Article pubs.acs.org/JAFC
Resveratrol Attenuates High-Fat Diet-Induced Disruption of the Blood−Brain Barrier and Protects Brain Neurons from Apoptotic Insults Huai-Chia Chang,†,‡ Yu-Ting Tai,§ Yih-Giun Cherng,∥ Jia-Wei Lin,⊥ Shing-Hwa Liu,# Ta-Liang Chen,‡ and Ruei-Ming Chen*,†,‡,§ †
Graduate Institute of Medical Sciences, Taipei Medical University; Comprehensive Cancer Center of Taipei Medical University, Taipei 11031, Taiwan ‡ Anesthetics Toxicology Research Center, Taipei Medical University Hospital, Taipei 11031, Taiwan § Brain Disease Research Center, Taipei Medical University Wan Fang Hospital, Taipei 11696, Taiwan ∥ Department of Anesthesiology, Taipei Medical University Shuang Ho Hospital, Taipei 23561, Taiwan ⊥ Department of Neurosurgery, Taipei Medical University Shuang Ho Hospital, Taipei 23561, Taiwan # Institute of Toxicology, College of Medicine, National Taiwan University, Taipei 10617, Taiwan ABSTRACT: The blood−brain barrier (BBB) maintains brain microenvironment. Our previous study showed that oxidized low-density lipoprotein (oxLDL) can damage the BBB by inducing apoptosis of cerebrovascular endothelial cells. This study was aimed at evaluating the effects of resveratrol on high-fat diet-induced insults to the BBB and brain neurons. Exposure of mice to a high-fat diet for 8 weeks increased levels of serum total cholesterol (146 ± 13) and LDL (68 ± 8), but resveratrol decreased such augmentations (119 ± 6; 45 ± 8). Permeability assays showed that a high-fat diet induced breakage of the BBB (88 ± 21). Meanwhile, resveratrol alleviated this interruption (16 ± 6). Neither resveratrol nor a high-fat diet caused the death of cerebrovascular endothelial cells. Instead, exposure to a high-fat diet disrupted the polymerization of occludin and zonula occludens (ZO)-1, but resveratrol significantly attenuated those injuries. Neither a high-fat diet nor resveratrol changed the levels of occludin or ZO-1 in brain tissues. Resveratrol protected brain neurons against high-fat diet-induced caspase-3 activation and genomic DNA fragmentation. This study shows that resveratrol can attenuate the high-fat diet-induced disruption of the BBB via interfering with occludin and ZO-1 tight junctions, and protects against apoptotic insults to brain neurons. KEYWORDS: resveratrol, high-fat diet, blood−brain barrier, tight junction, brain neurons
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occludens (ZO)-1.10 The presence of the BBB results in the nearly complete separation of the CNS from the rest of the body. Loss of BBB integrity is recognized as a cause of profound brain alterations, including coma and other pathological events.11 There is a body of hazardous materials present in the blood, which can damage the BBB. Our previous study showed that oxidized low-density lipoprotein (oxLDL) can induce apoptotic insults to cerebrovascular endothelial cells through a mitochondrion-dependent mechanism.12 In apolipoprotein E-knockout mice, chronic exposure to a high-fat diet induced a dramatic extravasation of immunoglobulins, indicating alterations in BBB functioning.13 Administration of a high-fat diet led to the formation of honeycomb-like foam cells and a narrowing of the lumen of arterioles in the brain cortex.14 Dietary fat may interfere with the BBB and further affect neuronal activities. Resveratrol (trans-3,4′,5-trihydroxystilbene) has attracted considerable attention due to its abundance in grapes and grape products such as wine, a long-standing component of the diet.15 Epidemiological studies showed an inverse correlation
INTRODUCTION The content of dietary fat may play important roles in the genesis of hypertension and atherosclerotic diseases.1,2 Statistically, coronary heart disease and high blood pressure are known to be two typical modifiable factors which induce ischemic stroke, a leading cause of morbidity and mortality in Western populations.3 Atherosclerosis, a chronic inflammatory disease, can cause obstruction of the arterial lumen, then disturb blood flow characterized by low and oscillatory shear stress, ultimately leading to the complications of cardiovascular or cerebrovascular diseases.4 Endothelial dysfunction and arterial stiffness were shown to be important risk factors in the etiology of atherosclerotic disease.5,6 Previous studies showed that highfat diet-induced hypercholesterolemia is associated with endothelial dysfunction in cerebral arterioles and promotes the development of atherosclerosis, resulting in pathological changes in the central nervous system (CNS).7,8 Thus, dietary fat is closely related to the clinical prevalence of vascular diseases. The blood−brain barrier (BBB), a unique feature of the CNS, is formed by specialized cerebrovascular endothelial cells that exist in brain microvasculature.9 The main structures responsible for the barrier properties are tight junctions, constructed by tight-junction proteins such as occludin and zonula © 2014 American Chemical Society
Received: Revised: Accepted: Published: 3466
August 19, 2013 March 31, 2014 April 2, 2014 April 2, 2014 dx.doi.org/10.1021/jf403286w | J. Agric. Food Chem. 2014, 62, 3466−3475
Journal of Agricultural and Food Chemistry
Article
Serum fractions were prepared by centrifugation of blood samples. Levels of serum total cholesterol, triglycerides, and high-density lipoprotein (HDL) were measured according to commercial kits provided by Randox Laboratories (Crumlin, Ireland, UK). Amounts of LDL were calculated by the formula LDL = total cholesterol − triglycerides/5 − HDL. Assays of the BBB Integrity. Integrity of the BBB was analyzed by assessing extravasation of Evans blue dye according to a previously described method.24 Briefly, after drug treatment, 0.1 mL of Evans blue dye (2%) was intravenously injected and allowed to circulate for 1 h. Subsequently, mice were intraperitoneally anesthetized with 80 mg sodium pentobarbital per kg body weight and perfused with PBS until a colorless perfusion fluid was obtained from the right atrium. Then, the brains were removed, weighed, and photographed for colorimetric blue signals. Confocal Microscopic Analysis of Evans Blue Dye. To further determine the effects of resveratrol and a high-fat diet on the BBB integrity, a confocal microscopic analysis of Evans blue was carried out as described previously.25 Briefly, after drug treatment, Evans blue dye was intravenously injected into mice. Following perfusion, the brain tissues were removed and sliced for the analysis of fluorescent signals excited by Evans blue in the brains using a confocal laser scanning microscope (model FV500, Olympus, Tokyo, Japan). Images were acquired and quantified using FLUOVIEW 4.0 software (Olympus). Confocal Microscopic Analyses of Occludin and ZO-1 Polymerization, Factor VIII, and Activated Caspase-3. Polymerization of occludin and ZO-1 in brain tissues was recognized by specific antibodies, and these were visualized using confocal microscopy following a previously described method.12 Briefly, after drug administration, brain tissues were removed, collected, and sliced. Tissues were fixed with a fixing reagent (acetone/methanol, 1:1) at −20 °C for 10 min. Following rehydration, the brains were incubated with 0.2% Triton X-100 at room temperature for 15 min. Rabbit polyclonal antibodies used in this study were respectively generated against human occludin and ZO-1 proteins (Invitrogen, Carlsbad, CA, USA). Immunodetection of occludin and ZO-1 in mouse brain tissues was carried out at 4 °C overnight. After washing, slices were sequentially reacted with the second antibodies and biotin-SP-conjugated AffiniPure goat antirabbit immunoglobulin G (IgG) (Jackson ImmunoResearch, West Grove, PA, USA) at room temperature for 1 h. After washing, the third antibody with Cy3-conjugated streptavidin (Jackson ImmunoResearch) was added to the brain slices and reacted at room temperature for 30 min. Localization of the BBB in brain tissues was recognized using an antibody against factor VIII protein (BioGenex, San Francisco, CA, USA). Activated caspase-3 was immunodetected using a polyclonal antibody against human cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA). A confocal laser scanning microscope (Olympus) was utilized for sample observation. The excitation wavelength was set to 568 nm, while a 585-nm long-pass filter was used to collect the emitted light. Images were acquired and quantified using FLUOVIEW software (Olympus). Immunodetection of Occludin and ZO-1. After drug treatment, the brains were removed and homogenized with lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1% NP-40 supplemented with 1 mM PMSF, 1 μM aprotinin, 1 μM leupeptin, 1 mM Na2VO4, and 1 mM NaF). Protein concentrations were quantified using a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins
between the intake of wine and death resulting from coronary heart disease.16 Since then, a lot of different targets have been found to be influenced by resveratrol treatment.17,18 Previous studies showed that red wine polyphenols contribute to improvements in endothelial dysfunction and ultimately reduce the blood pressure and associated cardiac and vascular diseases.19,20 Our previous studies have shown that resveratrol can protect cerebrovascular endothelial cells from oxLDLinduced apoptotic insults through Lox-1 receptor-mediated signal-transducing events.21,22 But to date, much less is known about the effects of the consumption of red wine on high-fat diet-influenced BBB damage and subsequent neuronal damage. Therefore, in this study, we attempted to evaluate the effects of resveratrol on high-fat diet-induced disruption of the BBB and its possible mechanisms.
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MATERIALS AND METHODS Chemicals. Resveratrol, Evans blue dye, sodium pentobarbital, methanol, and Triton-X were purchased from Sigma (St. Louis, MO, USA). The purity of resveratrol used in this study was 99%. Acetone and ethanol were bought from Mallinckrodt Baker (Paris, KY, USA). Phosphate-buffered saline buffer (PBS) was prepared by mixing 0.14 M sodium chloride, 2.6 mM potassium chloride, 8 mM sodium phosphate, and 1.5 mM potassium phosphate. Sodium chloride, potassium chloride, sodium phosphate, and potassium phosphate were purchased from Sigma. Animals. All procedures were performed according to the National Institutes of Health Guidelines for the Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Taipei Medical University, Taipei, Taiwan. Male ICR mice weighing 20−25 g were purchased from the Animal Center of the College of Medicine, National Taiwan University, Taipei, Taiwan. Before the experiments began, mice were allowed to acclimatize for 1 week in their animal quarters with air conditioning and an automatically controlled photoperiod of 12 h of light daily. Animals were allowed free access to rodent laboratory chow (Purina Mills, St. Louis, MO, USA). Experimental Design. Animals were randomly divided into (1) normal diet-, (2) normal diet + resveratrol-, (3) highfat diet-, and (4) high-fat diet + resveratrol-treated groups. The normal standard diet contained 5.1% fat (unsaturated fatty acids), 23.5% protein, and 50.3% complex carbohydrates. The composition of the high-fat diet included 20.1% fat (saturated fatty acids), 18.3% protein, and 51.2% carbohydrates. Our previous studies have shown that resveratrol at 10 μM can protect cerebrovascular endothelial cells from oxLDL-induced apoptotic insults.21,22 Provinciali et al. reported that when a mouse drank 1 mg/L resveratrol daily, the serum concentrations of resveratrol were about 4 μM.23 Thus, in the present study, resveratrol was dissolved in methanol and diluted with tap water to a concentration of 2.5 mg/L. The respective dose of resveratrol daily exposed to a mouse was 0.4 mg/kg body weight. Mice were administered a high-fat diet and resveratrol for 8 weeks. Control animals received 0.02% ethanol. The tap water with resveratrol was refreshed every day. The amounts of water and food consumed and the body weights were measured. At the end of the experiments, animals were sacrificed. Blood samples were collected and centrifuged to obtain serum. The brains were removed, weighed, and collected. Analysis of Serum Cholesterol and LDL. After drug treatment, ICR mice were sacrificed and the blood was collected. 3467
dx.doi.org/10.1021/jf403286w | J. Agric. Food Chem. 2014, 62, 3466−3475
Journal of Agricultural and Food Chemistry
Article
(50 μg/well) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes as described previously.21 Membranes were blocked with 5% nonfat milk at 37 °C for 1 h. Occludin and ZO-1 proteins were immunodetected using polyclonal antibodies (Invitrogen). β-Actin was immunodetected using a mouse monoclonal antibody against mouse β-actin (Sigma) as an internal control. Intensities of the immunoreactive protein bands were determined using an UVIDOCMW vers. 99.03 digital imaging system (UVtec, Cambridge, UK). Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay. A TUNEL assay was performed using the In Situ Cell Death Detection Kit (Roche Diagnostics, Mannheim, Germany), following the manufacturer’s instructions. In brief, brain sections were fixed in 4% paraformaldehyde for 20 min and incubated with 0.1% Triton X-100 for 10 min. A TUNEL labeling reaction with fluoresceinconjugated dUTP was carried out at 37 °C for 60 min. These samples were then double-stained with a monoclonal antibody that specifically recognizes a vertebrate nervous system- and neuron-specific nuclear protein (NeuN; Millipore, Billerica, MA, USA) and endothelium-specific factor VIII, and observed under a confocal microscope (Olympus) as described previously.22 Statistical Analyses. The statistical significance of differences between the control and drug-treated groups was evaluated using Student’s t-test, and differences were considered statistically significant at p values of
