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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 10, 2006 as doi:10.1096/fj.05-4943fje. |
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,1
,2
* Gastroenterologia, Università di Napoli "Federico II," Naples, Italy;
Endocannabinoid Research Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Pozzuoli, Italy;
Gastroenterologia, "A. O. Rummo," Benevento, Italy
2Correspondence: E-mail: vdimarzo{at}icmib.na.cnr.it;
SPECIFIC AIMS
The primary aim of the present study was to assess whether the colon levels of the two major endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG), are elevated during colon inflammation in animal models of inflammatory bowel diseases (IBDs) and in colon biopsies of patients with IBDs. We also aimed to understand the function of this potential up-regulation and to establish the basis for its possible therapeutic exploitation by assessing the effect of inhibitors of endocannabinoid inactivation on colon inflammation.
PRINCIPAL FINDINGS
1. Colon inflammation in rats
After 1 wk, treatment of rats with 2,4,6-trinitrobenzene sulfonic acid (TNBS) caused a significant inflammation of the colon as assessed according to macroscopic and microscopic evaluation and the MPO assay. It also caused an almost 3-fold enhancement of colon anandamide (but not 2-AG levels), which was limited to the submucosa, with no effect on mucosal endocannabinoid levels (data not shown).
2. Colon inflammation in mice
Treatment of mice with 2,4-dinitrobenzene sulfonic acid (DNBS) caused a significant inflammation of the colon as assessed according to macroscopic and microscopic evaluation and the MPO assay (Fig. 1
A–C). It also caused a robust enhancement of colon anandamide (but not 2-AG levels) that was already significant after 3 days and reached its maximum after 1 wk (4-fold elevation) (Fig. 1D
).
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3. Effect of inhibitors of endocannabinoid inactivation on colon inflammation
Chronic, daily treatment of DNBS-treated mice with 5 mg/kg VDM-11 (s.c.) or 1.4 mg/kg 5-ASA (i.r.) caused a complete reversion of colon inflammation as assessed according to macroscopic and microscopic evaluation and the MPO assay at both 3 and 7 days. Treatment with 10 mg/kg AA-5-HT (s.c.) ameliorated the inflammatory scores and MPO activity to a significantly lower extent than VDM-11 (Fig. 1A-C
).
4. Effect of inhibitors of endocannabinoid inactivation on endocannabinoid levels
In addition to reversal of inflammation, daily treatment of DNBS-treated mice with 5 mg/kg VDM-11 (s.c.) or 1.4 mg/kg 5-ASA (i.r.) caused a strong enhancement of the colon levels of anandamide at 3 and 7 days after the induction of colitis. 2-AG levels were not affected, except for a slight increase produced by 5-ASA at 7 days (Fig. 2D
). In contrast, analogous treatment with 10 mg/kg AA-5-HT (s.c.) did not affect either anandamide or 2-AG levels at any time point (data not shown).
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5. Ulcerative colitis in humans
A more than 2-fold elevation of anandamide, but not 2-AG levels, was found in mucosal biopsies from patients with untreated ulcerative colitis with respect to controls. Anandamide levels significantly correlated with clinical activity of the disease (r2=0.64, P=0.04, data not shown), while no correlation was found between endocannabinoid levels and endoscopic and histologic activities.
CONCLUSIONS AND SIGNIFICANCE
IBDs are chronic recurrent inflammatory disorders of the gastrointestinal tract that are of unknown etiology. They are characterized by a pronounced infiltration of neutrophils into colon mucosal lesions, accompanied by epithelial cell necrosis and ulceration. Most prevailing therapies for IBDs include glucocorticosteroids and sulfasalazine, which possess limited beneficial action. Corticosteroids are effective in the short-term treatment of acute flares of either form of IBDs (ulcerative colitis and Crohn’s disease), but they are not suitable for maintenance therapies due to a variety of systemic adverse reactions. Sulfasalazine and its derivative 5-aminosalicylic acid (5-ASA) are only effective in mild to moderate phases of the disease and in preventing relapse. Among the experimental animal models of IBDs developed to date, the intrarectal administration of DNBS or TNBS has been extensively used to study the mechanisms of colonic inflammation and to test antiinflammatory drugs.
Endocannabinoids (i.e., the endogenous agonists of cannabinoid CB1 and CB2 receptors) play several functions in the gastrointestinal tract, particularly in the colon where they tonically control propulsion. Endocannabinoids, and N-arachidonoyl-ethanolamine (anandamide) are involved more often than 2-arachidonoylglycerol (2-AG) in a series of pathological conditions of the small and large intestine, including inflammation. Their intestinal actions are usually mediated by the CB1 receptor subtype, but CB2 receptors and vanilloid TRPV1 channels have also been involved. In intestinal inflammatory conditions, in particular, anandamide exerts a protective effect against croton oil-induced small intestine hypermotility. In 2,4-dinitrobenzene sulfonic acid (DNBS) -induced inflammation (an animal model of IBDs) it was shown that CB1 receptors participate in controlling the inflammatory response and that genetic inactivation of the fatty acid amide hydrolase (FAAH), the enzyme mostly responsible for anandamide inactivation, affords protection against DNBS-induced inflammation. Although DNBS treatment increased CB1 receptor expression in myenteric neurons, no data were presented regarding the possible up-regulation of anandamide levels in DNBS-treated wild-type mice, a phenomenon that has been shown in all intestinal pathological states where this endocannabinoid has been involved. Although genetic deletion of FAAH does cause 15-fold elevation of anandamide levels in the brain, the same applies to the intestine only to a smaller extent, if at all, thus pointing to the need of studying the effect of pharmacological, in addition to genetic, blockade of FAAH on inflammation in IBDs. We recently obtained evidence that FAAH inhibitors do elevate anandamide levels in the small intestine of the healthy mouse, but there are no data available from animals with intestinal inflammation. Finally, the effect of anandamide cellular uptake inhibitors, which exert important effects during other pathological conditions affecting the intestine, has never been assessed in animal colitis. With the present study, we aimed at providing answers to these open questions.
We have shown here that in animal models as well as in patients with ulcerative colitis, colon inflammation is accompanied by enhanced anandamide, but not 2-AG levels. This enhancement seems to concern only the colon submucosa in the rat, whereas in humans it is seen in biopsies from the mucosa. These findings, together with earlier data on protective actions afforded by cannabinoid CB1 receptor stimulation in DNBS-treated mice, open the way to new therapeutic strategies against IBDs. Our data indicate that not only "direct" agonists of CB1 receptors, which may produce undesired psychotropic effects, but also "indirect" agonists (i.e., compounds capable of enhancing the lifespan of anandamide) would be able to exert a protective action against inflammation. Obvious examples of these compounds are inhibitors of anandamide inactivation through cellular reuptake or enzymatic hydrolysis. Such compounds act preferentially where there is an ongoing synthesis and degradation of anandamide, as we suggested here for the colon during colitis, thus producing fewer unwanted effects than "direct" agonists. By acting through up-regulation of anandamide, another advantage of this therapeutic strategy over the use of "direct" agonists is that it might produce beneficial actions by activating "indirectly" and in concert several targets of this compound, (i.e., not only CB1, but also CB2 and TRPV1 receptors) that are expressed in the colon. Stimulation of CB2 and activation followed by immediate desensitization of TRPV1, afford protection against colon inflammation and its consequences (Fig. 2
).
Acordingly, we showed here that pharmacological blockade of anandamide reuptake is as efficacious as the widely used antiinflammatory agent 5-ASA against DNBS-induced inflammation in mice. VDM-11 was used at a concentration (given i.p.) found to be active in mice in various intestinal physiological and pathological conditions. In agreement with its mechanism of action through prolongation of anandamide life span, VDM-11 concomitantly elevated anandamide, but not 2-AG levels, in the colon aliquots taken from the same mice used from inflammatory score determination. We did not determine through which of its many possible targets (see above) the pharmacologically elevated anandamide was acting, because we thought this was outside the scope of this study, which aimed only at demonstrating enhanced colon anandamide levels in IBDs and their possible therapeutic exploitation. Furthermore, multiple administrations to mice of DNBS, VDM-11 and, for example, a CB1 antagonist, would have posed technical problems.
Unlike VDM-11, the FAAH inhibitor AA-5-HT did not completely reverse colitis in DNBS-treated mice. This finding, according to the mechanism outlined above for VDM-11, might be due to the fact that this FAAH inhibitor did not significantly elevate the levels of either anandamide or 2-AG in the colon of these animals. Since FAAH was found to be up-regulated in the croton oil model of intestinal inflammation, it is possible that this enzyme in the intestine is less amenable than anandamide cellular reuptake to pharmacological inhibition under inflammatory conditions. In agreement with our findings, genetic inactivation of FAAH in mice was recently found to cause a much lower elevation of anandamide levels in the small intestine than in the brain, yet it results in protection against DNBS-induced inflammation. It is possible that these protective effects observed with either genetic or pharmacological inactivation of FAAH are mediated by FAAH substrates other than anandamide. Indeed, transgenic mice lacking FAAH only in peripheral tissues exhibit an antiinflammatory phenotype that is not blocked by cannabinoid receptor antagonists and has been suggested to be due to other peripherally elevated FAAH substrates.
Another intriguing finding of the present study was that 5-ASA, under the same conditions leading to protection from DNBS-induced colitis, enhances colon anandamide levels, thus raising the possibility that part of the antiinflammatory actions of this compound are due to anandamide. We tested 5-ASA on FAAH and anandamide reuptake in vitro and found no activity up to 50 µM (data not shown), thus excluding that this compound may act through inhibition of these two inactivating mechanisms. However, anandamide and 2-AG are also subject to oxidation by cycloxygenase-2 (COX-2) and further processing by prostaglandin synthases to form prostaglandin-ethanolamides. Since COX-2 is expressed in the colon of DNBS-treated animals and is inhibited by 5-ASA, it is possible that this compound raises anandamide levels (and after 7 days, it also raises 2-AG levels) by inhibiting COX-2-mediated endocannabinoid oxidation. However, no evidence has been presented here to support this hypothesis, which will have to be investigated in future studies.
We have provided evidence for the protective role of anandamide in colitis and IBDs, and a possible new strategy for the treatment of these disorders through the use of anandamide uptake inhibitors. During the preparation of this manuscript, Wright and colleagues reported that cannabinoid CB2 receptors are up-regulated in the colon of patients with IBDs and that stimulation of cannabinoid CB1 receptors contributes to epithelial wound closure. This finding, together with our present data, provides another rationale to test in clinical studies inhibitors of endocannabinoid degradation against the consequences of IBDs. It will be interesting to determine whether other intestinal inflammatory conditions are accompanied by elevated anandamide concentrations and whether they are likewise treatable with inhibitors of endocannabinoid reuptake.
FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4943fje;
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