Materials
Sarpogrelate obtained from Shanghai Linebon Ltd. Shanghai, China. Bromocriptine was from Novartis, Italy. Cabergoline was from Pfizer, Italy. Domperidone was from Jamjoom pharmaceutical company, KSA. Alloxan obtained from Sigma‐Aldrich (St Louis, MO, USA). Urea; Creatinine; Alkaline Phosphatase; Aspartate Aminotransferase Activity; LDH-1 assay kits; and TNF alpha ELISA Kit obtained from Abcam company. Troponin I test kit from Encode Medical Engineering Company. 2,3,5-Triphenyltetrazolium chloride from Gold Biotechnology, USA.
Animals
Wister albino male rats 5–7 weeks of age weighing 150–200g obtained from the animal house of Batterjee medical college. The animals were housed three per cage under controlled standard laboratory conditions in monitored ventilated cages and spontaneously given food and water. The steps of the research were approved by the ethical committee of research of Batterjee medical college. Tutelage was taken, particularly with relevant housing conditions, to avoid or minimize the animals' discomfort. The animals kept on solid floored cages with a deep layer of sawdust, and cages changed daily. Three laboratory technicians administered the treatment, and two investigators collected the data randomly. Data was coded prior to analysis so that the treatment group cannot be identified before analysis is completed. At the end of the study, all animals were euthanized by thiopental (intravenous injection, 150 mg/kg) for tissue collection.
Induction of diabetes by alloxan
Alloxan monohydrate dissolved in sterile normal saline. Diabetes was induced in 30 rats (150-200 g) by a single intraperitoneal injection of alloxan (5%) 150 mg/kg. The rats kept fasting for 12 h before the injection of alloxan. Fasting plasma glucose measured by obtaining tail blood samples. The rats which showed a plasma glucose level of 200 mg/dl or more were considered diabetic and taken in the study [22].
Experimental design
Tests took place four weeks after the induction of diabetes. The rats divided randomly into six diabetic groups and one healthy control group, each group of 6 rats. The sampling calculation is done based on resource equation method [23]. Once there was a stable rising in the urea and creatinine levels in the blood, drugs injected.
- The normal control group (Saline, IP)
- Diabetic control group (Saline, IP)
- Diabetic group treated with bromocriptine (4 mg/kg, IP)
- Diabetic group treated with cabergoline (0.6 mg/kg ,IP)
- Diabetic group treated with sarpogrelate (50 mg/kg, IP)
- Diabetic group treated with a combination between bromocriptine and sarpogrelate by the same doses.
- Diabetic group treated with a combination between cabergoline and sarpogrelate by the same doses.
Determination of blood glucose level
Blood glucose levels tested on the 0 day, 1st, 7th, 14th, and 21st days from the start of the experiment. Blood samples collected from the tail of the fasting animals. One millimeter of its end cut and a drop of blood used for blood glucose tests using an advanced glucometer (Roche, USA). The accuracy of glucometer checked with the O-toluidine method [24].
Blood Pressure Recording
Basal blood pressure was measured employing a non-invasive blood pressure recorder apparatus (Ugobasile instruments, Italy).
Each rat placed in an exceeding restrainer and appropriate cuff sensor mounted on its tail and warmed to about 33- 35 °C. The tail cuff was inflated to a pressure above 200 mmHg, systolic blood pressure; diastolic blood pressure was measured directly by the tail-cuff and pulse sensor two hours after treatment of drugs [25].
Estimation of liver & kidney functions
Alkaline phosphatase and aspartate aminotransferase activities determined using the method described by King and King (1954) [26]. The procedure of Tietz et al. (1994) [27] used to determine serum creatinine concentration, while the serum urea concentration determined by the method of Kaplan (1965) [28]. After the end of the study, the kidneys washed with normal saline and weighed. The kidney hypertrophy index calculated by determining the kidney weight and body weight ratio (g/g) x103.
Estimation of serum biomarkers of myocardial injury
The blood was withdrawn through the retro-orbital venous plexus method, kept at 37°C for 30 mins, and centrifuged at 4°C, 3000 rpm for 10 mins. Then the separated serum stored at -20°C for various biochemical analyses. The severity of the cardiac injury assessed by the estimation of lactate dehydrogenase (LDH-1) and cardiac troponin I (cTnI) in serum. LDH-1 and troponin levels analyzed by spectrophotometric methods using commercially available diagnostic kits consistent with the methods of Nieland, 1955 [29].
Serum TNF-α1 concentrations
Serum TNF-alpha cytokines level was identified by the ELISA technique using a quantitative sandwich enzyme immunoassay technique (Abcam Company). The test was done according to the company's instructions. The ELISA reader (optical density at 405nm immediately) calculated and applied on a standard curve to sort out the concentration of the cytokines.
Evaluation of Myocardial Infarct Size by TTC
For evaluation of the myocardial infarcted area, the hearts were removed, washed in phosphate-buffered saline, frozen and stored at −20°C, the frozen hearts sliced into 1 mm sections along the long axis from apex to base. Triphenyl tetrazolium chloride (TTC) staining accustomed to assess myocardial tissue viability and determine myocardial infarction size. The tissue slices incubated in 1% TTC PBS solution, pH 7.4, at 37°C for 20 min. Tissues fixed in 10% PBS-buffered formalin overnight at 2°C–8°C. Either side of every TTC-stained tissue slice was photographed with the photographic camera to differentiate the red-stained viable and also the white-unstained necrotic tissues [30]. Digital photographs downloaded to a computer. Areas stained in white and red were measured using SigmaScan software (SPSS Science) in trace-measurement mode. That mode was wont to measure either the ischemic or the infarcted areas, which may be a sum of calibrated pixels during a defined region. This was done manually by drawing an image layer on the photograph [31]. The following equation calculated the infarction size percentage:
The percentage of Infarct volume = Infarct volume / Total volume of slice X 100
Statistical analysis
The data and statistical analysis done comply with the recommendations on experimental design and analysis in pharmacology [32]. The results expressed as mean± SE. The significance of the differences between the values performed by a one-way ANOVA test and Dunnett's Multiple Comparison Test using GraphPad Prism software. P < 0.05 was considered to be a significant difference.