Ethical Committee Approval
This study was carried out with the decision of Fırat University Animal Experiments Ethics Committee dated 04.04.2013 and numbered 44.
Funding: Our study was supported by Fırat University Scientific Research Projects Coordination Unit (FÜBAP) (Project no: TF.1327)
Animals
In our study, 28 Wistar Albino male rats, 8-10 weeks old and weighing 250-270 g, were used. animals were divided into 4 groups (7 in each group)and had at libitum access to food and water. They were kept under a photoperiod of 12 hours of light and 12 hours of darkness in controlled temperature conditions of (20-24ºC). The experimental phase was design as 8 weeks.
Groups:
Control Group: No action was taken during the experiment phase.
NAC group: 100 mg/kg of NAC was administered intraperitoneally every day during the experimental period.
DM Group: A single dose of 50mg/kg Streptozotocin (STZ) was dissolved in 0.1 M sodium citrate Buffer (pH: 4.5) and administered intraperitoneally (i.p). After 72 hours, blood samples were taken from the tail veins of rats fasted for 12 hours. Blood glucose was measured in a glucometer device and rats whose fasting blood glucose level exceeded 250 mg/dl were considered diabetic.
DM + NAC group: In addition to the diabetes group, 100mg/kg NAC was administered i.p. every day during the experimental period.
At the end of the experiment, rats in all groups were anesthetized by i.p administration of ketamine (75 mg/kg) + xylazine (10 mg/kg), and testicular tissues were rapidly removed. Tissue samples were stored under suitable conditions for histological and biochemical studies.
Malondialdehyde (MDA) levels
Firstly, the tissues were homogenized. Then it was centrifuged and the supernatant was taken into another 1 ml tube. 1ml 10% TCA, 1ml 0.6% TBA, 1ml distilled water and 0.5ml 4% HCl were added to the tube and mixed. The prepared mixture was incubated at 90-95 oC for 120 minutes. After incubation, 3 ml of butanol was added and vortexed. After that the tubes were centrifuged at 5000 rpm for 5 min and the butanol phase was read at 532 nm against butanol in the spectrophotometer. Absorbance value (X) was calculated with the formula (X+0.0344)/0.0492). The value found was multiplied by 5 (because tissue homogenate was prepared in 5ml buffer).
Determination of apoptosis
Tunel method was used to determine apoptosis in testicular tissue. Sections of 5-6 μm thickness from paraffin blocks were taken on polylysine slides. Cells undergoing apoptosis were determined using the ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon, cat no: S7101, USA) in accordance with the manufacturer's instructions. Preparations were examined and photographed under the Novel N-800M microscope. In staining with Harris hematoxylin, blue stained nuclei were considered normal and brown stained nuclei were considered apoptotic. At least 500 normal and apoptotic cells were counted under 10x magnification in randomly selected areas. Apoptotic index (AI) was calculated by dividing the apoptotic cells by the total number of cells. Scale bar: 50µm.
Determination of TRPM2 immunoreactivity
Avidin-Biotin-Peroxidase Complex method was applied to determine TRPM2 immunoreactivity in testis tissue. Deparaffinized tissues were incubated with primary antibody (Rabbit Anti-TRPM2 antibody, ab101738, Abcam, Cambridge, UK) for 60 minutes. Then the tissues were incubated with secondary antibody (biotinylated Goat Anti-Polyvalent (anti-mouse / rabbit IgG), TP–125-BN, Lab Vision Corporation, USA) for 30 minutes in a humid environment at room temperature. Fast Red Substrate System (TA-125-AF, Lab Vision Corporation, USA) solution was dripped onto the tissues. Then staining was done with Mayer's hematoxylin and observed under a light microscope. Positive and negative controls were made according to the manufacturer instructions. The preparations were examined and photographed under the Novel N-800M microscope. Evaluation of immunohistochemical staining was based on the intensity of staining. The intensity of immunostaining was scored semi-quantitatively with numbers from 0 to +3.
Real Time-Polymerase Chain Reaction
RNA isolation
PureLink™ RNA Mini Kit was used for RNA isolation from testicular tissue. Lysis buffer solution was obtained by mixed 1 ml of lysis buffer and 10 μl of 2-mercaptoethanol in the falcon tube. Testicular tissue, homogenizer beads and 600 µl lysis buffer solution were placed in Eppendorf tubes. The tissues were lysed in a homogenizer for 6 minutes and then centrifuged at 12.000xg for 2 minutes at room temperature. All of the RNA-containing liquid phase was taken to a new eppendorf tube and isolations were performed according to the recommended protocols of manufacturer. Total RNA was stored at -80 °C until use.
cDNA synthesis
In our study, Qubit® RNA Assay Kit was used for RNA measurement. RNA amounts were measured spectrophotometrically and equalized for cDNA synthesis. cDNA synthesis was carried out using 10µl of RNA sample, 2 µl of 10XRT random primer, 2 µl of 10XRT buffer, 0.8 µl of 25XdNTP mix, 4.2 µl of nuclease-free water and 1µl of MultiScribe™Reverse Transcriptase enzyme in Termal cycler device. cDNA samples were stored at -20 °C until use.
Real-time PCR
For the determination of TRPM2 expression levels, TRPM2 primer (catalog number: Rn-01429417-m1), TaqMan Master Mix were used and reactions were implemented in Applied Biosystems 7500 Real-Time PCR system. For the normalization of mRNA expression levels, GAPDH (catalog number: Rn-01775763-g1) was used as Housekeeping Gene.
Statistical analysis
Obtained data were determined as mean ± standard deviation. Graphpad software was used for statistical analysis. Groups were analyzed using One-way ANOVA and posthoc tukey test. p<0.05 values were considered statistically significant.