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Molecular Nutrition & Food Research
Pea (Pisum sativum L.) seed albumin extracts show anti-inflammatory effect in the DSS model of mouse colitis
Mª Pilar Utrilla1, Mª Jesus Peinado2, Raquel Ruiz2, Alba Rodriguez-Nogales1, Francesca Algieri1, Mª Elena Rodriguez-Cabezas1, Alfonso Clemente2, Julio Galvez 1,#, Luis A. Rubio 2,# 1
CIBER-EHD, Department of Pharmacology, ibs.GRANADA, Center for Biomedical
Research (CIBM), University of Granada, Granada, Spain. 2
Physiology and Biochemistry of Animal Nutrition (EEZ, CSIC), Granada, Spain.
#
Both authors contributed equally to the supervision of the study
Correspondence: Julio Gálvez, Department of Pharmacology, Center for Biomedical Research, University of Granada, Avenida del Conocimiento s/n 18100-Armilla, Granada, Spain. E-mail: [email protected] Tel: +34-958-241793. Fax: +34-958-248964.
Abbreviations: AF-PSE, albumin fraction from pea seed extract; BBI, Bowman-Birk inhibitor; CIU, chymotrypsin inhibitory units; COX, cyclooxygenase; DAI, disease activity index; DSS, dextrane sodium sulphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IBD, inflammatory bowel disease; ICAM, intercellular adhesion molecule; IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; MCP, monocyte chemotactic protein; MMP, metalloproteinase; NSP, non-starch polysaccharides; PAMPs, pathogen-associated molecular patterns; PRRs, pattern recognition receptors; PSE, pea seed extract; RT-qPCR, real time-quantitative polymerase chain reaction; TLR, toll like receptors; TNF, tumor necrosis factor; ZO zonulae occludens.
Key words: Bowman-Birk inhibitor, mouse DSS experimental colitis, Intestinal microbiota, Pea protein fractions, Pisum sativum.
Received: 08-Sep-2014; Revised: 15-Jan-2015; Accepted: 16-Jan-2015. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/mnfr.201400630. This article is protected by copyright. All rights reserved.
www.mnf-journal.com
Page 2
Molecular Nutrition & Food Research
Abstract Scope. This study investigates the preventive effects of two pea (Pisum sativum) seed albumin extracts, either in the presence (pea seed extract) (PSE) or absence (albumin fraction from pea seed extract) (AF-PSE) of soluble polysaccharides, in the dextran sodium sulfate (DSS)-induced colitis in mice. Methods and results. Male C57BL/6J mice were assigned to five groups: one non-colitic and four colitic. Colitis was induced by incorporating DSS (3.5%) in the drinking water for four days, after which DSS was removed. Treated groups received orally PSE (15 g/kg•day), or AF-PSE (1.5 g/kg•day), or pure soy Bowman-Birk inhibitor (BBI) (50 mg/kg•day), starting two weeks before colitis induction, and maintained for nine days after. All treated groups showed intestinal anti-inflammatory effect, evidenced by reduced microscopic histological damage in comparison with untreated colitic mice. The treatments ameliorated the colonic mRNA expression of different pro-inflammatory markers: cytokines, inducible enzymes, metalloproteinases, adhesion molecules, and toll-like receptors, as well as proteins involved in maintaining the epithelial barrier function. Furthermore, the administration of PSE, AF-PSE or soy BBI restored bacterial counts, partially or totally, to values in healthy mice. Conclusion. PSE and AF-PSE ameliorated DSS-induced damage to mice, being their effects due, at least partially, to the presence of active BBI.
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Molecular Nutrition & Food Research
1. Introduction Inflammatory bowel disease (IBD) comprises chronic inflammatory conditions of the small and large intestine, and include two major diseases: Crohn’s disease and ulcerative colitis. They are chronic relapsing inflammatory diseases characterized by chronic diarrhea, abdominal pain and rectal bleeding that impair the patients’ quality of life. Although their etiology is not fully understood, both genetic and environmental factors contribute to IBD risk. Thus, it is generally accepted that these intestinal conditions occur in genetically susceptible individuals who have a dysfunctional intestinal epithelium barrier with increased tight junction permeability. These individuals develop an exacerbated and uncontrolled immune response in the digestive tract towards a dysbiotic intestinal microbiota, which leads to chronic intestinal inflammation [1]. IBD affects up to 0.5% of the human population in developed countries, and numbers are increasing not only in the Western countries, but also in regions where IBD had been less common [2]. Dietary components have been reported to affect gut microbiota, and even influence gut homeostasis directly [3]. Thus, a “westernized” diet characterized by low fiber, high sugar, high animal fat, and high total protein intake, mainly from animal origin, has been proposed to confer susceptibility to IBD [4, 5]. On the contrary, high fiber, fruit and vegetable intakes have been associated with a decreased risk of developing these intestinal conditions [6]. The main goals in IBD treatment include firstly to induce the remission of symptoms during the acute flare, and secondly to control chronic inflammation in order to avoid or delay the occurrence of new flares. In consequence, down-regulation of the exacerbated immune response is essential for the treatment of IBD patients. In fact, this is the main target of the drugs used nowadays in the pharmacological therapy of intestinal inflammation, which include aminosalicylates, immunosuppressants (glucocorticoids, azathioprine, metothrexate and cyclosporine A) and biologicals (infliximab or adalimumab). However, and despite their This article is protected by copyright. All rights reserved .
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Molecular Nutrition & Food Research
efficacy, the onset of adverse effects is very frequent, which in some cases may limit the required long-term use [7]. As a consequence, the development of new therapies that combine efficacy and less side effects is a very important issue in human IBD therapy. In this context, diet has been proposed to have an important role in the treatment of IBD [8]. Dietary components may have a direct effect on gut homeostasis, either by modulating the intestinal oxidative stress that occurs in IBD, or by affecting the expression of transcription factors involved in the inflammatory response [9]. Inflammation may be also affected indirectly by diet through the modification of gut microbiota [10], as it has been proposed to occur after the intake of high-fiber diets, and after the use of probiotics and/or prebiotics [11]. These dietary modifications have been reported to restore the balance of the intestinal microbiota composition, which in intestinal inflammation is characterized by low microbial biodiversity and relative predominance of “aggressive” bacteria and an insufficient concentration of “protective” species [12, 13]. In rodent models, several studies showed that DSS treated rats had higher total counts of Escherichia coli/Shigella and Enterobacteriaceae [14, 15]. As for rats, Verma et al. found a significant decrease in the microbiota in DSS-treated mice, with specific reductions in Lactobacillus, Ruminococcus, Bacteroides and Bifidobacteria [16]. These changes are similar to those found in humans by numerous investigators, who have used molecular techniques to demonstrate changes in the composition of the mucosal-associated and fecal microbiota in patients with Crohn’s disease, ulcerative colitis, and pouchitis. Most studies demonstrate decreased microbial diversity in active IBD, increased numbers of Enterobacteriaceae, including E. coli, and decreased Firmicutes, with selectively decreased Clostridium species [17]. A comprehensive study of 190 resected tissue samples by Frank et al. [12] showed decreased numbers of the phyla Firmicutes and Bacteroidetes with concomitant increases in Proteobacteria and Actinobacteria. The decreases in Firmicutes were largely due to decreases in Clostridium XIVa and IV groups within the Lachnospiraceae subgroups.
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Molecular Nutrition & Food Research
The nutritional status of IBD patients is significantly and very frequently compromised [18] due to poor food intake (anorexia, abdominal pain, nausea, vomiting or restricted diets), decreased absorption of nutrients (reduced absorptive surface due to inflammation, resection, bypass and fistulae), increased intestinal loss due to gastrointestinal disorders, and drug–nutrient interactions with corticosteroids, sulfasalazine, immunosuppressants or antimicrobials [19]. Accordingly, a rational nutritional therapeutic strategy to treat IBD patients should include dietary modifications and nutritional supplementations to provide calories, reduce food antigenic stimulation, regulate inflammatory and immune response, and stimulate the mucosal regeneration [20], thus promoting the reduction of symptoms, and the induction and maintenance of clinical remission. In this regard, the administration of selected vegetable extracts could have a positive effect on all these functions and be beneficial for the management of human IBD. This can be the case of protein extracts containing bioactive components including Bowman-Birk inhibitors (BBI), which are abundant in all legumes including soybeans, peas, lentils and chickpeas [21]. These bioactive compounds, present in the albumin fraction of legumes, are not altered by gastric acid or proteolytic enzymes after their oral intake [22], thus reaching the large intestine in significant amounts in the active form. A growing body of evidence suggests that dietary BBI may exert anti-inflammatory properties within the gastrointestinal tract [23-25]. Soybean BBI and an extract enriched in soybean BBI (BBIC) appears to show a potent beneficial effect when assessed in the dextran sulfate sodium (DSS) model of experimental colitis [26]. Histological studies and mortality rates show that the DSS treatment induced a severe inflammatory condition in mice that was reduced in extent and severity by soybean BBIC. These preclinical studies suggest that soybean BBI might be active in inflammatory processes. In order to evaluate safety and efficacy of soybean BBI in patients with active ulcerative colitis, a randomized double-blind placebo-controlled trial was
This article is protected by copyright. All rights reserved .
www.mnf-journal.com
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Molecular Nutrition & Food Research
performed [27]. After 12 weeks of therapy, the BBIC treatment exerted a potential benefit over placebo in terms of clinical response and induction of remission in patients with active ulcerative colitis, as assessed by the Sutherland Disease Activity Index (an index that consist of four major criteria as follows: stool frequency, rectal bleeding, mucosal appearance, and physician rating of disease activity). After BBIC treatment, no adverse side-effects or apparent toxicity were observed in ulcerative colitis patients. Approximately 50% of patients responded clinically and 36% showed remission of disease; in contrast, only 29% and 7.1% on the placebo group achieved a partial response or remission, respectively. The anti-carcinogenic and anti-inflammatory properties of Bowman-Birk inhibitors have been associated with their intrinsic ability to inhibit serine proteases [24]. Indeed, the ability of soybean BBI to inhibit several serine proteases involved in inflammatory processes, such as cathepsin G, elastase and mast cell chymase has been previously proposed [25]. In addition, several human serine proteases, associated with function of inflammatory or colon cancer cells, such as proteasome or matriptase, are inhibited by soybean BBI/BBIC [28]. Similar effects could be ascribed to a pea BBI concentrate that has been previously shown to exert anti-proliferative effects on human colon cancer cells [29], most probably due to the presence of active BBI. The present study was designed to investigate the preventive effects of pea albumin proteins in the mouse model of DSS-induced colitis, and the contribution of pure soybean BBI in this experimental model of colitis.
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Molecular Nutrition & Food Research
2. Material and methods This study was carried out in accordance with the regulations and requirements of the European Union concerning the protection of animals used for scientific purposes, and the experimental protocol was approved by the Ethic Committee of Laboratory Animals of the University of Granada (Spain) (Permit Number CEEA-2010-286). 2.1. Chemicals All chemicals, including soy BBI, were obtained from Sigma-Aldrich Química S.L. (Madrid, Spain), unless otherwise stated. 2.2. Pea seed fractionation procedure The fractionation procedure to obtain both pea seed extract (PSE) and the albumin fraction from pea seed extract (AF-PSE) was previously described [30]. Briefly, pea (Pisum sativum cv. Bilbo) seeds were ground in a laboratory hammer mill, the defatted meal extracted with 0.2 M borate buffer pH 8.0 containing 0.5 M NaCl, and centrifuged (30,074 x g, 30 min, 4ºC). The supernatant was adjusted to pH 4.5 with glacial acetic acid in the cold, stirred for 30 min, centrifuged, dialyzed extensively against distilled water, and centrifuged. Part of the supernatant was freeze-dried (PSE), and another part treated with (NH4)2SO4 (608 g/L) to eliminate soluble non-starch polysaccharides (NSP), stirred for 2 h in the cold, and centrifuged. The sediment was dialyzed extensively against distilled water and freeze-dried (AF-PSE). The compositions of PSE and AF-PSE have been previously described [25]: PSE is an albumin plus soluble NSP extract containing 411.4 mg protein/g freeze-dried material; AF-PSE is an albumin extract purified from PSE which contained 813.7 mg protein/g freezedried material. The inhibitory activity of the tested compounds was evaluated in terms of their ability to inhibit chymotrypsin. One chymotrypsin inhibitor unit was defined as that which gives a reduction in absorbance at 256 nm of 0.01, relative to chymotrypsin control reactions, in 5 min in a defined assay volume of 10 ml, by using BTEE as specific substrate [30]. The Chymotrypsin Inhibitor activities of these fractions were 6.4 and 60.9 CIU/mg of freeze dried material for PSE and AF-PSE, respectively.
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2.3. DSS model of mouse colitis Male C57BL/6J mice (7-9 weeks old) obtained from Janvier (St Berthevin Cedex, France) were randomly assigned to five different groups (n=10): one non-colitic control, and four DSS colitic groups. Mice were fed ad libitum throughout the whole experimental period on AIN-93G growth purified diet (TestDiet, St. Louis, MO, USA). The colitis was induced by adding 3.5% (w/v) DSS (36-50 KDa, MP Biomedicals, Ontario, USA) in the drinking water for four days (Garrido-Mesa et al., 2011). Three of these groups were orally treated by gavage with pea seed extract (PSE) (15 g/kg·day), its purified albumin fraction (AF-PSE) (1.5 g/kg·day), or pure soy BBI (soy BBI) (50 mg/ kg·day) over the whole experimental period, starting two weeks before colitis induction. The remaining group received the vehicle (phosphate buffer saline) used to administer the products. The amounts of BBI, AF-PSE and PSE in the gavage solution were adjusted so that 2700-3000 chymotrypsin inhibitory units (CIU) of inhibitory activity, equivalent to those found in 10 ml of commercial soymilk [31], were administered per mouse and day in all treatments. Soy BBI was used as control because our initial hypothesis was that the protease inhibitory effect present in both PSE and AF-PSE was responsible for the potential anti-inflammatory effect of these fractions. The different oral treatments were maintained until the mice were killed 23 days after the beginning of the assay, that is, nine days after the first day of colitis induction with DSS. The time schedule was: 14 days before colitis induction, 4 days DSS colitis induction, and 5 days recovery period. The experiments were done twice, and the data for each experimental group were pooled and presented as a single group since they did not differ significantly. Animal body weight, the presence of gross blood in the feces and stool consistency were individually evaluated daily by an observer unaware of the treatment. Each parameter was assigned a score according to the criteria proposed previously [32] and used to calculate an average daily disease activity index (DAI) (Table 1). Once the animals were sacrificed, the caecum was aseptically extracted, and the contents collected in sterile vials and stored at 80ºC for microbiological studies. The colon was weighed, its length measured, and representative specimens (0.5 cm length) containing all wall layers were taken from the distal inflamed region and fixed in 4% buffered formaldehyde for the histological studies; equivalent colonic segments were also obtained from the non-colitic group. The remaining colonic tissue was subsequently sectioned in different longitudinal fragments to be used for RNA isolation.
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2.4. Histological studies Cross-sections were selected and embedded in paraffin. Full-thickness sections of 5 µm were obtained at different levels and stained with hematoxylin and eosin. The histological damage was evaluated by a pathologist observer, who was blinded to the experimental groups, according to the criteria previously described [33] that takes into account the presence of ulceration, infiltration, edema and the condition of the crypts (Table 2). The colonic tissue is evaluated focusing on the previous features and a score ranging from 0 (healthy tissue) to 3 or 4 (severe damage), depending on the item, is assigned to each one. The sum gives the total score for each sample. 2.5. Gene expression analysis in colonic tissue The analysis of gene expression in the colonic samples was performed by real time quantitative PCR (RT-qPCR). For this purpose total RNA from colonic samples was isolated using TRI Reagent® following the manufacturer's protocol. All RNA samples were quantified with the Thermo Scientific NanoDrop™ 2000 Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA USA) and 2 μg of RNA were reverse transcribed using oligo(dT) primers (Promega, Southampton, UK). RT-qPCR amplification and detection was performed on optical-grade 48well plates in a EcoTM Real-Time PCR System (Illumina, San Diego, CA, USA) with 20 ng of cDNA, the KAPA SYBR® FAST qPCR Master Mix (Kapa Biosystems, Inc., Wilmington, MA, USA) and specific primers at their annealing temperature (Ta) (Table 3). To normalize mRNA expression, the expression of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was measured. The mRNA relative quantitation was calculated using the ∆∆Ct method.
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2.6. Microbial analysis of the caecal contents Total DNA was isolated from freeze-dried caecal samples (40 mg) using the QIAamp DNA stool kit (Qiagen, Hilden, Germany) by following manufacturer´s instructions. Only caecal samples could be taken due to the very low amounts of colonic contents in the current experiment. In order to increase its effectiveness, the lysis temperature was increased to 95ºC and an additional step with lysozyme (10mg/mL, 37 ºC, 30 min) incubation was added. Eluted DNA was treated with RNase and the DNA concentration assessed spectrophotometrically by using a NanoDrop ND-100 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Purified DNA samples were stored at -20°C until use (Ruiz and Rubio, 2009). Bacterial log10 number of copies was determined in caecal samples by using q-PCR. The 16S rRNA gene-targeted primers and PCR conditions used in this study were as previously reported [34]. The selected bacterial groups here quantified were chosen because they represent the main groups within the intestinal contents [12]. 2.7. Statistical analysis All results are expressed as the mean ± SEM. Differences between means were tested for statistical significance using a one-way analysis of variance (ANOVA) with Tukey post-hoc test. Nonparametric data (macroscopic and microscopic scores) were analyzed by the Kruskal-Wallis test. All statistical analyses were carried out with the GraphPad Prism version 5.0 (GraphPad Software Inc., La Jolla, CA, USA), with statistical significance set at P
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Molecular Nutrition & Food Research
Pea (Pisum sativum L.) seed albumin extracts show anti-inflammatory effect in the DSS model of mouse colitis
Mª Pilar Utrilla1, Mª Jesus Peinado2, Raquel Ruiz2, Alba Rodriguez-Nogales1, Francesca Algieri1, Mª Elena Rodriguez-Cabezas1, Alfonso Clemente2, Julio Galvez 1,#, Luis A. Rubio 2,# 1
CIBER-EHD, Department of Pharmacology, ibs.GRANADA, Center for Biomedical
Research (CIBM), University of Granada, Granada, Spain. 2
Physiology and Biochemistry of Animal Nutrition (EEZ, CSIC), Granada, Spain.
#
Both authors contributed equally to the supervision of the study
Correspondence: Julio Gálvez, Department of Pharmacology, Center for Biomedical Research, University of Granada, Avenida del Conocimiento s/n 18100-Armilla, Granada, Spain. E-mail: [email protected] Tel: +34-958-241793. Fax: +34-958-248964.
Abbreviations: AF-PSE, albumin fraction from pea seed extract; BBI, Bowman-Birk inhibitor; CIU, chymotrypsin inhibitory units; COX, cyclooxygenase; DAI, disease activity index; DSS, dextrane sodium sulphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IBD, inflammatory bowel disease; ICAM, intercellular adhesion molecule; IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; MCP, monocyte chemotactic protein; MMP, metalloproteinase; NSP, non-starch polysaccharides; PAMPs, pathogen-associated molecular patterns; PRRs, pattern recognition receptors; PSE, pea seed extract; RT-qPCR, real time-quantitative polymerase chain reaction; TLR, toll like receptors; TNF, tumor necrosis factor; ZO zonulae occludens.
Key words: Bowman-Birk inhibitor, mouse DSS experimental colitis, Intestinal microbiota, Pea protein fractions, Pisum sativum.
Received: 08-Sep-2014; Revised: 15-Jan-2015; Accepted: 16-Jan-2015. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/mnfr.201400630. This article is protected by copyright. All rights reserved.
www.mnf-journal.com
Page 2
Molecular Nutrition & Food Research
Abstract Scope. This study investigates the preventive effects of two pea (Pisum sativum) seed albumin extracts, either in the presence (pea seed extract) (PSE) or absence (albumin fraction from pea seed extract) (AF-PSE) of soluble polysaccharides, in the dextran sodium sulfate (DSS)-induced colitis in mice. Methods and results. Male C57BL/6J mice were assigned to five groups: one non-colitic and four colitic. Colitis was induced by incorporating DSS (3.5%) in the drinking water for four days, after which DSS was removed. Treated groups received orally PSE (15 g/kg•day), or AF-PSE (1.5 g/kg•day), or pure soy Bowman-Birk inhibitor (BBI) (50 mg/kg•day), starting two weeks before colitis induction, and maintained for nine days after. All treated groups showed intestinal anti-inflammatory effect, evidenced by reduced microscopic histological damage in comparison with untreated colitic mice. The treatments ameliorated the colonic mRNA expression of different pro-inflammatory markers: cytokines, inducible enzymes, metalloproteinases, adhesion molecules, and toll-like receptors, as well as proteins involved in maintaining the epithelial barrier function. Furthermore, the administration of PSE, AF-PSE or soy BBI restored bacterial counts, partially or totally, to values in healthy mice. Conclusion. PSE and AF-PSE ameliorated DSS-induced damage to mice, being their effects due, at least partially, to the presence of active BBI.
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Molecular Nutrition & Food Research
1. Introduction Inflammatory bowel disease (IBD) comprises chronic inflammatory conditions of the small and large intestine, and include two major diseases: Crohn’s disease and ulcerative colitis. They are chronic relapsing inflammatory diseases characterized by chronic diarrhea, abdominal pain and rectal bleeding that impair the patients’ quality of life. Although their etiology is not fully understood, both genetic and environmental factors contribute to IBD risk. Thus, it is generally accepted that these intestinal conditions occur in genetically susceptible individuals who have a dysfunctional intestinal epithelium barrier with increased tight junction permeability. These individuals develop an exacerbated and uncontrolled immune response in the digestive tract towards a dysbiotic intestinal microbiota, which leads to chronic intestinal inflammation [1]. IBD affects up to 0.5% of the human population in developed countries, and numbers are increasing not only in the Western countries, but also in regions where IBD had been less common [2]. Dietary components have been reported to affect gut microbiota, and even influence gut homeostasis directly [3]. Thus, a “westernized” diet characterized by low fiber, high sugar, high animal fat, and high total protein intake, mainly from animal origin, has been proposed to confer susceptibility to IBD [4, 5]. On the contrary, high fiber, fruit and vegetable intakes have been associated with a decreased risk of developing these intestinal conditions [6]. The main goals in IBD treatment include firstly to induce the remission of symptoms during the acute flare, and secondly to control chronic inflammation in order to avoid or delay the occurrence of new flares. In consequence, down-regulation of the exacerbated immune response is essential for the treatment of IBD patients. In fact, this is the main target of the drugs used nowadays in the pharmacological therapy of intestinal inflammation, which include aminosalicylates, immunosuppressants (glucocorticoids, azathioprine, metothrexate and cyclosporine A) and biologicals (infliximab or adalimumab). However, and despite their This article is protected by copyright. All rights reserved .
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Page 4
Molecular Nutrition & Food Research
efficacy, the onset of adverse effects is very frequent, which in some cases may limit the required long-term use [7]. As a consequence, the development of new therapies that combine efficacy and less side effects is a very important issue in human IBD therapy. In this context, diet has been proposed to have an important role in the treatment of IBD [8]. Dietary components may have a direct effect on gut homeostasis, either by modulating the intestinal oxidative stress that occurs in IBD, or by affecting the expression of transcription factors involved in the inflammatory response [9]. Inflammation may be also affected indirectly by diet through the modification of gut microbiota [10], as it has been proposed to occur after the intake of high-fiber diets, and after the use of probiotics and/or prebiotics [11]. These dietary modifications have been reported to restore the balance of the intestinal microbiota composition, which in intestinal inflammation is characterized by low microbial biodiversity and relative predominance of “aggressive” bacteria and an insufficient concentration of “protective” species [12, 13]. In rodent models, several studies showed that DSS treated rats had higher total counts of Escherichia coli/Shigella and Enterobacteriaceae [14, 15]. As for rats, Verma et al. found a significant decrease in the microbiota in DSS-treated mice, with specific reductions in Lactobacillus, Ruminococcus, Bacteroides and Bifidobacteria [16]. These changes are similar to those found in humans by numerous investigators, who have used molecular techniques to demonstrate changes in the composition of the mucosal-associated and fecal microbiota in patients with Crohn’s disease, ulcerative colitis, and pouchitis. Most studies demonstrate decreased microbial diversity in active IBD, increased numbers of Enterobacteriaceae, including E. coli, and decreased Firmicutes, with selectively decreased Clostridium species [17]. A comprehensive study of 190 resected tissue samples by Frank et al. [12] showed decreased numbers of the phyla Firmicutes and Bacteroidetes with concomitant increases in Proteobacteria and Actinobacteria. The decreases in Firmicutes were largely due to decreases in Clostridium XIVa and IV groups within the Lachnospiraceae subgroups.
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Page 5
Molecular Nutrition & Food Research
The nutritional status of IBD patients is significantly and very frequently compromised [18] due to poor food intake (anorexia, abdominal pain, nausea, vomiting or restricted diets), decreased absorption of nutrients (reduced absorptive surface due to inflammation, resection, bypass and fistulae), increased intestinal loss due to gastrointestinal disorders, and drug–nutrient interactions with corticosteroids, sulfasalazine, immunosuppressants or antimicrobials [19]. Accordingly, a rational nutritional therapeutic strategy to treat IBD patients should include dietary modifications and nutritional supplementations to provide calories, reduce food antigenic stimulation, regulate inflammatory and immune response, and stimulate the mucosal regeneration [20], thus promoting the reduction of symptoms, and the induction and maintenance of clinical remission. In this regard, the administration of selected vegetable extracts could have a positive effect on all these functions and be beneficial for the management of human IBD. This can be the case of protein extracts containing bioactive components including Bowman-Birk inhibitors (BBI), which are abundant in all legumes including soybeans, peas, lentils and chickpeas [21]. These bioactive compounds, present in the albumin fraction of legumes, are not altered by gastric acid or proteolytic enzymes after their oral intake [22], thus reaching the large intestine in significant amounts in the active form. A growing body of evidence suggests that dietary BBI may exert anti-inflammatory properties within the gastrointestinal tract [23-25]. Soybean BBI and an extract enriched in soybean BBI (BBIC) appears to show a potent beneficial effect when assessed in the dextran sulfate sodium (DSS) model of experimental colitis [26]. Histological studies and mortality rates show that the DSS treatment induced a severe inflammatory condition in mice that was reduced in extent and severity by soybean BBIC. These preclinical studies suggest that soybean BBI might be active in inflammatory processes. In order to evaluate safety and efficacy of soybean BBI in patients with active ulcerative colitis, a randomized double-blind placebo-controlled trial was
This article is protected by copyright. All rights reserved .
www.mnf-journal.com
Page 6
Molecular Nutrition & Food Research
performed [27]. After 12 weeks of therapy, the BBIC treatment exerted a potential benefit over placebo in terms of clinical response and induction of remission in patients with active ulcerative colitis, as assessed by the Sutherland Disease Activity Index (an index that consist of four major criteria as follows: stool frequency, rectal bleeding, mucosal appearance, and physician rating of disease activity). After BBIC treatment, no adverse side-effects or apparent toxicity were observed in ulcerative colitis patients. Approximately 50% of patients responded clinically and 36% showed remission of disease; in contrast, only 29% and 7.1% on the placebo group achieved a partial response or remission, respectively. The anti-carcinogenic and anti-inflammatory properties of Bowman-Birk inhibitors have been associated with their intrinsic ability to inhibit serine proteases [24]. Indeed, the ability of soybean BBI to inhibit several serine proteases involved in inflammatory processes, such as cathepsin G, elastase and mast cell chymase has been previously proposed [25]. In addition, several human serine proteases, associated with function of inflammatory or colon cancer cells, such as proteasome or matriptase, are inhibited by soybean BBI/BBIC [28]. Similar effects could be ascribed to a pea BBI concentrate that has been previously shown to exert anti-proliferative effects on human colon cancer cells [29], most probably due to the presence of active BBI. The present study was designed to investigate the preventive effects of pea albumin proteins in the mouse model of DSS-induced colitis, and the contribution of pure soybean BBI in this experimental model of colitis.
This article is protected by copyright. All rights reserved .
www.mnf-journal.com
Page 7
Molecular Nutrition & Food Research
2. Material and methods This study was carried out in accordance with the regulations and requirements of the European Union concerning the protection of animals used for scientific purposes, and the experimental protocol was approved by the Ethic Committee of Laboratory Animals of the University of Granada (Spain) (Permit Number CEEA-2010-286). 2.1. Chemicals All chemicals, including soy BBI, were obtained from Sigma-Aldrich Química S.L. (Madrid, Spain), unless otherwise stated. 2.2. Pea seed fractionation procedure The fractionation procedure to obtain both pea seed extract (PSE) and the albumin fraction from pea seed extract (AF-PSE) was previously described [30]. Briefly, pea (Pisum sativum cv. Bilbo) seeds were ground in a laboratory hammer mill, the defatted meal extracted with 0.2 M borate buffer pH 8.0 containing 0.5 M NaCl, and centrifuged (30,074 x g, 30 min, 4ºC). The supernatant was adjusted to pH 4.5 with glacial acetic acid in the cold, stirred for 30 min, centrifuged, dialyzed extensively against distilled water, and centrifuged. Part of the supernatant was freeze-dried (PSE), and another part treated with (NH4)2SO4 (608 g/L) to eliminate soluble non-starch polysaccharides (NSP), stirred for 2 h in the cold, and centrifuged. The sediment was dialyzed extensively against distilled water and freeze-dried (AF-PSE). The compositions of PSE and AF-PSE have been previously described [25]: PSE is an albumin plus soluble NSP extract containing 411.4 mg protein/g freeze-dried material; AF-PSE is an albumin extract purified from PSE which contained 813.7 mg protein/g freezedried material. The inhibitory activity of the tested compounds was evaluated in terms of their ability to inhibit chymotrypsin. One chymotrypsin inhibitor unit was defined as that which gives a reduction in absorbance at 256 nm of 0.01, relative to chymotrypsin control reactions, in 5 min in a defined assay volume of 10 ml, by using BTEE as specific substrate [30]. The Chymotrypsin Inhibitor activities of these fractions were 6.4 and 60.9 CIU/mg of freeze dried material for PSE and AF-PSE, respectively.
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2.3. DSS model of mouse colitis Male C57BL/6J mice (7-9 weeks old) obtained from Janvier (St Berthevin Cedex, France) were randomly assigned to five different groups (n=10): one non-colitic control, and four DSS colitic groups. Mice were fed ad libitum throughout the whole experimental period on AIN-93G growth purified diet (TestDiet, St. Louis, MO, USA). The colitis was induced by adding 3.5% (w/v) DSS (36-50 KDa, MP Biomedicals, Ontario, USA) in the drinking water for four days (Garrido-Mesa et al., 2011). Three of these groups were orally treated by gavage with pea seed extract (PSE) (15 g/kg·day), its purified albumin fraction (AF-PSE) (1.5 g/kg·day), or pure soy BBI (soy BBI) (50 mg/ kg·day) over the whole experimental period, starting two weeks before colitis induction. The remaining group received the vehicle (phosphate buffer saline) used to administer the products. The amounts of BBI, AF-PSE and PSE in the gavage solution were adjusted so that 2700-3000 chymotrypsin inhibitory units (CIU) of inhibitory activity, equivalent to those found in 10 ml of commercial soymilk [31], were administered per mouse and day in all treatments. Soy BBI was used as control because our initial hypothesis was that the protease inhibitory effect present in both PSE and AF-PSE was responsible for the potential anti-inflammatory effect of these fractions. The different oral treatments were maintained until the mice were killed 23 days after the beginning of the assay, that is, nine days after the first day of colitis induction with DSS. The time schedule was: 14 days before colitis induction, 4 days DSS colitis induction, and 5 days recovery period. The experiments were done twice, and the data for each experimental group were pooled and presented as a single group since they did not differ significantly. Animal body weight, the presence of gross blood in the feces and stool consistency were individually evaluated daily by an observer unaware of the treatment. Each parameter was assigned a score according to the criteria proposed previously [32] and used to calculate an average daily disease activity index (DAI) (Table 1). Once the animals were sacrificed, the caecum was aseptically extracted, and the contents collected in sterile vials and stored at 80ºC for microbiological studies. The colon was weighed, its length measured, and representative specimens (0.5 cm length) containing all wall layers were taken from the distal inflamed region and fixed in 4% buffered formaldehyde for the histological studies; equivalent colonic segments were also obtained from the non-colitic group. The remaining colonic tissue was subsequently sectioned in different longitudinal fragments to be used for RNA isolation.
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2.4. Histological studies Cross-sections were selected and embedded in paraffin. Full-thickness sections of 5 µm were obtained at different levels and stained with hematoxylin and eosin. The histological damage was evaluated by a pathologist observer, who was blinded to the experimental groups, according to the criteria previously described [33] that takes into account the presence of ulceration, infiltration, edema and the condition of the crypts (Table 2). The colonic tissue is evaluated focusing on the previous features and a score ranging from 0 (healthy tissue) to 3 or 4 (severe damage), depending on the item, is assigned to each one. The sum gives the total score for each sample. 2.5. Gene expression analysis in colonic tissue The analysis of gene expression in the colonic samples was performed by real time quantitative PCR (RT-qPCR). For this purpose total RNA from colonic samples was isolated using TRI Reagent® following the manufacturer's protocol. All RNA samples were quantified with the Thermo Scientific NanoDrop™ 2000 Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA USA) and 2 μg of RNA were reverse transcribed using oligo(dT) primers (Promega, Southampton, UK). RT-qPCR amplification and detection was performed on optical-grade 48well plates in a EcoTM Real-Time PCR System (Illumina, San Diego, CA, USA) with 20 ng of cDNA, the KAPA SYBR® FAST qPCR Master Mix (Kapa Biosystems, Inc., Wilmington, MA, USA) and specific primers at their annealing temperature (Ta) (Table 3). To normalize mRNA expression, the expression of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was measured. The mRNA relative quantitation was calculated using the ∆∆Ct method.
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2.6. Microbial analysis of the caecal contents Total DNA was isolated from freeze-dried caecal samples (40 mg) using the QIAamp DNA stool kit (Qiagen, Hilden, Germany) by following manufacturer´s instructions. Only caecal samples could be taken due to the very low amounts of colonic contents in the current experiment. In order to increase its effectiveness, the lysis temperature was increased to 95ºC and an additional step with lysozyme (10mg/mL, 37 ºC, 30 min) incubation was added. Eluted DNA was treated with RNase and the DNA concentration assessed spectrophotometrically by using a NanoDrop ND-100 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Purified DNA samples were stored at -20°C until use (Ruiz and Rubio, 2009). Bacterial log10 number of copies was determined in caecal samples by using q-PCR. The 16S rRNA gene-targeted primers and PCR conditions used in this study were as previously reported [34]. The selected bacterial groups here quantified were chosen because they represent the main groups within the intestinal contents [12]. 2.7. Statistical analysis All results are expressed as the mean ± SEM. Differences between means were tested for statistical significance using a one-way analysis of variance (ANOVA) with Tukey post-hoc test. Nonparametric data (macroscopic and microscopic scores) were analyzed by the Kruskal-Wallis test. All statistical analyses were carried out with the GraphPad Prism version 5.0 (GraphPad Software Inc., La Jolla, CA, USA), with statistical significance set at P
