http://www.nature.com/jcbfm/journal/v30/n12/full/jcbfm201053a.html
Epidemiologic studies have shown that foods rich in polyphenols, such as flavanols, can lower the risk of ischemic heart disease; however, the mechanism of protection has not been clearly established. In this study, we investigated whether epicatechin (EC), a flavanol in cocoa and tea, is protective against brain ischemic damage in mice. Wild-type mice pretreated orally with 5, 15, or 30 mg/kg EC before middle cerebral artery occlusion (MCAO) had significantly smaller brain infarcts and decreased neurologic deficit scores (NDS) than did the vehicle-treated group. Mice that were posttreated with 30 mg/kg of EC at 3.5 hours after MCAO also had significantly smaller brain infarcts and decreased NDS. Similarly, WT mice pretreated with 30 mg/kg of EC and subjected to N-methyl-D-aspartate (NMDA)-induced excitotoxicity had significantly smaller lesion volumes. Cell viability assays with neuronal cultures further confirmed that EC could protect neurons against oxidative insults. Interestingly, the EC-associated neuroprotection was mostly abolished in mice lacking the enzyme heme oxygenase 1 (HO1) or the transcriptional factor Nrf2, and in neurons derived from these knockout mice. These results suggest that EC exerts part of its beneficial effect through activation of Nrf2 and an increase in the neuroprotective HO1 enzyme.
Keywords:
epicatechin; heme oxygenase 1; MCAO; Nrf2; stroke; NF-E2-related factor-2
Introduction
Numerous epidemiologic studies have revealed a strong inverse correlation between ischemic heart disease and consumption of red wine and certain fruits and vegetables that contain high levels of flavonoids and other polyphenols (Simonyi et al, 2005). Flavanols (e.g., epicatechin (EC) and catechin) and their monomers/oligomers/polymers, known as procyanidins, compose a major category of secondary polyphenolic plant metabolites. Mounting evidence suggests a protective role for various polyphenols and flavonoids in cerebral ischemia (Lee et al, 2004; Lee et al, 2000; Shah et al, 2005; Shin et al, 2006; Simonyi et al, 2005). It is noteworthy also that flavanols have been shown to improve vasorelaxation (Duarte et al, 1993; Karim et al, 2000). The simple chemical structure of flavanols may interact with specific cellular and molecular targets, thereby mediating a wide range of biologic activities.
Studies have revealed that induction of phase II antioxidant enzymes through transcriptional activation is mediated through transcriptional factor Nrf2 and antioxidant-response elements (AREs). In these paradigms, polyphenols or electrophylic agents can target a specific set of genes that encode phase II enzymes, which include heme oxygenase 1 (HO1), nicotinamide adenine dinucleotide phosphate (NADPH) quinone oxidoreductase 1, and γ-glutamyl cystein ligase. These enzymes provide protection by regulating and maintaining intracellular redox states (Gong et al, 2002; Itoh et al, 2004). Of these, HO1 has been reported to have the most AREs on its promoter, making it a highly effective therapeutic target for protection against neurodegenerative diseases. It offers protection in part by degrading its pro-oxidant substrate, heme, and generating the antioxidants biliverdin and bilirubin (Doré et al, 1999). The reaction also releases one molecule of iron from the core of the heme porphyrin rings. This iron may then increase the cellular levels of ferritin, which can prevent additional generation of free radicals. Carbon monoxide (CO), another by-product of this reaction, may also have vasodilatory actions along with reported antiapoptotic and antiinflammatory properties at low concentrations; we have recently shown it to offer beneficial effects in transient ischemia (Zeynalov and Doré, 2009).
Increasing evidence suggests that HO1 can be upregulated through the Nrf2/ARE-mediated pathway (Satoh et al, 2006; Zhao et al, 2006). We have shown that Nrf2, a key regulator of the HO1 gene, has an important role in protection against cerebral ischemic stroke (Shah et al, 2007). Considering the beneficial properties of polyphenols and the possible role of the Nrf2/HO1 pathway, in this study we used in vivo and in vitro ischemic paradigms to analyze the protective properties of the flavanol (−)-EC. We hypothesized that EC would provide neuroprotection against brain injury induced by transient middle cerebral artery occlusion (MCAO) or N-methyl-D-aspartate (NMDA), and that the protection would occur through activation of the Nrf2/HO1 pathway.
Materials and methods
Animals
All animal protocols were approved by the institutional animal care and use committee of Johns Hopkins University. HO1 knockout (HO1−/−) mice were originally generated by Drs Poss and Tonegawa (Poss and Tonegawa, 1997). Mouse genotype was assessed by PCR and additionally confirmed by standard Western blot analysis. Male HO1−/−, Nrf2−/−, and wild-type (WT) mice (20 to 25 g; 7 to 8 weeks old) had access to food and water ad libitum and were housed under controlled conditions (23°C±2°C; 12-h light/dark periods). Animals were provided Teklad Global 18% Protein Rodent Diet (Harlan Holding, Inc., Wilmington, DE, USA), formula 2018S, which is a fixed-formula, autoclavable pellet chow that contains no nitrosamines and a low level of natural phytoestrogens, with 18% protein (non-animal) and 5% fat for consistent growth, gestation, and lactation. All mice were randomly assigned to the different experimental groups.
Gavage Administration of Epicatechin
Epicatechin was administered orally (per kilogram of body weight) by gavage with efforts made to minimize stress to the mice. For pretreatment studies, one dose of EC (2.5, 5, 15, or 30 mg/kg) or distilled water (control) was administered 90 minutes before MCAO; in posttreatment experiments, 30 mg/kg EC or distilled water was administered at 3.5 or 6 hours after MCAO. We selected these doses based on previous findings in which a 30-mg daily dose resulted in 7.3 ng/mg tissue (wet weight) (−)EC and 16.0 ng/mg tissue (wet weight) 3′-O-methyl-(−)EC in brain tissues (Cuevas et al, 2009; van Praag et al, 2007). In addition, a 30-mg dose of EC was observed to be neuroprotective in hippocampal toxicity caused by β-amyloid in rats (Cuevas et al, 2009).
Induction of Transient MCAO and Measurement of Infarct Size, Neurologic Deficits, Blood Gases, and Physiologic Parameters
Mice were subjected to MCAO as described previously (Shah et al, 2006). In brief, mice were anesthetized with halothane (3% initial, 1% to 1.5% maintenance) in O2 and air (80%:20%). Relative cerebral blood flow (CBF) was measured by laser-Doppler flowmetry (DRT4; Moor Instruments Ltd, Devon, UK) through a microfiber affixed to the skull over the area of parietal cortex approximately 6 mm lateral and 1 mm posterior of the bregma. Through a midline incision in the neck, a silicone-coated 7-0 Ethilon nylon filament (Ethicon, Inc., Somerville, NJ, USA) was advanced into the internal carotid artery through the severed external carotid artery to block the blood circulation to the middle cerebral artery, or Circle of Willis. A drop in CBF of 
80% was considered to be a successful occlusion. Mice not attaining the required decrease in CBF were excluded from the study. Cortical perfusion values were expressed as a percentage relative to baseline. Mice were moved to a 32°C humidity/temperature-controlled chamber to maintain a body temperature of 37°C during the 90-minute occlusion. With the mice anesthetized, reperfusion was initiated by withdrawing the filament. Mice were returned to the humidity/temperature-controlled chamber for 2 hours before being returned to their respective cages. The stroke was considered successful if no subarachnoid hemorrhage was observed, a lesion was produced, and the mouse survived to the required end point. To evaluate motor deficits, sensorimotor performance was evaluated on a 4-point neurologic deficit severity scale as described previously (Shah et al, 2006). Mice were assessed for neurologic deficits at 24 hours (pretreatment group) or 72 hours (posttreatment group) after occlusion by the following scale: 1, no deficit; 2, forelimb weakness; 3, inability to bear weight on the affected side; and 4, no spontaneous motor activity. After being tested, the mice were anesthetized, and their brains were removed and cut into 2-mm coronal sections, which were stained with 2,3,5-triphenyltetrazolium chloride (TTC; Sigma, St Louis, MO, USA). The sections were scanned individually by a video imaging system and analyzed using image analysis software (SigmaScan pro 4 and 5; Systat, Inc., Point Richmond, CA, USA). Measurements of pH, PaO2, PaCO2, and CBF were made in a separate cohort of mice.
Induction of NMDA-Induced Acute Excitotoxicity and Quantification of the Lesion Volume
Mice were administered a single oral dose of either saline or 30 mg/kg EC 90 minutes before unilateral intrastriatal NMDA injection. After body weight and rectal temperature were recorded, mice were anesthetized and placed on a stereotaxic stand. Then, 15 nmol of NMDA were injected slowly into the right striatum. The injection needle was slowly withdrawn, the hole was blocked with bone wax, and the skin was sutured. On recovery from anesthesia in a thermoregulated chamber, mice were transferred to their home cages. Throughout the experimental procedure, the rectal temperature of each mouse was monitored and maintained at 37.0°C±0.5°C. After 48 hours, mice were deeply anesthetized and transcardially perfused and fixed. Brains were equilibrated in 30% sucrose, frozen, and cut sequentially into 25-μm sections on a cryostat. The sections were stained with Cresyl Violet to estimate the lesion volume (Ahmad et al, 2006).
Neuronal Cell Cultures, Cell Survival, and Caspase Assays
Embryonic cortical neuronal cells were isolated from 17-day embryos of timed pregnant mice, and postnatal cortical neurons were obtained from 1- to 2-day-old mice, as previously detailed (Shah et al, 2007). Neurons (5 × 105 cells/well) were plated in serum-free Neurobasal medium supplemented with 1 mmol/L Glutamax (Invitrogen, Carlsbad, CA, USA) and B27 supplement onto 24-well plates coated with poly-D-lysine. Cells were maintained at 37°C in a 95% air and 5% CO2 humidified atmosphere. All experiments were performed after 14 days in vitro.
Neuronal survival was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay, as described (Shah et al, 2007). Cell viability was also measured by the lactate dehydrogenase (LDH) assay, which assesses the membrane integrity of cells. After experimental treatment, the culture medium was collected and mixed with substrate, enzyme, and dye solution. After 30 minutes of incubation in the dark, the reaction was terminated by adding 1:10 volume of 1 N hydrochloric acid. Absorbance was measured at 490 nm.
The caspase 3/7 activity of neuronal samples was measured according to the manufacturer’s instructions (Promega, Madison, WI, USA). Homogeneous caspase reagent was added to the cells, which were then incubated at room temperature for 18 hours in the dark. The fluorescence of each sample was measured at excitation and emission wavelengths of 485 and 530 nm, respectively. Experiments were repeated with at least three separate batches of cultures.
Western Blot Analysis
Cytosolic and nuclear fractions were isolated from the cortical neurons as described previously (Shah et al, 2007). Protein concentration was determined by a BCA kit (Pierce, Rockford, IL, USA). Equivalent amounts of protein per sample were resolved through sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% gels. The proteins were electrophoretically transferred to a nitrocellulose membrane, which was blocked for 1 hour at 22°C with 5% dried milk and then exposed to the primary antibody overnight at 4°C and to the secondary antibody in 5% dried milk for 1 hour at 22°C. Immunocomplexes were visualized by enhanced chemiluminescence detection (ECL; Amersham, Piscataway, NJ, USA). Each Western blot shown is a representative of at least three separate experiments.
Immunocytofluorescence Staining
After being treated with EC, neurons were permeabilized for 2 minutes with 0.5% Triton X-100 and then fixed with 3% paraformaldehyde for 20 minutes. Cells were first incubated with normal goat serum to block nonspecific binding and then with primary antibodies to NeuN (a neuronal marker; Chemicon, Temecula, CA, USA) or Nrf2 (Sigma) for 30 minutes. Cells were washed and then incubated with rhodamine-conjugated, affinity-purified donkey anti-rat IgG (H+L) and fluorescein isothiocyanate (FITC)-conjugated, affinity-purified goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) secondary antibodies for 30 minutes. Images were captured with a CoolSNAP HQ camera (Image Processing Solutions, Inc., North Reading, MA, USA) using OpenLab software (Improvision Inc., Boston, MA, USA).
Statistical Analysis
Data, expressed as mean±s.e.m., were analyzed using Student’s t-test, analysis of variance (ANOVA), or Newman–Keuls multiple range test. Neurologic deficit scores were analyzed using the nonparametric Kruskal–Wallis analysis of ranks and are presented as medians with interquartile ranges (25th and 75th percentiles). Statistical significance was set at P<0.05.
Results
Protective Effect of Epicatechin Pretreatment in Transient MCAO
Pretreatment of WT mice with EC 90 minutes before MCAO significantly and dose-dependently protected against neurologic deficit and brain injury. Infarct volumes of mice pretreated with EC were significantly smaller than those of vehicle-treated mice. Infarct volumes were 40.0%±3.2% (n=10) for the vehicle-treated group, 31.3%±2.6% (n=10; P<0.05) for the 5 mg/kg group, 30.9%±2.3% (n=9; P<0.04) for the 15 mg/kg group, and 27.6%±1.9% (n=11; P<0.002) for the 30 mg/kg group. However, there was no significant difference in the infarct volume of the 2.5 mg/kg group (37.6%±3.2%; n=12; Figure 1B). Similarly, NDS measured at 24 hours decreased with increasing concentration of EC. Vehicle-treated mice had a mean NDS of 3.1±0.1, whereas the NDS of mice that received 2.5, 5, 15, and 30 mg/kg averaged 2.8±0.2, 2.4±0.2, 2.2±0.4, and 1.8±0.2, respectively (Figure 1C). When the data were analyzed using the nonparametric Kruskal–Wallis analysis of ranks, we observed significant improvement of NDS only with 30 mg/kg pretreatment.
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