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Diplacone and mimulone ameliorate dextran sulfate sodium-induced colitis in rats
FITOTE-03110; No of Pages 8 Fitoterapia xxx (2015) xxx–xxx
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Fitoterapia
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Zora Vochyánová a, Ladislava Bartošová b, Veronika Bujdáková a, Petr Fictum c, Roman Husník d,e, Pavel Suchý b, Karel Šmejkal a, Jan Hošek f,⁎ a
Department of Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, 612 42 Brno, Czech Republic Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, 612 42 Brno, Czech Republic c Department of Pathological Morphology and Parasitology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences Brno, 612 42 Brno, Czech Republic d Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70808, USA e International Clinical Research Center (ICRC), St. Anne's University Hospital Brno, Pekařská 53, 656 91 Brno, Czech Republic f Department of Molecular Biology and Pharmaceutical Biotechnology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, 612 42 Brno, Czech Republic b
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Diplacone and mimulone ameliorate dextran sulfate sodium-induced colitis in rats
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journal homepage: www.elsevier.com/locate/fitote
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Article history: Received 28 November 2014 Accepted in revised form 14 January 2015 Available online xxxx
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Chemical compounds studied in this article: Diplacone (PubChem CID: 14539948) Mimulone (PubChem CID: 5716903) Sulfasalazine (PubChem CID: 5353980)
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Keywords: Diplacone Mimulone Geranylated flavanones Dextran sulfate sodium (DSS) Ulcerative colitis In vivo
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1. Introduction
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Inflammatory bowel diseases (IBD), such as Crohn's disease (CD) and ulcerative colitis (UC), are chronic inflammatory
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Diplacone (1) and mimulone (2), two geranylated flavanones, have previously shown antiinflammatory and antiradical activity in vitro. The present study aimed to evaluate their activity in vivo on a model of colitis induced in Wistar rats by an oral administration of dextran sulfate sodium (DSS). Diplacone (1) and mimulone (2) were administered at a bolus dose of 25 mg/kg by gastric gavage 48 and 24 h prior to the induction of colitis by DSS and every 24 h on the following days of the experiment. The effect of the treatment was assessed by monitoring the disease activity index (DAI), histopathological examination, evaluation of the weight and length of the colon and by analysis of the levels and activities of cyclooxygenase-2 (COX-2), matrix metalloproteinase-2 (MMP2), superoxide dismutase-2 (SOD2), and catalase (CAT) in the inflamed tissue. Administration of the test compounds prior and after induction of colitis ameliorated the symptoms of colitis (diarrhea, presence of the blood in the stool) and delayed their onset. The ability of compounds 1 and 2 to reduce the levels of COX-2 and to increase the ratio of pro-MMP2/ MMP2 activity correlates with the values of the DAI. The lowering of the levels of the antioxidant enzymes SOD2 and CAT reflects the ability of the test compounds to scavenge reactive oxygen species. © 2015 Published by Elsevier B.V.
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Abbreviations: CAT, catalase; CD, Crohn's Disease; COX-2, cyclooxygenase-2; DSS, dextran sulfate sodium; IBD, Inflammatory Bowel Disease; LPS, lipopolysaccharide; MMP2, matrix metalloproteinase 2; NF-κB, nuclear factor κB; PVP, polyvinylpyrollidone; ROS, reactive oxygen species; SAS, sulfasalazine; SOD2, superoxide dismutase-2; UC, ulcerative colitis. ⁎ Corresponding author. Tel.: +420 541562839; fax: +420 541240606. E-mail address: [email protected] (J. Hošek).
illnesses of the gastrointestinal tract with intermittent periods of relapse and with the progression of the disease difficult to predict. Their incidence in Western populations has been increasing in recent years. The etiology of chronic inflammatory illnesses of the gastrointestinal tract remains unclear and there is therefore no causal treatment. Current therapy is not completely successful and it is frequently connected with adverse effects [1]. Plant products may provide an option for the alternative or supplementary treatment of the patients, and their structures can serve to provide leads or inspiration for the synthesis of new anti-inflammatory agents.
http://dx.doi.org/10.1016/j.fitote.2015.01.012 0367-326X/© 2015 Published by Elsevier B.V.
Please cite this article as: Vochyánová Z, et al, Diplacone and mimulone ameliorate dextran sulfate sodium-induced colitis in rats, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.012
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2. Materials and methods
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2.1. Test compounds
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Diplacone (1) and mimulone (2) were isolated from the unripe fruits of Paulownia tomentosa (Thunb.) Steud. (Paulowniaceae) and characterized as previously described [7]. The identification was carried out using ESIMS, 1H and 13C NMR analyses (see Supplemental Data Fig. S1–S7); the purity was checked via HPLC analysis and exceeded 95% [7]. The chemical structures are shown in Fig. 1. Sulfasalazine was purchased from Sigma-Aldrich (Steinheim, Germany).
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2.2. Experimental animals
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Male Wistar rats (180–220 g) were supplied by AnLab, Ltd. (Prague, Czech Republic). The animals were kept under standard conditions (temperature 22 ± 2 °C, relative humidity 50 ± 10%, alternating 12 hour light/dark cycle). They were fed a standard diet and given water ad libitum. The experimental protocol was approved by the Expert Committee for the Welfare of Experimental Animals of the University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic (Approval No. 26-2013). To minimize the suffering of the laboratory animals, the number of pharmacological interventions was limited to the necessary minimum.
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2.3. Induction of colitis
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Rats were randomly divided into five groups (n = 8 for each group) after a one week period of acclimation. Colitis was induced by using 10% (w/v) DSS (MP Biomedicals, IllkirchGraffenstaden, France; molecular weight 36,000–50,000 Da) in the drinking water, as previously described [10], with some
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2.5. Disease activity index
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The rats were monitored daily for loss of body weight, stool consistency, and rectal bleeding in order to calculate the disease activity index (DAI) for each animal. The DAI, which reflects the severity of colitis, was modified according to Oh et al. (2006). The loss of body weight was assigned 0–2 points; it did not exceed 10% by the end of the experiment. The change in stool consistency was 0 points for normally formed stool, 1 point for loose stool, and up to 4 points for diarrhea according to the intensity of the changes. The presence of blood in the feces was assigned 0, 2, or 4 points (Table S1). The total DAI was then the sum of the points obtained from all of the parameters monitored. In the case of death during the experiment, the animal was assigned 10 points to enable the statistical evaluation.
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2.6. Histological evaluation
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Samples about 1.5 cm long were taken from the proximal and distal colon and fixed in 10% neutral buffered formalin then embedded in paraffin. Sections 3-μm-thick were stained with hematoxylin and eosin (H&E) and examined microscopically by a veterinary pathologist. The loss of epithelial surface, destruction of crypts and inflammatory cell infiltration into mucosa was assessed according to the criteria by Araki et al. (2000). Each parameter was assigned as follows: 0 = no change, 1 = localized and mild, 2 = localized and moderate, 3 = extensive and moderate, and 4 = extensive and severe. Total histological score is the sum of the points from all of the parameters [11].
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2.7. Preparation of protein for immunodetection
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The tested compounds 1 and 2, and the positive control sulfasalazine were administrated prophylactically, 48 and 24 h before the induction of colitis and in the following days concomitantly by gastric gavage under light isoflurane anesthesia. The substances were suspended in a 4% gel of PVP K90 (polyvinylpyrrolidone K90, Sigma-Aldrich) and were given at a bolus dose of 25 mg/kg of diplacone (1, 589 mmol/kg), mimulone (2, 612 mmol/kg), or sulfasalazine (SAS, 628 mmol/kg). Animals in the untreated group (DSS group) and the intact group were treated with the vehicle only (4% gel of PVP). On day five of the experiment, the animals were sacrificed by using an overdose of T61 (Intervet International B. V., Boxmeer, Netherlands), a veterinary euthanasia drug. Immediately after the euthanasia, the entire colon was removed, including the caecum; it was opened and cleaned with physiological saline, measured and weighed. Samples for histological evaluation were obtained and the remaining parts of the colons were frozen in liquid nitrogen and stored at − 80 °C for the next processing.
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modifications. The rats in the intact group received drinking 115 water only instead of DSS solution. 116
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In vitro and in vivo studies have shown that flavonoids are appropriate candidates for new anti-inflammatory drugs. Their ability to inhibit the activity and expression of phospholipase A2, cyclooxygenases, lipoxygenases, and inducible synthase of nitric oxide leads to diminished production of the mediators of inflammation [2]. Scavenging the reactive oxygen species (ROS) moderates the damage to tissue and affects the signal transduction pathways [3]. Furthermore, the intervention in the signal cascade of nuclear factor κB (NF-κB) changes the expression of a series of genes connected with the inflammatory response and apoptosis [4]. This study focused on two geranylated flavanones diplacone (1) and mimulone (2). Compounds 1 and 2 have shown anti-inflammatory [5,6], antiradical, cytoprotective [7,8], and antibacterial activities [9] in previous studies. The aim of the present study was to determine the biological activity of these compounds in vivo in the model of dextran sulfate sodium (DSS)-induced colitis in rats. The efficacy of compounds 1 and 2 was compared to sulfasalazine (SAS), a conventional drug used in IBD therapy. Besides the histological and physical examinations, the levels of the antioxidant enzymes superoxide dismutase-2 (SOD2) and catalase (CAT), the levels of cyclooxygenase-2 (COX-2), and the activity of matrix metalloproteinase-2 (MMP2) were investigated to elucidate the anti-inflammatory effects of these compounds.
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Using a bench blender, approximately 2 g of colonic tissue 166 was homogenized in lysis buffer [50 mM Tris–HCl (pH 7.5), 167
Please cite this article as: Vochyánová Z, et al, Diplacone and mimulone ameliorate dextran sulfate sodium-induced colitis in rats, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.012
Z. Vochyánová et al. / Fitoterapia xxx (2015) xxx–xxx
2.9. MMP2 activity
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MMP2 activity (pro-MMP2, MMP2) was evaluated using gelatin zymography, as described previously [12]. Twenty micrograms of the native proteins (without denaturation in the presence of β-mercaptoethanol) was loaded onto 10% SDSpolyacrylamide gel impregnated with 0.1% gelatin. After electrophoresis the gel was washed twice for 15 min in 2.5% (v/v) Triton X-100 to remove SDS. The gel was then incubated for 15 min at room temperature and subsequently overnight (about 16 h) at 37 °C in the development buffer [50 mM Tris– HCl (pH 8.8), 5 mM calcium chloride, 3 mM sodium azide, 0.5% (v/v) Triton X-100]. After that, the gel was stained with Coomassie blue for 2 h and destained until the bands were clearly visible. The intensity of the digested bands was determined by densitometric analysis using AlphaEase FC 4.0.0 software (Alpha Innotech Corporation). The ratio between the pro-MMP2 form and the active MMP2 form was calculated.
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Fig. 1. Chemical structures of diplacone (1) and mimulone (2).
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3. Results
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Protein samples were denatured in the presence of βmercaptoethanol and SDS at 70 °C for 5 min. The denatured proteins (80 μg for SOD2 and CAT, 120 μg for COX-2) were loaded onto 12% SDS-polyacrylamide gel. After electrophoresis, the proteins were transferred to a nitrocellulose membrane (0.2 μm, Bio-Rad, Hercules, USA), which was subsequently blocked with 5% BSA (Sigma-Aldrich) in TBST buffer [10 mM Tris–HCl (pH 7.5), 150 mM sodium chloride, 0.1% (v/v) Tween20] for 1 h. The membrane was then incubated with the primary mouse anti-COX-2 antibody at a dilution of 1:200 (Abcam, Cambridge, UK), the rabbit anti-SOD2 antibody at a dilution of 1:1000 (Sigma-Aldrich), the mouse anti-CAT antibody at a dilution of 1:1000 (Sigma-Aldrich), or the mouse anti-β-actin antibody at a dilution of 1:5000 (Abcam) overnight at 4 °C. After washing with distilled water, the membrane was incubated with the secondary antibody (antimouse IgG or anti-rabbit IgG, Sigma-Aldrich) at a dilution of 1:2000 for 1 h at room temperature. The secondary antibody was detected by colorimetric analysis using an Opti-CNTM Substrate Kit (Bio-Rad). The intensity of the protein bands was evaluated by densitometric analysis using AlphaEase FC 4.0.0 software (Alpha Innotech Corporation, San Leandro, USA).
The results are expressed as the mean values with error bars representing the standard error (SE) of the mean. One-way ANOVA followed by Tukey's post hoc test, two-way ANOVA followed by Bonferroni's post hoc test, and Kaplan–Meier analysis were used for the statistical evaluation. The specific analyses are referenced with the particular results. Values of p b 0.05 were considered to be statistically significant. GraphPad Prism 5.01 (GraphPad Software Inc., San Diego, USA) was used for the analysis.
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3.1. Disease activity index
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Administration of 10% DSS in the drinking water led to the loss of body weight, changes in the consistency of the stool, and rectal bleeding in the treated animals. The loss of body weight was comparable in all experimental groups. Changes in the consistency of the stool were observed in the untreated DSS group on day 3 of the experiment, on day 4 in the group treated with 1 and 2, and on day 3 or 4 in the SAS group. Blood appeared in the stool of all experimental groups treated with DSS one day after changes in the consistency of the stool. None of the described changes were observed in the intact group. The test compounds ameliorated the symptoms of colitis (diarrhea, presence of blood in the stool) and delayed their onset. The groups treated with diplacone (1) and mimulone (2) showed the lowest DAI on the last day of the experiment (p N 0.001 compared to the DSS-only group). Fig. 2A shows the development of the disease activity index for each group in the course of experiment. Data are also presented in Table S2. Four animals in the DSS group, one in the group treated with 1 and 2, and two in the SAS group died during the last day of the experiment. No mortality was observed in the intact group (Fig. 2B).
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3.2. Weight/length ratio of the colon
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The administration of 10% DSS for five days caused the weight/length ratio of the colon to increase by 30.4% compared to the intact group. Although the ratio increased less in the treated groups (20.4% for treatment with 1, 16.2% for treatment with 2, and 17.7% for treatment with SAS, as compared to the intact group), the differences were not statistically significant. The increase in the weight/length ratio of the colon was caused by shortening of the colon (Fig. S8).
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3.3. Histopathological examination
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Administration of DSS for five days induced pathological changes involving shortening or entire loss of the crypts, erosion of the epithelium, atrophy of the mucosa, infiltration of the lamina propria with neutrophils and macrophages, fibroplasia, and ulcerations. These changes were less severe in the proximal part of the colon. They were not observed in the intact group of animals. In the treated groups of animals, the intensity of the changes was similar to that observed in the untreated DSS group. Only two samples showed less severe lesions compared to the DSS group: one from the diplacone (1)
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2.10. Statistical analysis
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1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M saccharose] at a ratio of 3 mL of buffer per gram of tissue. After centrifugation at 9000 rpm for 15 min at 4 °C, the supernatant was stored. The protein concentration was measured using a Bradford-method protein assay kit (Amresco, Solon, USA) according to the manufacturer's instructions.
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Please cite this article as: Vochyánová Z, et al, Diplacone and mimulone ameliorate dextran sulfate sodium-induced colitis in rats, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.012
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3.5. Effect of treatment on MMP2 activity
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A marked decrease in the ratio of pro-MMP2/MMP2 was observed in the untreated DSS group compared to the intact group (p b 0.01). As shown in Fig. 6, the test compounds increased this ratio (by 50.7% in the case of 1, by 43.3% in the case of 2, and by 37.6% for SAS, as compared to the DSS group).
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4. Discussion
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Pharmacotherapy (aminosalicylates, glucocorticoids, immunosuppressives) and surgical procedures are currently used as the main instruments in the therapy of inflammatory bowel diseases. Contemporary approaches are not always completely effective; they are associated with a number of adverse effects, and they can negatively affect the quality of the patient's life. Therefore, there is an urgent need for a new strategy, either alternative or supplementary, for treating IBD. The beneficial properties of flavonoids (the ability to scavenge reactive oxygen species and to reduce the expression of proinflammatory markers) can also be utilized in the treatment of chronic inflammatory diseases, such as CD and UC. Diplacone (1) and mimulone (2) belong to the group of geranylated flavonoids. The lipophilic chain formed by the attachment of a geranyl unit at C-6 of the flavanone skeleton modifies their observed biological activities, as compared to their nongeranylated analogs [9,13,14]. This observation is connected with the increased affinity of such compounds for different biological membranes, with possible higher bioavailability and therefore greater therapeutic potential or potential toxicity than the parent non-geranylated compounds [15]. Nonetheless, only one study describes the anti-inflammatory activity of flavonoids substituted with a prenyl chain in animal models of colitis [16]. However, naringenin, the parent compound of 2, without a geranyl side chain, has been shown to ameliorate DSS-induced colitis in mice [17] and acetic acid-induced colitis in rats [18]. DSS-induced colitis was chosen as the optimal model for determining the benefits of prophylactic and curative administration of the geranylated flavanones diplacone (1) and mimulone (2). The administration of 10% DSS in drinking water caused acute colitis, localized especially in the distal part of the colon. In the pilot study preceding this experiment, the administration of lower concentrations of DSS (5% and 7%) was not effective—apart from the loss of body weight, no symptoms of colitis were observed (unpublished data), in contrast to the published studies carried out using Wistar rats [10]. Colitis induced by the administration of 10% DSS for five days was characterized by the loss of body weight, diarrhea, bloody stool and histologically observable damage (shortening or entire loss of the crypts, erosion of the epithelium, atrophy of the mucosa, infiltration of the lamina propria with neutrophils and macrophages, fibroplasia, and ulcerations). Other studies have also described the shortening of the colon in DSS-induced colitis [16,19]. We confirmed this fact, but the shortening was not significant to be compared to the intact group, probably because of the short-term administration of DSS. All of the compounds tested ameliorated and delayed changes in the consistency of the stool and rectal bleeding. Diplacone (1) showed the greatest therapeutic effect (i.e. the lowest DAI on the last day of experiment) when all of the compounds tested
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group and one from the SAS. Evaluation of colonic damage is shown in Fig. S9. The differences were not statistically significant. Representative samples are shown in Fig. 3.
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Fig. 2. Effect of the test compounds on DAI and survival in DSS-induced rat colitis. Colitis was induced by administration of 10% DSS in the drinking water for five days. Administration of diplacone (1), mimulone (2), and sulfasalazine (SAS) at a dose of 25 mg/kg or the vehicle (the DSS group and the intact group) was started two days before the induction of colitis. Colitis was not induced in the intact group. (A) Changes in the disease activity index (DAI), statistical evaluation: two-way ANOVA followed by Bonferroni's post hoc test. Diplacone (1) vs. DSS: ### p b 0.001, mimulone (2) vs. DSS: ** p b 0.01, *** p b 0.001, sulfasalazine vs. DSS: + p b 0.05. (B) Kaplan–Meier survival analysis.
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3.4. Effect of treatment on protein production
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The levels of SOD2 and CAT were not significantly modified by the induction of colitis. Treatment with compound 1 reduced the levels of the tested enzymes the most (Fig. 4). The administration of DSS led to a significant increase in the expression of COX-2 as compared to the intact group (p b 0.05). All of the test compounds lowered the levels of COX-2 in comparison to the untreated DSS group (Fig. 5). The levels of COX-2 were reduced in the SAS group the most (by 69.8% compared to the DSS group). The administration of compounds 1 and 2 reduced the expression of COX-2 by 55.9% and 48.7%, respectively.
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Please cite this article as: Vochyánová Z, et al, Diplacone and mimulone ameliorate dextran sulfate sodium-induced colitis in rats, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.012
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Fig. 3. Histological findings (H&E, magnification ×100). (A) Distal colon after five days of administration of 10% DSS in the drinking water. (B) Distal colon of the intact group, without the induction of colitis. (C) Distal colon after five days of administration of 10% DSS in the drinking water and treatment with diplacone (1) at a dose of 25 mg/kg, which was started two days before the induction of colitis. (D) Distal colon after five days of administration of 10% DSS in the drinking water and treatment with mimulone (2) at a dose of 25 mg/kg, which was started two days before the induction of colitis. (E) Distal colon after five days of administration of 10% DSS in the drinking water and treatment with sulfasalazine at a dose of 25 mg/kg, which was started two days before the induction of colitis.
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were compared. Conversely, the application of SAS demonstrated the lowest therapeutic effect. Unfortunately, none of the test compounds (1, 2, SAS) were able to fully protect the colon from histological damage and marked shortening. However, diplacone (1) has shown some cytoprotective effect in a study on alloxan-induced diabetes in mice [8]. The ability of 1 and 2 to ameliorate colitis can be associated with their antioxidant activity. Reactive oxygen
species (ROS) are responsible for tissue damage in DSSinduced colitis as well as in IBD. These are produced largely by neutrophils and macrophages that infiltrate the mucosa [20]. Generally, many flavonoids are able to scavenge ROS and to interact with antioxidant enzymes [3]. SOD2 and CAT are enzymes involved in the antioxidant defensive mechanisms of cells. SOD catalyzes the dismutation of superoxide to hydrogen peroxide and molecular oxygen; CAT then converts
Please cite this article as: Vochyánová Z, et al, Diplacone and mimulone ameliorate dextran sulfate sodium-induced colitis in rats, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.012
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SOD2 and CAT observed could be caused by a lower level of ROS, which are scavenged by 1. Compound 2 showed several times lower antiradical activity, probably because of the presence of only one hydroxyl group (para-hydroxy) on ring B [8]. In harmony with the mentioned studies, diplacone (1) exhibited the greater lowering of the levels of SOD2 and CAT. The inflammatory response is mediated by prostaglandins produced in large amounts by COX-2. Diplacone (1) has previously shown the ability to reduce the expression of
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the hydrogen peroxide to water and molecular oxygen. The levels and activities of these enzymes are related to the concentrations of their substrates [21]. Diplacone (1) and mimulone (2) have shown antiradical activity in previous studies [6,8]. The most potent activity is associated with the following structural conditions: 5,7-dihydroxy substitution on ring A, 4-oxo substitution on ring C, and 3′,4′-dihydroxy substitution on ring B (catechol moiety) of the flavonoid skeleton. Compound 1 acts as an efficient scavenger of superoxide and hydrogen peroxide [6]. The lower levels of
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Fig. 4. Effect of the test compounds on the levels of SOD2 and CAT. Colitis was induced by administration of 10% DSS in the drinking water for five days. Administration of diplacone (1), mimulone (2), or sulfasalazine (SAS) at a dose of 25 mg/kg or the vehicle (the DSS group and the intact group) was started two days before the induction of colitis. Colitis was not induced in the intact group. (A) SOD2/β-actin ratio. (B) CAT/β-actin ratio. Values were obtained from Western blot analysis as described in Materials and methods. Representative blots are shown.
Fig. 5. Effect of the test compounds on the levels of COX-2. Colitis was induced by administration of 10% DSS in the drinking water for five days. Administration of diplacone (1), mimulone (2), or sulfasalazine (SAS) at a dose of 25 mg/kg or the vehicle (the DSS group and the intact group) was started two days before the induction of colitis. Colitis was not induced in the intact group. The graph indicates the COX-2/β-actin ratio. Values were obtained from Western blot analysis as described in Materials and methods. A representative blot is shown.
Fig. 6. Effect of the test compounds on the activity of MMP2. Colitis was induced by administration of 10% DSS in the drinking water for five days. Administration of diplacone (1), mimulone (2), or sulfasalazine (SAS) at a dose of 25 mg/kg or the vehicle (the DSS group and the intact group) was started two days before the induction of colitis. Colitis was not induced in the intact group. The graph indicates the pro-MMP2/MMP2 ratio. The activity of (pro-)MMP2 was detected by zymography as described in Materials and methods. A representative zymogram is shown.
Please cite this article as: Vochyánová Z, et al, Diplacone and mimulone ameliorate dextran sulfate sodium-induced colitis in rats, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.012
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The authors have no conflicts to disclose.
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Acknowledgment
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The authors would like to thank Dr. F. Thomas Campbell for language correction of the manuscript and P. Balgová for technical assistance. This work was supported by the Internal Grant Agency of the University of Veterinary and Pharmaceutical Sciences Brno (No. 85/2013/FaF to Z.V.), by the European program “Operational Programme Education for Competitiveness” (registration number CZ.1.07/2.3.00/30.0014 to J. H.), and by the European Regional Development Fund, the European Social Fund and the State Budget of the Czech Republic (project FNUSA-ICRC No. CZ.1.05/1.1.00/02.0123 to R. H.).
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COX-2 in the lipopolysaccharide (LPS)-stimulated murine macrophage cell line [6]. In our experiment, both diplacone (1) and mimulone (2) were able to reduce the increased levels of COX-2 in colonic tissue after the administration of DSS in vivo. The mechanism by which compounds 1 and 2 affect the levels of COX-2 is the subject of further testing. Matrix metalloproteinases, particularly MMP2 and MMP9 (gelatinases 2 and 9), play an important role in the pathogenesis of IBD. These proteins are extracellular-matrix-degrading endopeptidases that affect the remodeling and destruction of tissue, the migration of immune cells, and the ulceration of the mucosa [22]. Increased levels and activities of both of these enzymes were found in the inflamed tissues of patients with CD and UC [23], as well as in an animal model of colitis [24]. Heimasaat et al. have demonstrated that the preventive use of a gelatinases blocker ameliorated acute DSS-induced colitis [25]. In addition, MMP2-deficient mice were protected from developing DSS-induced colitis [26]. Our results confirm the greater activity of MMP2 (smaller pro-MMP2/MMP2 ratio) in the untreated DSS group compared to the intact group. Prophylactic and curative administration of the test compounds (1, 2, and SAS) showed an increase in the ratio of the pro-form to the active form of MMP2. ROS are also responsible for converting MMPs to the active form [27]. The antiradical activity of the test compounds may be one of the possible mechanisms that reduce the activation of MMP2. The large number of natural compounds was evaluated on the DSS model of colitis [28], of the flavonoids namely hesperidin [29], chrysin [19], luteolin [30], icariin [16] and EGCG [31]. All of mentioned compounds apart from luteolin were also able to lower the activity of disease as compounds 1 and 2. In addition, they protected colon against histological damage. In conclusion, diplacone (1) and mimulone (2) exhibited greater effects than sulfasalazine following prophylactic/therapeutic administration. That the lowest disease activity index was achieved by 1 is probably associated with the greater antioxidant activity given by the presence of the catechol moiety. This statement is supported by previous in vitro and in vivo studies [6,8]. The ability of compounds 1 and 2 to increase the ratio of pro-MMP2/MMP2 activity and reduce the levels of COX-2 correlates with the values of DAI.
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[28] Vochyánová Z, Sikorová K, Šmejkal K, Hošek J. Plants in therapy of inflammatory bowel disease. Gastroenterol Hepatol 2014;68:248–54. [29] Xu L, Yang ZL, Li P, Zhou YQ. Modulating effect of Hesperidin on experimental murine colitis induced by dextran sulfate sodium. Phytomedicine 2009;16:989–95. [30] Karrasch T, Kim JS, Jang BI, Jobin C. The flavonoid luteolin worsens chemical-induced colitis in NF-kappaB(EGFP) transgenic mice through blockade of NF-kappaB-dependent protective molecules. PLoS One 2007; 2:e596. [31] Brückner M, Westphal S, Domschke W, Kucharzik T, Lügering A. Green tea polyphenol epigallocatechin-3-gallate shows therapeutic antioxidative effects in a murine model of colitis. J Crohns Colitis 2012;6: 226–35.
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Zora Vochyánová a, Ladislava Bartošová b, Veronika Bujdáková a, Petr Fictum c, Roman Husník d,e, Pavel Suchý b, Karel Šmejkal a, Jan Hošek f,⁎ a
Department of Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, 612 42 Brno, Czech Republic Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, 612 42 Brno, Czech Republic c Department of Pathological Morphology and Parasitology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences Brno, 612 42 Brno, Czech Republic d Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70808, USA e International Clinical Research Center (ICRC), St. Anne's University Hospital Brno, Pekařská 53, 656 91 Brno, Czech Republic f Department of Molecular Biology and Pharmaceutical Biotechnology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, 612 42 Brno, Czech Republic b
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Diplacone and mimulone ameliorate dextran sulfate sodium-induced colitis in rats
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Article history: Received 28 November 2014 Accepted in revised form 14 January 2015 Available online xxxx
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Chemical compounds studied in this article: Diplacone (PubChem CID: 14539948) Mimulone (PubChem CID: 5716903) Sulfasalazine (PubChem CID: 5353980)
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Keywords: Diplacone Mimulone Geranylated flavanones Dextran sulfate sodium (DSS) Ulcerative colitis In vivo
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1. Introduction
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Inflammatory bowel diseases (IBD), such as Crohn's disease (CD) and ulcerative colitis (UC), are chronic inflammatory
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Diplacone (1) and mimulone (2), two geranylated flavanones, have previously shown antiinflammatory and antiradical activity in vitro. The present study aimed to evaluate their activity in vivo on a model of colitis induced in Wistar rats by an oral administration of dextran sulfate sodium (DSS). Diplacone (1) and mimulone (2) were administered at a bolus dose of 25 mg/kg by gastric gavage 48 and 24 h prior to the induction of colitis by DSS and every 24 h on the following days of the experiment. The effect of the treatment was assessed by monitoring the disease activity index (DAI), histopathological examination, evaluation of the weight and length of the colon and by analysis of the levels and activities of cyclooxygenase-2 (COX-2), matrix metalloproteinase-2 (MMP2), superoxide dismutase-2 (SOD2), and catalase (CAT) in the inflamed tissue. Administration of the test compounds prior and after induction of colitis ameliorated the symptoms of colitis (diarrhea, presence of the blood in the stool) and delayed their onset. The ability of compounds 1 and 2 to reduce the levels of COX-2 and to increase the ratio of pro-MMP2/ MMP2 activity correlates with the values of the DAI. The lowering of the levels of the antioxidant enzymes SOD2 and CAT reflects the ability of the test compounds to scavenge reactive oxygen species. © 2015 Published by Elsevier B.V.
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Abbreviations: CAT, catalase; CD, Crohn's Disease; COX-2, cyclooxygenase-2; DSS, dextran sulfate sodium; IBD, Inflammatory Bowel Disease; LPS, lipopolysaccharide; MMP2, matrix metalloproteinase 2; NF-κB, nuclear factor κB; PVP, polyvinylpyrollidone; ROS, reactive oxygen species; SAS, sulfasalazine; SOD2, superoxide dismutase-2; UC, ulcerative colitis. ⁎ Corresponding author. Tel.: +420 541562839; fax: +420 541240606. E-mail address: [email protected] (J. Hošek).
illnesses of the gastrointestinal tract with intermittent periods of relapse and with the progression of the disease difficult to predict. Their incidence in Western populations has been increasing in recent years. The etiology of chronic inflammatory illnesses of the gastrointestinal tract remains unclear and there is therefore no causal treatment. Current therapy is not completely successful and it is frequently connected with adverse effects [1]. Plant products may provide an option for the alternative or supplementary treatment of the patients, and their structures can serve to provide leads or inspiration for the synthesis of new anti-inflammatory agents.
http://dx.doi.org/10.1016/j.fitote.2015.01.012 0367-326X/© 2015 Published by Elsevier B.V.
Please cite this article as: Vochyánová Z, et al, Diplacone and mimulone ameliorate dextran sulfate sodium-induced colitis in rats, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.012
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2. Materials and methods
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2.1. Test compounds
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Diplacone (1) and mimulone (2) were isolated from the unripe fruits of Paulownia tomentosa (Thunb.) Steud. (Paulowniaceae) and characterized as previously described [7]. The identification was carried out using ESIMS, 1H and 13C NMR analyses (see Supplemental Data Fig. S1–S7); the purity was checked via HPLC analysis and exceeded 95% [7]. The chemical structures are shown in Fig. 1. Sulfasalazine was purchased from Sigma-Aldrich (Steinheim, Germany).
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Male Wistar rats (180–220 g) were supplied by AnLab, Ltd. (Prague, Czech Republic). The animals were kept under standard conditions (temperature 22 ± 2 °C, relative humidity 50 ± 10%, alternating 12 hour light/dark cycle). They were fed a standard diet and given water ad libitum. The experimental protocol was approved by the Expert Committee for the Welfare of Experimental Animals of the University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic (Approval No. 26-2013). To minimize the suffering of the laboratory animals, the number of pharmacological interventions was limited to the necessary minimum.
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Rats were randomly divided into five groups (n = 8 for each group) after a one week period of acclimation. Colitis was induced by using 10% (w/v) DSS (MP Biomedicals, IllkirchGraffenstaden, France; molecular weight 36,000–50,000 Da) in the drinking water, as previously described [10], with some
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The rats were monitored daily for loss of body weight, stool consistency, and rectal bleeding in order to calculate the disease activity index (DAI) for each animal. The DAI, which reflects the severity of colitis, was modified according to Oh et al. (2006). The loss of body weight was assigned 0–2 points; it did not exceed 10% by the end of the experiment. The change in stool consistency was 0 points for normally formed stool, 1 point for loose stool, and up to 4 points for diarrhea according to the intensity of the changes. The presence of blood in the feces was assigned 0, 2, or 4 points (Table S1). The total DAI was then the sum of the points obtained from all of the parameters monitored. In the case of death during the experiment, the animal was assigned 10 points to enable the statistical evaluation.
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2.6. Histological evaluation
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Samples about 1.5 cm long were taken from the proximal and distal colon and fixed in 10% neutral buffered formalin then embedded in paraffin. Sections 3-μm-thick were stained with hematoxylin and eosin (H&E) and examined microscopically by a veterinary pathologist. The loss of epithelial surface, destruction of crypts and inflammatory cell infiltration into mucosa was assessed according to the criteria by Araki et al. (2000). Each parameter was assigned as follows: 0 = no change, 1 = localized and mild, 2 = localized and moderate, 3 = extensive and moderate, and 4 = extensive and severe. Total histological score is the sum of the points from all of the parameters [11].
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The tested compounds 1 and 2, and the positive control sulfasalazine were administrated prophylactically, 48 and 24 h before the induction of colitis and in the following days concomitantly by gastric gavage under light isoflurane anesthesia. The substances were suspended in a 4% gel of PVP K90 (polyvinylpyrrolidone K90, Sigma-Aldrich) and were given at a bolus dose of 25 mg/kg of diplacone (1, 589 mmol/kg), mimulone (2, 612 mmol/kg), or sulfasalazine (SAS, 628 mmol/kg). Animals in the untreated group (DSS group) and the intact group were treated with the vehicle only (4% gel of PVP). On day five of the experiment, the animals were sacrificed by using an overdose of T61 (Intervet International B. V., Boxmeer, Netherlands), a veterinary euthanasia drug. Immediately after the euthanasia, the entire colon was removed, including the caecum; it was opened and cleaned with physiological saline, measured and weighed. Samples for histological evaluation were obtained and the remaining parts of the colons were frozen in liquid nitrogen and stored at − 80 °C for the next processing.
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In vitro and in vivo studies have shown that flavonoids are appropriate candidates for new anti-inflammatory drugs. Their ability to inhibit the activity and expression of phospholipase A2, cyclooxygenases, lipoxygenases, and inducible synthase of nitric oxide leads to diminished production of the mediators of inflammation [2]. Scavenging the reactive oxygen species (ROS) moderates the damage to tissue and affects the signal transduction pathways [3]. Furthermore, the intervention in the signal cascade of nuclear factor κB (NF-κB) changes the expression of a series of genes connected with the inflammatory response and apoptosis [4]. This study focused on two geranylated flavanones diplacone (1) and mimulone (2). Compounds 1 and 2 have shown anti-inflammatory [5,6], antiradical, cytoprotective [7,8], and antibacterial activities [9] in previous studies. The aim of the present study was to determine the biological activity of these compounds in vivo in the model of dextran sulfate sodium (DSS)-induced colitis in rats. The efficacy of compounds 1 and 2 was compared to sulfasalazine (SAS), a conventional drug used in IBD therapy. Besides the histological and physical examinations, the levels of the antioxidant enzymes superoxide dismutase-2 (SOD2) and catalase (CAT), the levels of cyclooxygenase-2 (COX-2), and the activity of matrix metalloproteinase-2 (MMP2) were investigated to elucidate the anti-inflammatory effects of these compounds.
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Using a bench blender, approximately 2 g of colonic tissue 166 was homogenized in lysis buffer [50 mM Tris–HCl (pH 7.5), 167
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MMP2 activity (pro-MMP2, MMP2) was evaluated using gelatin zymography, as described previously [12]. Twenty micrograms of the native proteins (without denaturation in the presence of β-mercaptoethanol) was loaded onto 10% SDSpolyacrylamide gel impregnated with 0.1% gelatin. After electrophoresis the gel was washed twice for 15 min in 2.5% (v/v) Triton X-100 to remove SDS. The gel was then incubated for 15 min at room temperature and subsequently overnight (about 16 h) at 37 °C in the development buffer [50 mM Tris– HCl (pH 8.8), 5 mM calcium chloride, 3 mM sodium azide, 0.5% (v/v) Triton X-100]. After that, the gel was stained with Coomassie blue for 2 h and destained until the bands were clearly visible. The intensity of the digested bands was determined by densitometric analysis using AlphaEase FC 4.0.0 software (Alpha Innotech Corporation). The ratio between the pro-MMP2 form and the active MMP2 form was calculated.
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Protein samples were denatured in the presence of βmercaptoethanol and SDS at 70 °C for 5 min. The denatured proteins (80 μg for SOD2 and CAT, 120 μg for COX-2) were loaded onto 12% SDS-polyacrylamide gel. After electrophoresis, the proteins were transferred to a nitrocellulose membrane (0.2 μm, Bio-Rad, Hercules, USA), which was subsequently blocked with 5% BSA (Sigma-Aldrich) in TBST buffer [10 mM Tris–HCl (pH 7.5), 150 mM sodium chloride, 0.1% (v/v) Tween20] for 1 h. The membrane was then incubated with the primary mouse anti-COX-2 antibody at a dilution of 1:200 (Abcam, Cambridge, UK), the rabbit anti-SOD2 antibody at a dilution of 1:1000 (Sigma-Aldrich), the mouse anti-CAT antibody at a dilution of 1:1000 (Sigma-Aldrich), or the mouse anti-β-actin antibody at a dilution of 1:5000 (Abcam) overnight at 4 °C. After washing with distilled water, the membrane was incubated with the secondary antibody (antimouse IgG or anti-rabbit IgG, Sigma-Aldrich) at a dilution of 1:2000 for 1 h at room temperature. The secondary antibody was detected by colorimetric analysis using an Opti-CNTM Substrate Kit (Bio-Rad). The intensity of the protein bands was evaluated by densitometric analysis using AlphaEase FC 4.0.0 software (Alpha Innotech Corporation, San Leandro, USA).
The results are expressed as the mean values with error bars representing the standard error (SE) of the mean. One-way ANOVA followed by Tukey's post hoc test, two-way ANOVA followed by Bonferroni's post hoc test, and Kaplan–Meier analysis were used for the statistical evaluation. The specific analyses are referenced with the particular results. Values of p b 0.05 were considered to be statistically significant. GraphPad Prism 5.01 (GraphPad Software Inc., San Diego, USA) was used for the analysis.
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Administration of 10% DSS in the drinking water led to the loss of body weight, changes in the consistency of the stool, and rectal bleeding in the treated animals. The loss of body weight was comparable in all experimental groups. Changes in the consistency of the stool were observed in the untreated DSS group on day 3 of the experiment, on day 4 in the group treated with 1 and 2, and on day 3 or 4 in the SAS group. Blood appeared in the stool of all experimental groups treated with DSS one day after changes in the consistency of the stool. None of the described changes were observed in the intact group. The test compounds ameliorated the symptoms of colitis (diarrhea, presence of blood in the stool) and delayed their onset. The groups treated with diplacone (1) and mimulone (2) showed the lowest DAI on the last day of the experiment (p N 0.001 compared to the DSS-only group). Fig. 2A shows the development of the disease activity index for each group in the course of experiment. Data are also presented in Table S2. Four animals in the DSS group, one in the group treated with 1 and 2, and two in the SAS group died during the last day of the experiment. No mortality was observed in the intact group (Fig. 2B).
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The administration of 10% DSS for five days caused the weight/length ratio of the colon to increase by 30.4% compared to the intact group. Although the ratio increased less in the treated groups (20.4% for treatment with 1, 16.2% for treatment with 2, and 17.7% for treatment with SAS, as compared to the intact group), the differences were not statistically significant. The increase in the weight/length ratio of the colon was caused by shortening of the colon (Fig. S8).
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Administration of DSS for five days induced pathological changes involving shortening or entire loss of the crypts, erosion of the epithelium, atrophy of the mucosa, infiltration of the lamina propria with neutrophils and macrophages, fibroplasia, and ulcerations. These changes were less severe in the proximal part of the colon. They were not observed in the intact group of animals. In the treated groups of animals, the intensity of the changes was similar to that observed in the untreated DSS group. Only two samples showed less severe lesions compared to the DSS group: one from the diplacone (1)
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1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M saccharose] at a ratio of 3 mL of buffer per gram of tissue. After centrifugation at 9000 rpm for 15 min at 4 °C, the supernatant was stored. The protein concentration was measured using a Bradford-method protein assay kit (Amresco, Solon, USA) according to the manufacturer's instructions.
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A marked decrease in the ratio of pro-MMP2/MMP2 was observed in the untreated DSS group compared to the intact group (p b 0.01). As shown in Fig. 6, the test compounds increased this ratio (by 50.7% in the case of 1, by 43.3% in the case of 2, and by 37.6% for SAS, as compared to the DSS group).
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Pharmacotherapy (aminosalicylates, glucocorticoids, immunosuppressives) and surgical procedures are currently used as the main instruments in the therapy of inflammatory bowel diseases. Contemporary approaches are not always completely effective; they are associated with a number of adverse effects, and they can negatively affect the quality of the patient's life. Therefore, there is an urgent need for a new strategy, either alternative or supplementary, for treating IBD. The beneficial properties of flavonoids (the ability to scavenge reactive oxygen species and to reduce the expression of proinflammatory markers) can also be utilized in the treatment of chronic inflammatory diseases, such as CD and UC. Diplacone (1) and mimulone (2) belong to the group of geranylated flavonoids. The lipophilic chain formed by the attachment of a geranyl unit at C-6 of the flavanone skeleton modifies their observed biological activities, as compared to their nongeranylated analogs [9,13,14]. This observation is connected with the increased affinity of such compounds for different biological membranes, with possible higher bioavailability and therefore greater therapeutic potential or potential toxicity than the parent non-geranylated compounds [15]. Nonetheless, only one study describes the anti-inflammatory activity of flavonoids substituted with a prenyl chain in animal models of colitis [16]. However, naringenin, the parent compound of 2, without a geranyl side chain, has been shown to ameliorate DSS-induced colitis in mice [17] and acetic acid-induced colitis in rats [18]. DSS-induced colitis was chosen as the optimal model for determining the benefits of prophylactic and curative administration of the geranylated flavanones diplacone (1) and mimulone (2). The administration of 10% DSS in drinking water caused acute colitis, localized especially in the distal part of the colon. In the pilot study preceding this experiment, the administration of lower concentrations of DSS (5% and 7%) was not effective—apart from the loss of body weight, no symptoms of colitis were observed (unpublished data), in contrast to the published studies carried out using Wistar rats [10]. Colitis induced by the administration of 10% DSS for five days was characterized by the loss of body weight, diarrhea, bloody stool and histologically observable damage (shortening or entire loss of the crypts, erosion of the epithelium, atrophy of the mucosa, infiltration of the lamina propria with neutrophils and macrophages, fibroplasia, and ulcerations). Other studies have also described the shortening of the colon in DSS-induced colitis [16,19]. We confirmed this fact, but the shortening was not significant to be compared to the intact group, probably because of the short-term administration of DSS. All of the compounds tested ameliorated and delayed changes in the consistency of the stool and rectal bleeding. Diplacone (1) showed the greatest therapeutic effect (i.e. the lowest DAI on the last day of experiment) when all of the compounds tested
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group and one from the SAS. Evaluation of colonic damage is shown in Fig. S9. The differences were not statistically significant. Representative samples are shown in Fig. 3.
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Fig. 2. Effect of the test compounds on DAI and survival in DSS-induced rat colitis. Colitis was induced by administration of 10% DSS in the drinking water for five days. Administration of diplacone (1), mimulone (2), and sulfasalazine (SAS) at a dose of 25 mg/kg or the vehicle (the DSS group and the intact group) was started two days before the induction of colitis. Colitis was not induced in the intact group. (A) Changes in the disease activity index (DAI), statistical evaluation: two-way ANOVA followed by Bonferroni's post hoc test. Diplacone (1) vs. DSS: ### p b 0.001, mimulone (2) vs. DSS: ** p b 0.01, *** p b 0.001, sulfasalazine vs. DSS: + p b 0.05. (B) Kaplan–Meier survival analysis.
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3.4. Effect of treatment on protein production
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The levels of SOD2 and CAT were not significantly modified by the induction of colitis. Treatment with compound 1 reduced the levels of the tested enzymes the most (Fig. 4). The administration of DSS led to a significant increase in the expression of COX-2 as compared to the intact group (p b 0.05). All of the test compounds lowered the levels of COX-2 in comparison to the untreated DSS group (Fig. 5). The levels of COX-2 were reduced in the SAS group the most (by 69.8% compared to the DSS group). The administration of compounds 1 and 2 reduced the expression of COX-2 by 55.9% and 48.7%, respectively.
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Fig. 3. Histological findings (H&E, magnification ×100). (A) Distal colon after five days of administration of 10% DSS in the drinking water. (B) Distal colon of the intact group, without the induction of colitis. (C) Distal colon after five days of administration of 10% DSS in the drinking water and treatment with diplacone (1) at a dose of 25 mg/kg, which was started two days before the induction of colitis. (D) Distal colon after five days of administration of 10% DSS in the drinking water and treatment with mimulone (2) at a dose of 25 mg/kg, which was started two days before the induction of colitis. (E) Distal colon after five days of administration of 10% DSS in the drinking water and treatment with sulfasalazine at a dose of 25 mg/kg, which was started two days before the induction of colitis.
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were compared. Conversely, the application of SAS demonstrated the lowest therapeutic effect. Unfortunately, none of the test compounds (1, 2, SAS) were able to fully protect the colon from histological damage and marked shortening. However, diplacone (1) has shown some cytoprotective effect in a study on alloxan-induced diabetes in mice [8]. The ability of 1 and 2 to ameliorate colitis can be associated with their antioxidant activity. Reactive oxygen
species (ROS) are responsible for tissue damage in DSSinduced colitis as well as in IBD. These are produced largely by neutrophils and macrophages that infiltrate the mucosa [20]. Generally, many flavonoids are able to scavenge ROS and to interact with antioxidant enzymes [3]. SOD2 and CAT are enzymes involved in the antioxidant defensive mechanisms of cells. SOD catalyzes the dismutation of superoxide to hydrogen peroxide and molecular oxygen; CAT then converts
Please cite this article as: Vochyánová Z, et al, Diplacone and mimulone ameliorate dextran sulfate sodium-induced colitis in rats, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.012
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SOD2 and CAT observed could be caused by a lower level of ROS, which are scavenged by 1. Compound 2 showed several times lower antiradical activity, probably because of the presence of only one hydroxyl group (para-hydroxy) on ring B [8]. In harmony with the mentioned studies, diplacone (1) exhibited the greater lowering of the levels of SOD2 and CAT. The inflammatory response is mediated by prostaglandins produced in large amounts by COX-2. Diplacone (1) has previously shown the ability to reduce the expression of
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the hydrogen peroxide to water and molecular oxygen. The levels and activities of these enzymes are related to the concentrations of their substrates [21]. Diplacone (1) and mimulone (2) have shown antiradical activity in previous studies [6,8]. The most potent activity is associated with the following structural conditions: 5,7-dihydroxy substitution on ring A, 4-oxo substitution on ring C, and 3′,4′-dihydroxy substitution on ring B (catechol moiety) of the flavonoid skeleton. Compound 1 acts as an efficient scavenger of superoxide and hydrogen peroxide [6]. The lower levels of
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Fig. 4. Effect of the test compounds on the levels of SOD2 and CAT. Colitis was induced by administration of 10% DSS in the drinking water for five days. Administration of diplacone (1), mimulone (2), or sulfasalazine (SAS) at a dose of 25 mg/kg or the vehicle (the DSS group and the intact group) was started two days before the induction of colitis. Colitis was not induced in the intact group. (A) SOD2/β-actin ratio. (B) CAT/β-actin ratio. Values were obtained from Western blot analysis as described in Materials and methods. Representative blots are shown.
Fig. 5. Effect of the test compounds on the levels of COX-2. Colitis was induced by administration of 10% DSS in the drinking water for five days. Administration of diplacone (1), mimulone (2), or sulfasalazine (SAS) at a dose of 25 mg/kg or the vehicle (the DSS group and the intact group) was started two days before the induction of colitis. Colitis was not induced in the intact group. The graph indicates the COX-2/β-actin ratio. Values were obtained from Western blot analysis as described in Materials and methods. A representative blot is shown.
Fig. 6. Effect of the test compounds on the activity of MMP2. Colitis was induced by administration of 10% DSS in the drinking water for five days. Administration of diplacone (1), mimulone (2), or sulfasalazine (SAS) at a dose of 25 mg/kg or the vehicle (the DSS group and the intact group) was started two days before the induction of colitis. Colitis was not induced in the intact group. The graph indicates the pro-MMP2/MMP2 ratio. The activity of (pro-)MMP2 was detected by zymography as described in Materials and methods. A representative zymogram is shown.
Please cite this article as: Vochyánová Z, et al, Diplacone and mimulone ameliorate dextran sulfate sodium-induced colitis in rats, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.012
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The authors have no conflicts to disclose.
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Acknowledgment
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The authors would like to thank Dr. F. Thomas Campbell for language correction of the manuscript and P. Balgová for technical assistance. This work was supported by the Internal Grant Agency of the University of Veterinary and Pharmaceutical Sciences Brno (No. 85/2013/FaF to Z.V.), by the European program “Operational Programme Education for Competitiveness” (registration number CZ.1.07/2.3.00/30.0014 to J. H.), and by the European Regional Development Fund, the European Social Fund and the State Budget of the Czech Republic (project FNUSA-ICRC No. CZ.1.05/1.1.00/02.0123 to R. H.).
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COX-2 in the lipopolysaccharide (LPS)-stimulated murine macrophage cell line [6]. In our experiment, both diplacone (1) and mimulone (2) were able to reduce the increased levels of COX-2 in colonic tissue after the administration of DSS in vivo. The mechanism by which compounds 1 and 2 affect the levels of COX-2 is the subject of further testing. Matrix metalloproteinases, particularly MMP2 and MMP9 (gelatinases 2 and 9), play an important role in the pathogenesis of IBD. These proteins are extracellular-matrix-degrading endopeptidases that affect the remodeling and destruction of tissue, the migration of immune cells, and the ulceration of the mucosa [22]. Increased levels and activities of both of these enzymes were found in the inflamed tissues of patients with CD and UC [23], as well as in an animal model of colitis [24]. Heimasaat et al. have demonstrated that the preventive use of a gelatinases blocker ameliorated acute DSS-induced colitis [25]. In addition, MMP2-deficient mice were protected from developing DSS-induced colitis [26]. Our results confirm the greater activity of MMP2 (smaller pro-MMP2/MMP2 ratio) in the untreated DSS group compared to the intact group. Prophylactic and curative administration of the test compounds (1, 2, and SAS) showed an increase in the ratio of the pro-form to the active form of MMP2. ROS are also responsible for converting MMPs to the active form [27]. The antiradical activity of the test compounds may be one of the possible mechanisms that reduce the activation of MMP2. The large number of natural compounds was evaluated on the DSS model of colitis [28], of the flavonoids namely hesperidin [29], chrysin [19], luteolin [30], icariin [16] and EGCG [31]. All of mentioned compounds apart from luteolin were also able to lower the activity of disease as compounds 1 and 2. In addition, they protected colon against histological damage. In conclusion, diplacone (1) and mimulone (2) exhibited greater effects than sulfasalazine following prophylactic/therapeutic administration. That the lowest disease activity index was achieved by 1 is probably associated with the greater antioxidant activity given by the presence of the catechol moiety. This statement is supported by previous in vitro and in vivo studies [6,8]. The ability of compounds 1 and 2 to increase the ratio of pro-MMP2/MMP2 activity and reduce the levels of COX-2 correlates with the values of DAI.
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