Background
The central nervous system (CNS) is comprised of several neurotransmitter receptors, with glutamate receptors being one of the major excitatory neurotransmitter receptors that have multiple functions, such as neuronal plasticity, outgrowth, and survival, as well as memory, learning, and behavior [
1]. Conversely, glutamate release is under the control of various glutamate transporters as exposure to elevated levels is harmful to neurons. The overstimulation of postsynaptic glutamate receptors causes neuronal injury/death, which is termed as glutamate excitotoxicity as is believed to be involved in amyotrophic lateral sclerosis (ALS) and several other CNS disorders [
2‐
4]. This glutamate excitotoxicity is believed to arise specifically from the entrance of a high rate of Ca
2+ into the neurons as a result of over-stimulated postsynaptic glutamate receptors [
5]. The excess amount of intracellular Ca
2+ levels can increase the burden of mitochondrial Ca
2+, thereby inducing mitochondrial damage and reactive oxygen species (ROS) generation in the mitochondria [
6‐
8]. Overproduction of ROS is thought to cause a diversity of diseases. There is a sophisticated antioxidant defense mechanism in cells that aides in coping with ROS levels under normal physiological conditions, but under certain conditions, such as excessive ROS and inflammation, excessive Ca
2+ can cause cellular dysfunction and remodeling [
9,
10].
NF-E2-related factor 2 (Nrf2) is a transcription factor that is bound to the antioxidant response element (ARE) and can induce the regulation of various antioxidant encoding genes, particularly heme oxygenase-1 (HO-1) [
11]. Previous reports have proven that the activation of Nrf2 in cells and tissues in response to oxidative stress protects against oxidative injury. Under normal conditions, Nrf2 is localized in the cytoplasm [
12], while under oxidative stress conditions, it translocates into the nucleus and transactivates its target genes through ARE. Various protein kinases have been reported to be involved in the ARE-mediated gene Nrf2 activation in response to the oxidative stress signals including 5′ AMP-activated protein kinase (AMPK) [
13]. AMPK primarily functions as an energy sensor [
14]. AMPK activation not only suppresses ATP-consuming metabolic pathways but also accelerates the energy-producing signaling pathways to offer cellular protection against any stress.
Natural polyphenolic compounds that are obtained from fruits and vegetables have received a great deal of interest during the last decade, due to their potential to inhibit oxidative stress, as reported in various studies [
15]. Anthocyanins are a group of natural phenolic compounds that confer the different colors of plants and fruits. Evidence suggests that anthocyanins are strong natural antioxidants [
16,
17]. Our research group has recently reported the anti-apoptotic, antioxidant, and anti-obesity effects of Korean black bean-derived anthocyanins in different experimental models [
18‐
22].
In the current study, we extended our line of investigation to elucidate the exact mechanism of the neuroprotection of Korean black bean-derived anthocyanins against glutamate-induced excitotoxicity, oxidative stress, neuroinflammation, and neurodegeneration in the hippocampus of the developing rat brain and in SH-SY5Y and BV2 cells.
Methods
Animals and drug treatment
Sprague-Dawley (SD) rat pups (18-g average body weight) on postnatal day 7 (
n = 5 rats/group) were randomly divided into the following eight groups (the treatment outlines are given in Additional file
1):
2.
Glutamate for 2 h (Glu 2 h)
3.
Glutamate for 3 h (Glu 3 h)
4.
Glutamate for 4 h (Glu 4 h)
5.
Glutamate for 4 h + anthocyanins (Glu 4 h + Anth)
6.
Glutamate for 4 h + compound C (Glu 4 h + CC)
7.
Glutamate for 4 h + compound C + anthocyanins (Glu 4 h + CC + Anth)
Glutamate (10 mg/kg), anthocyanins (100 mg/kg), and compound C (10 mg/kg) [
23] in saline solution were intraperitoneally (i.p.) injected. Glutamate was administered for 2, 3, or 4 h. The control animals received 0.9 % saline solution, and all the rats were decapitated after 2, 3, or 4–12 h. All the experimental procedures were approved by local ethical committee for animals of the Department of Biology, Division of Applied Life Sciences, Gyeongsang National University South Korea.
Cell culturing and drug treatment
Murine BV2 microglia and human neuroblastoma SH-SY5Y cells were maintained in 10 % FBS- and 1 % penicillin/streptomycin-supplemented DMEM (Dulbecco’s modified Eagle’s medium) medium in a humidified 5 % CO2 incubator at 37 °C. The cells were treated with glutamate (30 mM), glutamate plus anthocyanins (30 mM + 20 μg/ml), glutamate plus AMPK siRNA (30 mM + 200 nM), glutamate plus AMPK siRNA plus anthocyanins (30 mM + 200 nM + 20 μg/ml), glutamate plus compound C (30 mM + 20 μM), and glutamate plus compound C and anthocyanins (30 mM + 20 μM + 20 μg/ml) for 3 h.
AMPK gene silencing
Small interfering RNA (siRNA) was purchased from Santa Cruz Biotechnology (sc-45312). Cultured SH-SY5Y cells were transfected with 200 nM siRNA using Lipofectamine 2000 reagent (Invitrogen) for 24 h [
24], then the DMEM medium was replaced with glutamate and anthocyanins as indicated in the respective “
Methods” section.
Cell viability assay
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed according to the manufacturer’s instructions (Sigma) to assess the SH-SY5Y cell viability after treatment. The cells were cultured in 96-well plates at a density of 1 × 104 cells per well containing 100 μl DMEM. When the cells were attached after 24 h, the medium was refreshed with the indicated concentration of glutamate (10, 20, and 30 mM) and anthocyanins (10, 20, and 30 μg/ml), while control cells received only DMEM medium. The cells were then incubated for an additional 3 h. After being cultured for 3 h, the cells were incubated with MTT solution for another 4 h at 37 °C. Subsequently, the medium was replaced with DMSO in each well. Finally, the absorbance was measured at 570 nm. All experiments were performed independently in triplicate.
Western blot analysis
Western blot analysis details were conducted as previously performed in our lab [
25]. Briefly, the animals were euthanized after 4 h following treatment of glutamate with or without anthocyanins. Then, the brains were carefully (hippocampus) collected and placed on dry ice for freezing tissue. Similarly, after treatment, the SH-SY5Y and BV2 cells were collected in PBS and centrifuged, and the supernatant was removed. The remaining pellet was dissolved in Pro Prep Protein Extraction Solution, according to the manufacturer’s protocol (iNtRON Biotechnology) to make cell lysates. The brain homogenates and cell lysates were quantified with Bio-Rad protein assay solution. The homogenates (20 μg protein) were fractionated by SDS-PAGE on 4–12 % (Bolt™ Mini Gels, Life Technologies). After transfer, membranes were blocked in 5 % skim milk (or BSA) and incubated overnight at 4 °C with primary antibody, and cross-reacting proteins were detected by ECL after reaction with horseradish peroxidase-conjugated secondary antibodies. The primary antibodies (1: 500 in Tris-buffered saline with Tween (TBST)) included rabbit-derived anti-COX2, anti-TNFα, anti-p-AMPKTh
172, anti-AMPK, anti-Nrf2, anti-caspase-3, anti-iNOS, anti-p-NF-
kB, mouse-derived anti-β-actin, anti-GFAP, anti-heme oxygenase-1 (HO-1), and goat-derived anti-Iba-1 from Santa Cruz Biotechnology (Santa Cruz, CA, USA). After using membrane-derived secondary antibodies (1: 1000 in TBST), ECL (Amersham Pharmacia Biotech, Uppsala, Sweden) detection reagent was used for visualization according to the manufacturer’s instructions. Densitometry analysis of the bands was performed using Sigma Gel software (SPSS, Chicago, IL, USA). Density values were calculated in arbitrary units (A.U.) relative to the untreated control.
Tissue collection and sample preparation
The animals were euthanized after 12 h of drug treatment to conduct morphological studies as we reported earlier [
26]. Briefly, the brain tissues from all the treated groups after 12 h were subjected to transcardial perfusion with 4 % ice-cold paraformaldehyde. After postfixing these brain tissues, 4 % paraformaldehyde was transferred to 20 % sucrose. The tissues were frozen in OCT (Tissue-Tek O.C.T. Compound Medium, Sakura Finetek USA, Inc., Torrance, CA, USA), sectioned into 14–16-μm sections in the coronal plane with a CM 3050S cryostat (Leica, Wetzlar, Germany). The sections were thaw-mounted on Probe-On positively charged slides (Thermo Fisher Scientific Inc., Waltham, MA, USA).
Fluoro-Jade B staining
Fluoro-Jade B staining was performed as reported earlier [
27]. Chamber slides were air-dried overnight. The slides were kept in a solution of 80 % ethanol and 1 % sodium hydroxide then in 70 % alcohol for 5 and 2 min, respectively, followed by immersion in distilled water for 2 min. The slides were placed for 10 min in 0.06 % potassium permanganate solution. The slides were then rinsed with distilled water and immersed in a solution containing 0.1 % acetic acid and 0.01 % Fluoro-Jade B for 20 min. After rinsing with distilled water and applying DAPI, the slides were dried, and glass cover slips were mounted on slides with mounting medium. Images were captured using an FITC filter on a confocal laser scanning microscope (FV 1000, Olympus, Japan).
Immunofluorescence
Immunofluorescence stainings were performed as we previously reported [
19]. Shortly, the slides were washed with 1× PBS and incubated with proteinase K solution at room temperature. After blocking in normal goat/rabbit serum, primary antibodies (1: 100 in PBS, mentioned in the “
Western blotting” section) were applied overnight at 4 °C. Fluorescence-based (FITC and TRITC from Santa Cruz Biotechnology) secondary antibodies (in PBS) were applied at room temperature. DAPI was used to stain the nucleus. The slides were mounted with glass coverslips, and the images were taken using a confocal microscope (FluoView FV 1000; Olympus, Tokyo, Japan).
TUNEL staining
To determine apoptotic cell death, TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling) staining was performed according to the manufacturer’s recommendations. An in situ cell death detection kit was purchased from Roche (Cat. No. 11684809910).
Oxidative stress (ROS) detection in vivo and in vitro
The ROS quantification assay in the brain homogenates of all treated groups was conducted as reported earlier in the literature and by our research group [
28,
29] with slight modification. Briefly, the brain homogenates from the respective groups were diluted 1:20 times with Locke’s buffer (ice-cold) to get 5 mg tissue/ml concentration. Then, the reaction mixture (1 ml) having Locke’s buffer of pH 7.4, 0.2 ml brain homogenate, and 10 ml of DCFH-DA (5 mM) was incubated for 15 min at room temperature to allow the DCFH-DA to be incorporated into any membrane-bound vesicles and the diacetate group cleaved by esterases. After 30 min of further incubation, the conversion of DCFH-DA to the fluorescent product DCF was measured using a spectrofluorometer with excitation at 484 nm and emission at 530 nm. ROS formation was quantified from a DCF-standard curve and data are expressed as pmol DCF formed/min/mg protein. Similarly, an in vitro ROS assay was performed with slight modification as previously described [
26]. Briefly; SH-SY5Y cells were sub-cultured in 96-well plates in 200 μl DMEM that was supplemented with 10 % FBS and 1 % penicillin/streptomycin in every well. The cells were incubated for 24 h at 37 °C in a humidified incubator having 5 % CO
2. The next day, the media was replaced by fresh media that contained Glu (30 mM), Glu plus anthocyanins (30 mM + 20 μg/ml), Glu plus compound C (30 mM + 20 μM), or Glu plus compound C and anthocyanins (30 mM + 20 μM + 20 μg/ml) for an additional 30 min. DCFDA (2′,7′-dichlorofluorescin diacetate) 600 μM dissolved in DMSO/PBS was added to each well and incubated for 30 min. The plates were then read in ApoTox-Glo™ (Promega) at 488/530 nm.
Glutamate assay
Glutamate assay kit (Cat. # KA1670) from Abnova (Taipei, Taiwan) was used to quantify glutamate in hippocampal brain tissue homogenates according to the manufacturer’s instruction.
Enzyme assays
The hippocampal rat brain homogenates and SH-SY5Y cell lysates of the experimental groups were evaluated using ELISA assays for total NF-
k
Bp65 (Life Technologies, Catalog #KHO0371) and Cyclex AMPK (Enzo Life Sciences), as per the manufacturer’s recommended protocols.
COX2 assay
An ELISA assay for COX2 (R&D Systems, Inc. 614 McKinley Place NE Minneapolis, MN 55413, USA) was performed. BV2 and SH-SY5Y cells were sub-cultured in 96-well plates, and treatment was performed in different groups, as described earlier, and then fixed with 4 % paraformaldehyde, as per the manufacturer’s recommendations.
GSH assays
The levels of total GSH and GSH/GSSG ratio were determined by using glutathione assay kit obtained from BioVision (BioVision Incorporated155 S. Milpitas Boulevard, Milpitas, CA 95035, USA), Fluorometric Assay Kit (Catalog #K264-100), according to the manufacturer’s instructions.
Statistical analysis
A computer-based Sigma Gel System (SPSS Inc., Chicago, IL) and the ImageJ program were used to analyze the density and integral optical density (IOD) of scanned X-ray films of Western blot and immunofluorescence images. Density values were expressed as the mean ± SEM. All data are presented as the mean ± SEM. A one-way ANOVA followed by Student’s t test (non-parametric Mann-Whitney and Wilcoxon tests) were used to determine the statistical significance (P < 0.05) of the obtained data.
Discussion
The current study demonstrates several important findings (as shown in detail in Fig.
7). First, it reports that exogenously administered glutamate in the developing rat can increase brain glutamate levels as well as activate and phosphorylate AMPK and NF-
k
B, which is accompanied by Nrf2 protein inhibition. These toxic effects that were induced by glutamate were observed following 2, 3, and 4 h of its administration, as measured and quantified by Western blot and ELISA assays. Those results indicate that glutamate can induce a maximal toxic effect after 4 h when compared to 2 and 3 h. Second, this study provides evidence that after 4 h, anthocyanins can reduce brain glutamate levels, AMPK activation, ROS production, and inflammation. Third, anthocyanins increased glutathione levels and stimulated Nrf2/HO-1 signaling, which has an important role in cellular defense against oxidants and toxic chemicals. Finally, we reported that blocking AMPK either with compound C or with siRNA not only negatively regulates glutamate-induced AMPK activation and inflammation but also can abolish the neuroprotective abilities of anthocyanins in young rats as well as in SH-SY5Y cells.
AMPK and oxidative stress have long been associated with neurodegenerative diseases. Oxidative stress disrupts mitochondrial respiration and damages mitochondria, which ultimately induce apoptotic cell death and degeneration [
30]. Mitochondria, a major regulator of apoptosis, have an important role in controlling cell life and death [
31]. Current findings indicate that anthocyanins decrease glutamate-induced ROS and energy reduction, thereby protecting against oxidative stress.
Here, for the first time, we found that AMPK signaling mediates a crucial role in the antioxidant activity of anthocyanins against glutamate-induced oxidative stress. This beneficial effect of anthocyanins, both in young rats and in cells, is associated with up-regulation of glutathione (both GSH levels and GSSG ratio) and the stimulation of Nrf2/HO-1 as an important antioxidant-signaling pathway. Our findings demonstrated that anthocyanins both inhibited glutamate-upregulated proinflammatory and neuroapoptotic markers as well as suppressed endogenous antioxidant molecules via activated AMPK reduction. Recently, anthocyanins have been shown to exert AMPK-mediated neuroprotection against kainic acid-induced excitotoxicity in cells [
22]. Anthocyanins are comprised of various flavonoids that have been reported to have beneficial effects against oxidative stress, inflammation, and neurodegeneration [
32‐
34]. These beneficial effects of anthocyanins are due to their antioxidative characteristics, as demonstrated by several researchers in their reports [
16,
17]. Similarly, numerous studies have indicated that cyanidin-3-O-glucoside (C3G), one of the active components of anthocyanins, is a potent neuroprotective agent against cerebral ischemia and β-amyloid-induced mitochondrial damage [
35,
36] and can block the release of apoptosis-inducing factor (AIF) in focal cerebral ischemia [
37]. The blueberry-enriched diet has been shown to reduce kainic acid-induced oxidative stress and excitotoxicity mediated memory impairment [
38].
Oxidative stress is one of the earliest pathological changes in neurodegenerative diseases such as Alzheimer’s disease (AD). The hippocampus and cortex sections of the brain are vulnerable to oxidative stress and are associated with the development of cognitive impairment as a feature of sporadic AD. Similarly, another risk factor for AD is aging, in which the endogenous system is unable to scavenge free radicals and ultimately produces oxidative stress in the brain [
39]. Additionally, the brain has a relatively weak antioxidant defense system compared to other organs of the body and so the activation of the endogenous antioxidant system is particularly vital for those tissues. In this regard, the stimulation of Nrf2-ARE signaling pathways in the brain has been considered as one of the major pharmaceutical strategies for the treatment and prevention of neurodegenerative disease and brain aging. Nrf2 is believed to be involved in regulating phase II antioxidant responses, which induce the activation of several free radical scavengers and beneficial enzymes. HO-1 is one of the important genes in the brain and is activated by Nrf2, as demonstrated by intensive studies that have a crucial role in providing shelter to neurons against cell death. Accordingly, numerous reports have described that Nrf2 activation [
40,
41] induces an increase in HO-1 in response to several neurodegenerative disorders [
42]. Many studies have shown that HO-1 activation is an important response to the threat of oxidative stress to neurons as it can rescue neurons from oxidative stress and cell death [
43,
44]. HO-1 has been strongly linked to reduced inflammation [
45]. Similarly, HO-1 knockout mice have been shown to develop inflammatory disease and are sensitive to experimental sepsis [
46], while the pharmacological activation of HO-1 has been found to be beneficial in the inflammation animal model [
47]. Excessive glutamate that is triggered by oxidative stress or ROS accumulation induces neuronal apoptosis and cell death. This process is similar in nature to numerous neurodegenerative diseases and some pathological conditions, such as ischemia and trauma [
3,
48,
49]. The literature has shown that glutamate can induce Nrf2 and HO-1 suppression, which is followed by the induction of inflammation and neurodegeneration in the postnatal brain as well as in cells. In the present study, we have demonstrated that a single dose of exogenously provided glutamate caused the induction of neuroinflammation and neurodegeneration in the postnatal rat brain. Additionally, glutamate treatment in SH-SY5Y and BV2 cells also induced the activation of proinflammatory and proapoptotic markers, such as NF-
k
B and caspase-3 expression. As per our results, these toxic effects of glutamate are AMPK dependent because our findings indicate that either compound C or siRNA-mediated silencing of AMPK can diminish the toxic effects of glutamate treatment both in vivo and in vitro.
Although some additional work is necessary to elucidate the mechanisms that are involved in this current network, the findings from this study have identified a major role of AMPK activation by anthocyanins in the attenuation of oxidative stress through the activation of Nrf2/HO-1 signaling. Furthermore, our study suggests that anthocyanins might be a pharmacological therapeutic agent for use in combination with other potential drugs in the treatment of various neurodegenerative diseases.
Acknowledgements
Not applicable (NA).