Animals
In this experimental study, 30 healthy adult male Wistar rats (Rattus norvegicus) with eight weeks old and weighing between 200 to 250 g were obtained from Pasteur Institute of Iran (Tehran, Iran). The rats were exclusively housed in plastic cages covered by sawdust bedding in the temperature of 20 ± 2 ° C and humidity about 60%. All rats had access to a standard diet and urban water. The. The lighting period was held at 12h light/12h dark cycle. All the procedures were approved and monitored by the ethics committee of laboratory animals of Islamic Azad University (Tehran, Iran). The procedures followed the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978) (Ethical code: IR.MUK.REC.1398.242). In this study, the animals were randomly assigned to 5 groups (n=6). Groups were as follows:
1) CO: control (rats were not received any treatments)
2) DFO group: rats were received deep-frying oil
3) DFO+EXE group: rats were received deep-frying oil deep-frying oil, then did aerobic exercise
4) DFO+OCT group: rats were received deep-frying oil deep-frying oil, then were prescribed octopamine.
5) DFO+EXE+OCT group: rats were received deep-frying oil deep-frying oil, then did aerobic exercise and were prescribed octopamine simultaneously.
Chemicals
Deep-frying oil Preparation
Deep-frying oil (DFO) was made by frying 5 kg catfish three times in 2.5 L cooking palm oil at 200ºC (measured with a cooking thermometer) for 15 min for 3-times [18-20]. After every frying, the oil was let to cool at room temperature for 5 hours [21]. For DFO, lipid hydroperoxide content was 90.94 (meq/kg), also total fatty acid composition represent in table1. DFO was gavage five days per week (10 ml/kg) for four weeks.
Octopamine
OCT (Sigma, UK) was dissolved in distilled water and then diluted with a Krebs-bicarbonate solution. OCT (100 µL in each injection) was injected intraperitoneally five days per week (10 mL/kg) for four weeks.
Aerobic exercise protocol
Rats of EXE group were introduced to treadmill running for a habituation period, during which each animal exercised on a motorized rodent treadmill at 9 m/min for 20 min per day (including 10 min at a prescribed speed, a 5-min warm-up, and 5-min cool-down). After the habituation period, rats were subjected to run at moderate exercise intensity for five days per week over four weeks (total duration of the exercise protocol and OCT supplementation). On the first day of exercise, the training began with the speed of 11 m/min for 10 min per day. The speed gradually increased to 20 m/min over the duration of the experiment. The exercise duration was also increased 2 min per day over the same period until it reached 26 min/day at the end of the second week.
Animal tissue sampling
Forty-eight hours after the last training session, all rats were sacrificed and their heart tissue were removed immediately and washed in saline normal 0.9%. The heart tissues (ventricle) were divided into two parts for freezing (-80 0C) and fixing (10% neutralized formalin). Bodyweight and heart weight also measure at the end of the protocol (Table 2).
Terminal transferase dUTP nick end labeling (TUNEL) assay
The apoptotic effects of aerobic exercise and octopamine on heart tissue samples in DFO treated rats were determined using the transferase-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick end labeling assay. Samples were fixed with 4 % (w/v) paraformaldehyde and processed using a commercial kit (In Situ Cell Death Detection Kit; Roche, Germany) in accordance with the manufacturer’s instructions. Cells were stained with Hoechst dye and imaged in a fluorescent microscope. The data were expressed as a percentage of the area of terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells in ten random fields. The number of TUNEL positive cells was measured carefully in 3 sections of the heart tissue per animal. The percentage of positively stained cells in the area was analyzed with Image J software v1.8 (NIH, USA).
Histopathological examination: H&E and Masson's trichrome staining
Histopathological examination of cardiac muscle tissue was done using hematoxylin and eosin (H&E) staining. To perfusion fixation, fresh heart tissues were placed into 10 % formalin and then dehydrated using alcohol. Finally, samples were embedded in paraffin, sectioned, and stained with H&E (Sigma-Aldrich, UK). H&E staining was performed according to the previously described methods [22]. Myofibers were counted in 10 fields of muscle sections for each rat.
Masson's trichrome stain (Histogenotech. Co., Tehran, Iran) were performed according to kit instructions except Aniline Blue-Solution incubation time which was 90 min in this study. The prolonged incubation is a standard procedure of trichrome staining for muscle. Briefly, heart tissue slides deparaffinized by submerging into three series of absolute xylene for 4 min. After that, the slides were washed with running tap water for 2 min. Then, slides were treated with the phosphomolybdic acid solution for another 10 min as a mordant. Next, slides immediately were submerged into methyl blue (Merck, Germany). Finally, slides were washed in running water for 2 min and treated with 1% acetic acid solution for 1 min.
RNA extraction
RNA were extracted from the heart tissue using TRizol reagent (Invitrogen, USA) according to the manufacturer’s instruction. The quality of the extracted RNA was determined based on the 260/280 absorbance ratio, measured by the NanoDrop spectrometer (Thermo Scientific, USA).
cDNA synthesis and quantitative RT-PCR analysis (qRT-PCR)
Synthesis of cDNA was carried out using a universal cDNA synthesis kit (Exiqon, Denmark) according to manufacturer's instructions. Specific primers for Caspase 3 and GAPDH (as housekeeping gene) genes were designed using Primer Premier 5 software (Premier Bio-Soft International, Palo Alto, CA, USA). Their sequences are presented in Table 3. Real-time quantitative PCR reactions were carried out in triplicate by using standard protocols with an ABI step one system (ABI, USA). Briefly, in a total volume of 10 μl, 20 ng/μl of cDNAs were added to a master mix comprising 10 pmol/μl of each primer (Caspase 3) and 5 mL of SYBR premix ExTaq II (Thermo Scientific, USA). The program for the run was set as follows: 95 °C for 15 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The PCR reaction was followed by a melting curve program (60–95 °C with a temperature transition rate of 1 °C s−1 and a continuous fluorescence reading). GAPDH was quantified as the reference to normalize differences in total RNA levels. The calculation was performed as follows:
Western blotting analysis
Heart tissue was lysed in lysis buffer (RIPA, Beyotime Institute of Biotechnology) supplemented with protease inhibitors (PMSF, Aladdin). The equal amounts of protein were separated by SDS-PAGE with 5%–12% Tris-Glycine gel (Invitrogen) and subjected to standard western blotting analysis. Western blotting was performed using Caspase 3 and pro-Caspase 3 antibodies (1:1000 dilution; Abcam) followed by treating with the corresponding secondary HRP-conjugated antibodies (1:2000 dilution; Abcam, USA). Finally, blots were developed using the ECL western blotting detection system (Amersham Life Sciences Inc., Arlington Heights, IL). The results were subjected to densitometry analysis using the ImageJ software in a blind analysis manner. To ensure that the same amount of proteins was loaded, the GAPDH protein was employed as an internal control.
Statistical analysis
The statistical analysis of obtained data was performed using the Graph Pad Prism statistical software version 5.01 (GraphPad, San Diego, CA, USA). The normality of the data was evaluated using the Kolmogorov–Smirnov test, two-way ANOVA. Tukey post-Hoc test was used to analyze the data from different groups. Data are expressed as the mean ± SD. The level of significance was set at P<0.05.