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- MATERIALS AND METHODS
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.
- Top of page
- MATERIALS AND METHODS
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.