Ethical statement
This study was approved by Adiyaman University Local Ethics Committee on Animal Experiments (Protocol No: 2018/008). The animals used in the experimental study were obtained from the Research Center unit of Adiyaman University. The experimental applications and maintenance were carried out there. After the experiments, the rat tissues were studied at Adiyaman University Faculty of Medicine in Histology and Embryology department, whereas sera were studied at the Faculty of Pharmacy in the biochemistry laboratory.
Induction of diabetes
Diabetes was induced via a single dose intraperitoneal (i.p.) injection of 50 mg/kg of Streptozocin (STZ) dissolved in 0.1 M buffer (pH: 4.5) of sodium citrate.
Chemicals and kits
STZ was purchased from a pharmaceutical chemical company, namely Sigma Chemical Co., St. Louis, Missouri, USA, and NAC was purchased from Husnu Arsan pharmaceutical industry.
Irisin was purchased from another pharmaceutical company, namely Irisin Rabbit Polyclonal H-067-17, Phoenix Pharmaceuticals, Inc., located in California, USA. Caspase-3 was purchased from Caspase-3, Rabbit Polyclonal IgG, Abcam, ab2302, situated in London, UK, and was measured using the immunohistochemical staining method. The highest quality solvents and chemicals available in the market were used in MDA and other measurement parameters. Total antioxidant status (TAS) and Total oxidant status (TOS) activation were studied in rats using ELISA kits (TAS ELISA kit: Diagnostics, Gaziantep Turkey, Lot No: 201912, TOS ELISA kit: Rel Assay Diagnostics, Gaziantep, Turkey, Lot No: 201925).
Animals
All the experiments were carried out following the ethical standards of the institution where the study was conducted. Twenty-eight male Wistar rats weighing 220–300 g and aged 10–12 weeks purchased from the Experimental Animal Unit of Adiyaman Application and Research Center (Adiyaman, Turkey) were used in the experiments. The experimental rats were divided into four groups, each containing 7 Wistar rats. Before carrying out the investigations, all the rats were kept under the same laboratory conditions for 14 days. The laboratory conditions were as follows: 55±5 % relative humidity, 22 ± 3 °C temperature, 12 hours day and 12 hours night period, fed with standard diet (DSA Poultry, Kirikkale, Turkey) and tap water ad libitum, respectively.
Experimental design
The male Wistar rats were divided into four groups randomly. There were 7 rats in each group and 30-d regimens of experimental or control groups. Animals were untreated in the control group. The treatments applied in the experimental groups were as follows: NAC treated group: 100mg/kg i.p. once daily, NAC (Sigma Chemical Company, USA) was administered intraperitoneally (i.p) to animals for 30 days starting on day 1 (Rana et al. 2006); and Diabetes-induced group: Single dose intraperitoneal injection of STZ (50mg/kg B.W.) which was dissolved in buffer (pH 4.5) containing 0.1M citrate was used to induce diabetes mellitus in overnight fasting Wistar rats (Baydas et al. 2003). By determining blood glucose concentration in STZ-induced rats 72 hours after injection of STZ, diabetes was assessed. The experimental animals detected to have blood glucose levels above 250 mg/dl were used for the experimental studies (Gozel et al. 2018; Pavana et al. 2009); DM + NAC group: STZ induced DM plus NAC as described previously. The doses, application methods, and duration of treatment of STZ and antioxidant agents (NAC) were arranged according to previous studies. Blood samples were taken by cardiac puncture at the end of the study before euthanasia. Then, by performing anesthesia using ketamine overdose (Ketalar; Eczacibasi Warner-Lambert, Levent, Istanbul, Turkey), all the animals were sacrificed, and their tissue samples were obtained accordingly. Blood was collected through a microcapillary tube from all rats, and biochemical analyses were performed to measure TAS, TOS, and MDA levels. Each liver sample was weighed, then prepared for histopathologic evaluation by light microscopy.
Taking liver samples
The experimental animals were anesthetized by intraperitoneal administration of ketamine ((75 mg/kg) + xylazine (10 mg/kg)) 24 hours after the last experiment. Then, the liver tissues of anesthetized animals were quickly removed and placed in a 10% formalin solution. For conducting an accurate study of TAS, TOS, and MDA parameters, some of the tissue samples were stored at – 80 0C, whereas some were placed in a 10% formalin solution and evaluated for 24 hours. Liver tissues were passed through a series of routine histological follow-ups and were eventually embedded in paraffin blocks. They were stored under suitable laboratory conditions for immunohistochemical and histopathological examinations.
TAS, TOS, and MDA measurements in tissue
After washing liver tissues with cold PBS buffer for tissue TAS and TOS levels, liver tissues were kept at -80 0C until analysis. Liver tissues obtained from rats were homogenized in ice-cold 0.1 M phosphate buffer with pH: 7.4 in a 1/10 ratio and were centrifuged at 1800 rpm. ELISA reader Bio-Tek ELX800 (BioTek Instruments, USA) was used in order to provide the reading for supernatants for TAS as Trolox equivalent/L µmol at 660 nm wavelength (Erel 2004) and Supernatants for TOS as µmol H2O2 Equiv./L at 530 nm wavelength (Erel 2005).
In the experimental setup, the rat liver tissues were homogenized in the buffer solution with a homogenizer for 3 minutes at 16000 rpm for MDA. (Ultra Turrax Type T25-B, IKA Labortechnic, Germany). Esterbauer and Cheeseman's 1990 method was used for this purpose. Malondialdehyde forms a pink-colored chromogen by reacting with thiobarbituric acid at 90-95 0C. The sample absorbance was observed to be cooled rapidly after fifteen minutes and was read at 532 nm spectrophotometrically (Esterbauer and Cheeseman 1990). The obtained results were expressed in nmol/g.
Hematoxylin-Eosin (H&E) staining method
Liver tissues were passed through a series of routine histological follow-ups and were eventually embedded in paraffin blocks. 4–6 mm thick sections from these blocks were prepared histopathologically after Hematoxylin-Eosin (H&E) staining. Using a Leica DM500 microscope (Leica DFC295), preparations were examined and photographed respectively.
Immunohistochemical staining method
4-6 mm thick sections taken from paraffin blocks were put into polylysine slides. Initially, deparaffinized tissues were passed through graded series of alcohol and were boiled accordingly in a citrate buffer solution in order to retrieve the antigen at pH: 6 in a microwave oven (750 W) for 7 + 5 minutes. After boiling the tissues that were kept at room temperature for about 20 minutes, they were washed with phosphate-buffered saline ((PBS), P4417, Sigma-Aldrich, USA) for 3 × 5 minutes. Then, in order to prevent endogen peroxidase activity, the tissues were incubated with a hydrogen peroxide block solution (Hydrogen Peroxide Block, TA-125-HP, Lab Vision Corporation, USA) for 5 minutes. After applying Ultra V Block solution to the tissues that were washed with PBS for 5 minutes to prevent floor staining, primary antibodies diluted at 1/200 were incubated with irisin and caspase-3 for each section in a humid environment for 60 minutes. Then, after being washed with PBS for 3 × 5 minutes, the tissues were incubated with a secondary antibody (Biotinylated Goat Anti-Polyvalent (anti-mouse/rabbit IgG), TP-125-BN, Lab Vision Corporation, USA) for 30 minutes at room temperature and in a humid environment. After incubating with a secondary antibody, the tissues were incubated with streptavidin peroxidase (TS-125-HR, Lab Vision Corporation, USA) for 30 minutes after being washed with PBS for three × 5 minutes and then taken into PBS. 3-amino-9-ethyl carbazole (AEC) Substrate and AEC Chromogen (AEC Substrate, TA-015 and HAS, AEC Chromogen, TA-002-HAC, Lab Vision Corporation, USA) solution were dripped on tissues, and the image was taken on a light microscope. After taking the image, the tissues were washed with PBS. After being passed through distilled water and PBS, the contrast-stained tissues with Mayer's hematoxylin were closed with an appropriate closure solution (Large Volume Vision Mount, TA-125-UG, Lab Vision Corporation, USA). Using a Leica DM500 microscope (Leica DFC295), the preparations were examined and photographed respectively.
Histoscore was calculated for immunoreactivity prevalence (0.1: < 25%, 0.4: 26-50%, 0.6: 51-75%, 0.9: 76-100%) and intensity (0: non-staining, + 0.5: very less, + 1: less, + 2: median, + 3: strong) in staining. The formulation for Histoscore calculation is as follows: Histoscore=prevalence x intensity.
Data analysis
SPSS 15.0 program was used for the statistical analyses. The normal distributions of immune variable groups and TAS, TOS, and MDA were assessed using the Kolmogorov-Smirnov test. One-way analysis of variance (ANOVA) was performed for group comparisons of TOS, TAS, MDA, and immunity variables. For the homogeneity test of variances, Levene’s statistic was applied accordingly. In order to identify the differences between significant variable groups, Tukey’s pair-wise multiple comparison tests were conducted. The obtained results were given as mean ± Standard Deviation (S.D.). The significance level was determined to be at least 0.05 (p< 0.05).