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Involvement of Kallikrein-Kinin System on Cardiopulmonary Alterations and Inflammatory Response Induced by Purified Aah I Toxin from Scorpion Venom Wafa Medjadba, Marie-France MartinEauclaire & Fatima Laraba-Djebari

Inflammation ISSN 0360-3997 Volume 39 Number 1 Inflammation (2016) 39:290-302 DOI 10.1007/s10753-015-0249-3

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Author's personal copy Inflammation, Vol. 39, No. 1, February 2016 ( # 2015) DOI: 10.1007/s10753-015-0249-3

Involvement of Kallikrein-Kinin System on Cardiopulmonary Alterations and Inflammatory Response Induced by Purified Aah I Toxin from Scorpion Venom Wafa Medjadba,1 Marie-France Martin-Eauclaire,2 and Fatima Laraba-Djebari1,3

Abstract—Bradykinins are released from kininogen by kallikrein. They increase capillary lung permeability after their binding to β1 and especially β2 receptors before being metabolized by kininase enzyme. This study was performed to evaluate cardiopulmonary damages and inflammatory response on injected rats with Aah I toxin of scorpion venom and the involvement of Kallikrein-Kinin system in this pathogenesis. Obtained results revealed that Aah I toxin induces inflammatory cell infiltration accompanied by cellular peroxidase activities, a release of cytokine levels, pulmonary and myocardial damage, with altered metabolic activities and imbalanced redox status. Administration of aprotinin (bradykinin inhibitor) and especially icatibant (bradykinin β2 receptor antagonist) seemed to be able to protect animals against the toxicity of Aah I; nevertheless, the use of captopril (kininase II inhibitor) reduced partially some cardiac disorders. These findings indicate that the kallikrein-kinin system may contribute to the physiopathological effect and lung edema formation induced by toxin, which suggests a potential use of drugs with significant anti-kinin properties. KEY WORDS: kallikrein-kinin system; lung permeability; inflammation; Aah I neurotoxin; cardiopulmonary damage.

species in Algeria, contains three alpha toxins (Aah I to III), which are responsible for the majority of the toxicity in mammals [4]. Previous studies reported that edema formation in the lung can be attributed to acute left ventricular failure [5, 6]. However, other authors attribute these symptoms to a non-cardiogenic origin [7, 8]; they assumed that it is due in part to an increase of pulmonary vascular permeability by activation and release of inflammatory mediators [9, 10]. The involved mechanisms in the inflammatory process and pulmonary edema induced by the Aah venom and its toxins have not been fully elicited. However, previous studies showed that A. australis hector venom and its Aah II toxin induce vascular permeability increase which may cause acute lung injury [11, 12]. Several experimental studies indicate that bradykinin, a major active component of kallikrein-kinin system, increases alveolo-capillary membrane permeability [13]. Bradykinin is cleaved by kallikrein enzyme from precursor named kininogen(s) and exerts its actions via G proteincoupled receptor B1 and B2 subtypes, with high affinity for the B2 receptor [14]. On the other hand, bradykinin is rapidly broken down to inactive fragments by kininases I

INTRODUCTION Scorpion stings represent a medical problem in many countries, and envenomation symptoms are associated with pulmonary edema (PE) and myocardial injury in the most severe cases [1]. These symptoms have been attributed to the neurotoxins’ pharmacological effects [2, 3]. Androctonus australis hector, the most dangerous scorpion

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USTHB, Faculty of Biological Sciences, Laboratory Cellular and Molecular Biology, Department Cellular and Molecular Biology, BP32, EL Alia, Bab Ezzouar, 16111 Algiers, Algeria 2 Aix-Marseille University, CNRS UMR 7286 CRN2M, IFR Jean-Roche, Faculté de Médecine Nord, Bd Pierre Dramard, 13916 Marseille, Cedex 20, France 3 To whom correspondence should be addressed at USTHB, Faculty of Biological Sciences, Laboratory Cellular and Molecular Biology, Department Cellular and Molecular Biology, BP32, EL Alia, Bab Ezzouar, 16111 Algiers, Algeria. E-mail: [email protected]; [email protected] Abbreviations: Aah, Androctonus australis hector; Aah I, Neurotoxins purified from Aah venom; ACE, Angiotensin-converting enzyme; ACEI, Angiotensin-converting enzyme inhibitor; Apro, Aprotinine; Cap, Captopril; Icati, lcatibant; IL-1β, Cytokines: Interleukin-1β; IL-6, Interleukin-6; I-10, Interleukin-10; MDA, Malondialdehyde

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Involvement of Kallikrein-Kinin System and especially kininase II named also angiotensinconverting enzyme (ACE), well known for its physiological role in the renin-angiotensin system [15]. Bradykinin produces local and systemic inflammation [16]. However, little is known about the probable promoting effect of bradykinin in induced pulmonary edema by scorpion toxins; thus, this study aims to better understand the molecular mechanisms underlying vascular permeability during inflammatory response and cardiopulmonary alterations induced by purified Aah I toxin from A. australis hector venom. We therefore explored the impact of the kallikrein-kinin system by inhibiting bradykinin formation, antagonizing its actions or inhibiting its degradation. Data that may contribute to an understanding of the mechanisms by which the cardio-pulmonary system is affected after scorpion envenomation.

Animals of the second group received subcutaneous injection of sublethal dose (10 μg/kg) of Aah I toxin; this group is divided into subgroups which were sacrificed at different time intervals following the Aah I injection. The third, fourth and fifth groups received, respectively, captopril (1.5 mg/kg by i.p. route), aprotinin (1 mg/ml of by i.v. route) and icatibant (100 mg / kg by i.p. route) 1 h prior to the sublethal dose of Aah I toxin. All pretreated animals were scarified 3 h after administration of Aah I toxin. Determination of Pulmonary Water Content Lungs collected from animals at the end of the experiments are used to evaluate pulmonary water content determined by the method described earlier [18]. Briefly, the lungs were weighted and dried to a constant weight in an oven (at 90 °C for 48 h). The difference between wet and dry weight was calculated to determine the water content.

MATERIALS AND METHODS Histological Analysis Non-biological Materials Chemical products and reagents used in these experiments were purchased from Sigma (USA), Merck (Darmstadt F.R.G.) and Prolabo (Darmstadt, Germany). Biological Materials Aah I Toxin Aah I toxin provided from Laboratory of Cellular and Molecular Biology, Faculty of Biological Sciences of USTHB was purified from A. australis hector venom (Aah), as described by Martin and Rochat (1986) [17]. Animals Wistar male rats (150–200 g body weight) were obtained from the animal breeding facility of Biological Sciences (USTHB). The animals were kept under controlled environment and received food and water ad libitum. Experiments were carried out in accordance with the European Community rules of ethical Committee for animals’ welfare. Methods In Vivo Protocol Twenty-eight rats were divided into groups of four rats each one. Untreated rats (control groups) received subcutaneous route saline solution (NaCl 0.9 %: w/v).

Organs (heart and lung) were extracted from scarified animals and immersed into a formol fixing solution (4 %) for 48 h, dehydrated in ethanol, cleared in xylen and embedded in paraffin. Histological sections (5 μm thick) were cut and stained with hematoxylin-eosin (H&E) for microscopic examination (Motic Digital Microscope PAL system). Evaluation of Metabolical Parameters Blood was collected; serum was separated after centrifugation at 10,000 g for 15 min. Phosphocreatine kinase (CPK) and lactate dehydrogenase (LDH) were evaluated in sera using Bayer commercial Kit according to the manufacturer’s instructions. The enzyme values were expressed in international units (IU/l). Evaluation of Inflammatory Mediators Cytokine Level Evaluation Blood were collected; the sera were collected and kept at 4 °C before use. Cytokine levels were evaluated by specific sandwich ELISA at 30 min, 3, 6 and 24 h in sera using cytokine Amersham kits for IL-1β, IL-6 and IL10 according to the manufacturer’s instructions. Cytokine concentrations were deduced from standard curves. The sensitivity for detection levels of the IL-6, IL-1β and IL-10 was, respectively, 10, 3 and 12 pg/ml.

Author's personal copy 292 Evaluation of Inflammatory Cell Infiltration in Bronchoalveolar Lavage Animals were sacrificed using an overdose of ether, the chest wall was humanely opened and the tracheas were cannulated. The airway lumina were washed in saline solution. Bronchoalveolar lavage (BAL) was recovered and analyzed to determine cell populations. Total cell counts were carried out using a hemocytometer (ADVIA, Hematology system). Leukocyte populations were identified and counted using Giemsa staining. Myeloperoxydase Activity Evaluation Neutrophil accumulation and activation were estimated by evaluation of myeloperoxidase (MPO) activity in lung tissue which was homogenized in Tris–HCl buffer 50 mM, pH 6.6 and then centrifuged at 6000 rpm for 30 min. The first supernatant (S1) was conserved at 4 °C, and the second supernatant (S2) was recovered after three freeze-thaw cycles of the pellet. One hundred microliters of supernatant (S1) and (S2) were added to 300 μL of chromogene substrate (0.167 mM O-dianisidine prepared in Tris–HCl 50 mM; pH 6.6 and H2O2 8.8 mM). Absorbance was read at 460 nm after 1 min of incubation at room temperature. Eosinophil Peroxidase Activity Evaluation Eosinophil accumulation in lung tissue was evaluated by assaying eosinophil peroxidase activity as previously described by Van Oosterhout and collaborators [19]. Lungs were weighed and homogenized with a manual grinder in 0.05 M Tris–HCl buffer pH 8 containing 0.1 % Triton ×100. Tissue homogenates were centrifuged at 500 g for 15 min; the supernatant was collected and kept at 4 °C until use. Aliquots (50 μl) were placed in the wells of an ELISA plate and 100 μl of 0.05 M Tris–HCl buffer pH 8–0.1 % Triton X-100 containing 4 mM of H 2 O 2 and ophenylenediamine (10 mM) were added. The plates were incubated for 1 h at room temperature in the dark. Absorbance was read at 490 nm using an ELISA reader. Results are expressed as changes in absorbance per 100 mg of tissue.

Medjadba, Martin-Eauclaire, and Laraba-Djebari copper-coated cadmium. Aliquots of sample were incubated with equal volumes of Griess reagent (1 % sulfanilamine, 0.1 % naphtyl ethylenediamine dihydrochloride and 2.5 % H3PO4). Absorbance was measured at 540 nm after 20 min of incubation in dark [20].

Estimation of Lipid Peroxidation (MDA) This assay is based on the formation of a complex between malondialdehyde with two molecules of thiobarbituric acid (TBA) in acid medium and in a hot temperature (95 °C for 45 min) according to Ohkawa et al. [21]. This complex can give pink color that will be extracted by the organic solvent butanol after centrifugation (2000 g during 20 min). MDA concentration is determined by measuring the absorbance at 530 nm.

Determination of Catalase Activity Evaluation of catalase activity was carried out according to the Aebi method [22]. Each sample (50 μl) was diluted (1/20) with phosphate buffer (50 mM, Ph 7). The reaction was thereafter initiated by 500 μl of H2O2 (0.2 %). Kinetics of degradation of H2O2 was followed during 2 min at 240 nm by spectrophotometer . The results were expressed in catalase unit (U) per 100 mg of tissue.

Statistical Analysis Statistical analysis was performed using Stat EL (AD Science, Paris, France, www.adscience.eu), a software operating on the spread sheet of Microsoft Excel (Microsoft, Redmond, Washington). Results are expressed as means ±SD and analyzed by ANOVA test; data were considered statistically significant if p values were p

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