activity in HT22 cells (Fig. 5). A. melanocarpa extract increased the GSH level. Exposure to glutamate increased the GSH level by 78.56 ± 6.26%, GR activity by 84.79 ± 0.29% and GPx activity by 89.04 ± 1.36%, whereas A. melanocarpa extract prevented the glutamate-induced depletion of GSH level (90.92 ± 12.19% at 100 μg/mL), GR activity (94.90 ± 2.68% at 100 μg/mL) and GPx activity (89.05 ± 1.36% at 100 μg/mL). Cyanidin-3-O-galactoside also significantly increased the GSH level, GR activity and GPx activity.

Fig. 5
figure 5

Glutathione (GSH) a, glutathione reductase (GR) b and glutathione peroxidase (GPx) c levels in HT22 cells. Data are means ± S.D. *p < 0.05 versus the glutamate-treated group

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Discussion

This study demonstrated that A. melanocarpa extract protected mouse hippocampal HT22neuronal cells against glutamate-induced death.

High levels of glutamate excitotoxicity can lead to neuronal cell death by oxidative stress. It is also involved in intercellular Ca2+ influx and reactive oxygen species (ROS) generation via NMDA receptor [17]. ROS, including hydroxyl radical (OH−), superoxide anion (O2 −) and hydrogen peroxide (H2O2), are generated in cells and lead to death due to DNA damage, protein oxidation, and lipid peroxidation [18]. Intercellular Ca2+ influx contributes to excessive ROS production and causes depolarization of the mitochondrial membrane. GSH is an important antioxidant involved in nutrient metabolism, DNA synthesis, signal transduction and cell proliferation in the CNS. GR and GPx are critical enzymes for the production of GSH. A high concentration of glutamate was involved in depletion of GSH by inhibiting cysteine uptake into cells [19]. Depletion of GSH or antioxidant enzymes, such as GR and GPx, leads to neuronal cell death [20]. Mitochondria play an important role in neuronal cell death. Mitochondrial dysfunction results in ROS production and cell apoptosis and is indicated by loss of the mitochondrial membrane potential [21]. A high glutamate level also induced a decrease in the mitochondrial membrane potential in cells in the CNS.

HT22 cells, an immortalized mouse hippocampal cell line, are used in vitro for mechanistic studies related to glutamate-induced cell death by oxidative stress [22].

Our results showed that A. melanocarpa extract protected HT22 cells against glutamate-induced cell death by inhibiting ROS generation and Ca2+ influx. A. melanocarpa extract also restored GSH, GPx and GR activity and increased the mitochondrial membrane potential. Therefore, the neuroprotective effect of A. melanocarpa extract against glutamate-induced cell death was likely mediated through attenuation of oxidative stress.

Cyanidin-3-O-galactoside is a major compound in A. melanocarpa extract. A previous study demonstrated that cyanidin-3-O-galactoside exerted an antioxidant effect and a cognitive effect on spatial memory, and regulates hippocampal ERK expression in senescence-accelerated mice [23]. Thus, cyanidin-3-O-galactoside may be involved in the neuroprotective effect of A. melanocarpa extract.

Conclusion

In conclusion, A. melanocarpa extract protected neuronal cells against glutamate-induced death due to its antioxidant activity. Therefore, A. melanocarpa extract may have therapeutic potential for neurodegenerative diseases, such as Alzheimer’s disease.