2.1. Materials
GLZ was prepared from Tehran Daru (Iran) at pharmaceutical grade. The biochemical reagents and solvents were purchased from Sigma (USA) and Merck (Germany) companies.
2.2. Animals and experimental design
Sixty-four male adult male BALB/c mice with a mean weight of 25–30 g were obtained from the Animal Center of Mazandaran University of Medical Sciences. All stages of this research work were performed according to the protocol of the Institutional Animal Ethics Committee. The animals were under suitable hygiene conditions, at a temperature of 23 ± 2°C, light/dark cycle 12-h, and humidity of 55 ± 5%. The animals were housed in individual cages and also had free ad libitum access to water and food. This study was approved by Ethical committee of Mazandaran University of Medical Sciences (ID#IR.MAZUMS.REC.1400.8619). The animal study were performed according to Health guide for the care and use of Laboratory animals approved by the Mazandaran University of Medical Sciences, Iran, Sari.
In the present research, animals were randomly distributed into eight groups (8 animals/group) as follows: Control group (I), mice received distilled water via gavage daily for 8 days consecutive; Groups II, III, and IV, mice were received GLZ at three doses of 5, 10 and 25 mg/kg/day; respectively) for 8 successive days via gavage; Group V (IR), lung and cardiac injuries were induced in animals at a single irradiation dose of 6 Gy on the 9th day of study. Group VI, VII, and VIII, mice were treated with GLZ (5, 10, and 15 mg/kg) with IR (6 Gy). The dose of IR was picked out based on the study of others [10, 22] and GLZ based on our previous study [23, 24].
2.3. Irradiation of mice
For animal irradiation, the box was designed in Plexiglas material with 18 separate houses for each mouse separately. On the 9th day of the study, mice without anesthesia were exposed whole body to X-ray at dose 6 Gy with 6 MeV X-ray beam that produced by a clinical linear accelerator (linear accelerator, Siemens, Primus, Germany) at a dose rate of 2 Gy/min. Immediately after irradiation exposure, mice were released from cages and kept under previous standard conditions. An overview of the experimental design is illustrated in Fig. 1.
2.4. Samples collection
One day after irradiation, half of the animals were anesthetized with ketamine (5 mg/kg) and xylazine (50 mg/kg) for biochemical evaluation. The thorax was opened and the lung and heart were removed, were immediately dissected and washed in phosphate buffer saline (PBS), and stored at − 80°C for evaluation of oxidative stress parameters. The other half was anesthetized a week later for histological assay. For histopathological examination, lung and heart were removed, fixed in 10% neutral buffered formalin.
2.5. Biochemical assays
Malondialdehyde (MDA) levels, as lipid peroxidation marker, were determined by the measurement of thiobarbituric acid (TBA) in the heart and lung tissues. Frozen heart and lung samples were homogenized in mannitol buffer at 4°C using a variable-speed homogenizer. After homogenization, samples were blended with 2-thiobarbituric acid, phosphoric acid, and distilled water. The mixture was boiled for 45 min, then was cooled, later n-butanol was added to extract the cold thiobarbituric acid reactants. Then, samples were centrifuged (3500 rpm, 10 min). The n-butanol layer optical density was distinguished by spectrophotometry. A standard curve of MDA was drawn. MDA content was expressed as nmol/gr tissue.
The glutathione, as an antioxidant, levels in the heart and lung were calculated using 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) reagent. The reaction between thiol and DTNAB produces yellow color that absorbance was read at 412 nm and introduced as µM [17].
The protein carbonyl (PC), as a biomarker of severe oxidative protein damage, levels in heart and lung supernatants were evaluated by using 2,4dinitrophenylhydrazine (DNPH) reagent. In this assay, 200 µL of sample supernatant was mixed with 300 µL of DNPH, and the mixture was heated in the bath at 37 0C for 30 min, then was centrifuged (10 min at 10000 ×g). After washing with ethanol-ethyl acetate, the mixture was centrifuged again. The precipitated proteins were dissolved in 600 µL guanidine hydrochloride, and then was heated at 37 0C for 15 min, and centrifuged. The absorbance of samples was read at wavelength 375 nm. Lung and heart protein carbonyl content was introduced as mM [25].
2.6. Histopathological evaluation
The fixed heart and lung were dehydrated in the alcohol series, clarified in the xylene, and embedded in the paraffin. The sections of 5µm thickness were stained with hematoxylin and eosin (H&E) and evaluated under a microscope (Nikon; Tokyo, Japan) with the magnification of ×40 in a blinded fashion. For the semi-quantitative assessment of sections, slides were then evaluated for heart and lung injury using a scoring system. Given the extent and severity of lung injury in each field, inflammatory cell infiltration (neutrophils, erythrocytes, macrophages, lymphocytes) in the alveolar sac and perivascular region, alveolar sac exudation, alveolar sac collapse, hyaline arteriosclerosis, thickened alveolar walls, edema, congestion and hemorrhage, lung damage was scored on a scale from 0 (normal lung), 1 (slight), 2 (mild), 3 (moderate), and 4 (severe) [26]. The heart injury based on the extent and severity of inflammation, interstitial proliferation, myocyte necrosis, myocyte vacuolization, edema, congestion, and hemorrhage in a field was scored on a 0–4 scale. Score 0 was assigned to normal heart, 1 to the lesion was minimal, 2 (mild), 3 (moderate), and 4 (severe) [27]. In the semi-quantitative evaluation, eight slides from each sample and five fields of each slide were evaluated with the light microscope and then, the average scores were considered for each lung and heart sample in the groups.
2.7. Statistical analysis
Data were analyzed by GraphPad Prism (version 6.07, USA) software. All the data are expressed as the mean ± SD. Different groups were compared using one-way ANOVA and Tukey tests to determine differences among the groups. The Kruskal–Wallis test, one of the nonparametric tests, was used for the analysis of the data obtained semiquantitatively in histopathological examinations. A p-value of < 0.05 was considered statistically significant.