Effect of quercitrin on the early stages of hapten induced colonic inflammation in the rat
Introduction
Inflammatory bowel disease (IBD), comprised of ulcerative colitis and Crohn's disease, is a chronic condition that affects primarily the distal part of the gut and courses with alternate periods of inflammatory bouts and remission of the symptoms. Despite having received a great deal of attention during the past few years, the etiology of IBD remains essentially unknown [1] and the pharmacological treatment is still unsatisfactory to date [2]. Therefore, the search of new lines of therapy is much warranted. Flavonoids are polyphenolic compounds of natural origin that are present in the normal diet in significant amounts [3]. While flavonoids are devoid of classical nutritional value, they are increasingly viewed as beneficial dietary components, namely in cardiovascular disease and cancer, considering their well-established antioxidant and antiradicalary properties. In addition, flavonoid compounds exert a plethora of biological effects, including enzyme inhibition (lipoxygenase, cyclooxygenase, nitric oxide synthase), immune cell modulation, etc. (reviewed in [3]).
The most common flavonoid moiety in nature is quercetin (3,5,7,3′,4′-pentahydroxyflavone), which is normally present as a glycoside, such as quercitrin (3-rutinoside) or rutin (3-rhamnoside). We have previously found that quercitrin and rutin exert a beneficial effect in experimental colonic inflammation [4], [5], [6]. Oral administration of either flavonoid 2 h prior to trinitrobenzene sulfonic acid (TNBS) administration and daily thereafter reduces neutrophil infiltration, counteracts glutathione depletion and protects the mucosa from ulceration. In addition, flavonoid treatment results in a marked improvement in colonic function, since fluid absorption, which is dramatically impaired by inflammation, is increased significantly compared with TNBS controls as early as 2 days postchallenge [4]. However, the mechanism of action of these flavonoids is poorly understood. Inflammation was induced by an enema of the hapten 2,4,6-trinitrobenzene sulfonic acid dissolved in 50% ethanol. This model was originally described by Morris et al. [7] as a simple, reproducible, and relatively inexpensive model of colonic inflammation extending up to 8 weeks and displaying signs of chronic inflammation following an initial strong acute inflammatory response. Thus chronic inflammation is evidenced by the presence of fibrosis, stenosis, granulomatous responses and the progressive substitution of neutrophils by lymphocytes and macrophages [8]. The inflammatory response to TNBS in rodents is driven by Th-1 lymphocytes [9]. This fact, coupled with the histological characteristics of TNBS colitis (i.e. transmural inflammation, fibrosis, granulomatosis) makes it a suitable model of Crohn's disease rather than ulcerative colitis. The TNBS model of colitis is characterized by an immediate intense acute inflammatory response with mucosal necrosis followed by a prolonged phase of healing of the mucosal lesions and progression to a chronic type of response [7], [10]. Our previous experiments examined the effect of the flavonoids once the inflammatory response had peaked and therefore could not discriminate between a preventive and curative action. Hence we designed the present study to verify whether quercitrin treatment, in identical conditions to those assayed previously, has an impact on the early stages of TNBS colitis.
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Animals
Female Wistar rats (200–250 g) supplied by the Laboratory Animal Service of the University of Granada were housed in makrolon cages (3–4 rats per cage) and provided with free access to tap water and food (Panlab A04). Animal quarters maintained a constant temperature (22–25 °C) and a 12 h light-dark cycle. Animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health.
Induction of colitis and assessment
The animals were fasted
Inflammatory response
As expected, colonic instillation of TNBS/ethanol triggered an intense inflammatory response that was restricted to the distal segment of the large bowel and characterized by extensive hemorrhage and bowel wall thickening of most of the distal colon [4], [5], [6], [7]. The median damage score was 9(8–10) (p < 0.05 vs. control group). Quercitrin treatment had no effect on macroscopic injury (damage score 8(7–10) and 9(7–10) for the Q1 and Q5 groups respectively, p > 0.05 vs. control group). In
Discussion
The objective of the present study was to ascertain whether quercitrin has a therapeutic effect in the early stages of TNBS colitis. Specifically, we wanted to know whether the previously established antiinflammatory effect of the flavonoid in later phases of the inflammatory reaction was attributable to an initial downregulation of the colonic insult brought about by the TNBS/ethanol enema or to an interference with the subsequent progression of the inflammatory reaction. In addition, the
Conclusion
In summary, we have demonstrated that quercitrin treatment has a beneficial impact on oxidative stress, alkaline phosphatase upregulation and nitric oxide synthesis, without significant antiinflammatory activity, in experimental colonic inflammation. Hence, it is likely that the beneficial effects of the flavonoid on trinitrobenzene sulfonic acid chronic colitis arise from an early downregulation of the inflammatory cascade that is associated with amelioration of the disturbances in
Acknowledgements
The authors want to thank Isabel Ballester and Dr. Mercedes González for their technical assistance. This study was supported by CICYT funds (SAF98-0157).
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