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Apple polyphenols extract (APE) improves colon damage in a rat model of colitis
Digestive and Liver Disease 44 (2012) 555–562
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Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld
Alimentary Tract
Apple polyphenols extract (APE) improves colon damage in a rat model of colitis夽 Giuseppe D’Argenio a,b,∗ , Giovanna Mazzone a , Concetta Tuccillo c , Maria T. Ribecco a , Giulia Graziani d , Antonietta G. Gravina c , Sergio Caserta e,f , Stefano Guido e,f , Vincenzo Fogliano d , Nicola Caporaso a , Marco Romano c a
Gastroenterology Unit, Department of Clinical and Experimental Medicine, Federico II University of Naples, Italy Institute of Protein Biochemistry, CNR, Naples, Italy c Gastroenterology Unit, Department of Clinical and Experimental Medicine, and CIRANAD, Second University of Naples, Italy d Department of Food Science, Federico II University, Naples, Italy e Department of Chemical Engineering, Federico II University, Naples, Italy f CEINGE – Advanced Biotechnologies, Naples, Italy b
a r t i c l e
i n f o
Article history: Received 7 October 2011 Accepted 17 January 2012 Available online 28 February 2012 Keywords: Apple polyphenols Calpain Rat colitis Tissue transglutaminase
a b s t r a c t Background and aim: Searching for alternative therapies that are effective, safe and less expensive of those currently used for ulcerative colitis, we investigated the efficacy of a polyphenol extract from apple in rat colitis. Methods: Rats with trinitrobenzensulphonic acid-induced colitis were treated daily with rectal administration of apple polyphenols 10−4 M for 14 days. COX-2, TNF-␣, tissue transglutaminase and calpain in colon mucosa samples were assessed by reverse transcription-polymerase chain reaction and western blot analyses. To ascertain the role of tissue transglutaminase in mucosal healing, wounded rat fibroblasts were incubated with cystamine (a tissue transglutaminase activity inhibitor). Results: Colitis was associated with increased COX-2, TNF-␣, calpain, and tissue transglutaminase mRNA. The protein expression of COX-2, TNF-␣ and calpain was increased whilst tissue transglutaminase was decreased. Apple extract treatment reduced the severity of colitis (p < 0.05) and restored all the considered biomarkers at the baseline level. Apple polyphenols reduced the degradation of tissue transglutaminase protein occurring through calpain action. Apple polyphenols-treated wounded fibroblast recovered within 24 h showing intense immunoreactivity for tissue transglutaminase. Conclusion: The efficacy of apple extract is mediated by its effects on COX-2 and TNF-␣. The unbalance between calpain and tissue transglutaminase may play a role in colonic damage and future therapeutic interventions in ulcerative colitis can target this mechanisms. © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction Ulcerative colitis (UC) is a chronic idiopathic inflammatory disease of the gastrointestinal tract that affects the large intestine causing frequent episodes of diarrhoea, abdominal pain, bloody stools and weight loss over the medium to long term. UC is characterized by marked lymphocyte and neutrophil infiltration confined to the colonic mucosa with neutrophilic infiltration of colonic crypts accompanied by ulceration and necrosis of epithelial cells
夽 This study has been supported by the University Research Fund, project code: A.10080.2.106.107. ∗ Corresponding author at: Dip. di Medicina Clinica e Sperimentale, Gastroenterologia, Facoltà di Medicina, Università Federico II, Via Pansini 5, 80131 Napoli, Italy. Tel.: +39 081 7462706. E-mail address: [email protected] (G. D’Argenio).
[1]. Treatment includes the use of topical or systemic drugs [2], but their beneficial effect is limited [3]. Corticosteroids affect the short-term treatment of acute inflammation, but their use in maintenance therapy is limited by the high incidence of side effects [4]. Sulphasalazin and 5-ASA exert beneficial effects only in moderately active disease and are used to prevent recurrence [5,6]. Studies of UC in humans do not allow to investigate the early stages of the disease, therefore, to better understand the pathogenic mechanisms responsible for initiation and maintenance of chronic bowel inflammation, animal models are needed that also allow us to test new treatments. Recently, much attention has been paid to naturally derived products from fruit or vegetables which may beneficially affect a number of pathologic conditions of the gastrointestinal tract. In particular, it has been demonstrated that dietary anti-oxidants play a crucial role in maintaining gastrointestinal homeostasis
1590-8658/$36.00 © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dld.2012.01.009
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by counteracting the gastrointestinal mucosa damaging effects of reactive oxygen species (ROS) [7]. The importance of nutrition with a significant intake of fresh fruits and vegetables has long been an assumption of medicine: the preventive and protective role of these foods is due to significant presence of phytochemicals which, on one hand prevent oxidative damage brought about by ROS inside the gastro intestinal tract, and the other hand stimulate the cell to “self-defence” stimulating intracellular mechanisms that counteract oxidative stress and inflammation [8]. Phenolic compounds represent an important part of phytochemicals in the diet and, in particular, the phenolic components of apple represent 22% of total polyphenols in the diet of U.S. consumers [9]. Polyphenols are secondary metabolic products of many plants characterized by the presence of an aromatic ring with one or more hydroxyl substituents. Phenolic compounds act both as antioxidants and as elicitors of different metabolic pathways [10]. Tissue transglutaminase (tTG) is a transamidating enzyme that catalyses a calcium-acyl transfer reaction between two different peptides [11]. Various transglutaminases isoforms have been claimed to play a role in colon inflammation [12]. Calpains are a family of proteases that require both calcium and a reduced environment for activity. The two homologous isoforms, -calpain and m-calpain, have been classically distinguished on differences in their in vitro calcium requirements for half-maximal activity with, -calpain requiring significantly less calcium than m-calpain [13]. The homologous isoforms of calpains have been shown to proteolyse several protein substrates. tTG was found to be an excellent in vitro substrate of -calpain, generating a large breakdown product [14]. However, in contrast to epidermal TG, cleavage of tTG by calpain resulted in inactivation. A recent study demonstrated that the degree of colitis caused by injection of DNBS was substantially reduced by treatment of rats with calpain inhibitor I [15], but the mechanisms of the anti-inflammatory effect of calpain inhibitor I are not entirely clear. Previous data indicated that tTG may be an endogenous substrate of calpain, and that calpain may be involved in the regulation of tTG transamidating activity in situ by modulating intracellular tTG levels [16]. We have previously demonstrated that apple polyphenols extract (APE) significantly counteracts gastric damage induced by ROS, indomethacin or acetyl salicylic acid in rats and that this protective effect is mainly due to the anti-oxidant activity of APE [7,17]. Therefore, this study was designed to investigate whether APE may accelerate healing of 2,4,6-trinitrobenzene-sulphonic acid (TNBS)-induced colitis in the rat. Also, in order to gain a better insight into the mechanism of action of APE, we investigated its effects on protein and gene expression of TNF-␣ and COX-2 and evaluated whether APE affected cell migration, the first step involved in gastrointestinal mucosal repair, in an in vitro wound healing model. Finally, we studied whether the balance between tTG and calpain might be involved in TNBS-induced colitis and in the therapeutic effect exerted by APE.
2. Materials and methods
2.2. TNBS-induced colitis in rats and APE treatment Male Wistar rats, weighing 200 ± 21 g, were obtained from Harlan Italy and were housed in a temperature-controlled environment with a 12 h light-dark cycle, and given free access to regular laboratory chow diet and water for at least one week. All animals received human care, and the study protocol was approved by the Committee of Laboratory Animals at Federico II University according to institutional guidelines. Rats then were given a single enema of TNBS solution (20 mg in 50% ethanol–water), via a rubber catheter inserted 8-cm lengths from the anus under light anaesthesia [18]. To evaluate the effect of APE on TNBS-induced colitis, 16 rats were divided in two groups (n = 8 in each group) and treated with rectal administration of 1 mL/die of APE 10−4 M or vehicle daily for 14 days, respectively. A further group of animals consisting of normal rats was used as controls. The weight of distal colon was measured, and inflammatory changes and ulceration in the distal colon were evaluated macroscopically (evaluating on a 0–5 scale hyperemia, fibrosis, ulcers, necrosis) [18]. Mucosa was separated from submucosal layer by gently scraping, and tissue specimens were than kept at −80 ◦ C until assayed. 2.3. Histology Colon biopsies were fixed in 10% formalin and embedded in paraffin, haematoxylin and eosin-stained sections eliminate were assessed histologically by light microscopy performed by a pathologist in a blinded fashion. According to previous reports [19,20], four parameters, each scored on a 0–5 scale of severity, were considered: extent of ulceration, submucosal infiltration, crypt abscesses, and wall thickness. The final total histological score (from 0 to 20) was determined by adding the sum of each assigned score and determined a rating of slight (1–5) moderate (6–10) or severe (11–20) colonic inflammation. 2.4. Protein extract preparations Frozen rat colon mucosa samples were homogenised in RIPA lyses buffer (0.1% sodium dodecyl sulphate (SDS), 0.5% deoxycholate, 1% Nonidet, 100 mM NaCl, 10 mM Tris–HCl (pH 7.4)) containing a protease inhibitor cocktail (Sigma, St Louis, Missouri, USA), 0.5 mM dithiotreitol, and 0.5% phenylmethylsulphonyl fluoride. After 30 min at 4 ◦ C, tissue lysates were clarified by centrifugation at 14,000 rpm for 10 min at 4 ◦ C. The cleared tissue lysates were collected and stored at −80 ◦ C and protein concentration of each sample was determined by Bradford assay (Coomassie brilliant blue protein assay; Bio-Rad, Melville, NY, USA). The antibodies used in this study were as follows: (1) goat polyclonal IgG anti-COX-2 (UPSTATE 07-693); (2) goat polyclonal IgG anti TNF-␣ (sc 1349 Santa Cruz Biotechnology); (3) mouse monoclonal IgG anti-tTG (CUB 7402 Neo-Markers); (4) mouse monoclonal anti Calpain-1 (sc 58323 Santa Cruz Biotechnology). The secondary antibodies were biotinylated anti goat IgG (Vector BA9500) and biotinylated anti mouse IgG (Vector BA200) as appropriate.
2.1. Preparation of apple extract and identification of phenolic compounds
2.5. Western blot analysis
Freeze dried apple flesh of “Annurca” variety was extracted with methanol as previously described [7]. Identification of phenolic compounds present in APE was performed by HPLC–ESI-MS analysis as described elsewhere [9,17]. The sum of the concentration of all compounds present in the extract was performed and the molecular weight of the catechin (CAT) was considered to obtain the molar concentration of APE. Therefore APE concentration was arbitrarily expressed in CAT equivalent.
Total protein extracts were subjected to SDS–PAGE (10% and 7% polyacrylamide) under reducing conditions. After electrophoresis, proteins were transferred to nitrocellulose membrane (pure nitrocellulose membrane, 0.45 m Bio-Rad Laboratories); complete transfer was assessed using pre-stained protein standards (Invitrogen LC5925). To block non-specific binding sites the membranes were treated for 1 h with blocking solution: 5% milk in TNT (10 mM Tris pH 8, 150 mM NaCl and 0.05% Tween-20), and then
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were incubated overnight at 4 ◦ C with the primary antibody: (1) anti-COX-2 (diluted 1:500); (2) anti-TNF-␣ (diluted 1:200) in TNT (0.05% Tween-20) 20% foetal calf serum (FBS); (3) anti-tTG (diluted 1:2000) in TNT (0.1% Tween-20) 5% milk; (4) anti-calpain (diluted 1:1000) in TNT (0.05% Tween-20) 20% FBS. After washing with TBS, membranes were incubated 2 h (at room temperature) with the appropriate biotinylated secondary antibody. Immunoreactive proteins were detected by development with the ABC Vectastain kit (Vector Laboratories, Burlingame, California, USA) according to the manufacturer’s instructions. 2.6. RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) analysis Total RNA was isolated from rat colon mucosa using TRIzol (Invitrogen, NY, USA) extraction reagent, according to the standard acid–guanidium–phenol–chloroform method [21]. RT-PCR analysis was performed on total RNA as described previously [22,23]. First-strand complementary DNA was prepared using 200 units of reverse transcriptase (Supertranscript RT, Life Technologies, Inc., Gaithersburg, MD), 1 g of total RNA as template, and 10 pmol/L of random hexamers in the presence of 0.1 mmol/L dithiothreitol, 0.5 mmol/L deoxynucleotide triphosphate-litium salt (Pharmacia, Milan, Italy), and 20 units of RNase inhibitor (Promega, Madison, USA). The reaction profile was 37 ◦ C × 10 min, followed by 42 ◦ C × 60 min. To control for contamination by genomic DNA, all RNA samples were run in duplicate with or without addition of reverse transcriptase. The sequences of primers used (PRIMM, Italy) and the annealing temperature are shown in Table 1 (supplementary file). PCR included the following steps: an initial denaturation at 95 ◦ C for 5 min; 35 amplification cycles consisting of denaturation at 94 ◦ C for 1 min; primer annealing for 1 min (see Table 1 for primer specific ◦ C) and extension at 72 ◦ C for 1 min. In all cases, the final extension step was at 72 ◦ C for 10 min. GAPDH primers were added after 6 cycles for TNF-␣, COX-2 and tTG and after 4 cycles for calpain. To test for contamination by genomic DNA, samples were run in duplicate with or without the addition of reverse transcriptase. PCR products were separated on 1.8% agarose gel electrophoresis and visualized by ethidium bromide staining. Sizes of the amplified fragments were estimated by comparison with migration of the 100-kb ladder molecular-weight marker (Life Technologies, Inc., Gaithersburg, MD). 2.7. NIH 3T3 fibroblast wound model The NIH 3T3 murine fibroblast scratch model is an established model system for detecting tTG. In this model, catalytically active tTG is hypothesized to provide mechanical stability to tissues by cross-linking ECM proteins after a wound has been inflicted [24]. In addition, fibroblasts are non-transformed cells as occurs for the other cell lines available for the migration studies. Fibroblasts were plated in a 24-multiwell plastic chamber and grown for 3–4 days in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 1% antibiotic–antimycotic solution (Life Technologies Inc., Gaithersburg, Maryland, USA) at 37 ◦ C in a humidified atmosphere of 5% CO2 , with a media change every 2 days, until the semi-confluence phase. Twenty-four hours before the wound, cells were starved in DMEM 0.1% FBS. Small scratches were made in the monolayer with a 0.1–10 L pipette tip, and the cells were incubated either with the specific tTG inhibitor cystamine (33 g/mL) or APE (10−4 M) and monitored for 30 h by time-lapse-microscopy (TLM). The experiments were done in triplicate. TLM experiments were performed by using an inverted microscope (Zeiss), with a long working distance 10× objective in phase
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contrast. The microscope is equipped with motorized stage and focus for automated sample positioning, and an incubator consisting of a plastic enclosure kept at 37 ± 0.1 ◦ C by a warm air flux, tuned by a PID controller. Furthermore a small amount of air, premixed with 5% CO2 , is blown trough a bubbling column for humidity saturation, and fed to a small chamber surrounding the sample to prevent water evaporation and pH changes in the cell culture medium. The images are captured by a cooled monochromatic CCD video camera (Hamamatsu). All the equipment are driven by a Labview macro that iteratively acquires images of selected regions every 30 min, for overall 30 h. After the experiment the cells were washed 3× with PBS and then fixed with cold (−20 ◦ C) methanol for 10 min. The cells were washed with PBS 2× for 5 min, blocked with 1% BSA in PBS for 5 min at room temperature, and then washed 2× using PBS. CUB 7402 Anti tTG antibody was diluted 1:500 in blocking buffer and incubated with the cells for 60 min at room temperature. The cells were washed 4× with PBS then incubated for 1 h at room temperature with the biotinylated secondary antibody (1:1000). Images of selected regions were acquired in a phase contrast microscope. 2.8. Statistical analysis As appropriate, the Student’s t test or the analysis of variance (ANOVA) followed by Bonferroni’s post hoc test were used for statistical evaluations. 3. Results 3.1. Effects of APE on TNBS colitis The rectal administration of TNBS induced the appearance of visible colonic damage extending to all the colon length with evident colonic wall thickness (Fig. 1A). Rectal administration of 1 mL/die of APE 10−4 M daily for 14 days ameliorated macroscopic injury to the rat colon (Fig. 1B). TNBS extensively damaged the superficial epithelium and caused intense inflammation and loss of epithelial cell layer in the region of the colonic mucosa (Fig. 1C). In contrast, APE treatment significantly decreased microscopic injury caused by induced colitis decreasing the extent of inflammatory infiltrate and the damage to the surface area (Fig. 1D). Quantitatively, APE treatment caused an approximate 60% decrease in the extent of macroscopic injury compared with control untreated rats (p < 0.05, Fig. 2A) and reduced the extent of microscopic injury by approximately 55% (p < 0.05, Fig. 2B). 3.2. RT-PCR To evaluate whether TNBS caused alterations in selected genes supposed to be involved in colitis inflammation and healing, we examined mRNA levels of COX-2, tTG, calpain, and TNF-␣. TNBS colitis was associated with increased mRNA expression of all tested genes (Fig. 3), whilst APE administration prevented the colitis associated increase of COX-2, TNF-␣, calpain and tTG (Fig. 3). 3.3. SDS–PAGE and western blot Administration of TNBS induced a significant increase in COX-2, TNF-␣, and calpain in inflamed tissue (Fig. 4, lanes 3–6). In contrast a strong reduction of tTG protein transcript was observed (Fig. 4, lanes 3–6). Treatment with APE counteracted TNBS-induced upregulation of COX-2, TNF-␣ and calpain protein expression as well as the TNBS-induced down-regulation of tTG protein expression (Fig. 4, lanes 7–9).
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Fig. 1. Macroscopic and microscopic appearance of rat colon with trinitrobenzensulphonic acid-induced colitis (A and C), and after treatment with apple polyphenols extract (B and D). In rats with colitis, the colon appears thickened and inflamed (A) and an abundant inflammatory infiltrate is also present (C). The treatment with apple polyphenols significantly reduced both wall thickness and mucosal inflammation (B and D).
3.4. Localization, activation and inhibition of tTG in a cell wound model In Fig. 5A a panel of images acquired during TLM experiments is reported. Control untreated, cystamine-treated and APE-treated samples are compared at 1, 20 and 30 h after the wound. In the supplementary material the complete movie of the TLM experiment is reported as avi file. The experiment demonstrated that APE significantly stimulated wound closure compared to control untreated and cystamine-treated wounded monolayers. In Fig. 5B a quantification of the difference in the wound closure between the three samples is shown. Cell density normalized respect to the initial value was plotted over the time of the experiment for control untreated, cystamine-treated and APE-treated samples. Each point is the average of three independent measurements on different samples. Data show that APE induces a
significant increase in cell density growth compared with control untreated and also compared with cystamine-treated samples. Furthermore, at immunohistochemistry, wounded monolayers showed a high density of tTG protein 30 h after APE treatment around the wounded area (Fig. 6A) compared to cells treated with cystamine (Fig. 6B) or untreated fibroblasts (Fig. 6C). 4. Discussion UC is a chronic, relapsing disease that causes inflammation and ulcerations of the colonic mucosa with a variable extent and severity. The aetiology of UC remains essentially unknown but the results from many studies in humans and animal models suggest that it is related to an abnormal immune response in the gastrointestinal tract, possibly associated with genetic and environmental, mainly microbial factors [25]. Several drugs have been mainly used for the
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Fig. 2. Macroscopic (A) and microscopic (B) damage of rat colon was scored as described in Section 2. Comparison between trinitrobenzensulphonic acid-induced colitis controls and apple polyphenols treated rats shows the efficacy of the treatment in reducing the mucosal damage. *p < 0.05 compared with controls.
treatment and maintenance of remission of UC, but the side effects or toxicity of these drugs are a major clinical problem [26]. For these reasons, products of natural origin that cover the gap between nutrients and pharmaceuticals (i.e. nutraceuticals) have become an alternative option in addition to the conventional therapies that are used to treat UC [27]. Growing evidences suggest that dietary fish oil and curcumin given together modulate colonic cytokinetics and gene expression in dextran sodium sulphate-treated mice [28], and that bovine colostrum is effective in the treatment of distal colitis [29]. In the present study, we demonstrated that APE has an anti-inflammatory effect on colonic injury provoked by TNBS in rats. TNBS-induced colitis is a well-established model for colitis that is phenotypically similar to human colon inflammation
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Fig. 3. Agarose gel electrophoresis of representative reverse transcriptionpolymerase chain reaction products of cyclooxygenase-2 (COX-2), tissue transglutaminase (tTG), calpain, and tumour necrosis factor-alpha (TNF-␣). Glyceraldehyde3-phosphate dehydrogenase (GAPDH) was chosen an internal control. The lanes are representative of different groups of treatments: normal rats, trinitrobenzensulphonic acid-induced colitis (TNBS) as controls, and rats with colitis treated with apple polyphenols extract (TNBS + APE).
[30]. A single rectal administration of TNBS, leads to colonic epithelial lesions and inflammation characterized by the presence of neutrophils and macrophages within damaged colon. APE was administered at a concentration that was demonstrated to be effective in rat gastric lesion and gastric cell line damaged by ROS [7]. The decrease in TNBS colitis induced by APE was accompanied by a lower weight loss of rats and a partial restoration of colon thickening, which is an indirect assessment of colon inflammation. Microscopic analysis confirmed clinical results, showing a protective action of APE measured as a decrease in ulceration, conservation of epithelial crypts, and a reduction of cell infiltration.
Fig. 4. Western blot analysis using antibodies against cyclooxygenase-2 (COX-2), tumour necrosis factor-alpha (TNF-␣), calpain, and tissue transglutaminase (tTG). In this context, COX-2, TNF-␣, and calpain were all increased by trinitrobenzensulphonic acid-induced colitis (TNBS), whilst tTG content was reduced, possibly degraded by calpain. Apple polyphenols extract (APE) reduced the protein content of COX-2, TNF-␣, and calpain and restored tTG protein level.
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Fig. 5. (A) Images acquired during time-lapse microscopy experiments, where control untreated, cystamine-treated and apple polyphenols extract (APE)-treated samples are compared at 1, 20 and 30 h after the wound. Quantification of the difference in the wound closure between the three samples is reported in (B).
Previous studies demonstrated that various transglutaminase isoforms, and in particular the tTG, are involved in remodelling matrices in the presence of continuous wound healing such as in UC [12]. Up-regulation of proteinases such as calpain, which is able to hydrolyse tTG [31], resulted in worsening of rat colitis [15]. Therefore, calpain-induced tTG degradation may account for the apparent discrepancy between increased tTG gene up-regulation and decreased tTG protein expression observed in the TNBS rats group. Accordingly, our results also showed that APE treatment inhibited TNBS-induced up-regulation of tTG expression and to a larger extent calpain gene expression. Moreover, it determined a reduction of tTG protein degradation as showed by western blot. This result suggests an important role for APE which is able to increase tTG availability for the colonic repairing process. Furthermore, the results of this study indicated that treatment with APE was able to reduce colon inflammation. The abnormal presence of inflammatory cells within the mucosa produces increased concentrations of inflammatory cytokines such as TNF-␣ [32], and
pro-inflammatory cytokines induced expression of genes associated with inflammation, such as COX-2. APE administration was able to inhibit TNF-␣ and COX-2 both at protein and mRNA levels, suggesting that APE could be useful in the prevention of relapse in patients with quiescent UC. On the other hand, TNF-␣ has been described as a key molecule in UC pathogenesis, and a monoclonal antibody against this molecule has proven to be effective in the treatment of moderate to severe UC [33]. This cytokine, recruiting leukocytes to inflammatory sites, stimulates monocytes and vascular endothelial cells to express cytokines, induces the cascade effects for other cytokines, and finally results in inflammatory lesions in tissues [34]. COX-2 is over-expressed in IBD and experimental colitis and COX-2 [35]. Whilst substantial evidence indicates exacerbation of IBD following the use of NSAIDs (i.e. nonselective COX inhibitors), it is not clear whether selective COX-2 inhibitors are safer than NSAIDs in IBD patients [36]. Also, the role of COX-2 inhibitors in rat colitis is controversial [35,37,38]. In our study, we confirm
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Fig. 6. Immunohistochemistry of NIH 3T3 cells fixed in methanol. Cells monolayer was wounded and incubated up to hours with cystamine, a tissue transglutaminase inhibitor, or with apple polyphenols extract (APE). The cells were than incubated with anti-tTG antibody: a marked staining of cells treated with APE (A), cystamine (B), control (C) was observed.
up-regulation of COX-2 in TNBS-induced colitis [38] and show that acceleration of mucosal healing induced by APE was associated with a decreased expression of COX-2. However, we cannot establish whether the decreased colonic damage in APE-treated animals is, at least in part, due to an APE-mediated decrease in pro-inflammatory COX-2 expression or is mediated by a COX-2 independent mechanism. The second block of experiments using an vitro wound healing model demonstrated that APE was able to modulate cell function through induction of chemotaxis. In fact, APE significantly influenced the restitution in a model of wounded fibroblast monolayers with a recruitment of large number of cells in the damaged area. Interestingly, these cells also expressed high levels of tTG, again confirming the role of this enzyme in the healing process. Moreover, the role of tTG in APE-related effect on restitution was further emphasized by the decrease in APE-induced stimulation of cell migration by the tTG inhibitor cystamine. In conclusion, the results of this study show that (a) APE significantly decreases gross and histological colon damage in TNBS colitis; (b) the clinical improvement was associated to a decrease in the up-regulation of COX-2, TNF-␣ and calpain induced by TNBS; (c) tTG protein concentration is decreased by TNBS at the protein level and this effect is counteracted by APE, likely through calpain inhibition; (d) APE accelerates in vitro healing of wounded fibroblast monolayers and this is associated with tTG over-expression; (e) the tTG inhibitor cystamine, counteracts APE-induced stimulation of cell migration. In this framework it can be hypothesized that the protection exerted by APE against TNBS colitis is mediated by its effects on COX-2, TNF-␣, and tTG. Moreover, data demonstrated that the unbalance between calpain and tTG may play a role in TNBSinduced colonic damage and future therapeutic interventions in UC can target this mechanisms.
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[27] Langmead L, Dawson C, Hawkins C, et al. Antioxidant effects of herbal therapies used by patients with inflammatory bowel disease: an in vitro study. Aliment Pharmacol Ther 2002;16:197–205. [28] Qian Jia, Ivanov I, Zlatomir Z, et al. Dietary fish oil and curcumin combine to modulate colonic cytokinetics and gene expression in dextran sodium sulphate-treated mice. Br J Nutr 2011;106:519–29. [29] Khan Z, Macdonald C, Wicks AC, et al. Use of the ‘nutriceutical’, bovine colostrum, for the treatment of distal colitis: results from an initial study. Aliment Pharmacol Ther 2002;16:1917–22. [30] Wirtz S, Neurath MF. Mouse models of inflammatory bowel disease. Adv Drug Deliv Rev 2007;59:1073–83. [31] Zhang J, Rodney P, Guttmann, et al. Tissue transglutaminase is an in situ substrate of calpain: regulation of activity. J Neurochem 1998;71:240–7. [32] Sandborn WJ, Targan SR. Biologic therapy of inflammatory bowel disease. Gastroenterology 2002;122:1592–608. [33] Ferrante M, Vermeire S, Katsanos KH, et al. Predictors of early response to infliximab in patients with ulcerative colitis. Inflamm Bowel Dis 2007;13:123–8. [34] Myers KJ, Murthy S, Flanigan A, et al. Antisense oligonucleotide blockade of tumor necrosis factor-alpha in two murine models of colitis. J Pharmacol Exp Ther 2003;304:411–24. [35] Martín AR, Villegas I, Alarcón de la Lastra C. The COX-2 inhibitor, rofecoxib, ameliorates dextran sulphate sodium induced colitis in mice. Inflamm Res 2005;54:145–51. [36] Kefalakes H, Stylianides TJ, Amanakis G, et al. Exacerbation of inflammatory bowel diseases associated with the use of nonsteroidal anti-inflammatory drugs: myth or reality? Eur J Clin Pharmacol 2009;65:963–70. [37] Cuzzocrea S, Mazzon E, Serraino I, et al. Celecoxib, a selective cyclo-oxygenase-2 inhibitor reduces the severity of experimental colitis induced by dinitrobenzene sulfonic acid in rats. Eur J Pharmacol 2001;431:91–102. [38] Paiotti APR, Miszputen SJ, Oshima CTF, et al. Effect of COX-2 inhibitor after TNBS-induced colitis in wistar rats. J Mol Hist 2009;40:317–24.
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Alimentary Tract
Apple polyphenols extract (APE) improves colon damage in a rat model of colitis夽 Giuseppe D’Argenio a,b,∗ , Giovanna Mazzone a , Concetta Tuccillo c , Maria T. Ribecco a , Giulia Graziani d , Antonietta G. Gravina c , Sergio Caserta e,f , Stefano Guido e,f , Vincenzo Fogliano d , Nicola Caporaso a , Marco Romano c a
Gastroenterology Unit, Department of Clinical and Experimental Medicine, Federico II University of Naples, Italy Institute of Protein Biochemistry, CNR, Naples, Italy c Gastroenterology Unit, Department of Clinical and Experimental Medicine, and CIRANAD, Second University of Naples, Italy d Department of Food Science, Federico II University, Naples, Italy e Department of Chemical Engineering, Federico II University, Naples, Italy f CEINGE – Advanced Biotechnologies, Naples, Italy b
a r t i c l e
i n f o
Article history: Received 7 October 2011 Accepted 17 January 2012 Available online 28 February 2012 Keywords: Apple polyphenols Calpain Rat colitis Tissue transglutaminase
a b s t r a c t Background and aim: Searching for alternative therapies that are effective, safe and less expensive of those currently used for ulcerative colitis, we investigated the efficacy of a polyphenol extract from apple in rat colitis. Methods: Rats with trinitrobenzensulphonic acid-induced colitis were treated daily with rectal administration of apple polyphenols 10−4 M for 14 days. COX-2, TNF-␣, tissue transglutaminase and calpain in colon mucosa samples were assessed by reverse transcription-polymerase chain reaction and western blot analyses. To ascertain the role of tissue transglutaminase in mucosal healing, wounded rat fibroblasts were incubated with cystamine (a tissue transglutaminase activity inhibitor). Results: Colitis was associated with increased COX-2, TNF-␣, calpain, and tissue transglutaminase mRNA. The protein expression of COX-2, TNF-␣ and calpain was increased whilst tissue transglutaminase was decreased. Apple extract treatment reduced the severity of colitis (p < 0.05) and restored all the considered biomarkers at the baseline level. Apple polyphenols reduced the degradation of tissue transglutaminase protein occurring through calpain action. Apple polyphenols-treated wounded fibroblast recovered within 24 h showing intense immunoreactivity for tissue transglutaminase. Conclusion: The efficacy of apple extract is mediated by its effects on COX-2 and TNF-␣. The unbalance between calpain and tissue transglutaminase may play a role in colonic damage and future therapeutic interventions in ulcerative colitis can target this mechanisms. © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction Ulcerative colitis (UC) is a chronic idiopathic inflammatory disease of the gastrointestinal tract that affects the large intestine causing frequent episodes of diarrhoea, abdominal pain, bloody stools and weight loss over the medium to long term. UC is characterized by marked lymphocyte and neutrophil infiltration confined to the colonic mucosa with neutrophilic infiltration of colonic crypts accompanied by ulceration and necrosis of epithelial cells
夽 This study has been supported by the University Research Fund, project code: A.10080.2.106.107. ∗ Corresponding author at: Dip. di Medicina Clinica e Sperimentale, Gastroenterologia, Facoltà di Medicina, Università Federico II, Via Pansini 5, 80131 Napoli, Italy. Tel.: +39 081 7462706. E-mail address: [email protected] (G. D’Argenio).
[1]. Treatment includes the use of topical or systemic drugs [2], but their beneficial effect is limited [3]. Corticosteroids affect the short-term treatment of acute inflammation, but their use in maintenance therapy is limited by the high incidence of side effects [4]. Sulphasalazin and 5-ASA exert beneficial effects only in moderately active disease and are used to prevent recurrence [5,6]. Studies of UC in humans do not allow to investigate the early stages of the disease, therefore, to better understand the pathogenic mechanisms responsible for initiation and maintenance of chronic bowel inflammation, animal models are needed that also allow us to test new treatments. Recently, much attention has been paid to naturally derived products from fruit or vegetables which may beneficially affect a number of pathologic conditions of the gastrointestinal tract. In particular, it has been demonstrated that dietary anti-oxidants play a crucial role in maintaining gastrointestinal homeostasis
1590-8658/$36.00 © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dld.2012.01.009
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by counteracting the gastrointestinal mucosa damaging effects of reactive oxygen species (ROS) [7]. The importance of nutrition with a significant intake of fresh fruits and vegetables has long been an assumption of medicine: the preventive and protective role of these foods is due to significant presence of phytochemicals which, on one hand prevent oxidative damage brought about by ROS inside the gastro intestinal tract, and the other hand stimulate the cell to “self-defence” stimulating intracellular mechanisms that counteract oxidative stress and inflammation [8]. Phenolic compounds represent an important part of phytochemicals in the diet and, in particular, the phenolic components of apple represent 22% of total polyphenols in the diet of U.S. consumers [9]. Polyphenols are secondary metabolic products of many plants characterized by the presence of an aromatic ring with one or more hydroxyl substituents. Phenolic compounds act both as antioxidants and as elicitors of different metabolic pathways [10]. Tissue transglutaminase (tTG) is a transamidating enzyme that catalyses a calcium-acyl transfer reaction between two different peptides [11]. Various transglutaminases isoforms have been claimed to play a role in colon inflammation [12]. Calpains are a family of proteases that require both calcium and a reduced environment for activity. The two homologous isoforms, -calpain and m-calpain, have been classically distinguished on differences in their in vitro calcium requirements for half-maximal activity with, -calpain requiring significantly less calcium than m-calpain [13]. The homologous isoforms of calpains have been shown to proteolyse several protein substrates. tTG was found to be an excellent in vitro substrate of -calpain, generating a large breakdown product [14]. However, in contrast to epidermal TG, cleavage of tTG by calpain resulted in inactivation. A recent study demonstrated that the degree of colitis caused by injection of DNBS was substantially reduced by treatment of rats with calpain inhibitor I [15], but the mechanisms of the anti-inflammatory effect of calpain inhibitor I are not entirely clear. Previous data indicated that tTG may be an endogenous substrate of calpain, and that calpain may be involved in the regulation of tTG transamidating activity in situ by modulating intracellular tTG levels [16]. We have previously demonstrated that apple polyphenols extract (APE) significantly counteracts gastric damage induced by ROS, indomethacin or acetyl salicylic acid in rats and that this protective effect is mainly due to the anti-oxidant activity of APE [7,17]. Therefore, this study was designed to investigate whether APE may accelerate healing of 2,4,6-trinitrobenzene-sulphonic acid (TNBS)-induced colitis in the rat. Also, in order to gain a better insight into the mechanism of action of APE, we investigated its effects on protein and gene expression of TNF-␣ and COX-2 and evaluated whether APE affected cell migration, the first step involved in gastrointestinal mucosal repair, in an in vitro wound healing model. Finally, we studied whether the balance between tTG and calpain might be involved in TNBS-induced colitis and in the therapeutic effect exerted by APE.
2. Materials and methods
2.2. TNBS-induced colitis in rats and APE treatment Male Wistar rats, weighing 200 ± 21 g, were obtained from Harlan Italy and were housed in a temperature-controlled environment with a 12 h light-dark cycle, and given free access to regular laboratory chow diet and water for at least one week. All animals received human care, and the study protocol was approved by the Committee of Laboratory Animals at Federico II University according to institutional guidelines. Rats then were given a single enema of TNBS solution (20 mg in 50% ethanol–water), via a rubber catheter inserted 8-cm lengths from the anus under light anaesthesia [18]. To evaluate the effect of APE on TNBS-induced colitis, 16 rats were divided in two groups (n = 8 in each group) and treated with rectal administration of 1 mL/die of APE 10−4 M or vehicle daily for 14 days, respectively. A further group of animals consisting of normal rats was used as controls. The weight of distal colon was measured, and inflammatory changes and ulceration in the distal colon were evaluated macroscopically (evaluating on a 0–5 scale hyperemia, fibrosis, ulcers, necrosis) [18]. Mucosa was separated from submucosal layer by gently scraping, and tissue specimens were than kept at −80 ◦ C until assayed. 2.3. Histology Colon biopsies were fixed in 10% formalin and embedded in paraffin, haematoxylin and eosin-stained sections eliminate were assessed histologically by light microscopy performed by a pathologist in a blinded fashion. According to previous reports [19,20], four parameters, each scored on a 0–5 scale of severity, were considered: extent of ulceration, submucosal infiltration, crypt abscesses, and wall thickness. The final total histological score (from 0 to 20) was determined by adding the sum of each assigned score and determined a rating of slight (1–5) moderate (6–10) or severe (11–20) colonic inflammation. 2.4. Protein extract preparations Frozen rat colon mucosa samples were homogenised in RIPA lyses buffer (0.1% sodium dodecyl sulphate (SDS), 0.5% deoxycholate, 1% Nonidet, 100 mM NaCl, 10 mM Tris–HCl (pH 7.4)) containing a protease inhibitor cocktail (Sigma, St Louis, Missouri, USA), 0.5 mM dithiotreitol, and 0.5% phenylmethylsulphonyl fluoride. After 30 min at 4 ◦ C, tissue lysates were clarified by centrifugation at 14,000 rpm for 10 min at 4 ◦ C. The cleared tissue lysates were collected and stored at −80 ◦ C and protein concentration of each sample was determined by Bradford assay (Coomassie brilliant blue protein assay; Bio-Rad, Melville, NY, USA). The antibodies used in this study were as follows: (1) goat polyclonal IgG anti-COX-2 (UPSTATE 07-693); (2) goat polyclonal IgG anti TNF-␣ (sc 1349 Santa Cruz Biotechnology); (3) mouse monoclonal IgG anti-tTG (CUB 7402 Neo-Markers); (4) mouse monoclonal anti Calpain-1 (sc 58323 Santa Cruz Biotechnology). The secondary antibodies were biotinylated anti goat IgG (Vector BA9500) and biotinylated anti mouse IgG (Vector BA200) as appropriate.
2.1. Preparation of apple extract and identification of phenolic compounds
2.5. Western blot analysis
Freeze dried apple flesh of “Annurca” variety was extracted with methanol as previously described [7]. Identification of phenolic compounds present in APE was performed by HPLC–ESI-MS analysis as described elsewhere [9,17]. The sum of the concentration of all compounds present in the extract was performed and the molecular weight of the catechin (CAT) was considered to obtain the molar concentration of APE. Therefore APE concentration was arbitrarily expressed in CAT equivalent.
Total protein extracts were subjected to SDS–PAGE (10% and 7% polyacrylamide) under reducing conditions. After electrophoresis, proteins were transferred to nitrocellulose membrane (pure nitrocellulose membrane, 0.45 m Bio-Rad Laboratories); complete transfer was assessed using pre-stained protein standards (Invitrogen LC5925). To block non-specific binding sites the membranes were treated for 1 h with blocking solution: 5% milk in TNT (10 mM Tris pH 8, 150 mM NaCl and 0.05% Tween-20), and then
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were incubated overnight at 4 ◦ C with the primary antibody: (1) anti-COX-2 (diluted 1:500); (2) anti-TNF-␣ (diluted 1:200) in TNT (0.05% Tween-20) 20% foetal calf serum (FBS); (3) anti-tTG (diluted 1:2000) in TNT (0.1% Tween-20) 5% milk; (4) anti-calpain (diluted 1:1000) in TNT (0.05% Tween-20) 20% FBS. After washing with TBS, membranes were incubated 2 h (at room temperature) with the appropriate biotinylated secondary antibody. Immunoreactive proteins were detected by development with the ABC Vectastain kit (Vector Laboratories, Burlingame, California, USA) according to the manufacturer’s instructions. 2.6. RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) analysis Total RNA was isolated from rat colon mucosa using TRIzol (Invitrogen, NY, USA) extraction reagent, according to the standard acid–guanidium–phenol–chloroform method [21]. RT-PCR analysis was performed on total RNA as described previously [22,23]. First-strand complementary DNA was prepared using 200 units of reverse transcriptase (Supertranscript RT, Life Technologies, Inc., Gaithersburg, MD), 1 g of total RNA as template, and 10 pmol/L of random hexamers in the presence of 0.1 mmol/L dithiothreitol, 0.5 mmol/L deoxynucleotide triphosphate-litium salt (Pharmacia, Milan, Italy), and 20 units of RNase inhibitor (Promega, Madison, USA). The reaction profile was 37 ◦ C × 10 min, followed by 42 ◦ C × 60 min. To control for contamination by genomic DNA, all RNA samples were run in duplicate with or without addition of reverse transcriptase. The sequences of primers used (PRIMM, Italy) and the annealing temperature are shown in Table 1 (supplementary file). PCR included the following steps: an initial denaturation at 95 ◦ C for 5 min; 35 amplification cycles consisting of denaturation at 94 ◦ C for 1 min; primer annealing for 1 min (see Table 1 for primer specific ◦ C) and extension at 72 ◦ C for 1 min. In all cases, the final extension step was at 72 ◦ C for 10 min. GAPDH primers were added after 6 cycles for TNF-␣, COX-2 and tTG and after 4 cycles for calpain. To test for contamination by genomic DNA, samples were run in duplicate with or without the addition of reverse transcriptase. PCR products were separated on 1.8% agarose gel electrophoresis and visualized by ethidium bromide staining. Sizes of the amplified fragments were estimated by comparison with migration of the 100-kb ladder molecular-weight marker (Life Technologies, Inc., Gaithersburg, MD). 2.7. NIH 3T3 fibroblast wound model The NIH 3T3 murine fibroblast scratch model is an established model system for detecting tTG. In this model, catalytically active tTG is hypothesized to provide mechanical stability to tissues by cross-linking ECM proteins after a wound has been inflicted [24]. In addition, fibroblasts are non-transformed cells as occurs for the other cell lines available for the migration studies. Fibroblasts were plated in a 24-multiwell plastic chamber and grown for 3–4 days in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 1% antibiotic–antimycotic solution (Life Technologies Inc., Gaithersburg, Maryland, USA) at 37 ◦ C in a humidified atmosphere of 5% CO2 , with a media change every 2 days, until the semi-confluence phase. Twenty-four hours before the wound, cells were starved in DMEM 0.1% FBS. Small scratches were made in the monolayer with a 0.1–10 L pipette tip, and the cells were incubated either with the specific tTG inhibitor cystamine (33 g/mL) or APE (10−4 M) and monitored for 30 h by time-lapse-microscopy (TLM). The experiments were done in triplicate. TLM experiments were performed by using an inverted microscope (Zeiss), with a long working distance 10× objective in phase
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contrast. The microscope is equipped with motorized stage and focus for automated sample positioning, and an incubator consisting of a plastic enclosure kept at 37 ± 0.1 ◦ C by a warm air flux, tuned by a PID controller. Furthermore a small amount of air, premixed with 5% CO2 , is blown trough a bubbling column for humidity saturation, and fed to a small chamber surrounding the sample to prevent water evaporation and pH changes in the cell culture medium. The images are captured by a cooled monochromatic CCD video camera (Hamamatsu). All the equipment are driven by a Labview macro that iteratively acquires images of selected regions every 30 min, for overall 30 h. After the experiment the cells were washed 3× with PBS and then fixed with cold (−20 ◦ C) methanol for 10 min. The cells were washed with PBS 2× for 5 min, blocked with 1% BSA in PBS for 5 min at room temperature, and then washed 2× using PBS. CUB 7402 Anti tTG antibody was diluted 1:500 in blocking buffer and incubated with the cells for 60 min at room temperature. The cells were washed 4× with PBS then incubated for 1 h at room temperature with the biotinylated secondary antibody (1:1000). Images of selected regions were acquired in a phase contrast microscope. 2.8. Statistical analysis As appropriate, the Student’s t test or the analysis of variance (ANOVA) followed by Bonferroni’s post hoc test were used for statistical evaluations. 3. Results 3.1. Effects of APE on TNBS colitis The rectal administration of TNBS induced the appearance of visible colonic damage extending to all the colon length with evident colonic wall thickness (Fig. 1A). Rectal administration of 1 mL/die of APE 10−4 M daily for 14 days ameliorated macroscopic injury to the rat colon (Fig. 1B). TNBS extensively damaged the superficial epithelium and caused intense inflammation and loss of epithelial cell layer in the region of the colonic mucosa (Fig. 1C). In contrast, APE treatment significantly decreased microscopic injury caused by induced colitis decreasing the extent of inflammatory infiltrate and the damage to the surface area (Fig. 1D). Quantitatively, APE treatment caused an approximate 60% decrease in the extent of macroscopic injury compared with control untreated rats (p < 0.05, Fig. 2A) and reduced the extent of microscopic injury by approximately 55% (p < 0.05, Fig. 2B). 3.2. RT-PCR To evaluate whether TNBS caused alterations in selected genes supposed to be involved in colitis inflammation and healing, we examined mRNA levels of COX-2, tTG, calpain, and TNF-␣. TNBS colitis was associated with increased mRNA expression of all tested genes (Fig. 3), whilst APE administration prevented the colitis associated increase of COX-2, TNF-␣, calpain and tTG (Fig. 3). 3.3. SDS–PAGE and western blot Administration of TNBS induced a significant increase in COX-2, TNF-␣, and calpain in inflamed tissue (Fig. 4, lanes 3–6). In contrast a strong reduction of tTG protein transcript was observed (Fig. 4, lanes 3–6). Treatment with APE counteracted TNBS-induced upregulation of COX-2, TNF-␣ and calpain protein expression as well as the TNBS-induced down-regulation of tTG protein expression (Fig. 4, lanes 7–9).
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Fig. 1. Macroscopic and microscopic appearance of rat colon with trinitrobenzensulphonic acid-induced colitis (A and C), and after treatment with apple polyphenols extract (B and D). In rats with colitis, the colon appears thickened and inflamed (A) and an abundant inflammatory infiltrate is also present (C). The treatment with apple polyphenols significantly reduced both wall thickness and mucosal inflammation (B and D).
3.4. Localization, activation and inhibition of tTG in a cell wound model In Fig. 5A a panel of images acquired during TLM experiments is reported. Control untreated, cystamine-treated and APE-treated samples are compared at 1, 20 and 30 h after the wound. In the supplementary material the complete movie of the TLM experiment is reported as avi file. The experiment demonstrated that APE significantly stimulated wound closure compared to control untreated and cystamine-treated wounded monolayers. In Fig. 5B a quantification of the difference in the wound closure between the three samples is shown. Cell density normalized respect to the initial value was plotted over the time of the experiment for control untreated, cystamine-treated and APE-treated samples. Each point is the average of three independent measurements on different samples. Data show that APE induces a
significant increase in cell density growth compared with control untreated and also compared with cystamine-treated samples. Furthermore, at immunohistochemistry, wounded monolayers showed a high density of tTG protein 30 h after APE treatment around the wounded area (Fig. 6A) compared to cells treated with cystamine (Fig. 6B) or untreated fibroblasts (Fig. 6C). 4. Discussion UC is a chronic, relapsing disease that causes inflammation and ulcerations of the colonic mucosa with a variable extent and severity. The aetiology of UC remains essentially unknown but the results from many studies in humans and animal models suggest that it is related to an abnormal immune response in the gastrointestinal tract, possibly associated with genetic and environmental, mainly microbial factors [25]. Several drugs have been mainly used for the
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Fig. 2. Macroscopic (A) and microscopic (B) damage of rat colon was scored as described in Section 2. Comparison between trinitrobenzensulphonic acid-induced colitis controls and apple polyphenols treated rats shows the efficacy of the treatment in reducing the mucosal damage. *p < 0.05 compared with controls.
treatment and maintenance of remission of UC, but the side effects or toxicity of these drugs are a major clinical problem [26]. For these reasons, products of natural origin that cover the gap between nutrients and pharmaceuticals (i.e. nutraceuticals) have become an alternative option in addition to the conventional therapies that are used to treat UC [27]. Growing evidences suggest that dietary fish oil and curcumin given together modulate colonic cytokinetics and gene expression in dextran sodium sulphate-treated mice [28], and that bovine colostrum is effective in the treatment of distal colitis [29]. In the present study, we demonstrated that APE has an anti-inflammatory effect on colonic injury provoked by TNBS in rats. TNBS-induced colitis is a well-established model for colitis that is phenotypically similar to human colon inflammation
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Fig. 3. Agarose gel electrophoresis of representative reverse transcriptionpolymerase chain reaction products of cyclooxygenase-2 (COX-2), tissue transglutaminase (tTG), calpain, and tumour necrosis factor-alpha (TNF-␣). Glyceraldehyde3-phosphate dehydrogenase (GAPDH) was chosen an internal control. The lanes are representative of different groups of treatments: normal rats, trinitrobenzensulphonic acid-induced colitis (TNBS) as controls, and rats with colitis treated with apple polyphenols extract (TNBS + APE).
[30]. A single rectal administration of TNBS, leads to colonic epithelial lesions and inflammation characterized by the presence of neutrophils and macrophages within damaged colon. APE was administered at a concentration that was demonstrated to be effective in rat gastric lesion and gastric cell line damaged by ROS [7]. The decrease in TNBS colitis induced by APE was accompanied by a lower weight loss of rats and a partial restoration of colon thickening, which is an indirect assessment of colon inflammation. Microscopic analysis confirmed clinical results, showing a protective action of APE measured as a decrease in ulceration, conservation of epithelial crypts, and a reduction of cell infiltration.
Fig. 4. Western blot analysis using antibodies against cyclooxygenase-2 (COX-2), tumour necrosis factor-alpha (TNF-␣), calpain, and tissue transglutaminase (tTG). In this context, COX-2, TNF-␣, and calpain were all increased by trinitrobenzensulphonic acid-induced colitis (TNBS), whilst tTG content was reduced, possibly degraded by calpain. Apple polyphenols extract (APE) reduced the protein content of COX-2, TNF-␣, and calpain and restored tTG protein level.
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Fig. 5. (A) Images acquired during time-lapse microscopy experiments, where control untreated, cystamine-treated and apple polyphenols extract (APE)-treated samples are compared at 1, 20 and 30 h after the wound. Quantification of the difference in the wound closure between the three samples is reported in (B).
Previous studies demonstrated that various transglutaminase isoforms, and in particular the tTG, are involved in remodelling matrices in the presence of continuous wound healing such as in UC [12]. Up-regulation of proteinases such as calpain, which is able to hydrolyse tTG [31], resulted in worsening of rat colitis [15]. Therefore, calpain-induced tTG degradation may account for the apparent discrepancy between increased tTG gene up-regulation and decreased tTG protein expression observed in the TNBS rats group. Accordingly, our results also showed that APE treatment inhibited TNBS-induced up-regulation of tTG expression and to a larger extent calpain gene expression. Moreover, it determined a reduction of tTG protein degradation as showed by western blot. This result suggests an important role for APE which is able to increase tTG availability for the colonic repairing process. Furthermore, the results of this study indicated that treatment with APE was able to reduce colon inflammation. The abnormal presence of inflammatory cells within the mucosa produces increased concentrations of inflammatory cytokines such as TNF-␣ [32], and
pro-inflammatory cytokines induced expression of genes associated with inflammation, such as COX-2. APE administration was able to inhibit TNF-␣ and COX-2 both at protein and mRNA levels, suggesting that APE could be useful in the prevention of relapse in patients with quiescent UC. On the other hand, TNF-␣ has been described as a key molecule in UC pathogenesis, and a monoclonal antibody against this molecule has proven to be effective in the treatment of moderate to severe UC [33]. This cytokine, recruiting leukocytes to inflammatory sites, stimulates monocytes and vascular endothelial cells to express cytokines, induces the cascade effects for other cytokines, and finally results in inflammatory lesions in tissues [34]. COX-2 is over-expressed in IBD and experimental colitis and COX-2 [35]. Whilst substantial evidence indicates exacerbation of IBD following the use of NSAIDs (i.e. nonselective COX inhibitors), it is not clear whether selective COX-2 inhibitors are safer than NSAIDs in IBD patients [36]. Also, the role of COX-2 inhibitors in rat colitis is controversial [35,37,38]. In our study, we confirm
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Fig. 6. Immunohistochemistry of NIH 3T3 cells fixed in methanol. Cells monolayer was wounded and incubated up to hours with cystamine, a tissue transglutaminase inhibitor, or with apple polyphenols extract (APE). The cells were than incubated with anti-tTG antibody: a marked staining of cells treated with APE (A), cystamine (B), control (C) was observed.
up-regulation of COX-2 in TNBS-induced colitis [38] and show that acceleration of mucosal healing induced by APE was associated with a decreased expression of COX-2. However, we cannot establish whether the decreased colonic damage in APE-treated animals is, at least in part, due to an APE-mediated decrease in pro-inflammatory COX-2 expression or is mediated by a COX-2 independent mechanism. The second block of experiments using an vitro wound healing model demonstrated that APE was able to modulate cell function through induction of chemotaxis. In fact, APE significantly influenced the restitution in a model of wounded fibroblast monolayers with a recruitment of large number of cells in the damaged area. Interestingly, these cells also expressed high levels of tTG, again confirming the role of this enzyme in the healing process. Moreover, the role of tTG in APE-related effect on restitution was further emphasized by the decrease in APE-induced stimulation of cell migration by the tTG inhibitor cystamine. In conclusion, the results of this study show that (a) APE significantly decreases gross and histological colon damage in TNBS colitis; (b) the clinical improvement was associated to a decrease in the up-regulation of COX-2, TNF-␣ and calpain induced by TNBS; (c) tTG protein concentration is decreased by TNBS at the protein level and this effect is counteracted by APE, likely through calpain inhibition; (d) APE accelerates in vitro healing of wounded fibroblast monolayers and this is associated with tTG over-expression; (e) the tTG inhibitor cystamine, counteracts APE-induced stimulation of cell migration. In this framework it can be hypothesized that the protection exerted by APE against TNBS colitis is mediated by its effects on COX-2, TNF-␣, and tTG. Moreover, data demonstrated that the unbalance between calpain and tTG may play a role in TNBSinduced colonic damage and future therapeutic interventions in UC can target this mechanisms.
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