Materials
Sarpogrelate was obtained from Shanghai Linebon Ltd. Shanghai, China. Bromocriptine was from Novartis, Italy. Cabergoline was purchased from Pfizer, Italy. Domperidone was purchased from Jamjoom pharmaceutical company, KSA. Alloxan was obtained from Sigma‐Aldrich (St Louis, MO, USA). Urea; Creatinine; Alkaline Phosphatase; Aspartate Aminotransferase Activity; LDH-1 assay kits; and TNF alpha ELISA Kit were obtained from Abcam company. Troponin I test kit was obtained from Encode Medical Engineering Company, Jeddah, KSA. 2,3,5-Triphenyltetrazolium chloride was obtained from Gold Biotechnology, USA.
Animals
Forty-two Wister albino male rats 5–7 weeks of age weighing 150–200 g were obtained from the animal house of Batterjee medical college, Jeddah, KSA. The animals were housed three per cage under controlled standard laboratory conditions in monitored ventilated cages and spontaneously given food and water. The ethical committee of research of Batterjee medical college approved the steps of the investigation. Tutelage was taken, particularly with relevant housing conditions, to avoid or minimize the animals' discomfort. The animals were kept on solid floored cages with a deep layer of sawdust, and the cages were changed daily. Data were 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 was 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 b.w. The rats kept fasting for 12 h before the injection of alloxan. Fasting plasma glucose was measured in blood samples collected from the tail vein. 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 diabetic rats divided randomly into six diabetic groups and one healthy control group, each group of 6 rats. The sampling calculation was done based on the resource equation method [23]. Once there was a stable rising in the urea and creatinine levels in the blood, drugs were injected once daily for one month.
- The normal control group (Saline, IP)
- Diabetic control group (Saline, IP)
- Diabetic group treated with bromocriptine (4 mg/kg b.w, IP) [24]
- Diabetic group treated with cabergoline (0.6 mg/kg b.w ,IP) [25]
- Diabetic group treated with sarpogrelate (50 mg/kg b.w, IP) [26]
- Diabetic group treated with a combination between bromocriptine and sarpogrelate at the same doses.
- Diabetic group treated with a combination between cabergoline and sarpogrelate at the same doses.
Determination of blood glucose level
Blood glucose levels were tested on the 7th, 14th, 21th, and 28th days from the beginning of the treatment. One millimeter of its end cut and a drop of blood was used for blood glucose tests using an advanced glucometer (Roche, USA). The accuracy of glucometer was checked with the O-toluidine method [27].
Blood Pressure Recording
Basal blood pressure was measured employing tail-cuff non-invasive blood pressure recorder apparatus (Ugobasile instruments, Italy). The measurement of the mean systolic blood pressure for each group of rats was done once time weekly for one month.
Each rat was placed in an exceeding restrainer, and an 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 [28].
Estimation of liver & kidney functions
At the end of the study, the blood sample was withdrawn through the retro-orbital venous plexus method, kept at 37°C for 30 min, and centrifuged at 4°C, 3000 rpm for 10 min. Then the separated serum was stored at -20°C for various biochemical analyses. Alkaline phosphatase and aspartate aminotransferase activities were determined using the method described by King and King (1954) [29]. The procedure of Tietz et al. (1994) [30] was used to determine serum creatinine concentration, while the serum urea concentration was determined by the method of Kaplan (1965) [31]. After the end of the study, all of the rats were sacrificed. The kidneys samples were collected, washed with normal saline, and weighed. The kidney hypertrophy index was calculated by determining the kidney weight and body weight ratio (g/g) x103.
Estimation of serum biomarkers of myocardial injury
The severity of the cardiac injury was assessed by estimating lactate dehydrogenase (LDH-1) and cardiac troponin I (cTnI) in serum. LDH-1 and troponin levels were analyzed by spectrophotometric methods using commercially available diagnostic kits consistent with the methods of Nieland [32].
Serum TNF-α concentration
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's optical density at 405nm was immediately calculated and applied on a standard curve to sort out the cytokines' concentration.
Evaluation of Myocardial Infarct Size by TTC
For assessment of the myocardial infarcted area, the hearts were removed, washed in phosphate-buffered saline, frozen, and stored at −20°C. The frozen hearts were sliced into 1 mm sections along the long axis from apex to base. Triphenyl tetrazolium chloride (TTC) staining is accustomed to assess myocardial tissue viability and determine myocardial infarction size. The tissue slices were incubated in 1% TTC PBS solution, pH 7.4, at 37°C for 20 min. Tissues were 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 white-unstained necrotic tissues [33]. Digital photographs are 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 [34]. The infarction size percentage was calculated using the following equation:
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 [35]. The results expressed as mean± SE. The significance of the differences between the values were 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.