Xanthohumol Modulates Inflammation, Oxidative Stress, and

Article pubs.acs.org/jnp

Xanthohumol Modulates Inflammation, Oxidative Stress, and Angiogenesis in Type 1 Diabetic Rat Skin Wound Healing Raquel Costa,† Rita Negraõ ,† Inês Valente,‡ Â ngela Castela,† Delfim Duarte,† Luísa Guardaõ ,§ Paulo J. Magalhaẽ s,‡ José A. Rodrigues,‡ Joaõ T. Guimaraẽ s,†,⊥ Pedro Gomes,∥ and Raquel Soares*,† †

Department of Biochemistry (U38-FCT), Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal § Animal House Department, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal ⊥ Department of Clinical Pathology, São João Hospital Center, 4200-319 Porto, Portugal ∥ Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal ‡

ABSTRACT: Type 1 diabetes mellitus is responsible for metabolic dysfunction, accompanied by chronic inflammation, oxidative stress, and endothelium dysfunction, and is often associated with impaired wound healing. Phenol-rich food improves vascular function, contributing to diabetes prevention. This study has evaluated the effect of phenol-rich beverage consumption in diabetic rats on wound healing, through angiogenesis, inflammation, and oxidative stress modulation. A wound-healing assay was performed in streptozotocin-induced diabetic Wistar rats drinking water, 5% ethanol, and stout beer with and without 10 mg/L xanthohumol (1), for a five-week period. Wounded skin microvessel density was reduced to normal values upon consumption of 1 in diabetic rats, being accompanied by decreased serum VEGF-A and inflammatory markers (IL-1β, NO, Nacetylglucosaminidase). Systemic glutathione and kidney and liver H2O2, 3-nitrotyrosine, and protein carbonylation also decreased to healthy levels after treatment with 1, implying an improvement in oxidative stress status. These findings suggest that consumption of xanthohumol (1) by diabetic animals consistently decreases inflammation and oxidative stress, allowing neovascularization control and improving diabetic wound healing.

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Additionally, multiple diabetic-associated conditions contribute to the disrupted healing capacity, including hyperglycemia, suppressed immune ability, chronic inflammation, hypoxia, and reactive oxygen species (ROS) generation.6 Cellular damage, disrupted redox signaling, and metabolic cascades are induced by ROS and involved in diabetic complications.7 Emerging evidence reveals that consumption of natural phenols is important in disease prevention, particularly in inflammation and oxidative stress associated diseases, such as diabetes.8 Xanthohumol (1) has multiple biological activities including anticarcinogenic, antibacterial, anti-inflammatory, and antioxidant properties,9 among others, and interest in this compound has increased due to its health-promoting properties. Xanthohumol may thus be a promising compound to act as a modulator of angiogenesis and related processes, namely, inflammation and oxidative stress.10

iabetes mellitus is a chronic metabolic disease with a high incidence and prevalence, affecting 170 million people worldwide.1 This number is expected to increase to pandemic proportions in the near future, being therefore a major burden to the healthcare system. Diabetes is characterized by a chronic hyperglycemic state, which constitutes a major risk factor for vascular disease development.2 In diabetic patients, a vascular paradox is often observed, characterized by the coexistence of increased vascularization in tissues, such as in the retina and kidney, and impaired vascularization in others, as in the skin and heart.3 Diabetes is associated with impaired wound healing, a major contributor to morbidity, and is characterized by exacerbated granulation tissue formation, delayed cellular infiltration, reduced collagen, and ineffective neovascularization.4 Furthermore, chronic inflammation and oxidative stress play important roles in diabetic vascular complications.5 Chronic inflammation is thought to impair regeneration and alter the synthesis of molecules that promote effective resolution of scars. © 2013 American Chemical Society and American Society of Pharmacognosy

Received: April 16, 2013 Published: November 7, 2013 2047

dx.doi.org/10.1021/np4002898 | J. Nat. Prod. 2013, 76, 2047−2053

Journal of Natural Products

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mellitus, in which impaired wound healing is a major complication and morbidity cause, as observed in the diabetic foot. In the current study, the main aim consisted in investigating the effect of consumption of xanthohumol (1) in the improvement of wound healing, as this compound is a potent antioxidant and anti-inflammatory agent,9b,11 two features exacerbated in type 1 diabetes and strongly implicated in diabetic vascular complications.12 Angiogenesis in Diabetic Rats Treated with Xanthohumol (1). Knowing that diabetic wound healing depends on angiogenesis, a wound-healing assay was established in the dorsal skin of type 1 diabetic rats. Seven days after skin injury, macroscopic evaluation of the external surface of the injured area showed that wound healing was delayed in diabetic animals as compared to healthy controls. Histological evaluation revealed that this delay in wound healing was accompanied by an increase in dermal granulation tissue (data not shown). Microvessel density (MVD) in the wound region of diabetic

Herein, the effect of the consumption of a 1-supplemented beer was assessed in the development of diabetic complications in a skin wound-healing assay, by evaluating angiogenesis, inflammation, and oxidative stress parameters.

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RESULTS AND DISCUSSION Considerable attention has been given to the potential of natural compounds for local or oral administration that may improve the wound-healing process. The use of phenolic molecules is of paramount interest in diseases such as diabetes

Figure 1. Angiogenesis evaluation using a skin wound-healing assay. Over a period of four weeks the following beverages were supplied ad libitum to Wistar rats: water (control, control of healthy animals; STZ-control, control of diabetic animals); 5% ethanol (STZ-ethanol, diabetic rats); Superbock stout beer (STZ-stout, diabetic rats); Superbock stout beer supplemented with 10 mg 1/L (STZ-stout with 1, diabetic rats). Longitudinal incisions were created on the dorsal surface of the rats, and beverages were supplied for more seven days. Then, wound tissue was collected for angiogenesis evaluation. (A) Wound tissue section micrographs were immunostained using anti-von-Willebrand factor (vWF) for evaluation of blood vessels (magnification: 200×). (B) Quantification of the blood vessels present in three tissue sections, for each animal, and normalized to the total area of the tissue section. Results are presented as means ± SEM (4 ≤ n ≤ 6). *p ≤ 0.05 vs control; #p ≤ 0.05 vs STZ-control; δp ≤ 0.05 vs STZethanol. 2048



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Figure 2. Evaluation of angiogenic and inflammatory markers in the plasma of skin-injured Wistar rats, after five weeks of beverage supplied ad libitum to the rats: Control, healthy rats treated with water; STZ-control, diabetic rats treated with water; STZ-ethanol, diabetic rats treated with 5% ethanol; STZ-stout, diabetic rats treated with Superbock stout beer; STZ-stout with 1, diabetic rats treated with Superbock stout beer supplemented with 10 mg 1/L. (A) VEGF-A levels; (B) IL1β levels; (C) N-acetylglucosaminidase (NAG) activity; (D) NO levels. Results are presented as means ± SEM (4 ≤ n ≤ 6). *p ≤ 0.05 vs control; #p ≤ 0.05 vs STZ-control; δp ≤ 0.05 vs STZ-ethanol; γp ≤ 0.05 vs STZ-stout.

Inflammation Mediators in Diabetic Rats upon Consumption of Xanthohumol (1). Since inflammation plays a relevant role in diabetes and 1 exerts anti-inflammatory effects, three markers of inflammation were further determined in rat serum: IL1β, NAG enzyme, and NO. IL1β serum levels were augmented significantly in diabetic controls when compared to healthy rats (20.94 ± 2.17 pg/mL vs 13.55 ± 1.35 pg/mL) (Figure 2B). A significant reduction in IL1β to normal serum levels was observed in STZ-stout supplemented with 1 (13.36 ± 0.92 pg/mL). Quantification of NAG enzyme, expressed in active macrophages, exhibited a similar profile, increasing in diabetic controls (372.7 ± 14.67 nmol/mL vs 280.7 ± 11.4 nmol/mL in healthy controls) (Figure 2C). Consumption of beer supplemented by 1 resulted in a significant reduction in NAG serum levels relative to other alcoholic beverages consumed (Figure 2C). NO was also increased significantly in the STZ-control (9.34 ± 0.18 μM) and in rats treated with STZ-ethanol (8.45 ± 0.20 μM) when compared to healthy controls (5.54 ± 0.62 μM) (Figure 2D). Consumption of stout beer with or without 1 led to a reduction in NO serum levels relative to healthy control levels. In the diabetic animals, consumption of supplemented beer led to the reduction of the inflammatory mediators (IL-1β, NO, and NAG) to the levels observed in the nondiabetic group. This reversal on the diabetic pro-inflammatory state by 1 may be explained by a reduced recruitment and activation of inflammatory cells and therefore improved resolution of the inflammatory phase, with a facilitated progression to the next phases. 17 Xanthohumol (1) acts through inflammatory cascades9b,10b,18 and inhibition of the Akt and NF-κB signaling pathways in the endothelium.19 Hence, beer supplemented with 1 likely favors the control of pro-inflammatory diseases, such as type 1 diabetes mellitus. Systemic and Local Oxidative Stress. Oxidative stress may play a key role in the pathogenesis of diabetic vascular

rats was reduced in comparison to healthy animals (Figure 1A and B). Stout containing 1 resulted in a significant decrease in MVD in diabetic rats (6.80 ± 0.23 vessels/mm2 vs 10.93 ± 0.46 vessels/mm2 in STZ-controls, 12.90 ± 2.08 vessels/mm2 in STZ-ethanol, and 8.12 ± 0.30 vessels/mm2 in STZ-stout) (Figure 1B). These findings confirm the antiangiogenic effect of 1 previously reported by our group.10 Diabetic rats presented reduced neovascularization in the injured area when compared to healthy animals. In the group treated with 1 MVD was even further reduced, in contrast to the STZ-ethanol group. Given the role of VEGF-A as a pro-angiogenic factor, the levels of this growth factor were quantified further in rat sera. VEGF-A serum levels revealed a pattern identical to the one found for MVD in the wound (Figure 2A). Accordingly, consumption of phenol compounds resulted in a significant reduction in systemic VEGF-A levels relative to healthy controls and STZ-ethanol (Figure 2A). An identical profile was obtained in the serum quantification of the main angiogenic promoter, VEGF-A. In diabetic patients, there is impaired wound healing accompanied by local reduced VEGFA levels. The present results show that the number of vessels in the wound areas of diabetic rats was decreased, and also reduced systemic VEGF-A was observed, in agreement with the study of Stepanovic and collaborators.13 In contrast, in this work, the increased levels of VEGF-A on ethanol consumption were reduced upon beer intake and that of beer supplemented by 1. Morrow and colleagues showed that moderate consumption of ethanol promotes vascularization through the Notch and angiopoietin 1 signaling pathways.14 Other researchers demonstrated that there is increased angiogenesis after consumption of 8 g/kg ethanol for 10 weeks.15 Compound 1 and other phenolic compounds are able to inhibit VEGF-A expression,16 explaining the observed inversion of the ethanol pro-angiogenic effect after supplemented beer consumption. 2049



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Figure 3. Evaluation of local and systemic oxidative stress of skin-injured Wistar rats, after five weeks of beverage supplied ad libitum to the rats: Control, healthy rats treated with water; STZ-control, diabetic rats treated with water; STZ-ethanol, diabetic rats treated with 5% ethanol; STZ-stout, diabetic rats treated with Superbock stout beer; STZ-stout with 1, diabetic rats treated with Superbock stout beer supplemented with 10 mg 1/L. (A) Chromatographic detection of plasma reduced (GSH) and oxidized (GSSG) glutathione; (B) kidney and liver release of H2O2. Results are means ± SEM (4 ≤ n ≤ 6) and are expressed as percentage of control. *p ≤ 0.05 vs control; #p ≤ 0.05 vs STZ-control.

liver and the kidney in diabetic control animals. In agreement with this observation, Banerjee and collaborators reported increased H2O2 levels in urine and suggested its use as a systemic marker of oxidative stress associated with several pathologies.22 The stout and especially the 1-supplemented beer led to a reduction of the H2O2 concentration to values similar to those found in the healthy group, which suggests a parallel “reversal” effect previously described for inflammation. 3-NT levels were then assessed in order to investigate the oxidative stress-derived protein damage. Expression of liver 3NT was significantly increased in diabetic controls when compared to healthy animals (5.52 ± 1.64 vs 1.95 ± 0.29), confirming the presence of oxidative protein damage in diabetic rats (Figure 4A). A significant reduction in 3-NT expression was found in liver from the STZ-stout and stout with 1 groups (1.38 ± 0.11) (Figure 4A). An identical pattern was found in the kidney, where 3-NT was augmented in STZ-C comparatively to healthy controls (3.76 ± 0.57 vs 2.06 ± 0.23). However, this value was reduced upon consumption of stout beer in both the presence (1.99 ± 0.30) and absence of 1 (1.51 ± 0.22) (Figure 4B). Irreversible oxidative protein damage was also evaluated by performing protein carbonylation assays by immunoblotting. Diabetic control animals exhibited significant increased protein carbonylation in the liver, in comparison to healthy control rats (13.80 ± 1.12 vs 4.63 ± 0.91) (Figure 4C). Interestingly, beer consumption alone (4.02 ± 0.96) or with 1 (4.67 ± 1.48) showed a reduction of hepatic protein carbonylation toward normal healthy levels, as compared with diabetic controls and STZ-ethanol (10.87 ± 0.69). Similarly, kidney protein carbonylation was increased significantly in STZ-controls (6.47 ± 0.45) relative to healthy animals (3.00 ± 0.23). This increase was reversed to healthy

complications, such as impaired wound healing. Therefore, glutathione levels were examined in rat serum. Although no significant differences were observed among the distinct groups, a tendency toward increasing levels in reduced/oxidized glutathione (GSH/GSSG) ratio was found in diabetic rats ingesting ethanol, stout, and stout supplemented with 1 (Figure 3A), indicating a decrease in oxidative stress, which accompanied the angiogenic and inflammatory pattern observed in rat serum. ROS, in turn, significantly contribute to the pathophysiology of type 1 diabetes mellitus through a wide variety of mechanisms,6 thereby decreasing wound-healing ability. The effect of 1-enriched beer was evaluated on the oxidative stress level. The consumption of 1-supplemented beer increased systemic reduced glutathione (GSH), one of the most important cellular antioxidant defenses.20 The GSH/GSSH ratio was also slightly increased in animals drinking beer. Moderate consumption of ethanol stimulates antioxidant defenses and increases reductase glutathione activity,21 which increases the GSH/GSSG ratio. Next, the release of H2O2, a ROS abundantly produced in oxidative stress conditions, was assessed in the kidney and liver. As compared to healthy controls, diabetic rats ingesting water (STZ-C) showed a significant increase in H2O2 release in the tissues evaluated (Figure 3B). In the liver, although there was no significant difference, a tendency toward an increase in H2O2 release was observed in rats consuming alcohol (STZ-ethanol, STZ-stout, STZ-stout with 1) as compared to healthy controls (Figure 3B). Conversely, in the kidney, H2O2 release decreased in rat groups with STZ-ethanol, STZ with stout, and STZ with stout supplemented with 1, when compared to diabetic controls (Figure 3B). Interestingly, this reduction was significant in the kidney of rats that drank stout supplemented with 1 (Figure 3B). An increased production of H2O2 was also observed in the 2050



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Figure 4. Evaluation of protein oxidative damage by Western blotting of skin-injured Wistar rats, after five weeks of beverage supplied ad libitum to the rats: Control, healthy rats treated with water; STZ-control, diabetic rats treated with water; STZ-ethanol, diabetic rats treated with 5% ethanol; STZ-stout, diabetic rats treated with Superbock beer; STZ-stout with 1, diabetic rats treated with Superbock stout beer supplemented with 10 mg 1/ L. 3-NT expression in (A) liver and (B) kidney. Protein carbonylation in (C) liver and (D) kidney. Results are means ± SEM (4 ≤ n ≤ 6) and are expressed as percentage of control. *p ≤ 0.05 vs control; #p ≤ 0.05 vs STZ-control; δp ≤ 0.05 vs STZ-ethanol.

Altogether, these findings suggest that protein damage was decreased to normal healthy levels after stout beer consumption and even further with stout supplemented with 1 intake. The observation made that antioxidant treatment of diabetic animals induced a decrease of carbonylated proteins corroborated these results.7b,24

levels in the STZ-ethanol, STZ-stout, and STZ-stout with 1 rats. Ingestion of stout and stout with 1 further resulted in a significant decrease in protein carbonylation relative to STZ controls and STZ-ethanol, suggesting an antioxidant effect (Figure 4D). Corroborating these findings, liver and kidney 3NT overexpression and protein carbonylation in diabetic controls, two markers of protein oxidation, were observed.7a,23 2051



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instructions, in a microplate reader (Thermo Fisher Scientific, Waltham, CA, USA). Local Oxidative Stress Assessment in Rat Tissues. The rate of H2O2 production by liver, kidney, and muscle was determined using an Amplex Red Hydrogen Peroxide Assay kit (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol.19 Fresh tissues were cut into square pieces (3 mm3) and incubated at 37 °C in Krebs-HEPES buffer (in mM: NaCl 118, KCl 4.5, CaCl2 2.5, MgCl2 1.20, K2HPO4 1.2, NaHCO3 25.0, Na-HEPES 25.0, and glucose 5; pH 7.4) for 90 min with constant shaking and oxygenation. Fluorescence intensity was measured in a multiplate reader (Spectromax Gemini; Molecular Devices, Sunnyvale, CA, USA). Tissue Protein Extraction. Kidney and liver tissue samples were processed for total protein extraction by mechanical homogenization in a Polytron with RIPA buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40 (IGEPAL); 0.25% Na-deoxycholate; 150 mM NaCl; 1 mM EDTA) containing protease inhibitors (1 mM PMSF; 1 μg/mL aprotinin and leupeptin each) and phosphatase inhibitors (1 mM Na3VO4 and 1 mM NaF). Lysates were incubated on ice for 60 min (vortexing every 10 min). After centrifugation (20.000g, 30 min, 4 °C), supernatants were collected and quantified with the BCA protein kit (Pierce, Thermo Scientific, Cramlington, UK). Western Blotting for 3-Nitrotyrosine (3-NT) Detection. Twelve micrograms of total protein was separated by electrophoresis in a 10% SDS-PAGE. Membranes were incubated with rabbit antihuman 3-NT (1:1000 dilution; clone 1A6, Upstate, Millipore, MA, USA) polyclonal antibody, followed by incubation with the respective secondary horseradish-peroxidase (HRP)-coupled antibody (1:2000; Santa Cruz Biotechnology, Heidelberg, Germany). The detection was performed using enhanced chemiluminescence (ECL kit; Amersham Biosciences, UK). After stripping, membranes were incubated with a rabbit anti-human β-actin antibody (1:3000; Abcam, Cambridge, UK). Mean relative intensities of 3-NT protein expression were quantified by densitometry (Vision Works LS software; UVP Inc., Upland, CA, USA) and normalized with the signal intensity of β-actin. Protein Carbonylation. Immunoblot quantification of protein carbonyl groups was performed. Briefly, equal amounts of protein samples (10 μg) were incubated with 10 mM 2,4-dinitrophenylhydrazine in 10% trifluoroacetic acid (TFA; VWR, Radnor, PA, USA) to produce carbonyl groups in 2,4-dinitrophenylhydrazone. Proteins were separated on 10% SDS-PAGE and transferred onto a Hybond nitrocellulose membrane. Then, membranes were balanced in TBS containing 20% methanol (Panreac, Barcelona, Spain) and washed in 10% TFA. Immunoblotting was carried out using a rabbit anti-DNP polyclonal antibody (1:5000, Sigma-Aldrich) followed by incubation with the goat anti-rabbit HRP-secondary antibody. Mean relative intensity of protein carbonylation was performed as described above. Glutathione Quantification in Rat Plasma. The chromatographic detection of the GSH/GSSG ratio was performed with a glutathione (GSH/GSSG) HPLC kit (Immunodiagnostik, Bensheim, Germany) using a reversed-phase HPLC VWR-Hitachi Elite LaChrom System (VWR International Gmb, Darmstadt, Germany), according to the manufacturer’s instructions. The chromatograms were analyzed using Agilent EZChrom Elite 3.3.2 software (Agilent Technologies, Santa Clara, CA, USA). Nitric Oxide (NO) Release. The detection of total NO levels, based on total amount of its metabolites, nitrate and nitrite, was performed in serum using the Griess reagent methods as previously described.10b N-Acetyl-ß-D-glucosaminidase Assay. NAG is a lysosomal enzyme highly expressed in activated macrophages, being an important inflammatory marker. The reaction was carried out as previously described.10b IL-1β Measurement. Interleukin (IL) 1β was quantified in rat plasma by ELISA using a commercial IL-1β kit (Biosource, Nivelles, Belgium), performed according to the manufacturer’s instructions in a spectrophotometer plate reader (Thermo Fisher Scientific). Statistical Analyses. Each determination was performed in at least three independent experiments. Statistical significance of difference groups was evaluated by ANOVA followed by the Bonferroni test. A

This work demonstrates that stout beer supplemented with 1 improved skin wound healing resolution, by consistently decreasing inflammation and oxidative stress and also allowing for a more controlled neovascularization. Altogether, these findings show that the systemic and local anti-inflammatory, antioxidant, and angiostatic properties of chronic consumption of stout beer are not as pronounced as those of stout beer supplemented with 1 by diabetic rats. The approach used in this study points to a possible preventive role of the beer supplemented by 1, targeting simultaneously inflammation, oxidative stress, and vascular processes, three intermingled processes playing relevant roles in diabetes.

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EXPERIMENTAL SECTION

General Experimental Procedures. Xanthohumol (1) was provided by Hopsteiner (Xantho-flav pure, Mainburg, Germany) and purified by the present coauthors from REQUIMTE, attaining 95% purity, as determined by HPLC. All other chemicals were obtained from standard commercial suppliers and were of analytical grade quality. The beer used in this study was Superbock stout beer (UNICER, Porto, Portugal), with the following phenol composition: polyphenols: 255 ± 13 mg/L; 1: traces (μM). The final concentration of 1 in the supplemented stout beer was 10 mg/L. Animals. Eight week-old healthy male Wistar rats were used in this study. Animals were maintained under controlled conditions of temperature (23 ± 5 °C), humidity (35 ± 5%), and 12 h light/dark cycles and were allowed access to regular chow diet and beverages ad libitum. Body weight, daily food intake, and beverage consumption were monitored. Beverages were renewed every two days and were kept in dark bottles to avoid degradation. All animal care and procedures were in accordance with the Portuguese Act 1005/92 (number 3, iii) and European Community guidelines (86/609/EEC) for the use of experimental animals. Induction of Type 1 Diabetes. Type 1 diabetes was induced by an intraperitoneal injection of 50 mg/kg of streptozotocin (STZ; Sigma-Aldrich, Sintra, Portugal) dissolved in 0.1 M citrate buffer (pH 4.5). Forty-eight hours after STZ injection, the diabetic condition was confirmed by measuring blood glucose content. Levels above 250 mg/ dL were included in this study.25 Experimental Design. Animals were divided randomly into five groups (n = 6): group 1 (normal rats drinking water), group 2 (diabetic rats drinking water), group 3 (diabetic rats treated with 5% ethanol), group 4 (diabetic rats treated with stout beer), and group 5 (diabetic rats treated with stout beer supplemented with 10 mg 1/L). After four weeks, a wound was inflicted in the dorsal skin. After general anesthesia with 0.1 mg/mL ketamine (Merial Laboratories, Barcelona, Spain) and 1 mg/mL domitor (Pfizer, New York, NY, USA), the dorsal skin was shaved and cleaned with 70% ethanol. A 1.5 cm thick longitudinal incision was created, and rats were examined daily for wound-healing progression. At day 7 postwounding, the animals were euthanized, and the blood was collected to assess inflammatory, biochemical, and oxidative stress markers. Skin wound tissue and the liver and the kidney were fixed in 10% neutral-buffered formalin, dehydrated, and embedded in paraffin. Three-micrometer-thick tissue sections were used for histological and immunohistochemistry analysis. Microvessel Density Evaluation. Immunohistochemistry was performed in three-micrometer-thick formalin-fixed skin tissues with anti-von Willebrand factor (vWF) antibody, as previously described.10a The numbers of vessels were counted in three tissue sections for each animal and normalized to the total area of the wound tissue section. Any positive-staining endothelial cell or cluster that was separated from adjacent microvessels was considered an individual vessel.26 Plasma VEGF-A Quantification. Plasma concentrations of VEGF-A were quantified using the Quantikine Rat VEGF ELISA kit (R&D Systems, Abingdon, UK) in accordance with the manufacturer’s 2052



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(16) (a) Stoclet, J. C.; Chataigneau, T.; Ndiaye, M.; Oak, M. H.; El Bedoui, J.; Chataigneau, M.; Schini-Kerth, V. B. Eur. J. Pharmacol. 2004, 500, 299−313. (b) Dell’Eva, R.; Ambrosini, C.; Vannini, N.; Piaggio, G.; Albini, A.; Ferrari, N. Cancer 2007, 110, 2007−2011. (17) Kagawa, S.; Matsuo, A.; Yagi, Y.; Ikematsu, K.; Tsuda, R.; Nakasono, I. Leg. Med. (Tokyo) 2009, 11, 70−75. (18) Biesalski, H. K. Curr. Opin. Clin. Nutr. Metab. Care 2007, 10, 724−728. (19) Albini, A.; Dell’Eva, R.; Vene, R.; Ferrari, N.; Buhler, D. R.; Noonan, D. M.; Fassina, G. FASEB J. 2006, 20, 527−529. (20) Masella, R.; Di Benedetto, R.; Vari, R.; Filesi, C.; Giovannini, C. J. Nutr. Biochem. 2005, 16, 577−586. (21) (a) Assuncao, M.; Santos-Marques, M. J.; Monteiro, R.; Azevedo, I.; Andrade, J. P.; Carvalho, F.; Martins, M. J. J. Agric. Food Chem. 2009, 57, 6066−6073. (b) Roig, R.; Cascon, E.; Arola, L.; Blade, C.; Salvado, M. J. Life Sci. 1999, 64, 1517−1524. (22) Banerjee, D.; Madhusoodanan, U. K.; Nayak, S.; Jacob, J. Clin. Chim. Acta 2003, 334, 205−209. (23) Nystrom, T. EMBO J. 2005, 24, 1311−1317. (24) Chis, I. C.; Ungureanu, M. I.; Marton, A.; Simedrea, R.; Muresan, A.; Postescu, I. D.; Decea, N. Diab. Vasc. Dis. Res. 2009, 6, 200−204. (25) Wei, M.; Ong, L.; Smith, M. T.; Ross, F. B.; Schmid, K.; Hoey, A. J.; Burstow, D.; Brown, L.. Heart, Lung Circ. 2003, 12, 44−50. (26) Soares, R.; Balogh, G.; Guo, S.; Gartner, F.; Russo, J.; Schmitt, F. Mol. Endocrinol. 2004, 18, 2333−2343.

difference between experimental groups was considered significant with a confidence interval of 95%, whenever p ≤ 0.05.

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AUTHOR INFORMATION

Corresponding Author

*(R. Soares) Tel/Fax: 351 225513624. E-mail: raqsoa@med. up.pt. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to UNICER for generously providing the stout beer used in this study. This study was partially funded by Fundaçaõ para a Ciência e Tecnologia, Portugal (FCT) (SFRM/BD/41888/2007; PTDC/SAU-OSM/102083/2008; SFRH/BD/69719/2010; PEst-C/EQB/LA0006/2011; PEstOE/SAU/UI0038/2011), COMPETE, ERAB (41/06), and iBeSa (Institute for Beverages and Health, Portugal; P 10-08).

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Xanthohumol Modulates Inflammation, Oxidative Stress, and

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