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orphanin FQ receptor-mediated effect in a rat model of experimental colitis
Pharmacological Research 70 (2013) 72–79
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Protective and worsening peripheral nociceptin/orphanin FQ receptor-mediated effect in a rat model of experimental colitis Carla Petrella a,∗,1 , Chiara Giuli a,1 , Maria Broccardo a , Helene Eutamene b , Christel Cartier b , Mathilde Leveque b , Andrea Bedini c , Santi Spampinato c , Lionel Bueno b , Vassilia Theodorou b , Giovanna Improta a , Simona Agostini b a
Department of Physiology and Pharmacology “V. Erspamer”, Sapienza University of Rome, Rome, Italy INRA, EI-Purpan, UMR 1331 TOXALIM Neuro-Gastroenterology and Nutrition Team, Toulouse, France c Department of Pharmacology, University of Bologna, Bologna, Italy b
a r t i c l e
i n f o
Article history: Received 15 October 2012 Received in revised form 9 January 2013 Accepted 15 January 2013 Keywords: N/OFQ NOP receptor Rat experimental colitis
a b s t r a c t Nociceptin/orphanin FQ (N/OFQ) and nociceptin orphanin peptide (NOP) receptors represent an endogenous system modulating gastrointestinal functions and inflammation. We investigated the peripheral effect of N/OFQ and of UFP-101, the NOP antagonist, in a model of colitis induced by TNBS (2,4,6 trinitrobenzenesulphonic acid; 60 mg/kg). Male rats received two intraperitoneal injections per day of N/OFQ, UFP-101 or saline for 3 days after colitis induction. Four days after TNBS, animals were sacrificed and colonic histological damage, myeloperoxidase (MPO) activity and cytokine (IL-1 and IL-10) levels were evaluated. N/OFQ plasmatic levels were assessed by radioimmunoassay. TNBS increased all the inflammatory variables considered. In colitic rats, N/OFQ (0.02 and 0.2 nmol/kg) improved microscopic damage, MPO activity and decreased IL-1 levels in comparison with TNBS group, whereas at the highest dose (20 nmol/kg) the peptide worsened colitis. UFP-101 at the dose of 1 nmol/kg, without pharmacological activity, antagonised the protective effect of N/OFQ (0.2 nmol/kg) on colitis, but at a dose level of 3 and 10 nmol/kg worsened inflammation, revealing the endogenous N/OFQergic system protective role. N/OFQ plasmatic levels were not modified in TNBS-treated rats compared with controls, whereas they were reduced in rats treated with the doses of UFP-101 aggravating colitis. In conclusion, peripheral low doses of N/OFQ have a beneficial effect on colonic inflammation in rats. In contrast, N/OFQ at a dose 100–1000-fold higher than those that protect worsens colitis, probably through different mechanisms. The peripheral N/OFQergic system can represent a new field of investigation in some intestinal inflammatory conditions. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Nociceptin/orphanin FQ (N/OFQ) and nociceptin orphanin peptide (NOP) receptors (the N/OFQergic system) are widely expressed in the brain and peripheral nervous system of mammals [1–4], as well as in peripheral tissues such as skeletal muscle, vas deferens, spleen and intestine [5]. In the gastrointestinal tract (GI), it is well established that the N/OFQergic system is distributed in peripheral and central sites modulating important GI functions both in
Abbreviations: TNBS, 2,4,6 trinitrobenzenesulphonic acid; MPO, myeloperoxidase; IL-, interleukin-; IBD, inflammatory bowel disease. ∗ Corresponding author at: Department of Physiology and Pharmacology “V. Erspamer”, Sapienza University of Rome, P.le Aldo Moro, 5-00185 Rome, Italy. Tel.: +39 0649912966; fax: +39 0649912487. E-mail addresses: [email protected], [email protected] (C. Petrella). 1 These authors are co-first authors. 1043-6618/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phrs.2013.01.004
physiological [6–11] and in pathological conditions [12–15]. In particular, it has been reported in rats that, by activation of peripheral and central NOP receptors, N/OFQ inhibits distal colon transit in the basal state and in experimental conditions of functional alteration (e.g. diarrhoea induced by castor oil, increased faecal output induced by CRF or restraint stress) [12]. NOP receptors and N/OFQ precursor (ppN/OFQ) mRNA are also expressed in several cells of the immune system, including monocytes, lymphocytes and polymorphonuclear cells [16–18]. Pro-inflammatory cytokines and other compounds can stimulate leukocytes to release N/OFQ [16,19]. However, the role of N/OFQ in the immune system modulation remains to be clarified. Controversial studies report exogenous N/OFQ to be both pro-inflammatory and anti-inflammatory, immunoenhancing and immunosuppressive [19–24]. Crohn’s disease and ulcerative colitis [25–27] are the major types of inflammatory bowel disease (IBD), a group of inflammatory conditions of the colon and small intestine in humans,
C. Petrella et al. / Pharmacological Research 70 (2013) 72–79
characterised by colonic dysmotility, visceral hypersensitivity, severe internal cramps/muscle spasms, diarrhoea, weakness, rectal bleeding, vomiting, weight loss. Crohn’s disease is associated with a local and systemic inflammatory response in the gut through the activation of Th-1- and Th-17-mediated responses that induce proinflammatory cytokine release. Increased IL-1 concentrations in the mucosa have been directly correlated with both the initiation and amplification of intestinal inflammation in animal models of colitis [28,29]. Crohn’s disease is also associated with a reduction of the suppressive cytokine signals, such as IL-10, that are normally responsible for turning off the immunologic and inflammatory protective processes [30]. Few studies exist about the role of the N/OFQergic system in experimental models of colonic inflammation that mimic IBD pathology. It has been shown that colitis induced by dextran sodium sulphate (DSS), an experimental model of ulcerative colitis, is significantly improved in NOP receptor-deficient mice compared to wild-type mice [23] and in mice treated with SB612111, a small molecule inhibitor of NOP receptors [31], and that N/OFQ expression is increased in the myenteric plexus and in some infiltrating cells [23]. On the other hand, peripheral administration of N/OFQ in rats has been shown to reduce visceral hypersensitivity to colorectal distension in a model of experimental colitis induced by 2,4,6 trinitrobenzenesulphonic acid (TNBS) in association with an overexpression of NOP receptors in the colon [32]. Taken together, these studies suggest a modulatory control of the N/OFQergic system in GI dysfunctions linked to dysmotility, pain and inflammation. To further investigate the role of the N/OFQergic system in gut inflammatory states, the aim of this study was to evaluate: (i) the effects of repeated peripheral administration of the NOP receptor agonist, N/OFQ (at doses active on colonic motility and visceral hypersensitivity [12,32]), and of the selective NOP receptor antagonist, UFP-101, on the principal inflammatory indexes (myeloperoxidase/MPO activity and histological damage), and on the major pro- and anti-inflammatory gut cytokines (IL-1 and IL-10, respectively) in an experimental model of colitis induced by TNBS in rats, that resembles Crohn’s disease; (ii) the N/OFQ plasmatic levels in healthy controls, in colitic and UFP-101-treated colitic rats. 2. Materials and methods 2.1. Drugs N/OFQ and UFP-101 were synthesised and purified at the University of Ferrara (Italy) by the group of Dr. Guerrini and Dr. Calò as previously described [33]. Drugs were freshly solubilised in saline solution (NaCl 0.9%) before each experiment. 2.2. Animals and induction of experimental colitis Male Wistar rats (200–250 g; Janvier SA, Le Genest St Isle, France) were individually housed in a temperature-controlled room (21 + 1 ◦ C). All the animals were maintained in a 12/12-h light/dark cycle and were provided food and water ad libitum. Treatments started at least 1 week after rat arrival. All experimental protocols were approved by the Local Animal Care and Use Committee of Institut National de la Recherche Agronomique. Overnight fasted animals were anaesthetised by intraperitoneal (IP) injection of acepromazine (0.6 mg/kg) (Calmivet, Vetoquinol, Lure, France) and ketamine (120 mg/kg) (Imalgene 1000, RhoneMerieux, Lyon, France) and colitis was induced by an intra-colonic (IC) administration of TNBS (Sigma Aldrich, France) at a dose of 60 mg/kg in 0.3 mL of 50% ethanol. TNBS was infused through a silicone catheter introduced in the proximal colon, 6 cm into the
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anus as previously described [34]. Control rats were infused with 0.3 mL of saline. 2.3. Experimental protocol All the experimental groups (8 rats for each one), reported below, were intraperitoneally injected twice a day for 3 consecutive days, 24 h after TNBS instillation. The injections were performed at 9.00 AM and 17.00 PM of each day of treatment. Control rats were injected with saline. TNBS-treated rats were injected with: saline (NaCl 0.9%), N/OFQ (0.02–0.2–2–20 nmol/kg) or the NOP receptor antagonist UFP-101 (1–3–10 nmol/Kg). In studies of antagonism, TNBS-treated rats were injected with: saline, UFP-101 (1 nmol/kg), N/OFQ (0.2 nmol/kg), and UFP-101 + N/OFQ or saline. UFP-101 was administered 15 min before saline or N/OFQ injections. Four days after TNBS instillation rats were euthanised. Samples of colon and colonic mucosa were collected to assess colonic damage (macroscopic and microscopic evaluation), myeloperoxydase activity (MPO) and pro- (IL-1) and anti- (IL-10) inflammatory colonic cytokine levels. Animal body weight and food intake were registered on the day of IC instillation of TNBS or saline and on the day of sacrifice, and their variations were expressed as percentage, with respect to the initial weight. 2.4. Histological evaluation 2.4.1. Macroscopic damage Immediately after sacrifice, colon samples were removed and rinsed with saline. Colonic damage was evaluated in double blind and expressed with a score that took into account the severity and extent of macroscopic lesions (hyperemia, ulcers), the presence and the extent of adhesions and the occurrence of diarrhoea, according to a modified Wallace et al. scale [35] (Table 1). 2.4.2. Microscopic damage Samples of distal colon near the core of the ulceration, were fixed in Duboscq-Brazil buffer, dehydrated and embedded in paraffin. Sections of 5 m were stained with eosin-haematoxylin, and examined by light microscopy. The extent of histological damage was expressed according to the criteria described by Fabia et al. [36]. Each parameter estimated was graded 0–3 depending upon the severity of the changes found: (0) no change, (1) mild, (2) moderated or (3) severe changes. Total damage was obtained by adding the individual scores. 2.5. Myeloperoxidase (MPO) activity assay The activity of MPO, a marker of polymorphonuclear primary granules, was determined in colon tissue according to a previously described technique [37]. Immediately after sacrifice, a distal colonic segment (1 cm long) was taken off at 5–6 cm from the anus. It was suspended in potassium phosphate buffer (KH2 PO4 44 mM, K2 HPO4 6 mM, pH 6.0), homogenised on ice with Polytron (PCU-2, Kinematica GmbH, Lucerne, Switzerland) and submitTable 1 Macroscopic score parameters. Stool consistency
Colon damage score
Tissue adhesions
0 = formed 1 = liquid
0 = none 1 = hyperemia 2 = slight erosion 3 = extensive erosion/ulceration 4 = extensive erosion/ulceration > 2 cm 5 = more sites of inflammation > 1 cm
0 = none 1 = mild 2 = strong
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ted to three cycles of freezing and thawing. Homogenates were then centrifuged at 9000 × g for 15 min at 4 ◦ C. The pellets were resuspended in hexadecyl trimethylammonium bromide buffer (0.5%, wt/vol) in potassium phosphate buffer to release MPO from polymorphonuclear neutrophil primary granules. These suspensions were sonicated (Büchi, Flawil, Switzerland) on ice and centrifuged at 9000 × g for 15 min at 4 ◦ C. Supernatant fractions were diluted in potassium phosphate buffer containing 0.167 mg Odianisidine dihydrochloride/mL and 0.00005% (vol/vol) H2 O2 . MPO from human neutrophils (Sigma, Saint Quentin Fallavier, France; 0.1 U/mL) was used as a standard. Changes in absorbance at 450 nm were recorded with a spectrophotometer (mc2UV, Safas, Monaco) every 10 s over 2 min. One unit of MPO activity was defined as the quantity of MPO degrading 1 mol H2 O2 min−1 mL−1 at 25 ◦ C. Protein concentrations (g/mL) were determined using a modified method of Lowry (Detergent Compatible Assay, BioRad, Ivry/Seine, France) and MPO activity was expressed as MPO units/g protein. 2.6. Colonic mucosa cytokine levels Tissue proteins were extracted with RIPA buffer (1% Igepal, 0.5% deoxycholic acid, and 0.1% sodium dodecyl sulphate in Trisbuffered saline 1×; pH 7.4) with protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Clear lysates were prepared by centrifugation at 10,000 × g for 10 min, and protein concentrations were assessed using the BC Assay Uptima kit (Interchim). Samples were then processed for ELISA using commercial kits to determine colonic contents of IL-1, and IL-10 (ELISA kits, Duoset R&D Systems, Lille, France). Data were expressed as concentration per mg of total protein. 2.7. N/OFQ plasmatic levels Blood samples from controls, TNBS saline-, TNBS + UFP-101 (1–3–10 nmol/kg)-treated rats were collected in tubes containing EDTA and Aprotinin (4 TIU/200 l), and immediately centrifuged at 1200 × g 4 ◦ C for 10 min. N/OFQ assay was performed as previously described [38]. The standard curve was constructed using B/B0 versus N/OFQ standard. The amount of N/OFQ in the unknown samples was extrapolated from the standard curve using GraphPad Prism version 3.0 (GraphPad Software, Inc., San Diego, CA, USA). 2.8. Statistical analysis All data are presented as means ± SEM. For statistical analysis Graph Pad Prism 4.0 (GraphPad, San Diego, CA) was used. Results were analysed by one way ANOVA followed by Dunnett’s Multiple Comparison Test. Statistical significance was set at p < 0.05. 3. Results 3.1. Histological evaluation 3.1.1. Macroscopic score The instillation of TNBS caused the appearance of extensive ulceration and necrotic zones of >1 to 5 cm, tissue adhesions and occasionally diarrhoea. The macroscopic score evaluated 4 days after TNBS instillation was not modified by the treatment with N/OFQ or its antagonist UFP-101, irrespective of the different administered doses (Table 2). 3.1.2. Microscopic Score TNBS instillation resulted in submucosal and mucosal infiltration of inflammatory cells and blood vessels dilatation, compared to control animals (Fig. 1A).
Table 2 Effect of different treatments on colonic macroscopic damage induced by TNBS colitis. Treatment
Macroscopic score (n.)
Controls TNBS+ Saline N/OFQ (0.02 nmol/kg) N/OFQ (0.2 nmol/kg) N/OFQ (2 nmol/kg) N/OFQ (20 nmol/kg) UFP-101 (1 nmol/kg) UFP-101 (3 nmol/kg) UFP-101 (10 nmol/kg)
0.0 ± 0.0
a
7.5 7.8 8.1 6.9 7.8 7.8 7.9 7.9
± ± ± ± ± ± ± ±
0.7a 1.0a 0.6a 1.1a 1.1a 0.9a 0.7a 1.1a
p < 0.01 vs controls.
In TNBS-induced colitis rats, repeated injections of N/OFQ induced a significant reduction of microscopic score when administered at doses of 0.02 and 0.2 nmol/kg compared with TNBS alone. N/OFQ, administered at 2 nmol/kg, did not affect microscopic damage induced by TNBS. Conversely, at the dose of 20 nmol/kg, N/OFQ significantly worsened microscopic scores compared with TNBS alone (Fig. 1A). Repeated UFP-101 treatment (3 and 10 nmol/kg) significantly worsened microscopic colonic damage induced by TNBS, whilst the UFP-101 dose of 1 nmol/kg did not show pharmacological activity (Fig. 1A). The pre-treatment with UFP-101 (1 nmol/kg) significantly antagonised the protective effect of N/OFQ (0.2 nmol/kg) on microscopic damage (Table 3). Representative histological samples of colonic sections from control and from TNBS-treated rats after saline, N/OFQ and UFP-101 administration are shown in Fig. 1B. 3.2. Body weight and food intake Treatment with TNBS caused a significant reduction of the animal’s weight and food intake) respect to control rats. None of these parameters underwent significant changes following treatment with either N/OFQ or UFP-101 (data not shown). 3.3. MPO activity The IC administration of TNBS significantly increased colonic MPO activity compared to controls. After TNBS-induced colitis, repeated IP injections of N/OFQ, at doses of 0.02 and 0.2 nmol/kg, significantly reduced MPO activity. Conversely, administered at the higher dose of 20 nmol/kg, N/OFQ induced a significant increase of colonic MPO activity in comparison with TNBS rats treated with saline (Fig. 2A). In addition, in TNBS rats, repeated administration of the selective NOP receptor antagonist, UFP-101, at doses of 3 and 10 nmol/kg, significantly increased colonic MPO activity compared to TNBS rats treated with saline (Fig. 2B). Table 3 Effect of pre-treatment with the NOP receptor antagonist, UFP-101, on the N/OFQinduced decrease of MPO activity and microscopic score, during TNBS colitis. Treatment
MPO activity (U/g of protein)
Microscopic score (n.)
Controls TNBS+ Saline N/OFQ (0.2 nmol/kg) UFP-101 (1 nmol/kg) UFP-101 + N/OFQ
155.1 ± 14.79
1.8 ± 0.36
589.9 ± 44.32a 261.1 ± 50.18b 588.5 ± 110.0 634.6 ± 107.6
5.0 ± 0.78a 2.0 ± 0.5b 4.75 ± 1.44 5.1 ± 1.00
a b
p < 0.01 vs controls. p < 0.01 vs TNBS saline-treated rats.
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Fig. 1. Effect of the treatment with N/OFQ and UFP-101 on histopathological alterations in TNBS-induced colitis in rats. Panel A: Histological score analysis of damage in colonic sections of rats treated with N/OFQ (0.02–0.2–2–20 nmol/kg), left panel, and with UFP-101 (1–3–10 nmol/kg), right panel. Each column is the mean ± SEM (n = 8 rats for each group). *p < 0.05 and **p < 0.01. The extent of histological damage was expressed according to the criteria described by Fabia et al. [36]. Panel B: Representative eosin and haematoxylin-stained colon sections from rats treated as above indicated. Scale bars = 50 m upper figures, 100 m lower figures.
When administered at the lower dose of 1 nmol/kg, UFP-101 did not show any significant effect per se (Fig. 2B), but abolished the inhibitory effect on MPO activity induced by N/OFQ (Table 3). 3.4. Colonic IL-1ˇ and IL-10 levels TNBS-treated rats showed an expected increase of proinflammatory IL-1 cytokine levels measured in colonic mucosa, compared to control animals. In rats with colitis, repeated treatment with N/OFQ (0.02 and 0.2 nmol/kg) significantly decreased IL-1 levels compared to TNBS animals injected with saline. Neither N/OFQ (20 nmol/kg;) nor UFP-101 (10 nmol/kg;) modified the pro-inflammatory cytokine levels after colitis (Fig. 3A). TNBS-treated rats showed an expected significant decrease in anti-inflammatory IL-10 cytokine levels measured in colonic mucosa in comparison with control animals. No administered doses of N/OFQ or UFP-101 affected IL-10 content after colitis induction (Fig. 3B). 3.5. N/OFQ plasmatic levels TNBS rats showed no difference in N/OFQ plasmatic levels in comparison with control animals. Three days of treatment with
UFP-101 (1–3–10 nmol/kg) after TNBS-induced colitis, significantly decreased N/OFQ plasmatic levels (Fig. 4). 4. Discussion This study shows that repeated peripheral administrations of low – but not high – doses of N/OFQ significantly decrease the colonic inflammatory profile in rat TNBS-induced colitis. Pretreatment with the selective NOP receptor antagonist UFP-101, at the dose devoid of modulatory effects on intestinal inflammation by itself, completely reversed the observed protective effect, indicating that the activation of peripheral NOP receptors mediates an anti-inflammatory action in this experimental model of colitis. Moreover, since UFP-101, administered at high doses, triggers pro-inflammatory effects and reduces N/OFQ plasmatic levels, we speculate that the endogenous peripheral N/OFQergic system has a protective role in the modulation of inflammation during TNBSinduced colitis. The beneficial effect of repeated injections of low doses of N/OFQ is shown by the reduction of colonic MPO activity and the improvement of colonic microscopic inflammatory damage evaluated by light microscopy. The reduction of TNBS-induced damage is not observed by evaluating the macroscopic scores (healing of ulcers, reduced hyperaemia) or the recovery of body weight and food intake. It is not easy to explain the lack of correspondence between
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Fig. 2. Colonic MPO activity measured in rats treated twice a day for 3 consecutive days with N/OFQ (0.02–0.2–2–20 nmol/kg; panel A) and UFP-101 (1–3–10 nmol/kg; panel B), 24 h after TNBS initiation. Each column is the mean ± SEM (n = 8 rats for each group). *p < 0.05 and **p < 0.01.
micro and macroscopic score. However, most of the studies showing that TNBS macroscopic score, MPO activity and microscopic scores are well correlated, use lower doses of TNBS [39] causing less severe damages, or evaluate the inflammatory indexes after a time period from the TNBS administration longer than that we have used [39,40]. Therefore, we cannot exclude that a less severe colitis or an extension of N/OFQ treatment may also induce an improvement of macroscopic scores. The protective effect of low doses of N/OFQ during TNBS-colitis is also associated with the reduction of one of the most significant signals of the colonic inflammation, the cytokine IL-1, while the anti-inflammatory IL-10 content does not change. The N/OFQ modulatory activity on IL-1 observed here is in line with that reported by other investigators, even if we cannot establish if N/OFQ affects the level of this cytokine through direct or indirect action. The hypothesis of a direct effect is supported by the evidence that NOP receptors are expressed on the surface of immune cells and mediate an anti-inflammatory action [21]. Both in vivo and in vitro studies have shown that N/OFQ suppresses the production of pro-inflammatory cytokines from rat splenocytes and human T cells [19,41], and decreases the expression of mRNA for IL-1 and other cytokines in astrocytes activated
Fig. 3. Effect of repeated injections of N/OFQ (0.02, 0.2 and 20 nmol/kg) and UFP101 (10 nmol/kg) on pro-inflammatory IL-1 (panel A) and anti-inflammatory IL-10 (panel B) mucosa colonic content. Levels of IL-1 and IL-10 were measured by ELISA kits 4 days after TNBS instillation. Each column is the mean ± SEM (n = 8 rats for each group). **p < 0.01.
in vivo by peripheral administration of inflammatory stimuli [42]. Furthermore, binding studies on immune system cells report a high affinity of N/OFQ binding sites in line with a direct action of a low concentration of the peptide [43]. As for an indirect modulatory role of N/OFQ on the immune system, it is well known that in the gut, signals associated with immune responses contribute to the pathophysiology of IBD by activating CRF1 (corticotrophin releasing factor) receptor pathways (i.e. mast cell activation), which are closely related to stress and inflammation [44,45]. Since N/OFQ has been shown to be a functional antagonist of CRF1 receptors [12,46], we cannot exclude that the peptide reduces the inflammatory process by the inhibition of peripheral CRF1 receptor pathways in colitic animals. We also report that peripheral repeated administration of a high dose of N/OFQ after colitis induction worsens some of the inflammatory parameters studied (MPO, histological scores), but do not modify the cytokine levels. The explanation for the opposite anti-inflammatory and pro-inflammatory effects induced by different dosages of N/OFQ remains to be determined. Certainly, the N/OFQ aggravating effect cannot be linked to the well-known antipropulsive colonic activity of the peptide, because both low and high doses of N/OFQ [12] evoke a reduction of colonic propulsion. On the other hand, there is a recent paper [47] showing that, in
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Fig. 4. N/OFQ plasmatic levels in control animals and in TNBS-induced colitis rats after saline or UFP-101 treatment. Plasmatic levels were measured with a radioimmunoassay kit for N/OFQ. Data are expressed as mean ± SEM (n = 8 rats for each group). *p < 0.05 and **p < 0.01.
healthy animals, N/OFQ at a high dose comparable to that used in our study, induces a pro-inflammatory effect due to the vasodilatation of mesenteric microcirculation, leading to a decrease in blood flow velocity, an increase in leucocyte rolling and adhesion, and to an increase in 2-integrin (CD18/CD11) expression on circulating rat neutrophils. Therefore, it is possible that the aggravating effect of the high-dose N/OFQ in TNBS-induced colitis is due to pathways controlling vascular permeability and chemotaxis. Although further studies will be necessary, we postulate that different mechanisms are involved in the protective and aggravating action of N/OFQ. In our study, repeated treatment with high doses of the NOP receptor antagonist, UFP-101, worsens colonic inflammation. This result together with the increase of visceral sensitivity to colorectal distension in rats treated with UFP-101, under similar experimental conditions [32], shows the pharmacological activity of the antagonist and reveals the possible protective role of the endogenous peripheral N/OFQergic system in colitis. The involvement of endogenous N/OFQergic system in experimental colitis has also been reported in mice [23,31]. However, the peripheral N/OFQ protective effect during TNBS-induced colitis in rats we have shown, is in contrast with the worsening role played by the NOP system in knockout mice and in mice treated with a NOP antagonist in a DSS model of colitis [23,31]. The NOP receptor system is known to mediate opposing effects (anxiogenic/anxiolytic; increase/decrease of locomotion; inhibition/stimulation of HPA) in mice and rats [48–53]. The reason why N/OFQ plays opposing roles in these different animal species is not yet understood. Regarding our results, the NOP wild-type rats we used represent an experimental model absolutely different from the NOP-deficient mice model [23], and thus is not easily comparable. Furthermore, the aetiopathogenetic mechanisms of the experimental models of colitis used in our and in Kato’s [23] studies are certainly different. In the TNBS model, which resembles Crohn’s disease in humans, TNBS – a hapten that results in change of the autologous molecules in the mucosa – when locally injected through intrarectal administration, leads to priming of antigen-specific T cells [54]. In the DSS model, which resembles human ulcerative colitis, DSS added to drinking water results in toxicity towards colonic mucosa causing
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defects in the epithelial barrier integrity and colonic permeability, increased dysplastic lesions and, at least in some cases, trough Th1-dependent pathway [55]. Finally, it is known that UFP-101, the NOP receptor antagonist used in our study, does not cross the blood-brain-barrier [56] and it is able to block only NOP peripheral sites, whilst SB612111, the small non-peptidic NOP antagonist used by Alt [31], can pass the barrier and block NOP central sites. Therefore, the opposite (protective and aggravating) roles shown in our and in Alt’s study [31] could be due to the involvement of different, peripheral and central, N/OFQ sites of action. In this regard, it’s largely described that N/OFQ mediates opposite central and peripheral effects [57]. To date, no studies correlate N/OFQ plasmatic levels and IBD in humans or in experimental models of colitis. Conversely, several studies correlate changes of N/OFQ plasmatic levels in other human diseases. Indeed, high levels of plasmatic N/OFQ have been associated with muscle-skeletal diseases, urinary stones and heart pain of unclear origin, as well as in patients affected by unstable angina [58]. On the other hand, low levels of N/OFQ have been linked to migraine [59], fibromyalgia [60], and chronic ischaemic cardiovascular diseases [61], while normal levels have been reported in women during labour [62]. To give further information on the N/OFQergic system role during experimental colitis, the N/OFQ plasmatic levels in TNBS-treated rats have been determined. The data obtained here confirm the presence of N/OFQ in rat plasma under physiological conditions, as previously observed [63], and show that N/OFQ plasmatic levels do not change in TNBS-treated rats. By blocking NOP receptors in TNBS-treated rats with UFP101, at doses that worsen colonic inflammatory damage, a decrease of N/OFQ plasmatic levels is induced in comparison with controls and with rats treated with TNBS alone. Such a prolonged block of the NOP receptors together with the decrease in N/OFQ plasmatic levels, probably due to an increased degradation and/or to a reduced synthesis of the peptide, put in evidence that a lower functionality of the endogenous peripheral N/OFQergic system during colitis results in a worsening of the inflammatory parameter studied. 5. Conclusions This paper shows that the peripheral NOP receptors mediate a dose-related protective and worsening effect on colitis induced by TNBS in the rat. These findings, together with the ability of N/OFQ to decrease colonic motility in a condition of altered functionality [12] and to reduce visceral hypersensitivity in an animal model well reproducing Crohn’s disease [32], offer an interesting insight on the modulatory role of the N/OFQergic system in some inflammatory conditions of the intestinal tract characterised by altered motility, visceral pain and mucosal lesions. Conflict of interest The authors have no competing interests to declare. Acknowledgments This work was supported by the Nexus Grant in memory of Prof. Marcello Tonini for a Research project in Gastroenterology [AstraZeneca Industries, Italy, Grant 2011]. The authors wish to thank Prof. Francesco Botrè, Dr. Monica Mazzarino and Francesca de Angelis (Federazione Medico Sportiva Italiana-Laboratorio Antidoping) for their help in performing the extraction of the peptide from rat plasma.
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Protective and worsening peripheral nociceptin/orphanin FQ receptor-mediated effect in a rat model of experimental colitis Carla Petrella a,∗,1 , Chiara Giuli a,1 , Maria Broccardo a , Helene Eutamene b , Christel Cartier b , Mathilde Leveque b , Andrea Bedini c , Santi Spampinato c , Lionel Bueno b , Vassilia Theodorou b , Giovanna Improta a , Simona Agostini b a
Department of Physiology and Pharmacology “V. Erspamer”, Sapienza University of Rome, Rome, Italy INRA, EI-Purpan, UMR 1331 TOXALIM Neuro-Gastroenterology and Nutrition Team, Toulouse, France c Department of Pharmacology, University of Bologna, Bologna, Italy b
a r t i c l e
i n f o
Article history: Received 15 October 2012 Received in revised form 9 January 2013 Accepted 15 January 2013 Keywords: N/OFQ NOP receptor Rat experimental colitis
a b s t r a c t Nociceptin/orphanin FQ (N/OFQ) and nociceptin orphanin peptide (NOP) receptors represent an endogenous system modulating gastrointestinal functions and inflammation. We investigated the peripheral effect of N/OFQ and of UFP-101, the NOP antagonist, in a model of colitis induced by TNBS (2,4,6 trinitrobenzenesulphonic acid; 60 mg/kg). Male rats received two intraperitoneal injections per day of N/OFQ, UFP-101 or saline for 3 days after colitis induction. Four days after TNBS, animals were sacrificed and colonic histological damage, myeloperoxidase (MPO) activity and cytokine (IL-1 and IL-10) levels were evaluated. N/OFQ plasmatic levels were assessed by radioimmunoassay. TNBS increased all the inflammatory variables considered. In colitic rats, N/OFQ (0.02 and 0.2 nmol/kg) improved microscopic damage, MPO activity and decreased IL-1 levels in comparison with TNBS group, whereas at the highest dose (20 nmol/kg) the peptide worsened colitis. UFP-101 at the dose of 1 nmol/kg, without pharmacological activity, antagonised the protective effect of N/OFQ (0.2 nmol/kg) on colitis, but at a dose level of 3 and 10 nmol/kg worsened inflammation, revealing the endogenous N/OFQergic system protective role. N/OFQ plasmatic levels were not modified in TNBS-treated rats compared with controls, whereas they were reduced in rats treated with the doses of UFP-101 aggravating colitis. In conclusion, peripheral low doses of N/OFQ have a beneficial effect on colonic inflammation in rats. In contrast, N/OFQ at a dose 100–1000-fold higher than those that protect worsens colitis, probably through different mechanisms. The peripheral N/OFQergic system can represent a new field of investigation in some intestinal inflammatory conditions. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Nociceptin/orphanin FQ (N/OFQ) and nociceptin orphanin peptide (NOP) receptors (the N/OFQergic system) are widely expressed in the brain and peripheral nervous system of mammals [1–4], as well as in peripheral tissues such as skeletal muscle, vas deferens, spleen and intestine [5]. In the gastrointestinal tract (GI), it is well established that the N/OFQergic system is distributed in peripheral and central sites modulating important GI functions both in
Abbreviations: TNBS, 2,4,6 trinitrobenzenesulphonic acid; MPO, myeloperoxidase; IL-, interleukin-; IBD, inflammatory bowel disease. ∗ Corresponding author at: Department of Physiology and Pharmacology “V. Erspamer”, Sapienza University of Rome, P.le Aldo Moro, 5-00185 Rome, Italy. Tel.: +39 0649912966; fax: +39 0649912487. E-mail addresses: [email protected], [email protected] (C. Petrella). 1 These authors are co-first authors. 1043-6618/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phrs.2013.01.004
physiological [6–11] and in pathological conditions [12–15]. In particular, it has been reported in rats that, by activation of peripheral and central NOP receptors, N/OFQ inhibits distal colon transit in the basal state and in experimental conditions of functional alteration (e.g. diarrhoea induced by castor oil, increased faecal output induced by CRF or restraint stress) [12]. NOP receptors and N/OFQ precursor (ppN/OFQ) mRNA are also expressed in several cells of the immune system, including monocytes, lymphocytes and polymorphonuclear cells [16–18]. Pro-inflammatory cytokines and other compounds can stimulate leukocytes to release N/OFQ [16,19]. However, the role of N/OFQ in the immune system modulation remains to be clarified. Controversial studies report exogenous N/OFQ to be both pro-inflammatory and anti-inflammatory, immunoenhancing and immunosuppressive [19–24]. Crohn’s disease and ulcerative colitis [25–27] are the major types of inflammatory bowel disease (IBD), a group of inflammatory conditions of the colon and small intestine in humans,
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characterised by colonic dysmotility, visceral hypersensitivity, severe internal cramps/muscle spasms, diarrhoea, weakness, rectal bleeding, vomiting, weight loss. Crohn’s disease is associated with a local and systemic inflammatory response in the gut through the activation of Th-1- and Th-17-mediated responses that induce proinflammatory cytokine release. Increased IL-1 concentrations in the mucosa have been directly correlated with both the initiation and amplification of intestinal inflammation in animal models of colitis [28,29]. Crohn’s disease is also associated with a reduction of the suppressive cytokine signals, such as IL-10, that are normally responsible for turning off the immunologic and inflammatory protective processes [30]. Few studies exist about the role of the N/OFQergic system in experimental models of colonic inflammation that mimic IBD pathology. It has been shown that colitis induced by dextran sodium sulphate (DSS), an experimental model of ulcerative colitis, is significantly improved in NOP receptor-deficient mice compared to wild-type mice [23] and in mice treated with SB612111, a small molecule inhibitor of NOP receptors [31], and that N/OFQ expression is increased in the myenteric plexus and in some infiltrating cells [23]. On the other hand, peripheral administration of N/OFQ in rats has been shown to reduce visceral hypersensitivity to colorectal distension in a model of experimental colitis induced by 2,4,6 trinitrobenzenesulphonic acid (TNBS) in association with an overexpression of NOP receptors in the colon [32]. Taken together, these studies suggest a modulatory control of the N/OFQergic system in GI dysfunctions linked to dysmotility, pain and inflammation. To further investigate the role of the N/OFQergic system in gut inflammatory states, the aim of this study was to evaluate: (i) the effects of repeated peripheral administration of the NOP receptor agonist, N/OFQ (at doses active on colonic motility and visceral hypersensitivity [12,32]), and of the selective NOP receptor antagonist, UFP-101, on the principal inflammatory indexes (myeloperoxidase/MPO activity and histological damage), and on the major pro- and anti-inflammatory gut cytokines (IL-1 and IL-10, respectively) in an experimental model of colitis induced by TNBS in rats, that resembles Crohn’s disease; (ii) the N/OFQ plasmatic levels in healthy controls, in colitic and UFP-101-treated colitic rats. 2. Materials and methods 2.1. Drugs N/OFQ and UFP-101 were synthesised and purified at the University of Ferrara (Italy) by the group of Dr. Guerrini and Dr. Calò as previously described [33]. Drugs were freshly solubilised in saline solution (NaCl 0.9%) before each experiment. 2.2. Animals and induction of experimental colitis Male Wistar rats (200–250 g; Janvier SA, Le Genest St Isle, France) were individually housed in a temperature-controlled room (21 + 1 ◦ C). All the animals were maintained in a 12/12-h light/dark cycle and were provided food and water ad libitum. Treatments started at least 1 week after rat arrival. All experimental protocols were approved by the Local Animal Care and Use Committee of Institut National de la Recherche Agronomique. Overnight fasted animals were anaesthetised by intraperitoneal (IP) injection of acepromazine (0.6 mg/kg) (Calmivet, Vetoquinol, Lure, France) and ketamine (120 mg/kg) (Imalgene 1000, RhoneMerieux, Lyon, France) and colitis was induced by an intra-colonic (IC) administration of TNBS (Sigma Aldrich, France) at a dose of 60 mg/kg in 0.3 mL of 50% ethanol. TNBS was infused through a silicone catheter introduced in the proximal colon, 6 cm into the
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anus as previously described [34]. Control rats were infused with 0.3 mL of saline. 2.3. Experimental protocol All the experimental groups (8 rats for each one), reported below, were intraperitoneally injected twice a day for 3 consecutive days, 24 h after TNBS instillation. The injections were performed at 9.00 AM and 17.00 PM of each day of treatment. Control rats were injected with saline. TNBS-treated rats were injected with: saline (NaCl 0.9%), N/OFQ (0.02–0.2–2–20 nmol/kg) or the NOP receptor antagonist UFP-101 (1–3–10 nmol/Kg). In studies of antagonism, TNBS-treated rats were injected with: saline, UFP-101 (1 nmol/kg), N/OFQ (0.2 nmol/kg), and UFP-101 + N/OFQ or saline. UFP-101 was administered 15 min before saline or N/OFQ injections. Four days after TNBS instillation rats were euthanised. Samples of colon and colonic mucosa were collected to assess colonic damage (macroscopic and microscopic evaluation), myeloperoxydase activity (MPO) and pro- (IL-1) and anti- (IL-10) inflammatory colonic cytokine levels. Animal body weight and food intake were registered on the day of IC instillation of TNBS or saline and on the day of sacrifice, and their variations were expressed as percentage, with respect to the initial weight. 2.4. Histological evaluation 2.4.1. Macroscopic damage Immediately after sacrifice, colon samples were removed and rinsed with saline. Colonic damage was evaluated in double blind and expressed with a score that took into account the severity and extent of macroscopic lesions (hyperemia, ulcers), the presence and the extent of adhesions and the occurrence of diarrhoea, according to a modified Wallace et al. scale [35] (Table 1). 2.4.2. Microscopic damage Samples of distal colon near the core of the ulceration, were fixed in Duboscq-Brazil buffer, dehydrated and embedded in paraffin. Sections of 5 m were stained with eosin-haematoxylin, and examined by light microscopy. The extent of histological damage was expressed according to the criteria described by Fabia et al. [36]. Each parameter estimated was graded 0–3 depending upon the severity of the changes found: (0) no change, (1) mild, (2) moderated or (3) severe changes. Total damage was obtained by adding the individual scores. 2.5. Myeloperoxidase (MPO) activity assay The activity of MPO, a marker of polymorphonuclear primary granules, was determined in colon tissue according to a previously described technique [37]. Immediately after sacrifice, a distal colonic segment (1 cm long) was taken off at 5–6 cm from the anus. It was suspended in potassium phosphate buffer (KH2 PO4 44 mM, K2 HPO4 6 mM, pH 6.0), homogenised on ice with Polytron (PCU-2, Kinematica GmbH, Lucerne, Switzerland) and submitTable 1 Macroscopic score parameters. Stool consistency
Colon damage score
Tissue adhesions
0 = formed 1 = liquid
0 = none 1 = hyperemia 2 = slight erosion 3 = extensive erosion/ulceration 4 = extensive erosion/ulceration > 2 cm 5 = more sites of inflammation > 1 cm
0 = none 1 = mild 2 = strong
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ted to three cycles of freezing and thawing. Homogenates were then centrifuged at 9000 × g for 15 min at 4 ◦ C. The pellets were resuspended in hexadecyl trimethylammonium bromide buffer (0.5%, wt/vol) in potassium phosphate buffer to release MPO from polymorphonuclear neutrophil primary granules. These suspensions were sonicated (Büchi, Flawil, Switzerland) on ice and centrifuged at 9000 × g for 15 min at 4 ◦ C. Supernatant fractions were diluted in potassium phosphate buffer containing 0.167 mg Odianisidine dihydrochloride/mL and 0.00005% (vol/vol) H2 O2 . MPO from human neutrophils (Sigma, Saint Quentin Fallavier, France; 0.1 U/mL) was used as a standard. Changes in absorbance at 450 nm were recorded with a spectrophotometer (mc2UV, Safas, Monaco) every 10 s over 2 min. One unit of MPO activity was defined as the quantity of MPO degrading 1 mol H2 O2 min−1 mL−1 at 25 ◦ C. Protein concentrations (g/mL) were determined using a modified method of Lowry (Detergent Compatible Assay, BioRad, Ivry/Seine, France) and MPO activity was expressed as MPO units/g protein. 2.6. Colonic mucosa cytokine levels Tissue proteins were extracted with RIPA buffer (1% Igepal, 0.5% deoxycholic acid, and 0.1% sodium dodecyl sulphate in Trisbuffered saline 1×; pH 7.4) with protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Clear lysates were prepared by centrifugation at 10,000 × g for 10 min, and protein concentrations were assessed using the BC Assay Uptima kit (Interchim). Samples were then processed for ELISA using commercial kits to determine colonic contents of IL-1, and IL-10 (ELISA kits, Duoset R&D Systems, Lille, France). Data were expressed as concentration per mg of total protein. 2.7. N/OFQ plasmatic levels Blood samples from controls, TNBS saline-, TNBS + UFP-101 (1–3–10 nmol/kg)-treated rats were collected in tubes containing EDTA and Aprotinin (4 TIU/200 l), and immediately centrifuged at 1200 × g 4 ◦ C for 10 min. N/OFQ assay was performed as previously described [38]. The standard curve was constructed using B/B0 versus N/OFQ standard. The amount of N/OFQ in the unknown samples was extrapolated from the standard curve using GraphPad Prism version 3.0 (GraphPad Software, Inc., San Diego, CA, USA). 2.8. Statistical analysis All data are presented as means ± SEM. For statistical analysis Graph Pad Prism 4.0 (GraphPad, San Diego, CA) was used. Results were analysed by one way ANOVA followed by Dunnett’s Multiple Comparison Test. Statistical significance was set at p < 0.05. 3. Results 3.1. Histological evaluation 3.1.1. Macroscopic score The instillation of TNBS caused the appearance of extensive ulceration and necrotic zones of >1 to 5 cm, tissue adhesions and occasionally diarrhoea. The macroscopic score evaluated 4 days after TNBS instillation was not modified by the treatment with N/OFQ or its antagonist UFP-101, irrespective of the different administered doses (Table 2). 3.1.2. Microscopic Score TNBS instillation resulted in submucosal and mucosal infiltration of inflammatory cells and blood vessels dilatation, compared to control animals (Fig. 1A).
Table 2 Effect of different treatments on colonic macroscopic damage induced by TNBS colitis. Treatment
Macroscopic score (n.)
Controls TNBS+ Saline N/OFQ (0.02 nmol/kg) N/OFQ (0.2 nmol/kg) N/OFQ (2 nmol/kg) N/OFQ (20 nmol/kg) UFP-101 (1 nmol/kg) UFP-101 (3 nmol/kg) UFP-101 (10 nmol/kg)
0.0 ± 0.0
a
7.5 7.8 8.1 6.9 7.8 7.8 7.9 7.9
± ± ± ± ± ± ± ±
0.7a 1.0a 0.6a 1.1a 1.1a 0.9a 0.7a 1.1a
p < 0.01 vs controls.
In TNBS-induced colitis rats, repeated injections of N/OFQ induced a significant reduction of microscopic score when administered at doses of 0.02 and 0.2 nmol/kg compared with TNBS alone. N/OFQ, administered at 2 nmol/kg, did not affect microscopic damage induced by TNBS. Conversely, at the dose of 20 nmol/kg, N/OFQ significantly worsened microscopic scores compared with TNBS alone (Fig. 1A). Repeated UFP-101 treatment (3 and 10 nmol/kg) significantly worsened microscopic colonic damage induced by TNBS, whilst the UFP-101 dose of 1 nmol/kg did not show pharmacological activity (Fig. 1A). The pre-treatment with UFP-101 (1 nmol/kg) significantly antagonised the protective effect of N/OFQ (0.2 nmol/kg) on microscopic damage (Table 3). Representative histological samples of colonic sections from control and from TNBS-treated rats after saline, N/OFQ and UFP-101 administration are shown in Fig. 1B. 3.2. Body weight and food intake Treatment with TNBS caused a significant reduction of the animal’s weight and food intake) respect to control rats. None of these parameters underwent significant changes following treatment with either N/OFQ or UFP-101 (data not shown). 3.3. MPO activity The IC administration of TNBS significantly increased colonic MPO activity compared to controls. After TNBS-induced colitis, repeated IP injections of N/OFQ, at doses of 0.02 and 0.2 nmol/kg, significantly reduced MPO activity. Conversely, administered at the higher dose of 20 nmol/kg, N/OFQ induced a significant increase of colonic MPO activity in comparison with TNBS rats treated with saline (Fig. 2A). In addition, in TNBS rats, repeated administration of the selective NOP receptor antagonist, UFP-101, at doses of 3 and 10 nmol/kg, significantly increased colonic MPO activity compared to TNBS rats treated with saline (Fig. 2B). Table 3 Effect of pre-treatment with the NOP receptor antagonist, UFP-101, on the N/OFQinduced decrease of MPO activity and microscopic score, during TNBS colitis. Treatment
MPO activity (U/g of protein)
Microscopic score (n.)
Controls TNBS+ Saline N/OFQ (0.2 nmol/kg) UFP-101 (1 nmol/kg) UFP-101 + N/OFQ
155.1 ± 14.79
1.8 ± 0.36
589.9 ± 44.32a 261.1 ± 50.18b 588.5 ± 110.0 634.6 ± 107.6
5.0 ± 0.78a 2.0 ± 0.5b 4.75 ± 1.44 5.1 ± 1.00
a b
p < 0.01 vs controls. p < 0.01 vs TNBS saline-treated rats.
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Fig. 1. Effect of the treatment with N/OFQ and UFP-101 on histopathological alterations in TNBS-induced colitis in rats. Panel A: Histological score analysis of damage in colonic sections of rats treated with N/OFQ (0.02–0.2–2–20 nmol/kg), left panel, and with UFP-101 (1–3–10 nmol/kg), right panel. Each column is the mean ± SEM (n = 8 rats for each group). *p < 0.05 and **p < 0.01. The extent of histological damage was expressed according to the criteria described by Fabia et al. [36]. Panel B: Representative eosin and haematoxylin-stained colon sections from rats treated as above indicated. Scale bars = 50 m upper figures, 100 m lower figures.
When administered at the lower dose of 1 nmol/kg, UFP-101 did not show any significant effect per se (Fig. 2B), but abolished the inhibitory effect on MPO activity induced by N/OFQ (Table 3). 3.4. Colonic IL-1ˇ and IL-10 levels TNBS-treated rats showed an expected increase of proinflammatory IL-1 cytokine levels measured in colonic mucosa, compared to control animals. In rats with colitis, repeated treatment with N/OFQ (0.02 and 0.2 nmol/kg) significantly decreased IL-1 levels compared to TNBS animals injected with saline. Neither N/OFQ (20 nmol/kg;) nor UFP-101 (10 nmol/kg;) modified the pro-inflammatory cytokine levels after colitis (Fig. 3A). TNBS-treated rats showed an expected significant decrease in anti-inflammatory IL-10 cytokine levels measured in colonic mucosa in comparison with control animals. No administered doses of N/OFQ or UFP-101 affected IL-10 content after colitis induction (Fig. 3B). 3.5. N/OFQ plasmatic levels TNBS rats showed no difference in N/OFQ plasmatic levels in comparison with control animals. Three days of treatment with
UFP-101 (1–3–10 nmol/kg) after TNBS-induced colitis, significantly decreased N/OFQ plasmatic levels (Fig. 4). 4. Discussion This study shows that repeated peripheral administrations of low – but not high – doses of N/OFQ significantly decrease the colonic inflammatory profile in rat TNBS-induced colitis. Pretreatment with the selective NOP receptor antagonist UFP-101, at the dose devoid of modulatory effects on intestinal inflammation by itself, completely reversed the observed protective effect, indicating that the activation of peripheral NOP receptors mediates an anti-inflammatory action in this experimental model of colitis. Moreover, since UFP-101, administered at high doses, triggers pro-inflammatory effects and reduces N/OFQ plasmatic levels, we speculate that the endogenous peripheral N/OFQergic system has a protective role in the modulation of inflammation during TNBSinduced colitis. The beneficial effect of repeated injections of low doses of N/OFQ is shown by the reduction of colonic MPO activity and the improvement of colonic microscopic inflammatory damage evaluated by light microscopy. The reduction of TNBS-induced damage is not observed by evaluating the macroscopic scores (healing of ulcers, reduced hyperaemia) or the recovery of body weight and food intake. It is not easy to explain the lack of correspondence between
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Fig. 2. Colonic MPO activity measured in rats treated twice a day for 3 consecutive days with N/OFQ (0.02–0.2–2–20 nmol/kg; panel A) and UFP-101 (1–3–10 nmol/kg; panel B), 24 h after TNBS initiation. Each column is the mean ± SEM (n = 8 rats for each group). *p < 0.05 and **p < 0.01.
micro and macroscopic score. However, most of the studies showing that TNBS macroscopic score, MPO activity and microscopic scores are well correlated, use lower doses of TNBS [39] causing less severe damages, or evaluate the inflammatory indexes after a time period from the TNBS administration longer than that we have used [39,40]. Therefore, we cannot exclude that a less severe colitis or an extension of N/OFQ treatment may also induce an improvement of macroscopic scores. The protective effect of low doses of N/OFQ during TNBS-colitis is also associated with the reduction of one of the most significant signals of the colonic inflammation, the cytokine IL-1, while the anti-inflammatory IL-10 content does not change. The N/OFQ modulatory activity on IL-1 observed here is in line with that reported by other investigators, even if we cannot establish if N/OFQ affects the level of this cytokine through direct or indirect action. The hypothesis of a direct effect is supported by the evidence that NOP receptors are expressed on the surface of immune cells and mediate an anti-inflammatory action [21]. Both in vivo and in vitro studies have shown that N/OFQ suppresses the production of pro-inflammatory cytokines from rat splenocytes and human T cells [19,41], and decreases the expression of mRNA for IL-1 and other cytokines in astrocytes activated
Fig. 3. Effect of repeated injections of N/OFQ (0.02, 0.2 and 20 nmol/kg) and UFP101 (10 nmol/kg) on pro-inflammatory IL-1 (panel A) and anti-inflammatory IL-10 (panel B) mucosa colonic content. Levels of IL-1 and IL-10 were measured by ELISA kits 4 days after TNBS instillation. Each column is the mean ± SEM (n = 8 rats for each group). **p < 0.01.
in vivo by peripheral administration of inflammatory stimuli [42]. Furthermore, binding studies on immune system cells report a high affinity of N/OFQ binding sites in line with a direct action of a low concentration of the peptide [43]. As for an indirect modulatory role of N/OFQ on the immune system, it is well known that in the gut, signals associated with immune responses contribute to the pathophysiology of IBD by activating CRF1 (corticotrophin releasing factor) receptor pathways (i.e. mast cell activation), which are closely related to stress and inflammation [44,45]. Since N/OFQ has been shown to be a functional antagonist of CRF1 receptors [12,46], we cannot exclude that the peptide reduces the inflammatory process by the inhibition of peripheral CRF1 receptor pathways in colitic animals. We also report that peripheral repeated administration of a high dose of N/OFQ after colitis induction worsens some of the inflammatory parameters studied (MPO, histological scores), but do not modify the cytokine levels. The explanation for the opposite anti-inflammatory and pro-inflammatory effects induced by different dosages of N/OFQ remains to be determined. Certainly, the N/OFQ aggravating effect cannot be linked to the well-known antipropulsive colonic activity of the peptide, because both low and high doses of N/OFQ [12] evoke a reduction of colonic propulsion. On the other hand, there is a recent paper [47] showing that, in
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Fig. 4. N/OFQ plasmatic levels in control animals and in TNBS-induced colitis rats after saline or UFP-101 treatment. Plasmatic levels were measured with a radioimmunoassay kit for N/OFQ. Data are expressed as mean ± SEM (n = 8 rats for each group). *p < 0.05 and **p < 0.01.
healthy animals, N/OFQ at a high dose comparable to that used in our study, induces a pro-inflammatory effect due to the vasodilatation of mesenteric microcirculation, leading to a decrease in blood flow velocity, an increase in leucocyte rolling and adhesion, and to an increase in 2-integrin (CD18/CD11) expression on circulating rat neutrophils. Therefore, it is possible that the aggravating effect of the high-dose N/OFQ in TNBS-induced colitis is due to pathways controlling vascular permeability and chemotaxis. Although further studies will be necessary, we postulate that different mechanisms are involved in the protective and aggravating action of N/OFQ. In our study, repeated treatment with high doses of the NOP receptor antagonist, UFP-101, worsens colonic inflammation. This result together with the increase of visceral sensitivity to colorectal distension in rats treated with UFP-101, under similar experimental conditions [32], shows the pharmacological activity of the antagonist and reveals the possible protective role of the endogenous peripheral N/OFQergic system in colitis. The involvement of endogenous N/OFQergic system in experimental colitis has also been reported in mice [23,31]. However, the peripheral N/OFQ protective effect during TNBS-induced colitis in rats we have shown, is in contrast with the worsening role played by the NOP system in knockout mice and in mice treated with a NOP antagonist in a DSS model of colitis [23,31]. The NOP receptor system is known to mediate opposing effects (anxiogenic/anxiolytic; increase/decrease of locomotion; inhibition/stimulation of HPA) in mice and rats [48–53]. The reason why N/OFQ plays opposing roles in these different animal species is not yet understood. Regarding our results, the NOP wild-type rats we used represent an experimental model absolutely different from the NOP-deficient mice model [23], and thus is not easily comparable. Furthermore, the aetiopathogenetic mechanisms of the experimental models of colitis used in our and in Kato’s [23] studies are certainly different. In the TNBS model, which resembles Crohn’s disease in humans, TNBS – a hapten that results in change of the autologous molecules in the mucosa – when locally injected through intrarectal administration, leads to priming of antigen-specific T cells [54]. In the DSS model, which resembles human ulcerative colitis, DSS added to drinking water results in toxicity towards colonic mucosa causing
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defects in the epithelial barrier integrity and colonic permeability, increased dysplastic lesions and, at least in some cases, trough Th1-dependent pathway [55]. Finally, it is known that UFP-101, the NOP receptor antagonist used in our study, does not cross the blood-brain-barrier [56] and it is able to block only NOP peripheral sites, whilst SB612111, the small non-peptidic NOP antagonist used by Alt [31], can pass the barrier and block NOP central sites. Therefore, the opposite (protective and aggravating) roles shown in our and in Alt’s study [31] could be due to the involvement of different, peripheral and central, N/OFQ sites of action. In this regard, it’s largely described that N/OFQ mediates opposite central and peripheral effects [57]. To date, no studies correlate N/OFQ plasmatic levels and IBD in humans or in experimental models of colitis. Conversely, several studies correlate changes of N/OFQ plasmatic levels in other human diseases. Indeed, high levels of plasmatic N/OFQ have been associated with muscle-skeletal diseases, urinary stones and heart pain of unclear origin, as well as in patients affected by unstable angina [58]. On the other hand, low levels of N/OFQ have been linked to migraine [59], fibromyalgia [60], and chronic ischaemic cardiovascular diseases [61], while normal levels have been reported in women during labour [62]. To give further information on the N/OFQergic system role during experimental colitis, the N/OFQ plasmatic levels in TNBS-treated rats have been determined. The data obtained here confirm the presence of N/OFQ in rat plasma under physiological conditions, as previously observed [63], and show that N/OFQ plasmatic levels do not change in TNBS-treated rats. By blocking NOP receptors in TNBS-treated rats with UFP101, at doses that worsen colonic inflammatory damage, a decrease of N/OFQ plasmatic levels is induced in comparison with controls and with rats treated with TNBS alone. Such a prolonged block of the NOP receptors together with the decrease in N/OFQ plasmatic levels, probably due to an increased degradation and/or to a reduced synthesis of the peptide, put in evidence that a lower functionality of the endogenous peripheral N/OFQergic system during colitis results in a worsening of the inflammatory parameter studied. 5. Conclusions This paper shows that the peripheral NOP receptors mediate a dose-related protective and worsening effect on colitis induced by TNBS in the rat. These findings, together with the ability of N/OFQ to decrease colonic motility in a condition of altered functionality [12] and to reduce visceral hypersensitivity in an animal model well reproducing Crohn’s disease [32], offer an interesting insight on the modulatory role of the N/OFQergic system in some inflammatory conditions of the intestinal tract characterised by altered motility, visceral pain and mucosal lesions. Conflict of interest The authors have no competing interests to declare. Acknowledgments This work was supported by the Nexus Grant in memory of Prof. Marcello Tonini for a Research project in Gastroenterology [AstraZeneca Industries, Italy, Grant 2011]. The authors wish to thank Prof. Francesco Botrè, Dr. Monica Mazzarino and Francesca de Angelis (Federazione Medico Sportiva Italiana-Laboratorio Antidoping) for their help in performing the extraction of the peptide from rat plasma.
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