Drugs and Chemicals
Sigma Chemical Co. provided the ISO and DZ (St. Louis, MO, USA). Agappe Diagnostics provided the aspartate transaminase (AST), alanine transaminase (ALT), creatine kinase (CK-MB), and lactate dehydrogenase (LDH) kits (Kerala, India). Commercially available kits (Qualigens Diagnostics) were used to test the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX), as well as reduced glutathione (GSH) and malondialdehyde (MDA) in cardiac tissues (Mumbai, India). R&D Systems provided ELISA kits for tumour tissue necrosis factor (TNF-α) and interleukin-6 (IL-6) research (Minneapolis, MN, USA).
The JSS College of Pharmacology sold 32 male Wistar-Albino rats weighing between 180 and 200 grammes. The animals were kept under typical laboratory settings, which included a 12 hr light/dark cycle with a temperature of 252°C and a humidity of 50±15%. They were given a week to acclimate to the circumstances of the animal home before the experiment and were given unlimited access to regular laboratory food and water. The experiment followed the rules set out by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), based in New Delhi, India. The Institutional Animal Ethics Committee of JSS College of Pharmacy in Ooty gave its approval to the experimental protocol (Approval No.06, dated,20-06-2019).
Induction of Experimental MI
To produce experimental MI, ISO was dissolved in normal saline and subcutaneously administered into rats (85 mg/ kg) at 24-hour intervals for two days. The ISO dosage was determined based on prior investigations [18, 19] and a pilot trial for fixation.
Animals were randomly assigned into four groups (each with eight rats) and treated as follows after a one-week acclimatisation period:
Sham: animals received distilled water (2 ml/kg) for 6 weeks, followed by injection with normal saline (1 ml) on the 43rd and 44th days.
ISO: animals received distilled water (2 ml/kg) for 6 weeks, followed by injection with ISO (85 mg/kg) on the 43rd and 44th days.
DZ10 + ISO: animals were orally pre-treated with DZ (10 mg/kg/ kg) by gastric gavage needle for 6 weeks, followed by injection with ISO (85 mg/kg) on the 43rd and 44th days.
DZ20 + ISO: animals were orally pre-treated with DZ (20 mg/kg/ kg) by gastric gavage needle for 6 weeks, followed by injection with ISO (85 mg/kg) on the 43rd and 44th days.
Previous investigations [20-22] were used to determine the DZ dosage. Rats were anaesthetized and slaughtered when the animal experiment was completed. The serum was isolated from blood samples using centrifugation. The heart samples were isolated from the surrounding tissues and cleaned twice with ice-cold phosphate buffer saline immediately after blood collection (PBS). To create about 10% w/v homogenates, the samples were homogenised in phosphate buffer (25 mM, pH 7.4). After centrifuging the homogenates at 1700 rpm for 10 minutes, the supernatant was collected and kept at 20°C until biochemical analysis. For histological investigation, some of the heart samples were kept in 10% formalin.
The cardiac apex was kept in 4% paraformaldehyde, processed in ethanol, and embedded in paraffin wax as soon as the blood was collected. The cardiac apex was stained with haematoxylin and eosin (H&E) and examined under a light microscope at a magnification of 100 times (Olympus, Tokyo, Japan).
Evaluation of Lipid Peroxidation and Antioxidant Enzyme Levels
After the experimental treatment, the homogenates of heart tissues were centrifuged at 16,000 rpm for 10 minutes. The supernatant was utilised to test MDA levels, SOD, CAT, and GPX activity, as well as GSH concentrations, using a microplate reader set at 560 and 532 nm, as directed by the manufacturer. The commercially available test kits were provided by Qualigens Diagnostics (Mumbai, India).
Measurement of MI markers in Serum
According to the manufacturer's instructions, commercial kits from Agappe Diagnostics (Kerala, India) were used to test the activities of AST, ALT, CK-MB, and LDH.
Determination of Pro-Inflammatory Cytokines in Heart
According to the manufacturer's instructions, ELISA kits were used to conduct an enzyme immunoassay of tumour tissue necrosis factor (TNF-α) and interleukin-6 (IL-6) in cardiac homogenate (Minneapolis, MN, USA). The colour intensity was measured at 450 nm using a microplate reader, and the cytokines levels were expressed as pg/ml of tissue (Infinite M200 Pro, Tecan, Switzerland).
All data were expressed as mean SD and analysed using one-way ANOVA followed by a post-hoc test to determine the significant difference between groups. The statistical analysis was carried out using GraphPad Prism 8. (GraphPad Software, Inc., San Diego, CA, USA). A p value of 0.05 or less was considered statistically significant in all tests.