PROTECTIVE EFFECT OF MELATONIN IN CARRAGEENAN-INDUCED ACUTE LOCAL INFLAMMATION

Pharmacological Research, Vol. 46, No. 2, 2002 doi:10.1016/S1043-6618(02)00089-0, available online at http://www.idealibrary.com on

PROTECTIVE EFFECT OF MELATONIN IN CARRAGEENAN-INDUCED ACUTE LOCAL INFLAMMATION ˙ IC ˙ I˙ a,∗ , E. AKPINAR a and A. KIZILTUNÇ b D. BIL a Department of Pharmacology, Medical School, Atatürk University, 25240 Erzurum, Turkey, b Department of Biochemistry, Medical School, Atatürk University, 25240 Erzurum, Turkey

Accepted 10 May 2002

The aim of the present study was to investigate the protective effect of the pineal hormone melatonin in a model of acute local inflammation (carrageenan-induced paw oedema). Inflammation was assessed by measurement of nitric oxide (NO), Malondialdehyde (MDA) and glutathione levels in the paw tissue in rats. The intraplantar injection of carrageenan elicited an inflammatory response that was characterised by a time-dependent increase in paw oedema, increased level of nitrite/nitrate and MDA, a lipid peroxidation product and decreased glutathione levels in the paw tissue. The maximal increase in paw volume was observed at 4 h after administration (maximal in paw volume 160 ± 3.34 ml). In addition, NO level and MDA were markedly increased in the carrageenan-treated paw (59.96 ± 6.58 and 19.33 ± 3.35 µmol g−1 , respectively), versus in the control paw glutathione level decreased in paw tissue (3.24 ± 0.24 ␮mol g−1 ). However, carrageenan-induced paw oedema was significantly reduced in a dose-dependent manner by treatment with melatonin (given at 5 and 10 mg kg−1 ) at 1, 2, 3, 4, 5 and 6 h after injection of carrageenan. Melatonin treatment also caused a significant reduction of the NO and MDA levels, while increasing glutathione level in the paw tissue. Our findings support the view that melatonin exerts anti-inflammatory effects. Part of these anti-inflammatory effect may be related to an inhibition of the NO and MDA production, while another part may be related to increase of the glutathione level in the paw tissue. © 2002 Elsevier Science Ltd. All rights reserved. Key wo rds: melatonin, nitric oxide, paw inflammation, glutathione, lipid peroxidation, rat.

INTRODUCTION Melatonin is an indole that is synthesised in and secreted from the pineal gland during the night [1]. Its lipophilicity ensures that melatonin rapidly enters cells, where it may accumulate in the nucleus [2]. Melatonin plays a modulatory role in physiological functions such as circadian rhythms, reproduction, sleep, ageing [1], mood, body temperature and behavioural performance [3]. On the other hand, melatonin is a potent scavenger of free radicals and stimulates other antioxidant activities by preventing hydroxyl (OH• ) and peroxyl (ROO• ) radical formation [2, 4]. Oxygen-derived free radicals and oxidants have been shown to play an important role in various forms of inflammation and reperfusion injury [5]. The role of melatonin as an immunomodulator is well established [6, 7]. Nitric oxide (NO) generated by the inducible form of nitric oxide synthase plays important roles in tissue ∗ Corresponding

author. Department of Pharmacology, Faculty of Medicine, Atatürk University, 25240 Erzurum, Turkey. E-mail: [email protected]

1043-6618/02/$ – see front matter

injury in a number of inflammatory diseases. One possible mechanism by which NO produces tissue injury is by interacting with superoxide anion (O2 − ) to form peroxynitrite (ONOO− ) [8]. Peroxynitrite is a potent oxidising molecule capable of eliciting lipid peroxidation and cellular damage [9]. The purpose of the present study was to investigate the anti-inflammatory effect of melatonin against carrageenan-induced paw oedema in rats. MATERIALS AND METHODS

Carrageenan-induced paw oedema Male Sprague–Dawley rat (175–200), which were obtained from the Atatürk University Pharmacology Laboratory, were used. Twelve hours prior to the experiments, the rats were deprived of food. All groups were homogeneous. The experiments are approved by the Institutional Animal Ethics Committee and experiments were performed with strict adherence to the ethical guidelines. Paw oedema was induced in rats by injecting 0.1 ml of carrageenan (1%, w/v) solution in distilled water into the © 2002 Elsevier Science Ltd. All rights reserved.

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subplantar region of the right hind paw [10]. The inflammatory agent was administered alone or in combination with either melatonin or indomethacin. Melatonin was dissolved in ethanol and further dissolved in saline solution (the final concentration of ethanol was 1%) and injected i.p. The injected volume was 1 ml. Indomethacine was given 1 ml by intragastric gavage. For these studies 30 male Sprague–Dawley rats were used. Animal were divided into five groups of six animal each. First group, used as control, received the same volume vehicle by i.p. injection. Melatonin was given at 5 and 10 mg kg−1 doses by i.p. injection 30 min before carrageenan administration. Indomethacine was dissolved in distilled water and given 10 mg kg−1 by gavage 1 h before carrageenan injection [11]. Paw volume (ml) was measured using a plethysmometer (model 7140; Ugo Basile, Milan, Italy) immediately before and six times with periods of 1 h following the injection of carrageenan. Paw oedema was expressed as the increase in paw volume (ml) after carrageenan injection with respect to the pre-injection value for each animal. The ratio of anti-inflammatory activity of melatonin was calculated by the following equation: anti-inflammatory activity (%) = (1−D/C)×100, where D represents the percentage of difference of the paw volume after melatonin-administered rats and C represents the percentage difference of paw volume in the control group.

Biochemical analysis Four grams of paw tissue was taken, rinsed in ice-cold distilled water, and immediately placed in three times their volume of cold 5 ml of 1.15% KCl containing 0.2% Triton X-100 and homogenised. Then the homogenate was centrifuged at 8000 g for 10 min and the supernatant was obtained. MDA assay. MDA was estimated according to the method of Satoh [12]. Briefly, 2.5 ml of 20% trichloroacetic acid and 1 ml of 0.67% thiobarbituric acid are added to 0.5 ml of the supernatant. Then the mixture is heated in boiling water for 30 min. The resulting chromogen is extracted with 4 ml of n-butyl alcohol and the absorbance of the organic phase is determined at 530 nm. Malondialdehyde-bis-diethyl-acetate was used as a standard and values were presented as µmol/g wet weight of tissue. NO assay. NO was measured by method of Moshage et al. [13]. The sum of nitrite (NO2 − ) and nitrate (NO3 − ) were evaluated as indicator of NO level. For nitrite determination; NO2 − was measured by using the Griess reaction [14]. Briefly, supernatant were diluted sixfold with distilled water and deproteinised with sulfosalicylic acid (35%). After centrifugation at 8000 g for 20 min at room temperature, 100 µl of supernatant was applied to a test tube, followed by 100 µl of Griess reagent (1 g/l sulfanilamide, 25 g/l phosphoric acid, and 0.1 g/l N-1-naphthylethylenediamine). After incubation

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for 10 min at room temperature, the colour of the product dye was developed and its absorbance was detected at 560 nm by spectrophotometer (Shimadzu, Japan). Calibration curves were made with sodium nitrite and sodium nitrate in distilled water (linear range 0–200 nmol/ml). For nitrate determination, NO3 − was measured as nitrite after enzymatic conversion by nitrate reductase (EC 1.6.6.2; Boehringer Mannheim, 20 U/mg). Supernatant was diluted sixfold with distilled water and deproteinised with sulfosalycylic acid (35%). To 100 µl of supernatant, 100 µl of 50 µmol/l NADPH, 100 µl of 5 µmol/l FAD and 20 µl of 200 U/l nitrate reductase were added and incubated for 20 min at 37 ◦ C, then 10 µl of 10 mg/l lactate dehydrogenase (Boehringer Mannheim) and 10 µl of 10 mmol/l sodium pyruvate was added and were further incubated for 5 min at 37 ◦ C for oxidation of NADPH and 35% sulfosalysilic acid was applied for deproteinisation and assayed with Griess reagent as described earlier. Values obtained by this procedure represent the sum of nitrite and nitrate. Nitrate concentration were obtained by subtracting nitrite concentration from the total nitrate + nitrite concentration. Values were presented as µmol/g wet weight of tissue. GSH assay. GSH in the supernatant was assessed according to the method of Griffith [15]. Sample (0.3 ml) taken from supernatant was mixed in a dilution reagent that contains 5% Triton X-100 and 1 mM EDTA. After centrifugation at 10,000 g for 10 min at 4 ◦ C, 200 µl from supernatant, 500 µl of 0.3 mM NADPH, 100 µl of 6 mM 5,5-dithio-bis-2-nitrobenzoic acid (DTNB) and 500 µl buffer (0.2 M sodium phosphate plus 10 mM EDTA, pH 7.5) were mixed carefully in a cuvette. Then 10 µl of glutathione reductase (E.C 1.6.4.2, 120 U/mg; Boehringer Mannheim) was added and incubated for 10 min at room temperature. The absorbance of colour developed was detected at 412 nm. The reference cuvette contained equal concentrations of DTNB, NADPH and enzyme but no sample. Exogenous GSH was used as a standard and values were presented as µmol/g wet weight of tissue.

Drugs and reagents Indomethacin obtained from Merck–Sharp–Dohne was dissolved in distilled water. Melatonin and all other reagents and compounds used were obtained from Sigma Chemical Company (St. Louis, MO, USA). Melatonin was dissolved in ethanol and further dissolved in saline solution (the final concentration of ethanol was 1%).

Statistical analysis All values in the figures and text are expressed as mean ± sem. The n represents the number of animals studied. Statistical analysis of data was performed by one-way analysis of variance, individual groups mean were then compared by using Mann–Whitney comparison non-parametric test. The P value less than 0.05 was considered significant.

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Fig. 1. Effect of melatonin (5 and 10 mg kg−1 ) and indomethacin (10 mg kg−1 ) on paw volume development elicited by carrageenan in the rat. The results are expressed as mean ± sem (n = 6) rats. Melatonin treatment significantly decreased paw volume in a dose-dependent manner at the all time.

RESULTS

Effects of melatonin on carrageenan-induced paw oedema Intraplantar injection of carrageenan in rats led to a time-dependent increase in paw volume (Fig. 1). The increase in paw volume was observed at 1 h and was maximal (160 ± 3.34 ml) at 4 h after administration. However, carrageenan-induced paw oedema was significantly reduced in a dose-dependent manner by treatment with melatonin at 1, 2, 3, 4, 5 and 6 h after injection of carrageenan (Fig. 1). The maximal anti-inflammatory effect of melatonin was observed at 10 mg kg−1 and 2 h after carrageenan injection (45.28, P < 0.005). Doses of melatonin (5 and 10 mg kg−1 ) and indomethacin (10 mg kg−1 ) showed 33.89, 45.28 and 47.07% inhibition, respectively, against carrageenan-produced oedema after 2 h (Table I). Indomethacin dosage of 10 mg kg−1 showed more activity than melatonin in carrageenan-induced oedema but there was not a significant difference between 10 mg kg−1 melatonin and indomethacin each statistically (P > 0.05). Melatonin inhibited inflammation almost such as indomethacin.

Effects of melatonin on biochemical parameter At 6 h following the intrapaw injection of carrageenan, paw tissues were also analysis for the bio-

chemical parameter such as NO, GSH and MDA levels. The corresponding paw tissue GSH levels was shown in Fig. 2. As shown in figure, carrageenan administration markedly decreased tissue GSH level (3.24 ± 0.24). When rats were pre-treated with melatonin, tissue GSH concentration was significantly increased to (5.80 ± 0.91 and 8.65 ± 1.39, P < 0.05), by 5 and 10 mg kg−1 melatonin, respectively. Indomethacin did not improve the loss in GSH level as well as melatonin (3.95 ± 0.41, P > 0.05). As shown in Fig. 3, NO level in paw tissue was significantly increased by carrageenan administration (59.96 ± 6.58). NO levels were significantly reduced in dose-dependent manner in rats treated with 5 and 10 mg kg−1 melatonin (36.0 ± 2.82, P < 0.05; 26.33 ± 2.48, P < 0.005). However, 10 mg kg−1 indomethacin did not reduce the NO level as well as melatonin (49.16 ± 7.11, P > 0.05). As shown in Fig. 4, MAD levels were significantly enhanced (19.33 ± 3.35) in the paw tissue at 6 h after carrageenan injection. MAD level was significantly decreased to (7.16 ± 2.63, P < 0.05; 2.50 ± 0.63, P < 0.005), respectively, by 5 and 10 mg kg−1 melatonin treatment in a dose-dependent manner. Indomethacin did not reduce the level as well as melatonin (8.30 ± 3.55, P > 0.05). The protection conferred by indomethacin

Table I The effect of melatonin and indomethacin to inflammation which is produced by carrageenan Drug

(5 mg kg−1 )

MEL MEL (10 mg kg−1 ) IND (10 mg kg−1 )

Paw volume before inflammation (ml)

Paw volume after inflammation (after 2 h)

90.33 ± 10.83 92 ± 8.39 98.16 ± 7.73

129 ± 5.96 125 ± 10.44 132.16 ± 7.35

MEL: melatonin; IND: indomethacin.

Difference volume of paw (ml)

(%)

39 33 34

43.33 35.86 34.69

% of anti-inflammatory effect

P

33.89 45.28 47.07

<0.05 <0.005 <0.005

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Fig. 2. Effect of melatonin (5 and 10 mg kg−1 ) and indomethacin (10 mg kg−1 ) on the tissue glutathione concentration at 6 h after carrageenan administration. Carrageenan administration significantly decreased glutathione concentration in the paw tissue. Melatonin treatment significantly reverted the effect of carrageenan. Data are shown as mean ± sem (∗ P < 0.05) versus control.

Fig. 3. Effect of melatonin (5 and 10 mg kg−1 ) and indomethacin (10 mg kg−1 ) on the NO level of paw tissue. NO concentration in the carrageenan-treated rats was significantly increased. Melatonin treatment significantly ameliorated the carrageenan-induced elevation of NO level. Values are means ± sem of six rats for each group. ∗ P < 0.05 versus control; ∗∗ P < 0.005 versus control.

Fig. 4. MDA concentration in the paw tissue at 6 h after carrageenan administration. MDA concentration was significantly increased in the paw tissue of the carrageenan-treated rats. Melatonin treatment (5 and 10 mg kg−1 ) significantly prevented the carrageenan-induced increase in MDA level. Values are means ± sem of six rats for each group. ∗ P < 0.05 versus control; ∗∗ P < 0.005 versus control.

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against carrageenan-induced biochemical changes was less effective than melatonin.

DISCUSSION The result of the present study indicates that melatonin plays a crucial role as a protective factor against the carrageenan-induced development of acute inflammation. Among the several models of acute inflammation, carrageenan-induced inflammation has been well established as a valid model to study free radical generation in paw tissue after inflammatory states. The cellular and molecular mechanism of the carrageenan-induced inflammation is well characterised, and these models of inflammation are standard models of screening for anti-inflammatory activity of various experimental compounds [16]. It appear that the early phase of the carrageenan oedema is related to the production of histamine, leukotrienes, platelet-activating factor and possibly cyclooxygenase products, while the delayed phase of the carrageenan-induced inflammatory response has been linked to neutrophil infiltration and the production of neutrophil-derived free radicals, such as hydrogen peroxide, superoxide and OH• radicals, as well as to the release of other neutrophil-derived mediators [17, 18]. In the recent years, l-arginine–NO pathway has been proposed to play an important role in the carrageenaninduced inflammatory response [19, 20]. Our present results also confirm that production of NO in the carrageenan-induced paw oedema model. The expression of the inducible isoform of NO synthase has been proposed as an important mediator of inflammation [21]. Pharmacological inhibitors of NOS and ablation of the gene for iNOS has been shown to reduce the development of the carrageenan-induced inflammatory response [19, 20]. The systemic inflammatory response is also associated with the production of oxygen-derived free radicals and there is now substantial evidence that much of the cytotoxicity is due to a concerted action of oxygenand nitrogen-derived free radicals and oxidants [10]. An important part of the oxidative injury associated with simultaneous production of NO and oxyradicals may be mediated by ONOO− , a toxic oxidant formed from the reaction of NO and superoxide [8]. In a number of pathophysiological condition, ONOO− has been proposed as an important mediator of cell damage under condition of inflammation and oxidant stress [21]. The ONOO− is cytotoxic via a number of independent mechanisms. Its cytotoxic effects include initiation of lipid peroxidation, inactivation of a variety of enzymes (most notably, mitochondrial respiratory enzymes), glutathione depletion [22]. Lipid peroxidation is implicated in the pathogenesis of inflammatory processes [23] In our study, MAD, a lipid peroxidation product, was elevated and glutathione level was decreased too. Glutathione is a known oxyradical scavenger. Cuzzocrea et al. [10] suggested that endogenous glutathione

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plays an important role against carrageenan-induced local inflammation. Melatonin is a hormone produced by the pineal gland during the dark phase of circadian cycle [1]. Melatonin is involved in many physiological function such as the control of sexual maturity and cycling, the immune response, the temperature regulation and there are indications that it is a key regulator of ageing and senescence [1]. Recently, the pineal hormone melatonin was reported to be a free radical scavenger and antioxidant [1, 2, 24, 25]. Melatonin reduces oxidative stress by several means. Thus, the indole is an effective scavenger of the OH• and the ROO• radicals [4, 26], and it may stimulate some important antioxidative enzymes like superoxide dismutase, glutathione peroxidase and glutathione reductase [2]. It was previously reported that melatonin has a protective role in lipopolysaccharide-induced septic shock and reverses adipsia and hyperthermia by suppressing pro-inflammatory cytokines, prostaglandins and NO production [27–29]. More recently, it has been shown that melatonin acts as a ONOO− scavenger and protects cultured cells against ONOO− -induced injury [30]. The cytotoxic effects of ONOO− are multiple and include protein oxidation, lipid peroxidation, inhibition of cellular metabolic pathways and signal transduction processes [31]. A more novel role of melatonin as an immunomodulator has also been proposed. Melatonin was known to modulate immune functions [7] and it was also demonstrated that melatonin binding sites are found on lymphocytes and macrophages [32]. Melatonin may exert certain biologic effects (such as the inhibition of tumour growth and counteraction of stress-induced immunodepression) by augmenting the immune response. Studies in mice have shown that melatonin stimulates the production of interleukin-4 in bone marrow T helper cells and of granulocyte–macrophage colony-stimulating factor in stomal cell [6], as well as protecting bone marrow cells from apoptosis induced by cytotoxic compounds [33]. In vitro studies on human peripheral blood mononuclear cells have shown that melatonin inhibits the production of tumour necrosis factor (TNF) and interferon-gamma [29, 34]. TNF is a pathogenic mediator of various infective and inflammatory diseases and anti-TNF antibodies are protective in animals models of septic or endotoxic shock [35]. Some study results support the hypothesis that melatonin inhibits TNF production through its antioxidant activity [28]. In fact, various antioxidant were reported to inhibit the synthesis of TNF, both in vivo and in vitro [36, 37]. Interestingly, melatonin also inhibits the transcription factor, nuclear factor-κB (NF κB) as do other antioxidants [28]. The NF κB induces many inflammatory genes that encode for pro-inflammatory cytokines, chemokines that selectively induce the inflammatory enzymes, such as inducible NO synthase, cyclooxygenase-2, adhesion molecules and inflammatory receptors (e.g. interleukin-2

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receptors) [29]. These released mediators contribute to the expression of inflammation and hyperalgesia. Cuzzocrea et al. [10] suggested that melatonin exerts potent anti-inflammatory effects. Part of these anti-inflammatory effects may be related to a reduction of prostaglandin production during the inflammatory process. In conclusion, we believe that melatonin effects on carrageenan-induced paw oedema could be due to a reduced production of the free radical. However, based on our observations, we are unable to show whether additional mechanisms are involved in such melatonininduced effects. Nevertheless, we discussed these additional mechanism in this study. Finally, we further support the idea that melatonin may exert anti-inflammatory action and its ability to readily penetrate the cell and its wide margin of safety, melatonin may find utility as a pharmacological agent in preventing and treating disease in which free radical formation is a pathogenic factor.

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