dehydrogenase ( LDH ) , acid phosphatase ( AcP ) , and alkaline phosphatase ( AlP ) by the methods of Bergmeyer

Website: www.phcog. com DOI: 10.4103/0973-1296.131024 Quick Response Code: P H C O G M A G . O R I G I N A L A R T I C L E Valcheva-Kuzmanova, et al.: Effect of AMFJ in amiodarone-induced pneumotoxicity Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 133 of the release of pro-infl ammatory cytokines such as tumor necrosis factor alpha and interleukins (interleukin IL-1 beta, IL-6, IL-8). [19,20] There are numerous pathologic similarities between human and rodent lungs and as such, rodent model presents an excellent tool to investigate pathologic changes in vivo. [21] The aim of the study was to investigate the effect of A. melanocarpa fruit juice (AMFJ) in a rat model of AD-induced pulmonary toxicity. MATERIALS AND METHODS Experimental substances AD hydrochloride and all other chemicals and reagents wereof analytical grade and were purchased from Sigma-Aldrich Company (Germany). The Quantikine Rat IL-6 and IL-10 immunoassay kits were from R and D Systems (USA). AMFJ was produced from A. melanocarpa Elliot fruits grown in the Balkan Mountains, Bulgaria. They were handpicked in September, crushed and squeezed. The juice was fi ltered, pasteurized at 80°C for 10 min and stored at 0°C until the experiment. The contents of phenolic substances in 100 mL AMFJ were: Total phenolics, 709.3 ± 28.1 mg as gallic acid equivalents, determined spectrophotometrically according to the Folin-Ciocalteu procedure;[22] total fl avonoids, 189.4 ± 8.6 mg as catechin equivalents, measured by a colorimetric assay developed by Zhishen et al.;[23] total anthocyanins, 106.8 ± 6.2 mg as cyanidin-3-glucoside equivalents, determined by a pH-differential spectrophotometry at pH 1.0 and pH 4.5;[24] quercetin, 11.8 mg, measured by a high-performance liquid chromatography method. [25] The values were the mean of duplicate determinations of three samples. Animals and experimental treatments The study was carried out on 96 male Wistar rats (weight 220-250 g, age 4 months). The animals were obtained from the Research and Laboratory Animal Breeding Center of Slivnitsa (Bulgaria) and were housed in the university animal quarters for 1 month at a temperature of 22°C ± 2°C and humidity of 50 ± 10%, given normal pelleted diet and water AD libitum. All procedures concerning animal treatment and experimentation were conducted in compliance with the national laws and policies, in conformity with the international guidelines (European Economic Community EEC Council Directive 86/609, IL 358, 1, December 12, 1987). The animals were divided into four groups of 24 rats. Each group was subdivided into four subgroups of six rats. One subgroup of each group was sacrifi ced on days 3, 5, 10, and 28 under thiopental anesthesia (50 mg/kg) after receiving the respective treatment in the course of 2, 4, 9, and 10 days, respectively. Group 1 (Control) received two intratracheal (i.t.) instillations of sterile distilled water (2 mL/kg) on days 0 and 2. The four subgroups of Group 1 received distilled water (10 mL/kg) orally through an orogastric cannula from day 1 to days 2, 4, 9, and 10, respectively. Group 2 (AD) received two i.t. instillations of AD (6.25 mg/kg, as a 3.125 mg/mL water solution) on days 0 and 2. [26] The subgroups of Group 2 received distilled water (10 mL/kg) orally through an orogastric cannula from day 1 to days 2, 4, 9, and 10, respectively. Group 3(AD + AMFJ5) was treated with AD i.t. on days 0 and 2, and from day 1 to days 2, 4, 9, and 10 the respective subgroups received AMFJ orally at a dose of 5 mL/kg diluted with distilled water to a total volume of 10 mL/kg. Group 4 (AD + AMFJ10) was treated with AD i.t. on days 0 and 2, and from day 1 to days 2, 4, 9, and 10 the respective subgroups received AMFJ orally at a dose of 10 mL/kg. AD was dissolved in distilled water at 60°C and allowed to cool to room temperature before the i.t. instillation. Lung weight coeffi cient The body weight and lung weight were measured for each animal. The lung weight coeffi cient (organ weight in mg/100 g body weight) was calculated. Bronchoalveolar lavage Bronchoalveolar lavage fl uid (BALF) was obtained on days 3, 5, and 10. Rats were sacrifi ced by exsanguination through cutting v. renalis. The chest was opened, and the lungs were perfused in situ via the right heart ventricle with saline (30 mL). Triple lavage of the left lung through the trachea with a total of 5 mL of saline was performed. The volume of fl uid recovered ranged from 80% to 90% of the fl uid introduced. Cytological assays of BALF One aliquot of the BAL F was used for total cell count ×105/L. The cells were removed by centrifugation at 300 × g for 10 min. The cell pellet was resuspended in 1 mL of saline. The cell differentials were analyzed using the procedure of Danos and Keebler, modifi ed by Saltini et al.[27] Cells were collected on nitrocellulose fi lters 25 mm in diameter with 5 μm pores (SWP-025-00 Catalogue number SMWP02500; Millipore Corp.) and were stained with H and E. Biochemical assays of BALF After centrifugation of BALF, the supernatant was used for the measurement of enzyme activities such as lactate Valcheva-Kuzmanova, et al.: Effect of AMFJ in amiodarone-induced pneumotoxicity 134 Pharmacognosy Magazine | April-June 2014 | Vol 10 | Issue 38 dehydrogenase (LDH), acid phosphatase (AcP), and alkaline phosphatase (AlP) by the methods of Bergmeyer et al.[28] Total protein content was measured by the method of Lowry et al.[29] The enzyme activities are represented in U/L, and total protein in mg/mL of BALF. Biochemical assays of lung homogenate Lung homogenate was obtained from the right lung. The tissue was homogenized with potassium chloride KCl (1.15%) in 1:10 ratio. The homogenate was centrifuged (9000 × g, 30 min), and the supernatant was stored on ice. Malondialdehyde (MDA) content i n nmol/g tissue was measured on days 3, 5, and 10 by the method of Ohkaw a et al.[30] Hydroxyproline (HP) levels (in μg/mL) were measured in lung homogenate of rats sacrifi ced on day 28 as described by Bergman and Loxley. [31] The method is based on the release of free HP from collagen by acid hydrolysis. Immunological assays of rat serum The serum of the experimental animals was used for the measurement of IL-6 and IL-10 in pg/mL on days 3, 5, and 10 by the ELISA method in accordance with the immunoassay kits manufacturer’s instructions. Statistical analysis Results are presented as mean ± SEM. The data were tested by one-way ANOVA, followed by Dunnett’s multiple comparison post-test to identify signifi cant difference. A level of P < 0.05 was considered signifi cant. All analyses were performed using GraphPad Prism Statistical Software.