Aug 21, 2012 - University School of Medicine, Erzurum, Turkey, 3Department of ... LEVO inhibited CAR-induced paw oedema and suppressed the production ...
Basic & Clinical Pharmacology & Toxicology, 2013, 112, 156–163
Doi: 10.1111/bcpt.12004
Beneficial Pharmacological Effects of Levosimendan on Antioxidant Status of Acute Inflammation Induced in Paw of Rat: Involvement in Inflammatory Mediators Emre Karakus1, Zekai Halici2,*, Abdulmecit Albayrak2, Yasin Bayir3, Ali Aydin4, Deniz Unal5, Elif Cadirci6, Irmak Ferah2 and Ersan Odaci7 Department of Pharmacology and Toxicology, Ataturk University School of Veterinary, Erzurum, Turkey, 2Department of Pharmacology, Ataturk University School of Medicine, Erzurum, Turkey, 3Department of Biochemistry, Ataturk University School of Pharmacy, Erzurum, Turkey, 5 Department of Histology and Embryology, Ataturk University School of Pharmacy, Erzurum, Turkey, 4Department of Orthopedic and Traumatology, Ataturk University School of Pharmacy, Erzurum, Turkey, 6Department of Pharmacology, Ataturk University School of Pharmacy, Erzurum, Turkey, and 7Department of Histology and Embryology, Karadeniz Technical University School of Medicine, Trabzon, Turkey
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(Received 26 June 2012; Accepted 21 August 2012) Abstract: Levosimendan (LEVO) is a new calcium sensitizer with positive inotropic and vasodilating properties that represents a new pharmacological class of inotropic drugs that stimulate elevated cardiac output. The purpose of this study was to examine anti-inflammatory effect and antioxidant activity of LEVO in a carrageenan (CAR)-induced inflammatory paw oedema rat model. The CAR-induced rat groups received LEVO 1, 2 and 3 mg/kg by intraperitonally and indomethacin (IND) 25 mg/kg by oral gavage. LEVO inhibited CAR-induced paw oedema and suppressed the production of TNF-a, IL-1 and IL-6 at doses of 2 and 3 mg/kg. In contrast to CAR-injected paws, 2 and 3 mg/kg doses of LEVO and IND increased superoxide dismutase (SOD) activity and also both doses of LEVO, and IND decreased the 8-isoprostaglandin F2a (8-ISO) level. A 2 mg/kg dose of LEVO produced 39%, 46%, 61% and 64.7% anti-inflammatory effects (p < 0.0001) for the 1st, 2nd, 3rd and 4th hours, respectively. Other results of our current study have shown that SOD and glutathione for CAR-injected groups were lower, and 8-ISO level was higher than those for the healthy rat group. LEVO may be provided as a pharmacological agent in the prevention or treatment of diseases in which acute or chronic inflammation occurs based on a pathogenic factor.
Inflammation and pain are two kinds of local defence reactions of living mammalian tissues in response to any injurious agent such as chemicals or physical injury [1]. There are various components to an inflammatory reaction, which can contribute to the associated symptoms and tissue injury. There are two phases of inflammation, acute and chronic. Acute inflammation is a short-term response that usually results in recovery and which includes oedema, an increase in blood vessel wall permeability [2], and leucocytes infiltration of the damaged region, which removes the stimulus and repairs the inflamed tissue. These complex phases and mediators that make up the inflammatory reaction comprise a well-defined defence mechanism that can induce or aggravate many reactions [3]. Persistent inflammation is associated with many diseases, including atherosclerosis, allergy, asthma, rheumatoid arthritis, Alzheimer’s disease and autoimmune diseases and also plays a role in various human cancers. All these inflammation-related diseases have attracted the attention of global scientific research. To counter the condition of inflammation, the standard mediAuthor for correspondence: Zekai Halici, Department of Pharmacology, Ataturk University School of Medicine, 25240 Erzurum, Turkey (fax +90 442 2360962, e-mail hzekai@atauni.edu.tr). *This research was conducted in the Research Laboratory of Basic and Clinical Medical Sciences and Drug Development at Ataturk University, School of Medicine, Department of Pharmacology, Erzurum, Turkey.
cal approach is to use anti-inflammatory drugs. Considering the frequent occurrence of the adverse effects of current drugs and opioids, there is a pharmacological interest in new drugs with minimal side effects [4–6]. A carrageenan (CAR)-induced inflammation model of animals is commonly used to determine the contribution of some new target anti-inflammatory drugs in resisting the biochemical changes associated with acute inflammation. CAR-induced acute inflammation is the best way to investigate the secrets of inflammation phases, which begin with the infiltration of phagocytes and the over-production of free radicals as well as the release of inflammatory mediators such as interleukin (IL)1b, IL-6, Nitric Oxide (NO) and tumour necrosis factor (TNF)-a [7]. Levosimendan (LEVO) is a new calcium sensitizer with positive inotropic and vasodilating properties that represents a new pharmacological class of inotropic drugs that stimulate elevated cardiac output. At higher concentrations of LEVO administration, intracellular cyclic adenosine monophosphate through phosphodiesterase III inhibition is elevated, which causes [8] vasodilatation in cerebrovascular, coronary and pulmonary beds [9–11]. With increasing frequency, the literature also demonstrates additional properties of LEVO, such as anti-inflammatory activities during ischaemia-reperfusion, acute heart failure or endotoxemia [12–14]. Morelli et al. [15] have already proven
© 2012 The Authors Basic & Clinical Pharmacology & Toxicology © 2012 Nordic Pharmacological Society
ANTI-INFLAMMATORY ACTIVITY OF LEVOSIMENDAN
the beneficial effects of LEVO on haemodynamics in the context of inflammation. Boost et al. [16] have described that LEVO attenuated the pro-inflammatory response in an experimental model of ventilator-induced lung injury. In addition, LEVO possibly exerts anti-inflammatory and anti-apoptotic effects in patients with severe heart failure [14,17]. Also, LEVO has previously been investigated in some sepsis models and provided promising results about its effects on inflammatory cytokines etc. [18,19]. So among these mechanisms, this study investigated the cytokines and oxidative stress in the basic mechanisms of inflammation. This is the first study investigating direct anti-inflammatory effect of LEVO on cytokine levels and oxidative stress in a CAR-induced paw oedema rat model. Previously, a patent by Haikala et al. [20] has reported an anti-inflammatory effect of LEVO in CARinduced paw oedema but that study has not been published as a scientific paper. This patent provides information only about anti-oedematous effects of LEVO but not about its effects on cytokines and oxidative stress. Our study first and foremost investigated the anti-inflammatory effect mechanism of LEVO by focusing on two possible pathways: cytokines and oxidative stress. In the present study, we examined the possibility of antiinflammatory and antioxidant activity of LEVO in CARinduced inflammatory paw oedema, which is a widely used model for investigating the acute inflammatory response and novel effects of anti-inflammatory drugs. Also, we measured the activities of superoxide dismutase (SOD) and level of glutathione (GSH) and 8-isoprostaglandin F2-a (8-ISO) in paw tissue as well as plasma level of IL-6, IL-1, and TNF-a at the 4th hour after CAR injection to understand the relationship between the anti-inflammatory mechanism of LEVO and antioxidant enzymes. Materials and Methods Animals. In this study, we used a total of 42 male Albino Wistar rats obtained from the Medical Experimental Research Centre, Ataturk University (ATADEM). The animals weighed between 180 and 200 g and were fed under normal temperature conditions (22°C) in separate groups before the experiments. Animal experiments were performed in accordance with the national guidelines for the use and care of laboratory animals, and the study was approved by the local animal care committee of Atatürk University. Chemicals. All chemicals for laboratory experimentation, including CAR, were purchased from Sigma Chemical (Munich, Germany). Indomethacin ( Endol 25 mg capsul) was purchased from Deva, Turkey; levosimendan (Simdax 2.5 mg/ml Daiichi Sankyo) and thiopental sodium (Pentothal Sodium 1 g) were purchased from Abbott Laboratories, Istanbul, Turkey. Carrageenan-induced paw oedema in rats. In the first series of experiments, the anti-inflammatory effects of LEVO and IND on CAR-induced paw oedema were studied in the rats. The rats were divided into seven groups (n = 6) for the experimental procedure. Three rat groups received LEVO 1, 2 and 3 mg/kg (intraperitonal) and IND 25 mg/kg (oral) [21]. IND was suspended in distilled water as a vehicle. Therefore, the control groups (1st and 3rd group) received an equal volume of distilled water as vehicle.
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The experimental groups can be summarised as:
Group Group Group Group Group Group Group
1: 2: 3: 4: 5: 6: 7:
Healthy Healthy + LEVO 3 mg/kg CAR (control group) CAR + IND 25 mg/kg CAR + LEVO 1 mg/kg CAR + LEVO 2 mg/kg CAR + LEVO 3 mg/kg
One hour after drug administration, 0.1 ml of 1% CAR was injected into the hind paw of each rat in each group. Before the CAR injection, the normal paw volumes of the rats were measured with a plethismometer. The CAR-induced increase in the paw volume was measured four times at 1-hr intervals. Namely, the paw volumes were measured every 60 min. during 4 hr after CAR injection. The effects of the drugs were determined by comparing the results of the drug-treated groups with those of the control group. The percent oedema inhibition was calculated according to the following formula: % oedema inhibition = Vc Vd/Vc 9 100 (Vc = the mean increase in paw volume in the absence of control group and Vd = the mean increase in paw volume after treatment with LEVO and IND). Data for the inflammation model were subjected to one-way analysis of variance (ANOVA) using SPSS 13.0 software (Prantice Hall, NJ, USA). Differences among the groups were obtained using the LSD option and were considered significant at p < 0.05. At the end of the experiment, paw tissues of all experimental animals were collected for biochemical examination. All of the paw tissues were immediately transferred to 80°C.
Biochemical investigations. Cytokine, IL-1b, IL-6 and TNF-a, serum measurements. Sera were separated from blood by allowing it to clot followed by centrifugation at 2860 9 g for 10 min. at 4°C and kept at 86°C until the samples were thawed for the assay. IL-1b, IL-6 and TNF-a from each sample were measured in duplicate with highly sensitive ELISA kits, InvitrogenKRC0011 (Camarillo, CA, USA), RayBiotech-ELR-IL6.001 (Norcross, GA, USA) and Invitrogen-KRC3011, respectively, specifically designed for rats, according to the manufacturer’s instructions. Biochemical investigation of paw tissues. After macroscopic analyses, rat paws were kept at 86°C. One hundred milligrams of tissues from each rat was firstly perfused with PBS/heparin and then homogenised in a specific homogenate tampon (appropriate buffer) on ice by UltraTurrax after grinding in liquid nitrogen. Then, they were centrifuged according to the manufacturer’s instructions. For biochemical investigation, SOD activity, 8-ISO and GSH levels from each supernatant were then measured in duplicate with highly sensitive ELISA kits, Cayman-706002 (Ann Arbor, MI, USA), Cell BiolabsSTA-337 (San Diego, CA, USA) and Cell Biolabs-STA-312, respectively, specifically designed for rat tissue, according to the manufacturer’s instructions. All the data are presented as the mean ± standard deviation results based on per mg protein. Protein determination. The protein concentrations were determined by the Lowry method using commercial protein standards (Total protein kit-TP0300-1KT; Sigma Aldrich, MO, USA). The differences in biochemical parameters in serum between groups were evaluated using one-way analysis of variance (ANOVA) with Tukey’s tests for post hoc comparisons and were considered significant at p < 0.05. Histopathological study. At the end of the experiment, the rats were killed and right hind paws were taken, fixed in 10% formalin solution for 24 hr, dehydrated by an increasing concentration alcohol series and cleared by xylene series. After histological processing, the tissues
© 2012 The Authors Basic & Clinical Pharmacology & Toxicology © 2012 Nordic Pharmacological Society
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were embedded in paraffin and sectioned to a thickness of 5 mm using a microtome (Leica RM2235; Leica Instruments, Nussloch, Germany) with disposable metal microtome blades (Leica 819, Leica Instruments, Nussloch, Germany). Sections were stained with haematoxylin and eosin (H&E) and deeper parts of skin were evaluated histopathologically concerning the inflammatory cell (IC) population under a light microscope (Olympus BX51, Tokyo, Japan) by a single, blind observer. Also, IC was scored in X20 magnification per four areas selected randomly as:
1 2 3 4
: : : :
IC IC IC IC
density density density density
is is is is
0–25% 25–50% 50–75% 75–100%
in the rats. The anti-inflammatory effects of 1 mg/kg dose of LEVO were determined as 6%, 23%, 24% and 27.40%, respectively (p < 0.0001), for the 1st, 2nd, 3rd and 4th hours. For the same hours, a 2 mg/kg dose of LEVO produced 39%, 46%, 61% and 64.7% anti-inflammatory effects (p < 0.0001), respectively. For the same hours, a 3 mg/kg dose of LEVO produced 67%, 77%, 80% and 84.3% anti-inflammatory effects (p < 0.0001), respectively. In comparison, the antiinflammatory effects of IND were 33%, 45%, 51% and 60.7%, respectively (p < 0.0001), for the same time intervals. Biochemical results for oxidant and antioxidant levels of paw tissue in rats.
Statistical analyses. Data for the inflammation model were subjected to one-way analysis of variance (ANOVA) using SPSS 13.0 software. Differences among the groups were obtained using the LSD option and were considered significant at p < 0.05. The differences in biochemical parameters in serum between groups were evaluated using one-way analysis of variance (anova) with Tukey’s tests for post hoc comparisons and were considered significant at p < 0.05. All data were expressed as mean ± standard deviation (S.D.).
Results Effects of IND and LEVO on CAR-induced paw oedema in rats. As seen in table 1 and fig. 1, both doses of LEVO and IND significantly decreased CAR-induced paw oedema formation
The activity of SOD and the level of GSH, as well as lipid peroxidation levels and the enzymatic activity of 8-ISO, were evaluated in all paw tissues. The results are presented in table 2. Table 2 shows that SOD and GSH for CAR-injected groups was lower, and 8-ISO level was higher than those for the healthy rat group. However, as compared with 8-ISO and SOD levels in LEVO and IND-administered rats, the opposite results were found for the levels of 8-ISO and SOD activities in CAR-injected tissues. In contrast to CAR-injected paws, 2 and 3 mg/kg doses of LEVO and IND increased SOD activity (p < 0.05) and also, both doses of LEVO and IND decreased the 8-ISO level (p < 0.05).
Table 1. Anti-inflammatory activity of levosimendan (LEVO) against carrageenan (CAR)-induced paw oedema in rats, n = 6. Rat paw volume (ml)
Drugs İndomethacin LEVO LEVO LEVO Control (CAR)
Dose (mg/kg) 25 1 2 3 –
N
Before inflammation
At the 4th hour of the CAR inflammation
6 6 6 6 6
0.97 0.91 1.00 0.99 0.92
1.17 1.28 1.18 1.07 1.43
Increase in rat paw volume (ml)
Anti-inflammatory effect (%)
p
± ± ± ± ±
60.7 27.4 64.7 84.3 –
