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2 and 5-lypoxygenase inhibitor in experimental colitis
European Journal of Pharmacology 789 (2016) 152–162
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
Use of a balanced dual cyclooxygenase-1/2 and 5-lypoxygenase inhibitor in experimental colitis Giovanni Pallio a, Alessandra Bitto a, Gabriele Pizzino a, Federica Galfo a, Natasha Irrera a, Letteria Minutoli a, Vincenzo Arcoraci a, Giovanni Squadrito b, Antonio Macrì b, Francesco Squadrito a,n, Domenica Altavilla c a
Department of Clinical and Experimental Medicine, Section of Pharmacology University of Messina, via Consolare Valeria 1 Messina, Italy Department of Human Pathology University of Messina, via Consolare Valeria 1 Messina, Italy c Department of of Biomedical and Dental Sciences and Morphofunctional Imaging, University of Messina, via Consolare Valeria 1 Messina, Italy b
art ic l e i nf o
a b s t r a c t
Article history: Received 8 June 2016 Received in revised form 18 July 2016 Accepted 20 July 2016 Available online 21 July 2016
Cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) play an important role in inflammatory bowel diseases (IBDs). We investigated the effects of flavocoxid, a dual COX/LOX inhibitor, in experimental colitis induced with either dinitrobenzenesulfonic acid (DNBS) or dextrane sulphate sodium (DSS) In the first model, colitis was induced in rats by a single intra-colonic instillation (25 mg in 0.8 ml 50% ethanol) of DNBS; after 24 h animals were randomized to receive orally twice a day, flavocoxid (10 mg/ kg), zileuton (50 mg/kg), or celecoxib (5 mg/kg). Sham animals received 0.8 ml of saline by a single intracolonic instillation. Rats were killed 4 days after induction and samples were collected for analysis. In the second model, colitis was induced in rats by the administration of 8% DSS dissolved in drinking water; after 24 h animals were randomized to the same above reported treatments. Sham animals received standard drinking water. Rats were killed 5 days after induction and samples were collected for analysis. Flavocoxid, zileuton and celecoxib improved weight loss, reduced colonic myeloperoxydase activity, macroscopic and microscopic damage, and TNF-α serum levels. Flavocoxid and celecoxib also reduced malondialdheyde, 6-keto PGF1α and PGE-2 levels while flavocoxid and zileuton decreased LTB-4 levels. In addition, flavocoxid treatment improved histological features and apoptosis as compared to zileuton and celecoxib; moreover only flavocoxid reduced TXB2, thus avoiding an imbalance in eicosanoids production. Our results show that flavocoxid has protective effect in IBDs and may represents a future safe treatment for inflammatory bowel diseases. & 2016 Elsevier B.V. All rights reserved.
Keywords: Baicalin Catechin LTB-4 PGE-2 Apoptosis
1. Introduction The pathogenesis of inflammatory bowel diseases (IBDs) is still poorly understood, however, increasing evidence sustains the interaction between environmental, genetic, and immunological factors (Podolsky, 2002). During IBD reactive metabolites of oxygen (ROS) stimulate: the release of chemokines; activation of lipid peroxidation; recruitment of neutrophils; the release of TNF-α (Pavlick et al., 2002; Rahimian et al., 2010); the activation of NF-κB and the apoptotic kinase JNK (Shaulian and Karin, 2012). Of the pro-inflammatory genes that are induced by ROS, it is noteworthy the expression of cyclooxygenase (COX-2) and 5-lipoxygenase (5n Correspondence to: Department of Clinical and Experimental Medicine, Section of Pharmacology, Torre Biologica 5th floor, c/o AOU Policlinico G. Martino, Via C. Valeria Gazzi, 98125 Messina, Italy. E-mail address: [email protected] (F. Squadrito).
http://dx.doi.org/10.1016/j.ejphar.2016.07.033 0014-2999/& 2016 Elsevier B.V. All rights reserved.
LOX). In IBDs COX-2 mediates the increase in PGE2, important for the development and maintenance of an inflammatory microenvironment (Sklyarov et al., 2011). Concomitantly, an increased activity of 5-lipooxygenase (5-LOX) lead to an enhanced formation of leukotriene A4 (LTA-4), further transformed by the LTA-4 hydrolase into LTB-4, especially in macrophages and polymorphonuclear leukocytes. LTB-4 attracts leukocytes to the site of inflammation, promoting their adhesion to the inflammed and damaged tissue (Singh et al., 2004), thus amplifying the inflammatory cascade in IBDs (Holma et al., 2007). Aspirin and NSAIDs are known to suppress COX expression/activity and eicosanoid production, however, inhibition of COXs may lead to cardiotoxicity due to an imbalanced production of pro-thrombotic eicosanoids (e.g. increased TxA2) and anti-thrombotic eicosanoids (e.g. decreased PGI2) (Bresalier et al., 2005; Solomon et al., 2005). Since the inhibition of one or both COX enzymes may shunt arachidonic acid metabolism to the 5-LOX pathway, zileuton, an
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Fig. 1. Colon tissues from sham (A), DNBS þ drug vehicle (B), DNBS þFlavocoxid (C), DNBSþ Zileuton (D), DNBS þCelecoxib (E) together with the graph representing macroscopic damage scores (F) and colon weight/length ratio (G). Values are expressed as the mean and S.E.M. *P o 0.0001 vs DNBS þdrug vehicle group. n ¼ 7 for each group.
active 5-lipoxygenase inhibitor, was compared in a trial versus mesalazine and placebo. However, zileuton was not superior to placebo in maintaining remission of symptoms in ulcerative colitis (Hawkey et al., 1997). Thus, it appears that a targeted inhibition of either COX or 5-LOX does not represent the proper therapeutic strategy to block the inflammatory response in IBDs. It can also be speculated that a balanced inhibition of both enzymes might have synergistic effects and could be the key point for an effective treatment of IBDs. Some natural compounds, termed medical foods, demonstrated their efficacy and safety in the dietary management of specific diseases. A well characterized medical food, flavocoxid is a proprietary blend of baicalin and catechin, concentrated and standardized to greater than 90% purity. Flavocoxid, has been also demonstrated to be a balanced dual inhibitor of the peroxidise moieties of COX and 5-LOX, blunting the proinflammatory response (Burnett et al., 2011), as demonstrated in LPS-stimulated macrophages (Altavilla et al., 2009), acute pancreatitis (Polito et al., 2010), cecal ligation and puncture (Bitto et al., 2012). In addition to COX and 5-LOX inhibition flavocoxid may reduce reactive oxygen species including hydroxyl radical, superoxide anion radical, and hydrogen peroxide, modulating in turn, NF-κB and TNF-α production (Bitto et al., 2014). To date, a dual inhibitor of both COX and 5-LOX pathways has not been investigated in experimental colitis. Therefore, we aimed at evaluating the potential therapeutic effect of Flavocoxid, in experimental colitis induced with either DNBS or DSS, and to compare its effect to the 5-LOX inhibitor Zileuton and to COX-2 inhibitor,
celecoxib.
2. Materials and methods 2.1. Animal and treatments All animal procedures were in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki), the ARRIVE Guidelines (McGrath et al., 2010), and authorised by the Animal Ethics Committee of the Department Clinical and Experimental Medicine (approval #04/14), and all efforts were made to minimize suffering. A total of 70 male Sprague–Dawley rats (250–300 g) were purchased from Charles River Laboratories (Calco, Milan, Italy). Animals were maintained in plastic cages under standard environmental conditions with water and food ad libitum. After 1 week of acclimation to the laboratory environment, animals were randomly assigned to 10 groups of 7 animals each. In preliminary experiments, flavocoxid dose was titrated against the effects on PGE-2 expression in 30 animals. The day after DNBS administration 6 animals/group received flavocoxid twice a day at 5, 10, 20, and 40 mg/kg (by gavage) and the treatments lasted 4 days. This experiment identified 20 mg/kg/day as the optimal dose to be used in the further experiments (Supplemental Figure 1). Zileuton and celecoxib dose and route of administration were chosen according to previously published
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Fig. 2. Food intake (A) in DNBS animals *P o 0.0001 vs DNBS þdrug vehicle at selected time points. Weight loss (B) in DNBS animals evaluated during the study period §P o 0.05, #Po 0.001 vs DNBS þdrug vehicle respectively. Food intake (C) in DSS animals *P o0.0001 vs DSS þ drug vehicle at selected time points. §P o 0.05 vs Zileuton at day 5 §§P o 0.05 vs Celecoxib at day 3 and day 4. Weight loss (D) in DSS animals evaluated during the study period ^P o 0.01,*P o 0.0001 vs DNBSþ drug vehicle respectively. #Po 0.001 vs Celecoxib, °P o0.0001 vs Zileuton n ¼ 7 for each group.
reports (Zingarelli et al., 1993, Cuzzocrea et al., 2001). In a first set of experiments, colitis was induced in 28 fasted rats that were lightly anesthetised with diethyl ether, by a single intra-colonic instillation of 2,4-Dinitrobenzenesulfonic acid (DNBS; Sigma-Aldrich, Milan, Italy; 25 mg in 0.8 ml 50% ethanol). The catheter was inserted into the colon via the anus up to the splenic flexure 8 cm from the anus. Thereafter, the animals were kept for 15 min in Trendelenburg position to avoid reflux, and allocated in single cages for all the duration of the experiment. After 24 h, colitis signs were evident and animals were randomized to receive orally twice a day either flavocoxid (20 mg/kg/ day; hereinafter DNBS þ Flavocoxid), zileuton (100 mg/kg/day; hereinafter DNBS þ Zileuton), celecoxib (10 mg/kg/day; hereinafter DNBS þCelecoxib) or vehicle (1:3 DMSO:0.1% methylcellulose solution; hereinafter DNBS þ drug vehicle). Animals in the Sham group (n¼ 7) received 0.8 ml of saline, in a single intra-colonic instillation. Animals were observed for clinical signs of colitis, as diarrhoea and appetite; 4 days after induction all rats were killed and enteric tissue and blood samples were collected for analysis. In a second set of experiments, colitis was induced in 28 rats by oral administration of 8% DSS (MP Biomedicals, Solon, OH, USA) through drinking water from Day 0 to Day 5. Sham animals (n ¼7) were allowed ad libitum access to water. To assess the extent of colitis, the body weight, stool consistency and blood in the stool were monitored daily. After 24 h, colitis signs were evident and
animals were randomized to receive orally twice a day either flavocoxid (20 mg/kg/day; hereinafter DSS þFlavocoxid), zileuton (100 mg/kg/day; hereinafter DSS þZileuton), celecoxib (10 mg/kg/ day; hereinafter DNBS þCelecoxib) or vehicle (1:3 DMSO:0.1% methylcellulose solution; hereinafter DSS þdrug vehicle). The day of sacrifice, the abdomen was opened by a midline incision, the descending colon was removed, opened along the anti-mesenteric border, rinsed and cut into 2 same pieces, one for histological assessments and the other for analysis of biochemical markers. Moreover, blood samples were collected from the heart to estimate the level of proinflammatory cytokines. Flavocoxid was a kind gift of Primus Pharmaceuticals, Inc. (Scottsdale, AZ, USA); zileuton was purchased from Tocris Bioscence (Bristol, UK); celecoxib was purchased from Sigma Aldrich (Milan, Italy). Drugs were dissolved in the vehicle above described, prepared fresh daily and administered starting from the day after DNBS or DSS administration, and every 12 h thereafter. 2.2. Evaluation of body weight and food intake Body weight and food intake were recorded every day between 9:00 and 10:00 a.m. from the day of colitis-induction to the end of the experiment. Body weight results were expressed as row data and compared with food intake, by using the following formula: food intake divided by body weight in grams and multiplied by 100.
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Fig. 3. Histological evaluations (H&E staining, original magnification 10) of sham (A), DNBS þ drug vehicle (B), DNBS þFlavocoxid (C), DNBSþ Zileuton (D), DNBSþ Celecoxib (E). The graph represent the microscopic damage score (F), values are expressed as the mean and S.E.M. *P o 0.0001 vs DNBS þ drug vehicle group; ^P o0.01vs DNBS þCelecoxib; °P o0.0001 vs DNBS þZileuton. n ¼7 for each group.
2.3. Macroscopic damage score Colon damage was evaluated by two independent observers as described previously (Wallace et al., 1992), according to the following criteria: 0 (no damage), 1 (localized hyperemia without ulcers), 2 (linear ulcers with no significant inflammation), 3 (linear ulcers with inflammation at one site), 4 (two or more major sites of inflammation and ulceration extending 1 cm along the length of the colon), and 5–8 (one point is added for each centimeter of ulceration beyond an initial 2 cm). Colon weight/length ratio was also evaluated. 2.4. Microscopic damage score For light microscopy, colon tissues were rapidly removed and fixed in 10% buffered formalin. Subsequently, specimens were embedded in paraffin, sectioned at 5-μm thickness and stained with hematoxylin and eosin (H&E), and observed with a Leica (Leica Microsystems, Milan, Italy) microscope. Assessment of tissue changes was carried out by two observers blinded to the experimental protocol. The following morphological criteria were considered: score 0 (no damage), score 1 (mild, focal epithelial oedema and necrosis), score 2 (moderate, diffuse swelling and necrosis of the villi), score 3 (severe, necrosis with presence of neutrophil infiltrate in the submucosa), score 4 (highly severe, widespread necrosis with massive neutrophil infiltrate and hemorrhage) as previously reported (Rahimian et al., 2010). 2.5. Terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) assay TUNEL assay was performed by using a TUNEL detection kit
according to the manufacturer's instructions (Genscript, Piscataway, NJ, USA). Briefly, paraffin embedded tissues were sectioned (5 mm), rehydrated and incubate with proteinase K solution, for 15 min at 21–37 °C. Tissues were then treated with a 3% H2O2 solution for 10 min at 15–25 °C to inactivate the endogenous peroxidase. Sections were immersed in TUNEL reaction mixture and incubated for 60 min at 37 °C under wet conditions, protected from light. After wash in PBS slides were incubate with streptavidin-HRP Solution for 30 min at 37 °C under wet conditions, protected from light. Positive staining of apoptotic and necrotic cells were visualized with diaminobenzidine tetra-hydrochloride (DAB; Sigma Aldrich). Slides were counterstained with hematoxylin, to highlight the nuclei, dehydrated, and mounted with coverslips. 2.6. Immunohistochemical evaluation of CD3 Paraffin-embedded tissues were sectioned (5 mm), rehydrated, and antigen retrieval was performed by using 0.05 M sodium citrate buffer (pH 6.0) in a microwave for 5 min. Tissues were treated with 1% hydrogen peroxide to block endogenous peroxidase activity, and with horse normal serum (Vector Laboratories, Burlingame, CA, USA) to prevent nonspecific staining. A primary antibody against CD3 (Abcam, Cambridge, UK) was used and the slides were kept overnight at 4 °C in a humid box. The slides were then washed in PBS, added with a secondary antibody and the ABC reagent was used (Vectastain Elite ABC kit, Vector Laboratories). The location of the reaction was visualized with diaminobenzidine tetra-hydrochloride (DAB; Sigma-Aldrich, Milan, Italy). Slides were counterstained with hematoxylin, dehydrated, and mounted with coverslips. As a part of the histologic evaluation, all slides were examined by a pathologist without knowledge of the previous
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Fig. 4. Histological evaluations (H&E staining, original magnification 10) of sham (A), DSS þ drug vehicle (B), DSSþ Flavocoxid (C), DSS þ zileuton (D), DSS þ Celecoxib (E). The graph represent the microscopic damage score (F), values are expressed as the mean and S.E.M. *P o 0.0001 vs DSS þ drug vehicle group. n ¼ 7 for each group.
treatment, by using masked slides from 5 to 40 magnification with a Leica microscope (Leica Microsystems, Milan, Italy). 2.7. Evaluation of LTB4, PGE2, TNF-α, 6-keto PGF1α and TXB2 in serum Serum samples were assayed in duplicate using a commercially available ELISA kit for PGE2 (Cusabio Biotech, Wuhan, China), LTB4 (Bioassay Technology, Shanghai, China), TNF-α (Abcam, Cambridge, UK), TXB2 and 6-keto PGF1α (Cusabio, Washington, Massachusetts). Results were obtained interpolating the absorbances with the standard curves and expressed as means and S.D. 2.8. Measurement of Myeloperoxidase activity Myeloperoxidase (MPO) activity, an indicator of polymorphonuclear leukocyte accumulation, was determined as previously described (Mullane et al., 1985). The colon was homogenized in a solution containing 0.5% hexa-decyl-trimethylammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7.0). Lysates were then centrifuged for 30 min at 25,000 g at 4 °C. An aliquot of the supernatant was allowed to react with a solution of 1.6 mM tetra-methyl-benzidine and 0.1 mM H2O2. The rate of change in absorbance was measured with spectrophotometry at 650 nm. MPO activity was defined as the quantity of enzyme degrading 1μmol hydrogen peroxide/min at 37° and was expressed in units per 1 g of tissue. 2.9. Malondialdehyde measurement The levels of malondialdehyde (MDA) in the colon were
determined as an indicator of lipid peroxidation. The colon was homogenized in 1.15% KCl solution, an aliquot of the homogenate 0,1 ml was added to a reaction mixture containing 0,2 ml of 8.1% SDS, 1,5 ml of 20% acetic acid, 1,5 ml of 0.8% thiobarbituric acid and 0.7 ml distilled water. Samples were boiled for 1 h at 95 °C and centrifuged at 3000g for 10 min. The absorbance of the supernatant was measured by spectrophotometer at 650 nm (Ohkawa et al., 1979). 2.10. Statistical analysis All quantitative data are expressed as mean 7S.D. for each group, and compared by using one-way ANOVA for non-parametric variables, with Tukey post-test for intergroup comparisons. Statistical significance was set at Po0.05. Graph were drawn using GraphPad Prism software version 5.0 for Windows (GraphPad Software Inc., La Jolla, CA, USA).
3. Results 3.1. Effects of flavocoxid on body weight, food intake, and macroscopic damage Colitis was successfully induced in rats following DNBS intracolonic administration as demonstrated by the appearance of diarrhoea as early as 6 h after challenge, with a significant loss in body weight at the end of the experiment (P o0.001 vs sham group). Treatment with flavocoxid, zileuton, or celecoxib resulted in solid feces after only 2 days of treatment, with a parallel recovery from weight loss. Four days after induction of colitis at a
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Fig. 5. Histological evaluations (TUNEL staining, original magnification 10) of sham (A), DNBS þ drug vehicle (B), DNBSþ Flavocoxid (C), DNBS þ Zileuton (D) or DNBS þCelecoxib (E).Tissues of DNBS þ drug vehicle group demonstrated a marked appearance of dark brown apoptotic cells and intercellular apoptotic fragments (see arrows). No apoptotic cells or fragments were observed in the. DNBS rats treated with Flavocoxid, while animals of Zileuton or Celecoxib group demonstrated residual apoptosis in the submucosal layer (see arrows). n ¼ 7 for each group.
macroscopic observation the colon mucosa of DNBS þ drug vehicle group appeared ulcerated, oedematous, and hyperaemic compared to sham group (P o0.0001; Fig. 1A–B). In the groups treated with flavocoxid, zileuton or celecoxib there was a significant reduction in the extent and severity of colon injury (P o0.0001; Fig. 1C–F). As shown in Fig. 1G, DNBS also caused a significant shortening of the colonic length, with a consequent increase in the weight/length ratio, compared to control animals (P o0.0001). Treatment of rats with flavocoxid, zileuton, or celecoxib significantly improved this condition. Body weight and food intake were recorded at baseline (day 0) and throughout the experimental period. Food intake is reported in Fig. 2A and raw body weight data are reported in Fig. 2B. Flavocoxid, celecoxib, or zileuton treatment increased food intake and caused weight gain during the treatment period (P o0.05 vs DNBS þdrug vehicle group, Po0.001 vs DNBS þ drug vehicle group at end of experiment). No difference was observed among the several treatments. DSS-administered animals showed diarrhoea and rectal bleeding after 24 h, while weight reduction was observed starting from day 4 (P o0.05 vs Sham). Treatment with flavocoxid, zileuton or celecoxib resulted in reduced bleeding and improved food intake and body weight (Fig. 2C–D). Flavocoxid treatment in DSS animals caused a greater improvement in food intake and body weight than either zileuton or celecoxib (Fig. 2C and D). 3.2. Flavocoxid reduces histological damage and apoptosis A normal appearance of the colonic mucosa with intact
epithelium was observed in sham group (Fig. 3A). The histological analysis, 4 days after induction of colitis, showed mucosal injury induced by DNBS administration that was characterized by necrosis of epithelium and massive infiltration of neutrophils and macrophages into the mucosa and submucosa layers. Thickening of the colon wall, transmural necrosis, loss of globet cells, and oedema were also observed (Fig. 3B). Treatment with flavocoxid significantly reduced the extent and severity of the histological alteration associated with DNBS administration by stimulating regeneration of the epithelium (Fig. 3C) with preservation of the crypts, and globet cells. Zileuton and celecoxib administration significantly reduced the histological signs of injury; however, these two drugs did not completely restore the epithelial layer, crypts and globet cells (Fig. 3D–E). Microscopic damage scores in DNBS þFlavocoxid, DNBS þZileuton and the DNBS þCelecoxib groups were significantly reduced than in DNBS þdrug vehicle group (Po0.0001; Fig. 3F). In addition, flavocoxid score was significantly improved compared to zileuton or celecoxib-treated groups (Po 0.01vs DNBS þ Celecoxib; Po 0.001 vs DNBS þ Zileuton Fig. 3F). Histological analysis of the DSS animals, carried 5 days after challenge, showed a flattening of the mucosa with a reduced presence of mucosal glands, a massive inflammatory infiltrate and oedema in the submucosal layer, with no evident signs of apoptosis (Fig. 4B), as compared to sham animals (Fig. 4A). Treatment with flavocoxid significantly reduced the extent and severity of the histological alteration associated with DSS administration by stimulating regeneration of the epithelium of the crypts, and globet cells (Fig. 4C). Zileuton or celecoxib administration significantly
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Fig. 6. Immunohistochemical evaluation of CD3 cells, (original magnification 40) of sham (A), DNBS þ drug vehicle (B), DNBS þ Flavocoxid (C), DNBSþ Zileuton (D) or DNBS þCelecoxib (E). The arrows indicate CD3-positive T cells. The graph (F) represent the number of positive cells in each group; values are expressed as the mean and S.E. M. *P o 0.0001 vs DNBS þ drug vehicle group. n ¼ 7 for each group.
reduced the histological signs of injury; however, they did not properly restored the architecture of the mucosa (Fig. 4D–E). The microscopic damage score is shown in the graph (Fig. 4F). Apoptosis evaluation, 4 days after DNBS administration, showed an intense staining in the remains of the mucosa and submucosa layers, with diffuse intercellular apoptotic fragments, especially in the DNBS þ drug vehicle group compared to sham (Fig. 5A-B). By contrast, specimens obtained from rats treated with flavocoxid, zileuton or celecoxib showed reduced apoptotic cells. (Fig. 5C-E). 3.3. Immunohistochemical evaluation of CD3 expression Colitis induced by both DNBS and DSS caused a marked colonic infiltrate of CD3 positive T cells when compared to sham group. The several treatments produced a significant decrease in the expression of CD3 positive cells and no difference was observed among the different groups (Fig. 6A–F; Fig. 7A–F).
3.4. Effects of Flavocoxid, zileuton or celecoxib on PGE-2, LTB-4, TNFα, 6-keto PGF1α, TXB2 levels Circulating levels of the main inflammatory eicosanoids, PGE-2, LTB-4, 6-keto PGF1α, and TXB2 and of the proinflammatory cytokine TNF-α were measured four days after DNBS administration. Serum PGE2 and LTB4 measurements were determined to prove flavocoxid efficacy in this experimental model. As shown in Fig. 8A and B a significant increase in both PGE-2 and LTB-4 was found in serum from DNBS þ drug vehicle animals compared to control animals (Po0.0001). PGE2 and LTB4 serum levels were significantly reduced by flavocoxid, likely as a direct consequence of COX-2 and 5-LOX inhibition induced by the dual COX/5LOX inhibitor (Fig. 8A–B. In contrast treatment with zileuton did not change PGE-2 levels (Fig. 8A–B), and celecoxib did not modify LTB4 levels (Fig. 8A–B). Serum levels of TNF-α were significantly augmented in the bloodstream of DNBS þdrug vehicle rats, compared to sham
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Fig. 7. Immunohistochemical evaluation of CD3 expression, (original magnification 40) of sham (A), DSS þ drug vehicle (B), DSS þFlavocoxid (C), DSS þZileuton (D) or DSS þ Celecoxib (E). The arrows indicate CD3-positive T cells. The graph (F) represent the number of positive cells in each group; values are expressed as the mean and S.E.M. *P o 0.0001 vs DSS þ drug vehicle group. n ¼ 7 for each group.
Fig. 8. PGE-2 (A), LTB-4 (B), and TNF-α (C) levels in serum of sham, DNBS þdrug vehicle DNBSþ Flavocoxid, DNBS þZileuton or DNBSþ Celecoxib at the end of the experiment. *Po 0.0001 vs DNBS þ drug vehicle; °P o 0.0001 vs DNBSþ zileuton; ^P o 0.0001 vs DNBSþ Celecoxib. n¼ 7 for each group.
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Fig. 9. 6-keto PGF1α (A), TXB2 (B), levels in serum of DNBS animals at the end of the experiment. *P o 0.0001 vs DNBS þ drug vehicle; °P o 0.0001 vs DNBSþ zileuton; ^P o 0.0001 vs DNBS þCelecoxib. n ¼7 for each group. 6-keto PGF1α (C), TXB2 (D), levels in serum of DSS animals at the end of the experiment. *Po 0.0001 vs DSS þ drug vehicle; °P o 0.0001 vs DSS þzileuton; ^P o0.0001 vs DSS þCelecoxib. n ¼7 for each group.
(Fig. 8C; P o0.0001). All treatment markedly blunted the serum concentration of TNF-α (Fig. 8C; Po0.0001 vs DNBS þdrug vehicle). Additionally, 6-keto PGF1α and TXB2 levels (the stable hydrolysis products of PGI2 and TXA2) were measured in serum four days after DNBS administration and five days after DSS administration. A significant increase of both was found in serum from DNBS þ drug vehicle animals compared to controls (Fig. 9A–B; Po0.0001). As a consequence of COX and 5-LOX inhibition by flavocoxid treatment, 6-keto PGF1α and TXB2 serum levels were significantly reduced (Fig. 9A–B, P o0.0001 vs DNBS þdrug vehicle). Celecoxib administration reduced only 6-keto PGF1α levels (Fig. 9A–B, P o0.0001 vs DNBS þdrug vehicle), while zileuton treatment did not modify 6-keto PGF1α and TXB2 levels. Similar results have been obtained from DSS-treated animals (Fig. C–D). 3.5. Effects of flavocoxid, zileuton and celecoxib on lipid peroxidation and neutrophil infiltration MDA levels were increased as expected in DNBS þ drug vehicle groups (P o0.0001 vs sham; Fig. 10A); of the three treatment used, only flavocoxid and celecoxib blunted lipid peroxidation (P o0.01; Fig. 10A), demonstrating a superior antioxidant activity. Accumulation of polymorphonuclear granulocytes determined as myloperoxidase activity was increased in DNBS þ drug vehicle group (P o0.0001 vs sham; Fig. 10B). All treatment significantly reduced MPO activity (P o0.0001; Fig. 10B). Flavocoxid treatment more significantly reduced the MPO activity compared to celecoxib group (P o0.01 Fig. 10B).
In the DSS þ drug vehicle group MDA levels were increased as expected (P o0.0001 vs sham; Fig. 10C), only flavocoxid (Po0.001 vs DSS þ Drug vehicle) and celecoxib (Po 0.01 vs DSS þ drug vehicle) blunted lipid peroxidation (Fig. 10C) demonstrating a superior antioxidant activity. Accumulation of neutrophils determined as MPO was increased in DSS þ drug vehicle group (Po0.0001 vs sham; Fig. 10D). All treatment significantly reduced MPO activity; moreover flavocoxid treatment had a greater efficacy in blunting MPO activity compared to celecoxib group (Po0.01 Fig. 10D).
4. Discussion Reactive species of oxygen and nitrogen have been linked to inflammatory bowel disease almost 20 years ago (Grisham, 1994). These species increase, among the others, the expression of the enzymes COX-2 and 5-LOX that produce crucial mediators for the development of colitis, in particular PGE-2 and LTB-4 levels (Sharon et al., 1978). The eicosanoids are able to further activate inflammatory cells and the oxidative stress with a consequent transcription of NFκB-related cytokines. In agreement with these previous data, our results show that flavocoxid, with a balanced inhibition of either COX and 5-LOX, was more effective than zileuton or celecoxib in reducing eicosanoid production during experimental colitis. The reduced PGE-2 and LTB-4 levels, together with decreased lipid peroxidation (as demonstrated by MDA levels) could be also related to the antioxidant properties of this flavonoid compound. Halting of eicosanoid production decreased
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Fig. 10. Effects of Flavocoxid, Zileuton and Celecoxib on Malondialdehyde levels (A) and Myeloperoxidase activity (B) in DNBS animals. Values are expressed as means and S. D. #P o0.01 vs DNBS þ drug vehicle group;*Po 0.0001 vs DNBS þ drug vehicle; ^P o 0.01 vs DNBS þ Celecoxib. Effects of Flavocoxid, Zileuton and Celecoxib on Malondialdehyde levels (C) and Myeloperoxidase activity (D) in DSS animals. Values are expressed as means and S.D. #P o0.01 vs DSS þ drug vehicle. ##Po 0.001 vs DSS þ drug vehicle group; *P o 0.0001 vs DSS þ drug vehicle; ^P o 0.01 vs DSS þ Celecoxib. n ¼ 7 for each group.
oxidative stress in the colon as demonstrated by the markedly visible improvement in the histological pictures. In fact, at a first glance, the colon appeared to be clean with no sign of hemorrhage, and at a microscopic observation we noticed a reduced presence of oedema with a preserved architectural structure of mucosal and submucosal layers, and absence of apoptotic/necrotic cells at the same level. As additional consequence of the reduced eicosanoid production also neutrophil infiltration was significantly reduced, and this is obvious because LTB-4 is more active than PGE2 as chemotactic substance. Interestingly, flavocoxid, zileuton and celecoxib succeeded in reducing MPO activity in colon specimens following DNBS or DSS induction. The reduced infiltrate of early inflammatory cells is in turn responsible for the reduction in TNFα production; it is indeed known that this proinflammatory cytokine is mainly produced during the first stages of the inflammatory process by neutrophils and macrophages and has the ability to activate apoptosis. All these positive effects observed in animals treated with flavocoxid were further confirmed by the reduction of diarrhoea and the consequent improvement in weight loss. Prostaglandins from COX-1 provide for the processes of water and electrolyte transport, vasodilatation, proliferation, and intercellular integration. During inflammatory activation, the considerable amount of prostaglandins synthesized by COX-2 enhance the activity of 5-LOX and stimulate the release of inflammatory leukotriens as LTB4, LTD4, and LTC4, further responsible for cell infiltration and oedema in submucosal layer (Sklyarov et al., 2011). Moreover cysteinyl leukotriens, induce local vasoconstriction, thus reducing blood flow and enhancing the susceptibility of gastric mucosa to injury. In light of these observations, leukotrienes may
play an important role in the development of the inflammatory process, and it is now clear that PGs and LTs have complementary effects (Martel-Pelletier et al., 2003). These effects are blunted by flavocoxid through a balanced peroxidase inhibiton of the COX and LOX enzymes, together with a specific block of phospholipase-A2 (PLA-2) activity (Burnett et al., 2011). Both effects are peculiar, in fact common non steroid antinflammatory drugs (NSAIDs) inhibit the cycloxygenase moiety of COX, preventing PGG2 formation from arachidonic acid. As previously shown celecoxib, a selective inhibitor of COX-2, ameliorates the severity of experimental colitis induced by DNBS (Cuzzocrea et al., 2001), and it was also effective in subjects with ulcerative colitis (Sandborn et al., 2006). However, large clinical studies clearly demonstrated increased cardiovascular risk for patients treated for long periods with either COX-2 inhibitors or NSAIDs (McGettigan and Henry, 2011), dramatically reducing the rationale for the use of these drugs in IBDs. In fact, the imbalanced inhibition of COX-2 and COX-1 (by selective COX-2 inhibitors) leads to a production of several AA metabolites responsible for an enhanced risk of cardiovascular accidents. In addition, the currently available NSAIDs and COX-2 inhibitors do not influence the 5-LOX pathway, but shunting the AA metabolism towards the 5-LOX pathway, increase the dangerous leukotrienes (LTs) that cause vasoconstriction and leukocyte attraction and accumulation. Indeed, flavocoxid and zileuton reduced T-cell infiltrate. However, additional mechanism involving COX metabolites may be operative: in fact celecoxib also succeeded in reducing T-cells influx. Enhanced levels of LTs are also measured in a variety of pathological conditions such as asthma, gastric ulceration, renal insufficiency, and cardiovascular complications. As a consequence, these evidence has driven the attention of researcher to different
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pathways involved in the arachidonic acid cascade, as the 5-LOX, to treat IBDs. Zileuton is used during asthma to prevent wheezing, shortness of breath, coughing, and chest tightness; its specific inhibitory effect on leukotriene production supports the rationale for using this drug in experimental colitis. The selective 5-lipoxygenase inhibitor was shown to accelerate mucosal healing in a rat model of colitis (Bertrán et al., 1996), but it wasnot effective in maintaining remission of symptoms in ulcerative colitis (Hawkey et al., 1997). In addition zileuton has been shown to increase the serum concentration or effects of theophylline, propranolol, and warfarin because is a minor substrate of CYP1A2, 2C8/9, 3A4, and a weak inhibitor of CYP1A2 (Lu et al., 2003). Indeed the protection against the microscopic damage induced by DNBS was greater with flavocoxid than with either zileuton or celicoxib. In addition, flavocoxid induced a more sustained improvement in food intake and weight gain than the other two drugs under study. Finally, flavocoxid was the only drug that achieved a significant reduction in both oxidative stress and leukocyte accumulation. For all the above reported reasons it is reasonable to speculate that a balanced inhibition of COX-1 and COX-2 together with the inhibition of 5-LOX achieved using flavocoxid could represent the option of choice for the treatment of colitis. The here reported results on Flavocoxid efficacy in two different experimental conditions candidate this natural dual inhibitor as a new therapeutic approach to IBDs. These data, however, deserve further confirmation in a clinical setting.
Acknowledgements This research received no grant from any funding agency in the public, commercial or not-for-profit sectors.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2016.07. 033.
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Cardiovascular events associated with rofecoxib in a colorectal adenoma chemoprevention trial. N. Engl. J. Med. 352, 1092–1102. Burnett, B.P., Bitto, A., Altavilla, D., Squadrito, F., Levy, R.M., Pillai, L., 2011. Flavocoxid inhibits phospholipase A2, peroxidase moieties of the cyclooxygenases (COX), and 5-lipoxygenase, modifies COX-2 gene expression, and acts as an antioxidant. Mediat. Inflamm. 2011, 385780. Cuzzocrea, S., Mazzon, E., Serraino, I., Dugo, L., Centorrino, T., Ciccolo, A., Sautebin, L., Caputi, A.P., 2001. Celecoxib, a selective cyclooxygenase-2 inhibitor reduces the severity of experimental colitis induced by dinitrobenzene sulfonic acid in rats. Eur. J. Pharmacol. 431, 91–102. Grisham, M.B., 1994. Oxidants and free radicals in inflammatory bowel disease. Lancet 344, 859–861. Hawkey, C.J., Dube, L.M., Rountree, L.V., Linnen, P.J., Lancaster, J.F., 1997. A trial of zileuton versus mesalazine or placebo in the maintenance of remission of ulcerative colitis. The European Zileuton study group for ulcerative colitis. Gastroenterology 112, 718–724. Holma, R., Salmenpera, P., Virtanen, I., Vapaatalo, H., Korpela, R., 2007. Prophylactic potential of montelucast against mild colitis induced by dextran sulfate sodium in rats. J. Physiol. Pharmacol. 58, 455–467. Lu, P., Schrag, M.L., Slaughter, D.E., Raab, C.E., Shou, M., Rodrigues, A.D., 2003. Mechanism-based inhibition of human liver microsomal cytochrome P450 1A2 by zileuton, a 5-lipoxygenase inhibitor. Drug Metab. Dispos. 31, 1352–1360. Martel-Pelletier, J., Lajeunesse, D., Reboul, P., Pelletier, J.P., 2003. Therapeutic role of dual inhibitors of 5-LOX and COX, selective and non-selective non-steroidal anti-inflammatory drugs. Ann. Rheum. Dis. 62, 501–509. McGettigan, P., Henry, D., 2011. Cardiovascular risk with non-steroidal anti-inflammatory drugs: systematic review of population-based controlled observational studies. PLoS Med. 8, 1001098. McGrath, J., Drummond, G., Kilkenny, C., Wainwright, C., 2010. Guidelines for reporting experiments involving animals: the ARRIVE guidelines. Br. J. Pharmacol. 160, 1573–1576. Mullane, K.M., Kraemer, R., Smith, B., 1985. Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemic myocardium. J. Pharmacol. Methods 14, 157–167. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358. Pavlick, K.P., Laroux, F.S., Fuseler, J., Wolf, R.E., Gray, L., Hoffman, J., Grisham, M.B., 2002. Role of reactive metabolites of oxygen and nitrogen in inflammatory bowel disease. Free Radic. Biol. Med. 33, 311–322. Podolsky, D.K., 2002. Inflammatory bowel disease. N. Engl. J. Med. 347, 417–429. Polito, F., Bitto, A., Irrera, N., Squadrito, F., Fazzari, C., Minutoli, L., Altavilla, D., 2010. Flavocoxid, a dual inhibitor of cyclooxygenase-2 and 5-lipoxygenase, reduces pancreatic damage in an experimental model of acute pancreatitis. Br. J. Pharm. 161, 1002–1011. Rahimian, R., Fakhfouri, G., Daneshmand, A., Mohammadi, H., Bahremand, A., Rasouli, M.R., Mousavizadeh, K., Dehpour, A.R., 2010. Adenosine A2A receptors and uric acid mediate protective effects of inosine against TNBS-induced colitis in rats. Eur. J. Pharmacol. 649, 376–381. Sandborn, W.J., Stenson, W.F., Brynskov, J., Lorenz, R.G., Steidle, G.M., Robbins, J.L., Kent, J.D., Bloom, B.J., 2006. Safety of celecoxib in patients with ulcerative colitis in remission: a randomized, placebo-controlled, pilot study. Clin. Gastroenterol. Hepatol. 4, 203–211. Sharon, P., Ligumsky, M., Rachmilewitz, D., Zor, U., 1978. Role of prostaglandins in ulcerative colitis. Enhanced production during active disease and inhibition by sulphasalazine. Gastroenterology 75, 638–640. Shaulian, E., Karin, M., 2012. AP-1 as a regulator of cell life and death. Nat. Cell Biol. 4, 131–136. Singh, V., Patil, C., Jain, N., Singh, A., Kulkarni, S.K., 2004. Effect of nimesulide on acetic acid- and leukotriene-induced inflammatory bowel disease in rats. Prostaglandins Other Lipid Mediat 71, 163–175. Sklyarov, A.Y., Panasyuk, N.B., Fomenko, I.S., 2011. Role of nitric oxide-synthase and cyclooxygenase/lypooxygenase systems in development of experimental ulcerative colitis. J. Physiol. Pharmacol. 62, 65–73. Solomon, S.D., McMurray, J.J., Pfeffer, M.A., Wittes, J., Fowler, R., Finn, P., Anderson, W.F., Zauber, A., Hawk, E., Bertagnolli, M., 2005. Cardiovascular risk associated with celecoxib in a clinical trial for colorectal adenoma prevention. N. Engl. J. Med. 352, 1071–1080. Wallace, J.L., Keenan, C.M., Gale, D., Shoupe, T.S., 1992. Exacerbation of experimental colitis by nonsteroidal anti-inflammatory drugs is not related to elevated leukotriene B4 synthesis. Gastroenterology 102, 18–27. Zingarelli, B., Squadrito, F., Graziani, P., Camerini, R., Caputi, A.P., 1993. Effects of zileuton, a new 5-lipoxygenase inhibitor, in experimentally induced colitis in rats. Agents Actions 39, 150–156.
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
Use of a balanced dual cyclooxygenase-1/2 and 5-lypoxygenase inhibitor in experimental colitis Giovanni Pallio a, Alessandra Bitto a, Gabriele Pizzino a, Federica Galfo a, Natasha Irrera a, Letteria Minutoli a, Vincenzo Arcoraci a, Giovanni Squadrito b, Antonio Macrì b, Francesco Squadrito a,n, Domenica Altavilla c a
Department of Clinical and Experimental Medicine, Section of Pharmacology University of Messina, via Consolare Valeria 1 Messina, Italy Department of Human Pathology University of Messina, via Consolare Valeria 1 Messina, Italy c Department of of Biomedical and Dental Sciences and Morphofunctional Imaging, University of Messina, via Consolare Valeria 1 Messina, Italy b
art ic l e i nf o
a b s t r a c t
Article history: Received 8 June 2016 Received in revised form 18 July 2016 Accepted 20 July 2016 Available online 21 July 2016
Cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) play an important role in inflammatory bowel diseases (IBDs). We investigated the effects of flavocoxid, a dual COX/LOX inhibitor, in experimental colitis induced with either dinitrobenzenesulfonic acid (DNBS) or dextrane sulphate sodium (DSS) In the first model, colitis was induced in rats by a single intra-colonic instillation (25 mg in 0.8 ml 50% ethanol) of DNBS; after 24 h animals were randomized to receive orally twice a day, flavocoxid (10 mg/ kg), zileuton (50 mg/kg), or celecoxib (5 mg/kg). Sham animals received 0.8 ml of saline by a single intracolonic instillation. Rats were killed 4 days after induction and samples were collected for analysis. In the second model, colitis was induced in rats by the administration of 8% DSS dissolved in drinking water; after 24 h animals were randomized to the same above reported treatments. Sham animals received standard drinking water. Rats were killed 5 days after induction and samples were collected for analysis. Flavocoxid, zileuton and celecoxib improved weight loss, reduced colonic myeloperoxydase activity, macroscopic and microscopic damage, and TNF-α serum levels. Flavocoxid and celecoxib also reduced malondialdheyde, 6-keto PGF1α and PGE-2 levels while flavocoxid and zileuton decreased LTB-4 levels. In addition, flavocoxid treatment improved histological features and apoptosis as compared to zileuton and celecoxib; moreover only flavocoxid reduced TXB2, thus avoiding an imbalance in eicosanoids production. Our results show that flavocoxid has protective effect in IBDs and may represents a future safe treatment for inflammatory bowel diseases. & 2016 Elsevier B.V. All rights reserved.
Keywords: Baicalin Catechin LTB-4 PGE-2 Apoptosis
1. Introduction The pathogenesis of inflammatory bowel diseases (IBDs) is still poorly understood, however, increasing evidence sustains the interaction between environmental, genetic, and immunological factors (Podolsky, 2002). During IBD reactive metabolites of oxygen (ROS) stimulate: the release of chemokines; activation of lipid peroxidation; recruitment of neutrophils; the release of TNF-α (Pavlick et al., 2002; Rahimian et al., 2010); the activation of NF-κB and the apoptotic kinase JNK (Shaulian and Karin, 2012). Of the pro-inflammatory genes that are induced by ROS, it is noteworthy the expression of cyclooxygenase (COX-2) and 5-lipoxygenase (5n Correspondence to: Department of Clinical and Experimental Medicine, Section of Pharmacology, Torre Biologica 5th floor, c/o AOU Policlinico G. Martino, Via C. Valeria Gazzi, 98125 Messina, Italy. E-mail address: [email protected] (F. Squadrito).
http://dx.doi.org/10.1016/j.ejphar.2016.07.033 0014-2999/& 2016 Elsevier B.V. All rights reserved.
LOX). In IBDs COX-2 mediates the increase in PGE2, important for the development and maintenance of an inflammatory microenvironment (Sklyarov et al., 2011). Concomitantly, an increased activity of 5-lipooxygenase (5-LOX) lead to an enhanced formation of leukotriene A4 (LTA-4), further transformed by the LTA-4 hydrolase into LTB-4, especially in macrophages and polymorphonuclear leukocytes. LTB-4 attracts leukocytes to the site of inflammation, promoting their adhesion to the inflammed and damaged tissue (Singh et al., 2004), thus amplifying the inflammatory cascade in IBDs (Holma et al., 2007). Aspirin and NSAIDs are known to suppress COX expression/activity and eicosanoid production, however, inhibition of COXs may lead to cardiotoxicity due to an imbalanced production of pro-thrombotic eicosanoids (e.g. increased TxA2) and anti-thrombotic eicosanoids (e.g. decreased PGI2) (Bresalier et al., 2005; Solomon et al., 2005). Since the inhibition of one or both COX enzymes may shunt arachidonic acid metabolism to the 5-LOX pathway, zileuton, an
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Fig. 1. Colon tissues from sham (A), DNBS þ drug vehicle (B), DNBS þFlavocoxid (C), DNBSþ Zileuton (D), DNBS þCelecoxib (E) together with the graph representing macroscopic damage scores (F) and colon weight/length ratio (G). Values are expressed as the mean and S.E.M. *P o 0.0001 vs DNBS þdrug vehicle group. n ¼ 7 for each group.
active 5-lipoxygenase inhibitor, was compared in a trial versus mesalazine and placebo. However, zileuton was not superior to placebo in maintaining remission of symptoms in ulcerative colitis (Hawkey et al., 1997). Thus, it appears that a targeted inhibition of either COX or 5-LOX does not represent the proper therapeutic strategy to block the inflammatory response in IBDs. It can also be speculated that a balanced inhibition of both enzymes might have synergistic effects and could be the key point for an effective treatment of IBDs. Some natural compounds, termed medical foods, demonstrated their efficacy and safety in the dietary management of specific diseases. A well characterized medical food, flavocoxid is a proprietary blend of baicalin and catechin, concentrated and standardized to greater than 90% purity. Flavocoxid, has been also demonstrated to be a balanced dual inhibitor of the peroxidise moieties of COX and 5-LOX, blunting the proinflammatory response (Burnett et al., 2011), as demonstrated in LPS-stimulated macrophages (Altavilla et al., 2009), acute pancreatitis (Polito et al., 2010), cecal ligation and puncture (Bitto et al., 2012). In addition to COX and 5-LOX inhibition flavocoxid may reduce reactive oxygen species including hydroxyl radical, superoxide anion radical, and hydrogen peroxide, modulating in turn, NF-κB and TNF-α production (Bitto et al., 2014). To date, a dual inhibitor of both COX and 5-LOX pathways has not been investigated in experimental colitis. Therefore, we aimed at evaluating the potential therapeutic effect of Flavocoxid, in experimental colitis induced with either DNBS or DSS, and to compare its effect to the 5-LOX inhibitor Zileuton and to COX-2 inhibitor,
celecoxib.
2. Materials and methods 2.1. Animal and treatments All animal procedures were in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki), the ARRIVE Guidelines (McGrath et al., 2010), and authorised by the Animal Ethics Committee of the Department Clinical and Experimental Medicine (approval #04/14), and all efforts were made to minimize suffering. A total of 70 male Sprague–Dawley rats (250–300 g) were purchased from Charles River Laboratories (Calco, Milan, Italy). Animals were maintained in plastic cages under standard environmental conditions with water and food ad libitum. After 1 week of acclimation to the laboratory environment, animals were randomly assigned to 10 groups of 7 animals each. In preliminary experiments, flavocoxid dose was titrated against the effects on PGE-2 expression in 30 animals. The day after DNBS administration 6 animals/group received flavocoxid twice a day at 5, 10, 20, and 40 mg/kg (by gavage) and the treatments lasted 4 days. This experiment identified 20 mg/kg/day as the optimal dose to be used in the further experiments (Supplemental Figure 1). Zileuton and celecoxib dose and route of administration were chosen according to previously published
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Fig. 2. Food intake (A) in DNBS animals *P o 0.0001 vs DNBS þdrug vehicle at selected time points. Weight loss (B) in DNBS animals evaluated during the study period §P o 0.05, #Po 0.001 vs DNBS þdrug vehicle respectively. Food intake (C) in DSS animals *P o0.0001 vs DSS þ drug vehicle at selected time points. §P o 0.05 vs Zileuton at day 5 §§P o 0.05 vs Celecoxib at day 3 and day 4. Weight loss (D) in DSS animals evaluated during the study period ^P o 0.01,*P o 0.0001 vs DNBSþ drug vehicle respectively. #Po 0.001 vs Celecoxib, °P o0.0001 vs Zileuton n ¼ 7 for each group.
reports (Zingarelli et al., 1993, Cuzzocrea et al., 2001). In a first set of experiments, colitis was induced in 28 fasted rats that were lightly anesthetised with diethyl ether, by a single intra-colonic instillation of 2,4-Dinitrobenzenesulfonic acid (DNBS; Sigma-Aldrich, Milan, Italy; 25 mg in 0.8 ml 50% ethanol). The catheter was inserted into the colon via the anus up to the splenic flexure 8 cm from the anus. Thereafter, the animals were kept for 15 min in Trendelenburg position to avoid reflux, and allocated in single cages for all the duration of the experiment. After 24 h, colitis signs were evident and animals were randomized to receive orally twice a day either flavocoxid (20 mg/kg/ day; hereinafter DNBS þ Flavocoxid), zileuton (100 mg/kg/day; hereinafter DNBS þ Zileuton), celecoxib (10 mg/kg/day; hereinafter DNBS þCelecoxib) or vehicle (1:3 DMSO:0.1% methylcellulose solution; hereinafter DNBS þ drug vehicle). Animals in the Sham group (n¼ 7) received 0.8 ml of saline, in a single intra-colonic instillation. Animals were observed for clinical signs of colitis, as diarrhoea and appetite; 4 days after induction all rats were killed and enteric tissue and blood samples were collected for analysis. In a second set of experiments, colitis was induced in 28 rats by oral administration of 8% DSS (MP Biomedicals, Solon, OH, USA) through drinking water from Day 0 to Day 5. Sham animals (n ¼7) were allowed ad libitum access to water. To assess the extent of colitis, the body weight, stool consistency and blood in the stool were monitored daily. After 24 h, colitis signs were evident and
animals were randomized to receive orally twice a day either flavocoxid (20 mg/kg/day; hereinafter DSS þFlavocoxid), zileuton (100 mg/kg/day; hereinafter DSS þZileuton), celecoxib (10 mg/kg/ day; hereinafter DNBS þCelecoxib) or vehicle (1:3 DMSO:0.1% methylcellulose solution; hereinafter DSS þdrug vehicle). The day of sacrifice, the abdomen was opened by a midline incision, the descending colon was removed, opened along the anti-mesenteric border, rinsed and cut into 2 same pieces, one for histological assessments and the other for analysis of biochemical markers. Moreover, blood samples were collected from the heart to estimate the level of proinflammatory cytokines. Flavocoxid was a kind gift of Primus Pharmaceuticals, Inc. (Scottsdale, AZ, USA); zileuton was purchased from Tocris Bioscence (Bristol, UK); celecoxib was purchased from Sigma Aldrich (Milan, Italy). Drugs were dissolved in the vehicle above described, prepared fresh daily and administered starting from the day after DNBS or DSS administration, and every 12 h thereafter. 2.2. Evaluation of body weight and food intake Body weight and food intake were recorded every day between 9:00 and 10:00 a.m. from the day of colitis-induction to the end of the experiment. Body weight results were expressed as row data and compared with food intake, by using the following formula: food intake divided by body weight in grams and multiplied by 100.
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Fig. 3. Histological evaluations (H&E staining, original magnification 10) of sham (A), DNBS þ drug vehicle (B), DNBS þFlavocoxid (C), DNBSþ Zileuton (D), DNBSþ Celecoxib (E). The graph represent the microscopic damage score (F), values are expressed as the mean and S.E.M. *P o 0.0001 vs DNBS þ drug vehicle group; ^P o0.01vs DNBS þCelecoxib; °P o0.0001 vs DNBS þZileuton. n ¼7 for each group.
2.3. Macroscopic damage score Colon damage was evaluated by two independent observers as described previously (Wallace et al., 1992), according to the following criteria: 0 (no damage), 1 (localized hyperemia without ulcers), 2 (linear ulcers with no significant inflammation), 3 (linear ulcers with inflammation at one site), 4 (two or more major sites of inflammation and ulceration extending 1 cm along the length of the colon), and 5–8 (one point is added for each centimeter of ulceration beyond an initial 2 cm). Colon weight/length ratio was also evaluated. 2.4. Microscopic damage score For light microscopy, colon tissues were rapidly removed and fixed in 10% buffered formalin. Subsequently, specimens were embedded in paraffin, sectioned at 5-μm thickness and stained with hematoxylin and eosin (H&E), and observed with a Leica (Leica Microsystems, Milan, Italy) microscope. Assessment of tissue changes was carried out by two observers blinded to the experimental protocol. The following morphological criteria were considered: score 0 (no damage), score 1 (mild, focal epithelial oedema and necrosis), score 2 (moderate, diffuse swelling and necrosis of the villi), score 3 (severe, necrosis with presence of neutrophil infiltrate in the submucosa), score 4 (highly severe, widespread necrosis with massive neutrophil infiltrate and hemorrhage) as previously reported (Rahimian et al., 2010). 2.5. Terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) assay TUNEL assay was performed by using a TUNEL detection kit
according to the manufacturer's instructions (Genscript, Piscataway, NJ, USA). Briefly, paraffin embedded tissues were sectioned (5 mm), rehydrated and incubate with proteinase K solution, for 15 min at 21–37 °C. Tissues were then treated with a 3% H2O2 solution for 10 min at 15–25 °C to inactivate the endogenous peroxidase. Sections were immersed in TUNEL reaction mixture and incubated for 60 min at 37 °C under wet conditions, protected from light. After wash in PBS slides were incubate with streptavidin-HRP Solution for 30 min at 37 °C under wet conditions, protected from light. Positive staining of apoptotic and necrotic cells were visualized with diaminobenzidine tetra-hydrochloride (DAB; Sigma Aldrich). Slides were counterstained with hematoxylin, to highlight the nuclei, dehydrated, and mounted with coverslips. 2.6. Immunohistochemical evaluation of CD3 Paraffin-embedded tissues were sectioned (5 mm), rehydrated, and antigen retrieval was performed by using 0.05 M sodium citrate buffer (pH 6.0) in a microwave for 5 min. Tissues were treated with 1% hydrogen peroxide to block endogenous peroxidase activity, and with horse normal serum (Vector Laboratories, Burlingame, CA, USA) to prevent nonspecific staining. A primary antibody against CD3 (Abcam, Cambridge, UK) was used and the slides were kept overnight at 4 °C in a humid box. The slides were then washed in PBS, added with a secondary antibody and the ABC reagent was used (Vectastain Elite ABC kit, Vector Laboratories). The location of the reaction was visualized with diaminobenzidine tetra-hydrochloride (DAB; Sigma-Aldrich, Milan, Italy). Slides were counterstained with hematoxylin, dehydrated, and mounted with coverslips. As a part of the histologic evaluation, all slides were examined by a pathologist without knowledge of the previous
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Fig. 4. Histological evaluations (H&E staining, original magnification 10) of sham (A), DSS þ drug vehicle (B), DSSþ Flavocoxid (C), DSS þ zileuton (D), DSS þ Celecoxib (E). The graph represent the microscopic damage score (F), values are expressed as the mean and S.E.M. *P o 0.0001 vs DSS þ drug vehicle group. n ¼ 7 for each group.
treatment, by using masked slides from 5 to 40 magnification with a Leica microscope (Leica Microsystems, Milan, Italy). 2.7. Evaluation of LTB4, PGE2, TNF-α, 6-keto PGF1α and TXB2 in serum Serum samples were assayed in duplicate using a commercially available ELISA kit for PGE2 (Cusabio Biotech, Wuhan, China), LTB4 (Bioassay Technology, Shanghai, China), TNF-α (Abcam, Cambridge, UK), TXB2 and 6-keto PGF1α (Cusabio, Washington, Massachusetts). Results were obtained interpolating the absorbances with the standard curves and expressed as means and S.D. 2.8. Measurement of Myeloperoxidase activity Myeloperoxidase (MPO) activity, an indicator of polymorphonuclear leukocyte accumulation, was determined as previously described (Mullane et al., 1985). The colon was homogenized in a solution containing 0.5% hexa-decyl-trimethylammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7.0). Lysates were then centrifuged for 30 min at 25,000 g at 4 °C. An aliquot of the supernatant was allowed to react with a solution of 1.6 mM tetra-methyl-benzidine and 0.1 mM H2O2. The rate of change in absorbance was measured with spectrophotometry at 650 nm. MPO activity was defined as the quantity of enzyme degrading 1μmol hydrogen peroxide/min at 37° and was expressed in units per 1 g of tissue. 2.9. Malondialdehyde measurement The levels of malondialdehyde (MDA) in the colon were
determined as an indicator of lipid peroxidation. The colon was homogenized in 1.15% KCl solution, an aliquot of the homogenate 0,1 ml was added to a reaction mixture containing 0,2 ml of 8.1% SDS, 1,5 ml of 20% acetic acid, 1,5 ml of 0.8% thiobarbituric acid and 0.7 ml distilled water. Samples were boiled for 1 h at 95 °C and centrifuged at 3000g for 10 min. The absorbance of the supernatant was measured by spectrophotometer at 650 nm (Ohkawa et al., 1979). 2.10. Statistical analysis All quantitative data are expressed as mean 7S.D. for each group, and compared by using one-way ANOVA for non-parametric variables, with Tukey post-test for intergroup comparisons. Statistical significance was set at Po0.05. Graph were drawn using GraphPad Prism software version 5.0 for Windows (GraphPad Software Inc., La Jolla, CA, USA).
3. Results 3.1. Effects of flavocoxid on body weight, food intake, and macroscopic damage Colitis was successfully induced in rats following DNBS intracolonic administration as demonstrated by the appearance of diarrhoea as early as 6 h after challenge, with a significant loss in body weight at the end of the experiment (P o0.001 vs sham group). Treatment with flavocoxid, zileuton, or celecoxib resulted in solid feces after only 2 days of treatment, with a parallel recovery from weight loss. Four days after induction of colitis at a
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Fig. 5. Histological evaluations (TUNEL staining, original magnification 10) of sham (A), DNBS þ drug vehicle (B), DNBSþ Flavocoxid (C), DNBS þ Zileuton (D) or DNBS þCelecoxib (E).Tissues of DNBS þ drug vehicle group demonstrated a marked appearance of dark brown apoptotic cells and intercellular apoptotic fragments (see arrows). No apoptotic cells or fragments were observed in the. DNBS rats treated with Flavocoxid, while animals of Zileuton or Celecoxib group demonstrated residual apoptosis in the submucosal layer (see arrows). n ¼ 7 for each group.
macroscopic observation the colon mucosa of DNBS þ drug vehicle group appeared ulcerated, oedematous, and hyperaemic compared to sham group (P o0.0001; Fig. 1A–B). In the groups treated with flavocoxid, zileuton or celecoxib there was a significant reduction in the extent and severity of colon injury (P o0.0001; Fig. 1C–F). As shown in Fig. 1G, DNBS also caused a significant shortening of the colonic length, with a consequent increase in the weight/length ratio, compared to control animals (P o0.0001). Treatment of rats with flavocoxid, zileuton, or celecoxib significantly improved this condition. Body weight and food intake were recorded at baseline (day 0) and throughout the experimental period. Food intake is reported in Fig. 2A and raw body weight data are reported in Fig. 2B. Flavocoxid, celecoxib, or zileuton treatment increased food intake and caused weight gain during the treatment period (P o0.05 vs DNBS þdrug vehicle group, Po0.001 vs DNBS þ drug vehicle group at end of experiment). No difference was observed among the several treatments. DSS-administered animals showed diarrhoea and rectal bleeding after 24 h, while weight reduction was observed starting from day 4 (P o0.05 vs Sham). Treatment with flavocoxid, zileuton or celecoxib resulted in reduced bleeding and improved food intake and body weight (Fig. 2C–D). Flavocoxid treatment in DSS animals caused a greater improvement in food intake and body weight than either zileuton or celecoxib (Fig. 2C and D). 3.2. Flavocoxid reduces histological damage and apoptosis A normal appearance of the colonic mucosa with intact
epithelium was observed in sham group (Fig. 3A). The histological analysis, 4 days after induction of colitis, showed mucosal injury induced by DNBS administration that was characterized by necrosis of epithelium and massive infiltration of neutrophils and macrophages into the mucosa and submucosa layers. Thickening of the colon wall, transmural necrosis, loss of globet cells, and oedema were also observed (Fig. 3B). Treatment with flavocoxid significantly reduced the extent and severity of the histological alteration associated with DNBS administration by stimulating regeneration of the epithelium (Fig. 3C) with preservation of the crypts, and globet cells. Zileuton and celecoxib administration significantly reduced the histological signs of injury; however, these two drugs did not completely restore the epithelial layer, crypts and globet cells (Fig. 3D–E). Microscopic damage scores in DNBS þFlavocoxid, DNBS þZileuton and the DNBS þCelecoxib groups were significantly reduced than in DNBS þdrug vehicle group (Po0.0001; Fig. 3F). In addition, flavocoxid score was significantly improved compared to zileuton or celecoxib-treated groups (Po 0.01vs DNBS þ Celecoxib; Po 0.001 vs DNBS þ Zileuton Fig. 3F). Histological analysis of the DSS animals, carried 5 days after challenge, showed a flattening of the mucosa with a reduced presence of mucosal glands, a massive inflammatory infiltrate and oedema in the submucosal layer, with no evident signs of apoptosis (Fig. 4B), as compared to sham animals (Fig. 4A). Treatment with flavocoxid significantly reduced the extent and severity of the histological alteration associated with DSS administration by stimulating regeneration of the epithelium of the crypts, and globet cells (Fig. 4C). Zileuton or celecoxib administration significantly
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Fig. 6. Immunohistochemical evaluation of CD3 cells, (original magnification 40) of sham (A), DNBS þ drug vehicle (B), DNBS þ Flavocoxid (C), DNBSþ Zileuton (D) or DNBS þCelecoxib (E). The arrows indicate CD3-positive T cells. The graph (F) represent the number of positive cells in each group; values are expressed as the mean and S.E. M. *P o 0.0001 vs DNBS þ drug vehicle group. n ¼ 7 for each group.
reduced the histological signs of injury; however, they did not properly restored the architecture of the mucosa (Fig. 4D–E). The microscopic damage score is shown in the graph (Fig. 4F). Apoptosis evaluation, 4 days after DNBS administration, showed an intense staining in the remains of the mucosa and submucosa layers, with diffuse intercellular apoptotic fragments, especially in the DNBS þ drug vehicle group compared to sham (Fig. 5A-B). By contrast, specimens obtained from rats treated with flavocoxid, zileuton or celecoxib showed reduced apoptotic cells. (Fig. 5C-E). 3.3. Immunohistochemical evaluation of CD3 expression Colitis induced by both DNBS and DSS caused a marked colonic infiltrate of CD3 positive T cells when compared to sham group. The several treatments produced a significant decrease in the expression of CD3 positive cells and no difference was observed among the different groups (Fig. 6A–F; Fig. 7A–F).
3.4. Effects of Flavocoxid, zileuton or celecoxib on PGE-2, LTB-4, TNFα, 6-keto PGF1α, TXB2 levels Circulating levels of the main inflammatory eicosanoids, PGE-2, LTB-4, 6-keto PGF1α, and TXB2 and of the proinflammatory cytokine TNF-α were measured four days after DNBS administration. Serum PGE2 and LTB4 measurements were determined to prove flavocoxid efficacy in this experimental model. As shown in Fig. 8A and B a significant increase in both PGE-2 and LTB-4 was found in serum from DNBS þ drug vehicle animals compared to control animals (Po0.0001). PGE2 and LTB4 serum levels were significantly reduced by flavocoxid, likely as a direct consequence of COX-2 and 5-LOX inhibition induced by the dual COX/5LOX inhibitor (Fig. 8A–B. In contrast treatment with zileuton did not change PGE-2 levels (Fig. 8A–B), and celecoxib did not modify LTB4 levels (Fig. 8A–B). Serum levels of TNF-α were significantly augmented in the bloodstream of DNBS þdrug vehicle rats, compared to sham
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Fig. 7. Immunohistochemical evaluation of CD3 expression, (original magnification 40) of sham (A), DSS þ drug vehicle (B), DSS þFlavocoxid (C), DSS þZileuton (D) or DSS þ Celecoxib (E). The arrows indicate CD3-positive T cells. The graph (F) represent the number of positive cells in each group; values are expressed as the mean and S.E.M. *P o 0.0001 vs DSS þ drug vehicle group. n ¼ 7 for each group.
Fig. 8. PGE-2 (A), LTB-4 (B), and TNF-α (C) levels in serum of sham, DNBS þdrug vehicle DNBSþ Flavocoxid, DNBS þZileuton or DNBSþ Celecoxib at the end of the experiment. *Po 0.0001 vs DNBS þ drug vehicle; °P o 0.0001 vs DNBSþ zileuton; ^P o 0.0001 vs DNBSþ Celecoxib. n¼ 7 for each group.
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Fig. 9. 6-keto PGF1α (A), TXB2 (B), levels in serum of DNBS animals at the end of the experiment. *P o 0.0001 vs DNBS þ drug vehicle; °P o 0.0001 vs DNBSþ zileuton; ^P o 0.0001 vs DNBS þCelecoxib. n ¼7 for each group. 6-keto PGF1α (C), TXB2 (D), levels in serum of DSS animals at the end of the experiment. *Po 0.0001 vs DSS þ drug vehicle; °P o 0.0001 vs DSS þzileuton; ^P o0.0001 vs DSS þCelecoxib. n ¼7 for each group.
(Fig. 8C; P o0.0001). All treatment markedly blunted the serum concentration of TNF-α (Fig. 8C; Po0.0001 vs DNBS þdrug vehicle). Additionally, 6-keto PGF1α and TXB2 levels (the stable hydrolysis products of PGI2 and TXA2) were measured in serum four days after DNBS administration and five days after DSS administration. A significant increase of both was found in serum from DNBS þ drug vehicle animals compared to controls (Fig. 9A–B; Po0.0001). As a consequence of COX and 5-LOX inhibition by flavocoxid treatment, 6-keto PGF1α and TXB2 serum levels were significantly reduced (Fig. 9A–B, P o0.0001 vs DNBS þdrug vehicle). Celecoxib administration reduced only 6-keto PGF1α levels (Fig. 9A–B, P o0.0001 vs DNBS þdrug vehicle), while zileuton treatment did not modify 6-keto PGF1α and TXB2 levels. Similar results have been obtained from DSS-treated animals (Fig. C–D). 3.5. Effects of flavocoxid, zileuton and celecoxib on lipid peroxidation and neutrophil infiltration MDA levels were increased as expected in DNBS þ drug vehicle groups (P o0.0001 vs sham; Fig. 10A); of the three treatment used, only flavocoxid and celecoxib blunted lipid peroxidation (P o0.01; Fig. 10A), demonstrating a superior antioxidant activity. Accumulation of polymorphonuclear granulocytes determined as myloperoxidase activity was increased in DNBS þ drug vehicle group (P o0.0001 vs sham; Fig. 10B). All treatment significantly reduced MPO activity (P o0.0001; Fig. 10B). Flavocoxid treatment more significantly reduced the MPO activity compared to celecoxib group (P o0.01 Fig. 10B).
In the DSS þ drug vehicle group MDA levels were increased as expected (P o0.0001 vs sham; Fig. 10C), only flavocoxid (Po0.001 vs DSS þ Drug vehicle) and celecoxib (Po 0.01 vs DSS þ drug vehicle) blunted lipid peroxidation (Fig. 10C) demonstrating a superior antioxidant activity. Accumulation of neutrophils determined as MPO was increased in DSS þ drug vehicle group (Po0.0001 vs sham; Fig. 10D). All treatment significantly reduced MPO activity; moreover flavocoxid treatment had a greater efficacy in blunting MPO activity compared to celecoxib group (Po0.01 Fig. 10D).
4. Discussion Reactive species of oxygen and nitrogen have been linked to inflammatory bowel disease almost 20 years ago (Grisham, 1994). These species increase, among the others, the expression of the enzymes COX-2 and 5-LOX that produce crucial mediators for the development of colitis, in particular PGE-2 and LTB-4 levels (Sharon et al., 1978). The eicosanoids are able to further activate inflammatory cells and the oxidative stress with a consequent transcription of NFκB-related cytokines. In agreement with these previous data, our results show that flavocoxid, with a balanced inhibition of either COX and 5-LOX, was more effective than zileuton or celecoxib in reducing eicosanoid production during experimental colitis. The reduced PGE-2 and LTB-4 levels, together with decreased lipid peroxidation (as demonstrated by MDA levels) could be also related to the antioxidant properties of this flavonoid compound. Halting of eicosanoid production decreased
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Fig. 10. Effects of Flavocoxid, Zileuton and Celecoxib on Malondialdehyde levels (A) and Myeloperoxidase activity (B) in DNBS animals. Values are expressed as means and S. D. #P o0.01 vs DNBS þ drug vehicle group;*Po 0.0001 vs DNBS þ drug vehicle; ^P o 0.01 vs DNBS þ Celecoxib. Effects of Flavocoxid, Zileuton and Celecoxib on Malondialdehyde levels (C) and Myeloperoxidase activity (D) in DSS animals. Values are expressed as means and S.D. #P o0.01 vs DSS þ drug vehicle. ##Po 0.001 vs DSS þ drug vehicle group; *P o 0.0001 vs DSS þ drug vehicle; ^P o 0.01 vs DSS þ Celecoxib. n ¼ 7 for each group.
oxidative stress in the colon as demonstrated by the markedly visible improvement in the histological pictures. In fact, at a first glance, the colon appeared to be clean with no sign of hemorrhage, and at a microscopic observation we noticed a reduced presence of oedema with a preserved architectural structure of mucosal and submucosal layers, and absence of apoptotic/necrotic cells at the same level. As additional consequence of the reduced eicosanoid production also neutrophil infiltration was significantly reduced, and this is obvious because LTB-4 is more active than PGE2 as chemotactic substance. Interestingly, flavocoxid, zileuton and celecoxib succeeded in reducing MPO activity in colon specimens following DNBS or DSS induction. The reduced infiltrate of early inflammatory cells is in turn responsible for the reduction in TNFα production; it is indeed known that this proinflammatory cytokine is mainly produced during the first stages of the inflammatory process by neutrophils and macrophages and has the ability to activate apoptosis. All these positive effects observed in animals treated with flavocoxid were further confirmed by the reduction of diarrhoea and the consequent improvement in weight loss. Prostaglandins from COX-1 provide for the processes of water and electrolyte transport, vasodilatation, proliferation, and intercellular integration. During inflammatory activation, the considerable amount of prostaglandins synthesized by COX-2 enhance the activity of 5-LOX and stimulate the release of inflammatory leukotriens as LTB4, LTD4, and LTC4, further responsible for cell infiltration and oedema in submucosal layer (Sklyarov et al., 2011). Moreover cysteinyl leukotriens, induce local vasoconstriction, thus reducing blood flow and enhancing the susceptibility of gastric mucosa to injury. In light of these observations, leukotrienes may
play an important role in the development of the inflammatory process, and it is now clear that PGs and LTs have complementary effects (Martel-Pelletier et al., 2003). These effects are blunted by flavocoxid through a balanced peroxidase inhibiton of the COX and LOX enzymes, together with a specific block of phospholipase-A2 (PLA-2) activity (Burnett et al., 2011). Both effects are peculiar, in fact common non steroid antinflammatory drugs (NSAIDs) inhibit the cycloxygenase moiety of COX, preventing PGG2 formation from arachidonic acid. As previously shown celecoxib, a selective inhibitor of COX-2, ameliorates the severity of experimental colitis induced by DNBS (Cuzzocrea et al., 2001), and it was also effective in subjects with ulcerative colitis (Sandborn et al., 2006). However, large clinical studies clearly demonstrated increased cardiovascular risk for patients treated for long periods with either COX-2 inhibitors or NSAIDs (McGettigan and Henry, 2011), dramatically reducing the rationale for the use of these drugs in IBDs. In fact, the imbalanced inhibition of COX-2 and COX-1 (by selective COX-2 inhibitors) leads to a production of several AA metabolites responsible for an enhanced risk of cardiovascular accidents. In addition, the currently available NSAIDs and COX-2 inhibitors do not influence the 5-LOX pathway, but shunting the AA metabolism towards the 5-LOX pathway, increase the dangerous leukotrienes (LTs) that cause vasoconstriction and leukocyte attraction and accumulation. Indeed, flavocoxid and zileuton reduced T-cell infiltrate. However, additional mechanism involving COX metabolites may be operative: in fact celecoxib also succeeded in reducing T-cells influx. Enhanced levels of LTs are also measured in a variety of pathological conditions such as asthma, gastric ulceration, renal insufficiency, and cardiovascular complications. As a consequence, these evidence has driven the attention of researcher to different
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pathways involved in the arachidonic acid cascade, as the 5-LOX, to treat IBDs. Zileuton is used during asthma to prevent wheezing, shortness of breath, coughing, and chest tightness; its specific inhibitory effect on leukotriene production supports the rationale for using this drug in experimental colitis. The selective 5-lipoxygenase inhibitor was shown to accelerate mucosal healing in a rat model of colitis (Bertrán et al., 1996), but it wasnot effective in maintaining remission of symptoms in ulcerative colitis (Hawkey et al., 1997). In addition zileuton has been shown to increase the serum concentration or effects of theophylline, propranolol, and warfarin because is a minor substrate of CYP1A2, 2C8/9, 3A4, and a weak inhibitor of CYP1A2 (Lu et al., 2003). Indeed the protection against the microscopic damage induced by DNBS was greater with flavocoxid than with either zileuton or celicoxib. In addition, flavocoxid induced a more sustained improvement in food intake and weight gain than the other two drugs under study. Finally, flavocoxid was the only drug that achieved a significant reduction in both oxidative stress and leukocyte accumulation. For all the above reported reasons it is reasonable to speculate that a balanced inhibition of COX-1 and COX-2 together with the inhibition of 5-LOX achieved using flavocoxid could represent the option of choice for the treatment of colitis. The here reported results on Flavocoxid efficacy in two different experimental conditions candidate this natural dual inhibitor as a new therapeutic approach to IBDs. These data, however, deserve further confirmation in a clinical setting.
Acknowledgements This research received no grant from any funding agency in the public, commercial or not-for-profit sectors.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2016.07. 033.
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