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):
Glutamate for 2 h (Glu 2 h)
Glutamate for 3 h (Glu 3 h)
Glutamate for 4 h (Glu 4 h)
Glutamate for 4 h + anthocyanins (Glu 4 h + Anth)
Glutamate for 4 h + compound C (Glu 4 h + CC)
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)  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 , 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 . 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-AMPKTh172, 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 . 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 . 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 stainings were performed as we previously reported . 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).
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 . 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 % CO2. 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 kit (Cat. # KA1670) from Abnova (Taipei, Taiwan) was used to quantify glutamate in hippocampal brain tissue homogenates according to the manufacturer’s instruction.
The hippocampal rat brain homogenates and SH-SY5Y cell lysates of the experimental groups were evaluated using ELISA assays for total NF-
Bp65 (Life Technologies, Catalog #KHO0371) and Cyclex AMPK (Enzo Life Sciences), as per the manufacturer’s recommended protocols.
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.
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.
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.