Morin, a bioflavonoid with antioxidant properties, shows intestinal anti-inflammatory activity in the acute phase of the trinitrobenzenesulphonic acid model of rat colitis.
Morin, a bioflavonoid with antioxidant properties, shows intestinal anti-inflammatory activity in the acute phase of the trinitrobenzenesulphonic acid model of rat colitis.
To assess the anti-inflammatory activity of morin in the chronic stages of trinitrobenzenesulphonic acid-induced rat colitis.
Rats were rendered colitic by a single colonic instillation of 30 mg of the hapten trinitrobenzenesulphonic acid dissolved in 0.25 mL of 50% ethanol. A group of colitic animals was given morin orally at doses of 25 mg/kg daily. Animals were sacrificed every week for 4 weeks. Colonic damage was evaluated macroscopically and microscopically. Different biochemical markers of colonic inflammation were also assayed, including myeloperoxidase activity, leukotriene B4 and interleukin-1β synthesis, glutathione and malonyldialdehyde levels and nitric oxide synthase activity.
The administration of morin facilitated tissue recovery during the 4 weeks following colonic insult with trinitrobenzenesulphonic acid, as demonstrated macroscopically and microscopically, as well as biochemically by a reduction in myeloperoxidase activity. The intestinal anti-inflammatory effect of morin was accompanied by a significant reduction in colonic leukotriene B4 and interleukin-1β levels, improvement in colonic oxidative stress and inhibition of colonic nitric oxide synthase activity.
Morin exerts a beneficial anti-inflammatory effect in the chronic phase of trinitrobenzenesulphonic acid-induced rat colitis through the down-regulation of some of the mediators involved in the intestinal inflammatory response, including free radicals, cytokines, leukotriene B4 and nitric oxide.
Inflammatory bowel disease is a chronic disease of the digestive tract, and usually refers to two related conditions, namely ulcerative colitis and Crohn’s disease. The aetiology of inflammatory bowel disease remains unknown, although it is believed that an alteration in the intestinal immune system contributes to the inflammation that occurs. As in other inflammatory processes, inflammatory bowel disease is characterized by an up-regulation in the synthesis and release of different pro-inflammatory mediators, including reactive oxygen and nitrogen metabolites, eicosanoids, platelet-activating factor and cytokines.1 All of these mediators contribute to the pathogenic cascade that initiates and perpetuates the inflammatory response of the gut. As a consequence, and until its aetiology has been completely elucidated, the best strategy to effectively down-regulate intestinal inflammation is to interfere with multiple stages of the inflammatory cascade, preferably with a single drug treatment. In fact, the drugs currently used for the management of human inflammatory bowel disease, i.e. compounds delivering 5-aminosalicylic acid and systemic or local glucocorticoids, exert their beneficial effects through a combination of different mechanisms.2, 3 Unfortunately, these drugs are not devoid of potentially serious side-effects, thus limiting their use.4 For this reason, the development of new drug treatments is an important goal in inflammatory bowel disease.
Flavonoids comprise a class of natural products which are found in fruits, vegetables, nuts, seeds, herbs, spices, stems, flowers, tea and red wine, and are consumed regularly as part of the human diet. Flavonoids are known to possess several biological activities, mainly related to their ability to inhibit enzymes and/or their antioxidant properties,5 which could justify their consideration as valid drugs in the pharmacological treatment of inflammatory bowel disease. In addition, several in vitro and in vivo studies have shown their ability to down-regulate the immune response, an effect that may also contribute to their potential beneficial influence in these intestinal conditions.5 In fact, previous studies have proven the efficacy of some of these compounds, including quercitrin, rutoside, morin, diosmin, hesperidin and the flavonoid derivate DA-6034, in several experimental models of rat colitis.6–10 Morin is a flavonoid found in the fig and other Moraceae, which are used as herbal medicines, and has been suggested to act as a food preservative.11 It has certain biological activities, including antioxidant and/or free radical scavenger properties, in different biological systems, such as the cardiovascular system and hepatic tissue,12–15 and an inhibitory effect on leukotriene B4 production via inhibition of lipoxygenase activity.16, 17 Both free radicals and leukotriene B4 have been postulated to play a key role in the pathogenesis of inflammatory bowel disease;18–20 indeed, the beneficial activity of the drugs currently used in inflammatory bowel disease therapy, such as aminosalicylates, has been ascribed to their antioxidant properties and/or inhibition of leukotriene B4 synthesis and release.2 In a previous study, we reported the preventative activity of morin in the acute stage of the trinitrobenzenesulphonic acid (TNBS) model of rat colitis,8 in which both its antioxidant activity and its ability to down-regulate leukotriene B4 synthesis seemed to be involved. However, the dosing protocol used, in which the flavonoid was administered for 5 days prior to colonic challenge, and the fact that the inflammatory status was examined 48 h after TNBS instillation, made it impossible to distinguish between a real anti-inflammatory effect and an artefact due to a physical interference in the damage effect of TNBS or to a delay in the appearance of colonic damage. In addition, the long-term effects of drugs intended for inflammatory bowel disease therapy are most relevant, given the chronic nature of this condition.
These facts led us to undertake the present experiments, in which we tested the most active dose of morin in the previous study, 25 mg/kg, in the chronic stage of TNBS-induced rat colitis. We followed a curative dosing protocol, in which the administration of the flavonoid was started 2 h after induction of colitis with TNBS, in contrast to the preventative dosing used in the previous study. With these experiments, we evaluated the ability of the flavonoid to accelerate mucosal repair once the colonic damage had been induced, and studied the probable mechanisms involved in its beneficial effect, with special attention given to its effect on colonic oxidative stress and on the production of some of the mediators involved in the inflammatory response, namely leukotriene B4, interleukin-1ß and nitric oxide.
This study was carried out in accordance with the ‘Guide for the Care and Use of Laboratory Animals’ as promulgated by the National Institutes of Health.
Colitis was induced by the method originally described by Morris et al.21 Briefly, female Wistar rats (180–220 g), obtained from the Laboratory Animal Service of the University of Granada (Granada, Spain), were randomly distributed into several experimental groups. The animals were housed in makrolon cages (3–4 rats per cage) and maintained in an air-conditioned atmosphere with a 12-h light–dark cycle; they were provided with free access to tap water and food (Panlab A.04). The animals were fasted overnight and anaesthetized with halothane. Under anaesthesia, the animals were given 30 mg of TNBS dissolved in 0.25 mL of 50% ethanol (v/v) by means of a Teflon cannula inserted 8 cm through the anus. During and after TNBS administration, the rats were kept in a head-down position until they recovered from the anaesthetic, and were then returned to their cage. The rats from the non-colitic (normal) group received 0.25 mL of phosphate-buffered saline.
Once the rats had been rendered colitic by TNBS administration, they were treated orally with 25 mg/kg of morin (suspended in 1 mL of water), starting 2 h after colitis induction and continuing once daily thereafter until the day before the animals were killed. This dose was chosen on the basis of a previous study, as it was the most active in preventing the TNBS-induced acute damage in the colonic tissue of the rat.8 The animals were killed at 1, 2, 3 and 4 weeks after induction of colitis. A TNBS control group and a normal (non-colitic) group were included for reference, and received only the vehicle orally (1 mL of water). The body weight and total food intake for each group were recorded daily. All experimental groups contained 10 rats per treatment and time point, except for the non-colitic groups that contained five rats.
Animals were sacrificed with an overdose of halothane, and the entire colon was removed. The colonic segments were placed on an ice-cold plate, cleaned of fat and mesentery and blotted on filter paper. Each specimen was weighed and its length measured under a constant load (2 g). The colon was longitudinally opened and scored for macroscopically visible damage on a 0–10 scale by two observers unaware of the treatment, according to the criteria described by Bell et al.22 (Table 1), which take into account the extent as well as the severity of colonic damage. The colon was subsequently divided longitudinally into four pieces for biochemical determination. Two fragments were frozen at – 30 °C for myeloperoxidase and malonyldialdehyde determination, and another sample was weighed and frozen in 1 mL of 5% (w/v) trichloroacetic acid for total glutathione content determination. The remaining sample was immediately processed for the measurement of leukotriene B4 and interleukin-1β synthesis. All biochemical measurements were completed within 1 week from the time of sample collection and were performed in duplicate. Myeloperoxidase activity was measured according to the technique described by Krawisz et al.;23 the results were expressed as myeloperoxidase units per gram of wet tissue and one unit of myeloperoxidase activity was defined as that degrading 1 μmol hydrogen peroxide/min at 25 °C. The total glutathione content was quantified with the recycling assay described by Anderson,24 and the results were expressed as nmol/g wet tissue. Colonic malonyldialdehyde contents were evaluated according to the method proposed by Esterbauer and Cheeseman,25 and expressed as nmol/g wet tissue. Colonic samples for leukotriene B4 and interleukin-1β synthesis determinations were immediately weighed, minced on an ice-cold plate and suspended in a tube with 10 mM sodium phosphate buffer (pH 7.4) (1:5 w/v). The tubes were placed in a shaking water bath (37 °C) for 20 min and centrifuged at 9000 g for 30 s at 4 °C; the supernatants were frozen at –80 °C until assay. Leukotriene B4 and interleukin-1β were quantified by enzyme-linked immunosorbent assay (Amersham, Barcelona, Spain), and the results were expressed as ng/g wet tissue.
Nitric oxide synthase activity was determined in rat cultured colonic explants. For this purpose, five additional rats from each group (non-colitic, control and morin-treated) and time point studied were used. Once the rats had been killed, colonic specimens (0.4 × 0.8 cm) were obtained from the most damaged distal area of the colon in both colitic groups and from the equivalent zone in the non-colitic group. The colonic explants were kept in NaCl (0.15 M) containing penicillin (300 U/mL) and streptomycin (300 μg/mL) at 4 °C for 15 min, and the organ culture was performed according to the method of Eastwood and Trier.26 In brief, the tissue was orientated on metal grids and cultured for 24 h at 37 °C, with 95% O2, 5% CO2, in RPMI1640 medium (Sigma, Spain) containing penicillin (100 U/mL) and streptomycin (100 μg/mL) in the presence of L-arginine (10–2 M).
Nitric oxide synthase activity was monitored by the quantification of citrulline content in the colonic explant, as previously described by Gaginella et al.27 For this purpose, the cultured colonic explants were weighed and homogenized (1:5 w/v) for 20 s in a buffer containing sucrose (0.32 M), ethylenediaminetetra-acetic acid (EDTA) (0.1 mM), dithiothreitol (1 mM), soybean trypsin inhibitor (10 μg/mL), leupeptin (10 μg/mL) and aprotinin (2 μg/mL). The homogenates were centrifuged at 10 000 g for 5 min at 4 °C and the supernatant was processed for colorimetric determination of citrulline.28 Nitric oxide synthase activity was expressed as μmol citrulline/g wet tissue.
These experiments were performed in colonic segments obtained from control colitic rats 2 weeks after TNBS instillation, when the greatest colonic nitric oxide synthase activity was obtained during the course of the present experiments (see ‘Results’ section). Colonic tissue was homogenized (1:5 w/v) for 60 s in 10 mMN-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES) (pH 7.4) containing sucrose (0.32 M), EDTA (100 μM), dithiothreitol (1 mM), phenylmethylsulphonyl fluoride (1 mg/mL) and leupeptin (10 μg/mL); the resulting homogenate was centrifuged at 10 000 g for 10 min at 4 °C, and the supernatants were assayed for protein content.29 In these studies, nitric oxide synthase activity was determined by monitoring the conversion of L-[3H]arginine to L-[3H]citrulline.30 Samples (40 μg protein) were incubated at room temperature for 30 min with 100 μL of the above buffer containing various concentrations of morin (1–100 μM in 1% dimethylsulphoxide), in the presence of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) (1 mM) and a mixture of unlabelled and L-[3H]arginine (10 μM, 1 μCi/mL). Incubations were terminated by the addition of 20 mM HEPES (1 mL, pH 5.5) containing 1 mM ethyleneglycoltetra-acetic acid and 1 mM EDTA. L-[3H]Citrulline was separated from arginine by adding 1.5 mL of a 1:1 suspension of Dowex (50W) in water. The radioactivity was measured in the supernatants by liquid scintillation counting and the percentage inhibition of nitric oxide synthase activity for each concentration of morin was calculated.
Five additional animals from each group were used for colonic histological studies. Representative whole gut specimens were taken from a region of the inflamed colon immediately adjacent to the gross macroscopic damage in the distal colon of each animal and were fixed in 4% buffered formaldehyde. Cross-sections were selected and embedded in paraffin. Equivalent colonic segments were also obtained from the non-colitic group. Sections, 5 μm thick, were obtained at different levels and stained with haematoxylin and eosin. The histological damage was scored on a scale from 0 to 6 according to criteria adapted from those reported by Zea-Iriarte et al.31 (Table 1), which are based on the presence of ulceration and/or inflammation, as well as on the depth of the intestinal layer affected by the inflammatory process. All the parameters of histological damage were recorded by a pathologist observer (AC) who was unaware of the treatment conditions.
Glutathione reductase was obtained from Boehringer Mannheim (Barcelona, Spain) and L-[2,3,4,5-3H]arginine monohydrochloride was purchased from Amersham Iberica (Barcelona, Spain). All other reagents, including morin, were provided by Sigma (Madrid, Spain).
All results are expressed as the mean ± S.E.M. Differences between means were tested for statistical significance using a one-way analysis of variance (ANOVA) and post hoc least significance tests. Non-parametric data (score) are expressed as the median (range) and were analysed using the Mann–Whitney U-test. Differences between proportions were analysed with the chi-squared test. Statistical significance was set at P < 0.05. Data from the non-colitic animals (non-colitic group), which did not differ significantly from one another, were pooled together and presented as a single group.
Intracolonic administration of 30 mg of TNBS in 50% ethanol resulted in colonic inflammation which persisted during the 4 weeks of the experiment, with signs of anorexia, diarrhoea and loss of weight in all the colitic animals, in accordance with previous studies.6, 32 Thus, the damage induced by TNBS was characterized by severe ulceration and/or inflammation of the colonic tissue, typically extending along the colon from 5–6 cm at 1 week to 3 cm at 4 weeks (Table 2). The existence of adhesions to adjacent organs was also noted in all the colitic animals from the TNBS control group during the first 2 weeks, and in 50% of the animals during the last 2 weeks (Table 3). In addition, the colonic segments showed bowel wall thickening; a significant increase in the colonic weight/length ratio in comparison with non-colitic animals was observed (P < 0.01, Table 2). All of these features were most severe at 1 week after TNBS administration, and then showed a time-dependent reduction in severity, but the lesions were still observed after 4 weeks (Table 2). Histological assessment of the colonic specimens from the TNBS control group revealed mucosal damage, which was accompanied by goblet cell depletion, severe transmural inflammation and disruption of the normal architecture of the colon during the 4 weeks following the TNBS insult (Table 4). At 1 and 2 weeks, extensive ulceration and inflammation involving all the intestinal layers of the colon were observed, even resulting, in some specimens, in perforation of the intestinal wall with peritoneal reaction (Figure 1). From this time point on, the histological sections showed a progressive restoration, although at 4 weeks the colonic histology was still altered, showing partially re-epithelialized ulcers and an improvement of the glandular structure; this was not complete, however, as the mucin content of goblet cells was not replenished, and the existence of glandular crypts with mitotic activity was still evident (Figure 2). The alteration in the colonic histology was also characterized by severe oedema, interstitial microhaemorrhages and diffuse leucocyte infiltration, and only after 4 weeks was there a slight decrease in the inflammatory cell infiltrate; only two of the five samples studied showed a patchy distribution in the infiltrate. The leucocyte infiltrate during the first 2 weeks was mainly composed of neutrophils and, to a lesser extent, histiocytes. However, this varied during the last 2 weeks, when no cell type was predominant, although lymphocytes, mast cells, macrophages and plasma cells were present (Figures 1 and 2).
The biochemical determinations revealed that TNBS instillation enhanced colonic myeloperoxidase activity (Figure 3) and increased colonic leukotriene B4 (Figure 4) and interleukin-1ß (Figure 5) levels. All peaked at 1 week and declined gradually with time, reaching normal values after 4 weeks, with the exception of leukotriene B4, which was still significantly higher than normal at 4 weeks (P < 0.05). As a consequence of the inflammatory process, an alteration in the colonic oxidative status was also observed. Thus, the rats from the TNBS control group showed increased levels of malonyldialdehyde throughout the study in comparison with the non-colitic animals (P < 0.01, Figure 6). In addition, a significant reduction in colonic glutathione levels was also noted after 1 week (680 ± 47 vs. 1325 ± 94 nmol/g tissue in non-colitic animals, P < 0.01), although these values were normalized thereafter (data not shown).
Treatment with 25 mg/kg of morin resulted in a significantly lower colonic damage score than in the control animals at all time points studied (P < 0.05, Table 2), with a reduction in the extent of colonic haemorraghic necrosis and/or inflammation. As a consequence of its anti-inflammatory activity, a reduction in the incidence of diarrhoea and of adhesions between the colon and adjacent organs was also noted, compared with non-treated colitic animals, especially during the first 2 weeks, when statistically significant differences were achieved (Table 3). However, this beneficial effect was not accompanied by a reduction in the colonic weight/length ratio (Table 2). The lack of an effect on this ratio is due to the severe and extensive damage induced by TNBS/ethanol, which is difficult to overcome by pharmacological treatment, as has been suggested previously.33 Histological analysis of the colonic specimens from rats treated with morin revealed a less damaged intestinal cytoarchitecture, which was already evident from the first week of treatment, with an almost complete restoration after 4 weeks of treatment (Table 4, Figures 1 and 2). The colonic ulcers were also present at 1 week, but to a lesser extent than in the control animals, and the transmural involvement of the lesions was progressively reduced with time, only affecting the mucosal layer at 4 weeks. At this time, there were zones with an almost completely restored epithelial cell layer. In addition, the presence of goblet cells was already evident after 2 weeks of treatment, in contrast with the non-treated control animals. The glandular structure was almost completely restored after 4 weeks, as the existence of intact, mucin-repleted goblet cells was noted, together with the absence of dilated crypts. The improvement in the colonic histology was accompanied by a reduction in the inflammatory infiltrate at all time points studied. Thus, the leucocyte infiltrate varied in the histological sections obtained from the animals treated with morin from extensive and diffuse in the first week, mainly composed of neutrophils, to slight and patchy after 4 weeks of treatment, with lymphocytes and histiocytes being the predominant cell types, although mast cells were also conspicuous. It is noteworthy that the latter are associated with post-inflammatory fibrosis in situations of tissue restoration or repair.34 The lower leucocyte infiltration was confirmed biochemically, by a reduction in colonic myeloperoxidase activity, which was significant at 1 and 3 weeks of treatment (Figure 3). This effect was accompanied by a significant decrease in colonic leukotriene B4 levels during the first 2 weeks (Figure 4) and in interleukin-1β content after 3 weeks (Figure 5). Colonic oxidative stress was only affected by morin treatment after 1 week, with a reduction in colonic malonyldialdehyde levels (Figure 6) and the prevention of glutathione depletion (1046 ± 109 vs. 680 ± 47 nmol/g tissue in TNBS control animals, P < 0.01).
Citrulline generation by cultured colonic explants obtained from control colitic rats was significantly increased during the 4 weeks of the experiments (Figure 7). However, and in contrast with most of the biochemical markers of inflammation studied, i.e. colonic myeloperoxidase activity, leukotriene B4 and interleukin-1β levels and plasma malonyldialdehyde content, which displayed the highest values at 1 week, citrulline levels peaked 2 weeks after TNBS instillation and declined at 4 weeks, although still showing higher values than those of non-colitic animals (P < 0.01).
Morin treatment applied to colitic rats resulted in an inhibition of colonic nitric oxide synthase activity at 3 and 4 weeks, as evidenced by a significant reduction in the citrulline content in the cultured colonic explants, compared with the corresponding TNBS controls (Figure 7).
In vitro assays revealed that morin inhibits colonic nitric oxide synthase activity, showing an IC50 value of 58.5 ± 3.7 μmol/L, with a maximum inhibitory effect of 78.3 ± 7.4% at a concentration of 100 μmol/L (mean ± S.E.M. from six separate experiments).
The results obtained in the present study confirm the therapeutic efficacy of morin when administered at a dose of 25 mg/kg in the chronic stage of the TNBS model of colitis. Morin was able to reduce macroscopic colonic damage, as scored according to the severity and extent of involved tissue, and also ameliorated the histological lesions that characterize this experimental model of colitis, i.e. epithelial sloughing, goblet cell depletion and intense granulocyte infiltration. This beneficial effect was achieved by acceleration of the healing process that occurred during the 4 weeks following colonic insult.
The anti-inflammatory effect exerted by morin was associated with a decrease in colonic myeloperoxidase activity, a marker of neutrophil infiltration, which has been previously described to be up-regulated in experimental colitis.35 Margination and extravasation of circulating granulocytes contributes markedly to the chronic injury in this model of inflammatory bowel disease. For this reason, myeloperoxidase activity has been widely used to detect and follow intestinal inflammatory processes, and a reduction in the activity of this enzyme can be interpreted as a manifestation of the anti-inflammatory activity of a given compound.33 The ability of morin to reduce granulocyte infiltration was confirmed histologically; the level of leucocyte infiltrate in the colonic mucosa was lower in animals treated with morin than in the corresponding TNBS control groups. This inhibitory effect on the infiltration of inflammatory cells into the colonic mucosa might account for the beneficial effect of this flavonoid against tissue injury, most probably through the combination of several mechanisms.
One of these mechanisms could be the inhibition of colonic leukotriene B4 synthesis in the inflamed colon, which was evident at all time points studied. These results confirm previous in vitro studies that revealed the ability of this flavonoid to down-regulate the synthesis of leukotriene B4 via inhibition of lipoxygenase activity.16, 17 Leukotriene B4 is a potent neutrophil chemotactic agent, which induces neutrophil adherence to the vascular wall and reinforces the effects of other mediators, such as platelet-activating factor, in promoting neutrophil migration across the endothelial monolayer, as well as increasing mesenteric vascular permeability.36 Indeed, leukotriene B4 synthesis has been shown to be up-regulated in the colonic mucosa of patients with ulcerative colitis and Crohn’s disease compared to normal tissues,18 and it has been proposed that inhibition of leukotriene B4 synthesis may contribute to the therapeutic effect exerted by different drugs used in the treatment of inflammatory bowel disease, such as sulfasalazine and other compounds delivering 5-aminosalicylic acid.2 However, the evident inhibition of colonic leukotriene B4 production observed after morin treatment was not correlated with a similar reduction in other biochemical parameters evaluated in the present study, such as myeloperoxidase activity or interleukin-1β levels, at least during the first 2 weeks after colonic challenge. This may be due to the enormous number of different mediators involved in the inflammatory response, many of which display chemotactic properties or promote cytokine synthesis and release by cells resident in the intestine, which are not properly down-regulated by this flavonoid.
Given the well-known antioxidant properties ascribed to this flavonoid, both in vitro and in vivo,12–15 a mechanism that may be crucial in the intestinal anti-inflammatory effect of morin is an inhibitory effect on free radical generation, a common feature in these intestinal conditions.37 Treatment with morin ameliorated the colonic oxidative stress that occurred after TNBS administration to rats,38 and thus contributed to tissue repair, at least in the early phases of colonic damage. This effect seems to be relevant, given that free radical production also stimulates the infiltration of leucocytes into colonic tissue, which then produce a large amount of free radicals and eicosanoids themselves, further increasing the concentration of free radicals. As a consequence, a rapid inhibition of free radical generation could contribute to a lower level of leucocyte infiltration into the inflamed tissue and, in turn, to a lower leukotriene B4 production, as these cells are considered to be the main site of arachidonic acid metabolism in inflammatory bowel disease.22
During the last decade, it has become increasingly clear that chronic colonic inflammation is associated with enhanced nitric oxide production, mainly via inducible nitric oxide synthase activity, in both humans and experimental animals.39–41 Although it is well documented that nitric oxide has homeostatic regulatory functions in the intestine,42 even showing anti-inflammatory properties,43, 44 nitric oxide overproduction by inducible nitric oxide synthase has been suggested to be deleterious to intestinal function,39, 40 thus contributing significantly to gastrointestinal immunopathology during the chronic inflammatory events that take place in inflammatory bowel disease. The important role attributed to nitric oxide in these intestinal conditions prompted us to study whether the beneficial effects of morin on TNBS-induced chronic colitis could be related to an effect on colonic nitric oxide production. For this purpose, colonic citrulline content was used as an indirect index of colonic nitric oxide synthase activity in the inflamed tissue, as previously proposed by other authors.27, 45 The results obtained in the present study reveal that colonic inflammation is associated with a higher citrulline production in cultured colonic explants in comparison with that in non-colitic samples. These results confirm previous observations reported both in the same experimental model of rat colitis40 and in human inflammatory bowel disease,41 which described enhanced nitric oxide production in the inflamed mucosa by colonic inducible nitric oxide synthase. The intestinal anti-inflammatory effect exerted by morin was associated with a significant inhibition of colonic nitric oxide synthase activity from the third week. As a consequence, an inhibition of nitric oxide production may also contribute to the beneficial effect shown by this flavonoid in the chronic stage of TNBS-induced colitis, thus preventing, at least partially, the deleterious activity ascribed to nitric oxide when it is produced in large amounts by inducible nitric oxide synthase. In fact, similar beneficial effects have been reported after nitric oxide synthase inhibition in different experimental models of intestinal inflammation.39, 40, 46 It is important to note that the greatest inhibition of colonic nitric oxide synthase in inflamed tissue, i.e. after 4 weeks of treatment, coincides with the most pronounced anti-inflammatory effect of morin, as demonstrated by the histological studies, when the colonic restoration in the intestinal cytoarchitecture was almost complete in comparison with that in the corresponding TNBS control animals, which still showed evident signs of severe inflammation. The ability of morin to inhibit nitric oxide production has been described previously in vitro in C6 astrocyte cell cultures.47 This effect may be related to the ability of morin, in common with the majority of flavonoids, to inhibit the activity of many different enzyme systems.5 This was confirmed in the present study, as morin was able to inhibit colonic nitric oxide synthase activity in vitro effectively. However, it is unlikely that an inhibitory effect of morin on inducible nitric oxide synthase expression plays a role in the inhibitory effect on nitric oxide synthase activity observed in vivo in the colitic animals treated with the flavonoid, based on the results recently reported by Raso et al.48 These authors described how morin, at concentrations between 0.5 and 50 μM, had no effect on lipopolysaccharide-induced inducible nitric oxide synthase expression in a macrophage cell line.
Finally, colonic interleukin-1β was also evaluated in this model of rat colitis, as this is a pro-inflammatory cytokine considered to be one of the primary triggers of intestinal inflammation, and its production has also been reported to be increased during active disease in both ulcerative colitis and Crohn’s disease,49, 50 as well as in experimental models of intestinal inflammation.51, 52 The present study confirms these observations: a significant increase in colonic interleukin-1β levels was observed in colitic control animals during the 4 weeks after TNBS challenge in comparison with that in non-colitic animals. The anti-inflammatory effect of morin was associated with an inhibition in the colonic production of this pro-inflammatory cytokine, which was evident after 3 weeks of treatment. There was a correlation between the decrease in colonic interleukin-1ß production and the inhibition of colonic nitric oxide synthase activity. In fact, it has been proposed that interleukin-1β may be one of the cytokines responsible for the induction of inducible nitric oxide synthase in enterocytes.53
In summary, morin treatment facilitates the recovery of the damaged tissue in the chronic phase of TNBS-induced rat colitis, an effect associated with an amelioration in the production of some of the mediators involved in the inflammatory response of the intestine, such as free radicals, leukotriene B4, nitric oxide and interleukin-1ß. Further studies are necessary to explore the effectiveness of morin in human inflammatory bowel disease, most probably through appropriate supplementation with this flavonoid. The reason for this is that, although morin can be found in fruits and vegetables in the human diet, the equivalent human dose of morin used in the present study on a weight to weight basis is much higher than that acquired exclusively through dietary intake.
The authors are grateful for the technical help provided by Dr Sánchez de Medina L.-H. This study was supported by the Spanish Ministry of Education and Culture with CICYT (SAF98-0157 and SAF98-0160) funds.