Male albino Wistar rats (70–80 g) were obtained from the Pharmacy College Animal Care Center at King Saud University. The animals were acclimatized for 10 days prior to starting the experiments. The rats were housed in standard conditions of 22 ± 1°C, 50–55% humidity, and 12-h day/night cycles. All experimental protocols, including euthanasia procedure, blood sampling, and final sacrifice followed National Institutes of Health guidelines on the care and use of laboratory animals (NIH, 1996), and this animal study was approved by the Ethical Committee of Pharmacy College, Animal Care Center, King Saud University.
HCD in pellet form was prepared by adding 1% cholesterol + 0.5% cholic acid to normal cholesterol rat chow powder (protein 20%, fat 4%, fiber 3.5%, ash 6%, total energy 2850 Kcal/kg). Six rats were fed normal cholesterol rat chow, and eighteen rats were fed HCF for 6 weeks. The rats had free access to water and food throughout the experimental period.
After six weeks, the HCD-fed rats were randomly divided into three groups (n = 6 rats in each group). The four treatment groups in this study were as follows: Group-1, rats fed normal rat chow and treated with vehicle (control group); Group-2, HCD-fed rats treated with vehicle; Group-3, HCD-fed rats treated with BL (25 mg/kg/day, orally, “low dose”) for four weeks; Group-4, HCD-fed rats treated with BL (50 mg/kg/day, orally, “high dose”) for four weeks. HCD feeding was continued during BL supplementation until the end of experiment. Body weight and general health conditions were carefully monitored weekly throughout the experimental period. Blood samples were collected by cardiac puncture under light ether anesthesia and were centrifuged at 4,000 rpm for 10 min; the serum samples were stored at –20°C until analysis. At the end of the experimental period, animals were decapitated and heart, liver, and kidneys were dissected, and weighed. A small portion of the tissues was immediately dipped into liquid nitrogen for 1 min and then stored at –80°C until analysis. Heart, liver, and kidney tissues were preserved in 10% formaldehyde for histopathological evaluations.
Total cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (LDL), high-density lipoprotein-cholesterol (HDL), creatinine, and blood urea nitrogen (BUN) levels were estimated using commercially available diagnostic kits (Human Diagnostics, Wiesbaden, Germany). The serum activities of creatine kinase-B (CK-B), lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), alanine aminotransferase (ALT), aspartate aminotransferase (AST) were measured using commercially available diagnostic kits (Human Diagnostics, Wiesbaden, Germany). Inflammatory biomarkers, including tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), prostaglandin E-2 (PGE-2), caspase 3, and nitric oxide (NO) were measured using ELISA kits for rats (R&D Systems, USA).
Organ’s (heart, liver and kidney) small portions were homogenized in physiological buffer (1:10, w/v) and TBARS and GSH levels were measured by using ELISA kits (Cayman Chemical Co., USA). In Post-mitochondria supernatants of heart, liver and kidney, enzymatic activities of SOD, CAT and GPx were measured by using ELISA kits (R&D systems Inc., USA).
Across sectional portion of a heart, liver and kidney tissues from each group of treatment were preserved in 10% buffered formalin. The samples were embedded in paraffin blocks and sections of thickness 5 mm were cut using a Leica CM3050 S Research Cryostat (Leica Bio-systems, USA). The sections were stained with H&E. Finally, they were examined under the microscope for histopathological changes by an observer who was blind with respect to the treatment groups.
Data are expressed as the mean ± standard error of the mean (SEM) and were analyzed using one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls multiple comparison tests (n = 6). Differences between groups were considered statistically significant when P ≤ 0.05. All statistical analyses were conducted using GraphPad Prism (v. 5) software.