2. Materials and methods
2.1. Chemicals Zingerone was obtained from Alfa Aesar, Heysham, UK. Cisplatin, Bradford reagent, trichloroacetic acid (TCA), thiobarbituric acid (TBA), reduced glutathione (GSH), glacial metaphosphoric acid (Gmpa), 5,5ˊ-dithiobis-(2-nitrobenzoic acid) (DTNB), proteinase K and agarose were purchased from Sigma-Aldrich Co, MO, USA. All other chemicals and solvents used were of the highest purity grade available.
2.2. Animals Sixty Adult male albino rats (180-200g) were obtained from the animal farm of the Egyptian Holding Company for Biological Products and Vaccines (VACSERA), Cairo, Egypt. Animals were maintained in a well-ventilated room with 12:12 h light/dark cycle in polypropylene cages and kept on a standard …show more content…
Additionally, plasma was used to determine cardiac troponin T (cTnT) and B-natriuretic peptide (BNP) levels using enzyme linked immunosorbent assay (ELISA) standard kits (MyBioSource Inc., San Diego, CA, USA) according to the manufacturer's instruction.
2.6. Assessment of cardiac oxidative stress Heart homogenates were used to estimate malondialdehyde (MDA) level as one of the main end products of lipid peroxidation (Yoshioka et al., 1979), reduced glutathione (GSH) content (Beutler et al., 1963), superoxide dismutase (SOD) (SOD assay kit, Sigma-Aldrich Co, St Louis, MO, USA) and catalase (CAT) activities (Sinha, 1972).
2.7. Assessment of cardiac inflammation Sera were used to estimate TNF-α concentration using commercially available ELISA kit (Boster Biological Technology, Pleasanton, CA, USA) while Myeloperoxidase (MPO) activity was determined in cardiac tissue homogenate using ELISA kit (Linco research inc., St. Charles, MO, USA) according to the manufacturer's instructions.
2.8. Western blot …show more content…
DNA fragmentation assay DNA extraction was done by a standard procedure using proteinase K digestion with phenol: chloroform: isoamyl alcohol extraction method and 10 μg of the extracted DNA were loaded onto 2% agarose gel containing 0.5 μg/ml ethidium bromide. DNA fragments were separated by gel electrophoresis at 80 V for 90 min in Tris-acetate-EDTA (TAE) buffer. Gels were illuminated with 300 nm UV light using UV transilluminator and a photographic record was made.
2.10. Histopathology Immediately after dissection, heart tissues (n=3/group) were excised and immersed in 10% neutral buffered formalin solution. Heart samples were processed (dehydrated in graded concentrations of alcohol, immersed in xylene) and embedded in paraffin. Sections were cut at 5μm thicknesses on a rotary microtome, mounted and stained with hematoxylin and eosin. These sections were evaluated for histological changes under light microscopy.
2.11. Statistical
2.1. Chemicals Zingerone was obtained from Alfa Aesar, Heysham, UK. Cisplatin, Bradford reagent, trichloroacetic acid (TCA), thiobarbituric acid (TBA), reduced glutathione (GSH), glacial metaphosphoric acid (Gmpa), 5,5ˊ-dithiobis-(2-nitrobenzoic acid) (DTNB), proteinase K and agarose were purchased from Sigma-Aldrich Co, MO, USA. All other chemicals and solvents used were of the highest purity grade available.
2.2. Animals Sixty Adult male albino rats (180-200g) were obtained from the animal farm of the Egyptian Holding Company for Biological Products and Vaccines (VACSERA), Cairo, Egypt. Animals were maintained in a well-ventilated room with 12:12 h light/dark cycle in polypropylene cages and kept on a standard …show more content…
Additionally, plasma was used to determine cardiac troponin T (cTnT) and B-natriuretic peptide (BNP) levels using enzyme linked immunosorbent assay (ELISA) standard kits (MyBioSource Inc., San Diego, CA, USA) according to the manufacturer's instruction.
2.6. Assessment of cardiac oxidative stress Heart homogenates were used to estimate malondialdehyde (MDA) level as one of the main end products of lipid peroxidation (Yoshioka et al., 1979), reduced glutathione (GSH) content (Beutler et al., 1963), superoxide dismutase (SOD) (SOD assay kit, Sigma-Aldrich Co, St Louis, MO, USA) and catalase (CAT) activities (Sinha, 1972).
2.7. Assessment of cardiac inflammation Sera were used to estimate TNF-α concentration using commercially available ELISA kit (Boster Biological Technology, Pleasanton, CA, USA) while Myeloperoxidase (MPO) activity was determined in cardiac tissue homogenate using ELISA kit (Linco research inc., St. Charles, MO, USA) according to the manufacturer's instructions.
2.8. Western blot …show more content…
DNA fragmentation assay DNA extraction was done by a standard procedure using proteinase K digestion with phenol: chloroform: isoamyl alcohol extraction method and 10 μg of the extracted DNA were loaded onto 2% agarose gel containing 0.5 μg/ml ethidium bromide. DNA fragments were separated by gel electrophoresis at 80 V for 90 min in Tris-acetate-EDTA (TAE) buffer. Gels were illuminated with 300 nm UV light using UV transilluminator and a photographic record was made.
2.10. Histopathology Immediately after dissection, heart tissues (n=3/group) were excised and immersed in 10% neutral buffered formalin solution. Heart samples were processed (dehydrated in graded concentrations of alcohol, immersed in xylene) and embedded in paraffin. Sections were cut at 5μm thicknesses on a rotary microtome, mounted and stained with hematoxylin and eosin. These sections were evaluated for histological changes under light microscopy.
2.11. Statistical