Received 6 August 2008; Accepted 15 January 2009

Central European Journal of Biology

Institute of Neurobiology, Bulgarian Academy of Sciences,1113 Sofia, Bulgaria

In vivo effects of N/OFQ(1-13)NH2 and its structural analogue [ORN9]N/OFQ(1-13)NH2

on carrageenan-induced inflammation: rat-paw oedema and antioxidant status

Rositsa Zamfirova*, Elina Tzvetanova, Albena Alexandrova, Lubomir Petrov, Polina Mateeva, Almira Pavlova, Margarita Kirkova, Simeon Todorov

Abstract: The effects of nociceptin(1-13)NH2 (N/OFQ(1-13)NH2) and its structural analogue [Orn9]N/OFQ(1-13)NH2 on acute carrageenan (CG)-induced peripheral inflammation and paw antioxidant status were studied. CG was injected intraplantarly in the right hind paw of rats and the volume of the inflamed paw was measured each 30 min for a period of 4h. When administered simultaneously with CG, N/OFQ(1-13)NH2 decreased the paw volume, whereas if injected 15 min before CG it had no effect. [Orn9]N/OFQ(1-13)NH2 produced the opposite effects at the same time-intervals of its administration. We also investigated whether these neuropeptides influence CG-induced changes in cell antioxidant system, especially at the 4th hour of CG administration. CG alone decreased the glutathione level and superoxide dismutase activity, as measured in post-nuclear homogenate of the inflamed paw. However, CG injection increased glutathione peroxidase and glucose-6-phospate dehydrogenase activities, while the activity of glutathione reductase was unchanged. The peptides themselves did not change all measured parameters. Moreover, neither N/OFQ(1-13)NH2 nor [Orn9]N/OFQ(1-13)NH2 modified CG-induced changes in the antioxidant status, regardless of the time of their injection (simultaneously or 15 min before CG). The present results suggest that N/OFQ(1-13)NH2 and [Orn9]N/OFQ(1-13)NH2 most likely affect the neuronal inflammation, rather than act as pro- or antioxidants.

© Versita Warsaw and Springer-Verlag Berlin Heidelberg.

Keywords: N/OFQ(1-13)NH2 • [Orn9]N/OFQ(1-13)NH2 • CG-induced inflammation • Paw-oedema • Glutathione level • Lipid peroxidation • Antioxidant enzymes

* E-mail: nikolova@bio.bas.bg

Research Article

1. IntroductionLocal inflammation is a patho-physiological process, involving increased arachidonic-acid metabolism and blood vessel permeability, neutrophil extravasation, oedema and release of pro-inflammatory mediators. Opioid receptors, including those for N/OFQ, are localised on primary sensory neurons projecting to most peripheral organs and tissues, and act as regulators of neurogenic inflammation. It is known that opioid peptides suppress neurogenic inflammation [1-3] based on their ability to inhibit transmitter release from the capsaicin-sensitive primary afferent nerve terminals. Like other

opioids, N/OFQ suppresses the release of substance P (SP) and calcitonine gene-related peptide (CGRP), whereas the non-neurogenic inflammation is unaffected [4]. As a lot of diseases are accompanied by inflammation (asthma, arthritis, gastritis, myocarditis, etc.), the inhibitory effects of N/OFQ are intensively investigated on virtually all systems of the organism: pulmonary [5-7], gastrointestinal [8,9], cardiovascular [10,11], immune system [12,13], etc. To the contrary, this peptide promotes processes that could increase the inflammatory response. Experiments on peritoneal [14] and brain [15] mast cells and on brain neurons [16] have shown that

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N/OFQ dose-dependently liberates histamine (HA), a well-known mediator of inflammation. Based on the same mechanism, the peptide evokes vasodilation and macromolecular leak from mesenteric blood vessels [11]. In acute inflamed knee joints, a topic application of N/OFQ leads to a secondary release of pro-inflammatory mediators (NK-1, SP, CGRP, VIP) after activation of N/OFQ/orphanin peptide (NOP)-receptors on synovial mast cells and leukocytes [17]. Based on data obtained in their experiments on rat paw oedema, Nemeth et al. concluded that chemically induced discharge of mediators from mast cells and from capsaicin-sensitive afferent nerve terminals were inhibited by nociceptin. This mechanism of action underlies the anti-inflammatory effect of this neuropeptide [18]. In general, present knowledge suggests that it would be hard to predict the effect of N/OFQ on inflammation in vivo because of its controversial effects on different systems involved in inflammation. The production of reactive oxygen species (ROS), and the defence systems against them, are well balanced in the body. Hence, the balance collapse (oxidative stress) evokes ROS toxicity, inducing oxidative injury in various tissues, and oxidative stress is assumed as a potential mechanism in the pathogenesis of different diseases, including inflammation. In primary astrocytes, Rosenberger et al. [19] have demonstrated that ROS dramatically induce expression of two neuropeptide genes, the opioid proenkephalin and the opioid-related proorphanin (also known as pro N/OFQ). The profound induction of these genes by oxidative stress may suggest a role for N/OFQ and enkephalin in injury and stress responses of the CNS and neuropathophysiological conditions, involving reactive oxygen species. N/OFQ has been found to reduce ethanol-induced gastric lesions [20]. The peptide shows protective activity and increases gastric mucosal resistance to ethanol by operating both in the central nervous system and in the periphery [8]. It has been found that N/OFQ, in concentrations present in cerebrospinal fluid after fluid percussion brain injury (FPI), increases O2

- production in a cyclooxygenase-dependent manner after FPI [21,22]. This peptide also increases PKC-dependent O2

- production after insult [23]. Recently, as a result of different modifications of N/OFQ sequence, many novel ligands of the NOP receptor have been synthesised [23-29]. N/OFQ and some of its structural analogues (highly potent NOP receptor agonists), might be favourable in treatment of pain, cough, oedema, drug dependence, stress induced anorexia, whereas the antagonists might be useful in management of depression, dementia, or parkinsonism. The high-potency, high-affinity, selectivity of action,

and in vivo effectiveness also make them useful pharmacological tools for future studies in the field of N/OFQ and its NOP receptor. In our study, we investigate and compare the in vivo effects of N/OFQ(1-13)NH2 and its structural analogue [Orn9]N/OFQ(1-13)NH2, a highly potent NOP-receptors agonist [30], on acute CG-induced peripheral inflammation. Because CG, when injected in rat paw, produces acute inflammation and pain via enhancement of ROS formation [31-33], we also studied the effects of both peptides on CG-induced changes in antioxidant status of tissues from inflamed paws as a possible mechanism of the peptide action.

2. Experimental Procedures

2.1.Materials2-thiobarbituric acid, riboflavine, NADP+, NADPH, reduced and oxidized glutathione, as well as γ-carrageenan were obtained from Sigma-Aldrich (Germany). All other reagents used were analytical grade. N/OFQ(1-13)NH2

and [ORN9]N/OFQ(1-13)NH2 were synthesised in the Department of Organic Chemistry, University of Chemical Technology and Metallurgy (Sofia, Bulgaria). Carrageenan (CG), as an 1%-solution, was prepared in 1% dimethylcellulose. The solutions of N/OFQ(1-13)NH2

and [Orn9]N/OFQ(1-13)NH2 were freshly prepared in saline before each experiment. All other solutions used in the study were prepared with over-glass re-distilled water.

2.2. AnimalsThe experiments were performed on male Wistar rats weighing 180-200 g and housed at 22-25oC. The animals were allowed an acclimation period, with free access to food and water, and a natural day/night light cycle. Prior to the experiments, the animals were fasted for 24 h with ad-libitum water-access. All animals were sacrificed under light ether anaesthesia. The experiments were performed according to the “Principles of laboratory animal care” (NIH publication No. 85-23, revised 1985), and the rules of the Ethics Committee of the Institute of Physiology, Bulgarian Academy of Sciences (registration FWA 00003059 by the US Department of Health and Human Services).

2.3.Treatment and tissue preparationsRats were divided into experimental groups as follows: (1) Controls (untreated animals); (2) CG-treated; (3) treated with N/OFQ(1-13)NH2 or [Orn9]N/OFQ(1-13)NH2, jointly with CG; (4) treated with

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N/OFQ(1-13)NH2 or [Orn9]N/OFQ(1-13)NH2, 15 min before CG; (5) treated with only N/OFQ (1-13)NH2 or [Orn9] N/OFQ(1-13)NH2. CG was intraplantarly (i.pl.) applied in a volume of 0.1 ml into the right hind paw; N/OFQ(1-13)NH2 and [Orn9]N/OFQ(1-13)NH2 (20 μg/ml) were intraperitoneally (i.p.) injected in a volume of 0.1 ml/100 g body weight. After CG injection, the volume of paw-oedema was measured with a plethysmomether (Ugo Basile, Varese, Italy) every 30 min for a period of 4 hours. The animals were sacrificed 4 h after CG treatment. Tissues from left and right paw were separated, homogenized and centrifuged (for 10 min at 600 g) to obtain a post-nuclear homogenates. An additional sonication (4x30”) of these homogenates was also applied.

2.4 Measuring of paw-oedema volumePaw volume was measured every 30 min for a period of 4 hours after CG injection. The moment of CG injection serves as time zero. To obtain a control (pre-CG value) the right hind-paw was also measured before the CG injection. Data are expressed as changes in the oedema (difference from the pre-CG volume).

2.5 The biochemical indices The biochemical indices were measured in tissue homogenates, obtained from left and right paw of the rats.

2.6 Analytical methodsProtein content was measured by the method of Lowry et al. [34]. Total glutathione (GSH) level (with DTNB, 5,5’-dithiobis(2-nitro-benzoic acid) (3,3’-6)) was measured at 412 nm according to Tietze [35] and was expressed in ng/mg protein, using GSSG as a reference standard. Glutathione peroxidase (GSH-Per) activity (with t-butylhydroperoxide as substrate) was measured by the method of Gunzler et al. [36]. The values, measured at 340 nm were expressed in nmoles NADPH oxidized per min per mg protein, using a molar extinction coefficient of 6.22 x 106 M-1cm-1. Glutathione reductase (GSSG-Red) activity was measured by the method of Pinto and Bartley [37]. The values, measured at 340 nm were expressed in nmoles NADPH oxidized per min per mg protein, using a molar extinction coefficient of 6.22 x 106 M-1cm-1. Glucose-6-phosphate dehydrogenase (Glu-6-P-DH) activity was determined after Cartier et al. [38]. The values, measured at 340 nm were expressed in nmoles NADP+ reduced per min per mg protein, using a molar extinction coefficient of 6.22 x 106 M-1cm-1.

Superoxide dismutase (Cu,Zn-SOD) activity was determined according to Beauchamp and Fridovich [39] in a riboflavin/methionin/ν medium. The values were expressed in U/mg protein (one unit of SOD activity is the amount of the enzyme, producing 50% inhibition of nitro-blue tetrazolium - reduction).

2.7 Statistical analysisThe data were statistically analysed by one-way ANOVA (Dunnett post hoc test), P<0.05 being accepted as the minimum level of statistical significance of the established differences.

3. ResultsIn CG-treated animals, the volume of the right rat paws increased with the time, the maximum being at 180-210 min, and remained stable to the end of the experiment (Figure 1 and Figure 2). In the group injected simultaneously with CG and N/OFQ(1-13)NH2, at the end of observation period (4 h), the maximal increase in the inflamed-paw volume was markedly lower compared to that in CG-treated animals (Figure 1). The first significant difference between the both experimental groups was found at the 90th min. In general, the dynamics of paw oedema increase in N/OFQ(1-13)NH2/CG-treated group was the same as in CG-treated. When applied 15 min before CG, this neuropeptide exerted no inhibitory effect on paw-oedema. On the contrary, a tendency toward an increase in inflamed paw volume was observed after the 150th min., compared to the CG-treated animals.

Figure 1. Effects of N/OFQ(1-13)NH2 on CG-induced acute inflammation. Values represent the mean ± SEM of 7-9 animals. The volume was measured each 30 min (for a period of 4 hours) after CG injection, considered as a zero time. Statistically significant differences versus controls at: *P<0.05; **P<0.01

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The structural N/OFQ(1-13)NH2 analogue tested, [Orn9]N/OFQ(1-13)NH2, alleviated the inflammatory process in the first 90 min regardless of when it was injected: simultaneously with CG or 15 min before it (Figure 2). As time proceeded (120-240 min), the paw oedema volume of the rats, injected jointly with CG and [Orn9]N/OFQ(1-13)NH2, did not differ from that of the CG-group. In the animals treated with [Orn9]N/OFQ (1-13)NH2 15 min before CG, the paw volume remained significantly smaller when compared to the group treated with CG only.

Based on the differences in the effects of N/OFQ(1-13)NH2 and [Orn9]N/OFQ(1-13)NH2, we also tested whether these neuropeptides are able to affect the changes in cell antioxidant system at 4th hour of CG administration. The effects of both peptides on the levels of cell non-enzyme (GSH) and enzyme (SOD, GSH-Px, GSSG-Red) antioxidants were estimated. The activity of Glu-6-P-DH, which is of importance for the antioxidant GSH-conjugated enzyme activities, was also measured under these experimental conditions. All parameters were defined in tissue homogenates from right (CG-treated) and left (untreated with CG) paws. The effects of both peptides in paw tissues from control (untreated with CG) animals were also studied. Four hours after CG-injection, GSH total level in right paw tissue decreased, while that in the left paw tissue remained unchanged (Figure 3). A decrease in SOD activity in right, but not in left paw tissue was also observed (Figure 4). The CG-treatment did not change the activity of GSSG-Red, but increased GSH-Px activity in the inflamed right

paw tissue. A very strong increase in Glu-6-P-DH activity was also observed (Table 1). The peptides alone did not significantly change all measured parameters. Moreover, neither N/OFQ(1-13)NH2 nor [Orn9]N/OFQ(1-13)NH2 modified CG-induced changes in the antioxidant status, regardless of the time of their injection (simultaneously or 15 min before CG).

In addition, the values of all parameters measured in left paw homogenates of CG-treated animals were similar to those in paw tissue homogenates of CG-untreated animals. Hence, the values measured in left paw tissue might be used as controls for those obtained in right paw tissue of CG-treated animals.

Figure 2. Effects of [ORN9]N/OFQ(1-13)NH2 on CG-induced acute inflammation. Values represent the mean ± SEM of 7-9 animals. The volume was measured each 30 min (for a period of 4 hours) after CG injection, considered as a zero time. Statistically significant differences versus controls at: *P<0.05; **P<0.01

Figure 3. Effects of N/OFQ(1-13)NH2 and [ORN9]N/OFQ(1-13)NH2 on GSH level in rat paw tissues after CG-induced acute inflammation. Values represent the mean ± SEM of 7-9 animals. The total glutathione (GSH) level was expressed in ng/mg protein. Statistically significant differences versus controls at: **P<0.01

Figure 4. Effects of N/OFQ(1-13)NH2 and [ORN9]N/OFQ(1-13)NH2 on SOD activity in rat paw tissues after CG-induced acute inflammation. Values represent the mean ± SEM of 7-9 animals. The enzyme activity was expressed in U/mg protein. Statistically significant differences versus controls at *P<0.05

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4. DiscussionAdministration of the mucopolysaccaride CG into the paw of the rat or mouse induces a local inflammation, characterized by enhanced arachidonic acid metabolism, increased levels of malondialdehyde, nitric oxide (NO) and prostaglandin E2, increased production of proinflammatory cytokines, as well as decreased glutathione levels in the swollen rat paws [40-43]. Measuring the concentration of endogenous antioxidants in plasma and in the inflammation exudate of rats with CG-induced granuloma, Muntane et al. [44] establish iron overload and increased malondialdehyde, urate and caeruloplasmin levels, as well as decreased levels of ascorbic acid and SH groups. According to Oyanagui [31], superoxide (O2

-) participates in the prostaglandin-phase swelling (2-4 hour) of CG-paw oedema. The attenuation of some of the inflammation parameters by M40403, a synthetic SOD, clearly indicates that O2

- plays a crucial role in the development of the inflammatory response by altering key components of the inflammatory cascade [32]. The importance of O2

-, and of peroxynitrite (NOOO-) and hydroxyl radical (.OH) formed with O2

- participation, for acute inflammation and inflammatory pain is discussed also by other authors [33,45-47]. In general, CG is accepted to produce both inflammation and pain via increased formation of reactive

oxygen species. Moreover, CG-induced inflammation is found to be useful model for testing the antioxidant efficacy of different substances, as celecoxib, local anesthetics, arrhythmic agents, flavonoids and many other including anti-inflammatory drugs [40,41,43,45-54]. For example, the anti-inflammatory effects of n-acetylcysteine may include: an inhibition of neutrophil infiltration and prevention of the activation of NADPH oxidase, responsible for O2

- production; inhibition of the activation of NF-kB and prevention of the expression of iNOS, responsible for NO.-production; inhibition of ONOO- formation, a result of NO./O2

- combination [47]. Taken all together, the CG-model appears most appropriate for our investigation. After intra-plantar injection of CG we observed a time-dependent increase in the volume of right rat paw, with the CG-induced oedema peaking in 3-4 hours. Similar results were reported by others authors [40,55]. Four hours after CG application we also found changes in endogenous cell antioxidants: GSH level and SOD activity decreased, and GSH-Px activity increased in tissues from right, but not from left paws in accordance with published data [40,51,53]. Additionally, we found a strong increase of Glu-6-P-DH activity. The higher activity of this enzyme, producing NADPH (a key electron-donor in the defence against oxidising agents and in reductive biosynthetic reactions), might be ascribed to an increase of its

Neuropeptides GSH-Px activity GSSG-Red activity Glu-6-P-DH activity

I. CONTROL ANIMALS

Controls 14,7 ± 1,36 8,29 ± 0,723 1,33 ± 0,252

N/OFQ(1-13)NH2 16,2 ± 2,22 8,43 ± 0,999 1,46 ± 0,375

[ORN9]N/OFQ(1-13)NH2 14,5 ± 0,78 7,78 ± 0,846 1,39 ± 0,355

II. CARRAGEENAN-TREATED ANIMALS

left paw right paw left paw right paw left paw right paw

14,5 ± 1,04 21,9 ± 1,83** 8,64 ± 0,759 8,43 ± 0,628 1,39 ± 0,224 6,50 ± 0,615***

III. CARRAGEENAN-TREATED ANIMALS (peptides are administered jointly with CG)

left paw right paw left paw right paw left paw right paw

N/OFQ(1-13)NH2 16,4 ± 2,69 19,1 ± 1,25* 7,74 ± 0,598 7,78 ± 0,355 1,43 ± 0,354 6,33 ± 0,812***

[ORN9]N/OFQ(1-13)NH2 15,1 ± 2,23 20,1 ± 1,42* 7,34 ± 0,713 8,10 ± 1,000 1,29 ± 0,299 6,01 ± 0,559***

IV. CARRAGEENAN-TREATED ANIMALS (peptides are administered 15 min before CG)

left paw right paw left paw right paw left paw right paw

N/OFQ(1-13)NH2 12,0 ± 1,63 23,1 ± 1,63** 7,80 ± 0,555 7,69 ± 0,334 1,19 ± 0,278 6,87 ± 0,472***

[ORN9]N/OFQ(1-13)NH2 12,2 ± 2,51 22,4 ± 1,62** 8,99 ± 0,415 8,82 ± 0,249 1,19 ± 0,181 6,23 ± 0,542***

Table 1. Effects of N/OFQ(1-13)NH2 and [ORN9]N/OFQ(1-13)NH2 on enzyme activities in rat paw tissues after CG-induced acute inflammation. Values represent the mean ± SEM of 7-9 animals. The enzyme activities were expressed in nmoles NADP(H)/min/mg protein, using molar

extinction coefficient of 6.22x106 M-1cm-1. Significant differences versus controls at: *P<0.05; **P<0.001; ***P<0.001.

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substrate (NADP+), as a result of CG-induction of NAD(P)H oxidases (NADPH + e- + O2 → NADP+ + O2

- + H+). In the present experiments we observed that in vivo N/OFQ(1-13)NH2 injected simultaneously with CG reduced the paw-oedema, most likely depressing the neurogenic component of CG-induced acute inflammation. Our suggestion is based on literature data [4] revealing that N/OFQ does not affect the no-neurogenic inflammation. Additionally, the weak effect of N/OFQ(1-13)NH2 injected before CG, suggests that the peptide in vivo does not exert protective or substantial pro-inflammatory action. Tested under our experimental conditions, N/OFQ(1-13)NH2 had no effect (0-120 min), or tended to increase the oedema volume after 120 min (the effect was not statistically significant). In this case, a probable explanation might be a secondary release of HA and other pro-inflammatory mediators by N/OFQ [14,15,17]. The lack of effect of pre-treatment with N/OFQ has also been reported by Sun et al. [56] on bee venom-induced increase in paw volume. A surprising finding was that [Orn9]N/OFQ(1-13)NH2

had a different time-course inhibitory effect on CG-induced inflammation than that expected on the basis of its structure. When the basic amino acid Lys9 in N/OFQ(1-13)NH2 was replaced by its structural analogue Orn9, the newly synthesised peptide showed an increased agonist activity compared to the native product [30]. We expected that N/OFQ(1-13)NH2 and [Orn9]N/OFQ(1-13)NH2 would produce similar effects in vivo. However, under identical experimental conditions, [Orn9]N/OFQ(1-13)NH2 had a suppressing effect on paw oedema volume when applied before CG, and had no effect on the inflammatory processes when injected jointly with CG. It is known that N/OFQ metabolism is time-dependent [57], and fragments with different length and activity progressively appear [58-60]. We presume that the same enzymes metabolise [Orn9]N/OFQ(1-13)NH2

and N/OFQ(1-13)NH2, since the only difference in the structure of both peptides is that the side-chain of

[Orn9 N/OFQ(1-13)NH2 is shortened with one CH2-group. Nevertheless, the peptides and the resulting fragments could differ in ionic interactions of the basic amino acids and/or to a conformation-related access of the enzyme-sensitive bounds to the active site. Accordingly, it could be speculated that [Orn9]N/OFQ (1-13)NH2 (or its active fragments) interact more slowly with NOP-receptors, as compared to N/OFQ (1-13)NH2. This could explain the more rapid N/OFQ (1-13)NH2 effects (applied simultaneously with CG) and the delay of [Orn9]N/OFQ (1-13)NH2

action (applied 15 min before CG). In general, the possible discrepancy in the kinetic association of the ligands can only be known by binding experiments. Thus, the experiments with N/OFQ(1-13)NH2 and [Orn9]N/OFQ (1-13)NH2, injected simultaneously or 15 min before CG, revealed differences in the effects of N/OFQ(1-13)NH2 and [ORN9]N/OFQ(1-13)NH2 on acute CG-induced peripheral inflammation. However, independent of the time of their administration, neither N/OFQ(1-13)NH2 nor [Orn9]N/OFQ(1-13)NH2 modified CG-induced changes in the antioxidant status. Moreover, both peptides alone did not change the tested antioxidant markers in paw of CG-untreated animals. It might be suggested that neither N/OFQ(1-13)NH2 nor its structural analogue [Orn9]N/OFQ(1-13)NH2 act by free-radicals mechanism. In conclusion, it might be assumed that N/OFQ(1-13)NH2 and [Orn9]N/OFQ(1-13)NH2 more likely affect only neuronal inflammation.

AcknowledgmentsThis study was supported by Grant L-1510 from the National Research Fund, Sofia, Bulgaria.

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