Sd 2521

Homogenization of heart tissues was carried out in a 10-fold volume of ice-cooled phosphate-buffered saline (PBS) (ice-cooled, pH 7.4). Following centrifugation at 10,000× g for 20 min and 4 °C, the supernatant was then collected for analysis of oxidative and inflammatory markers. ELISA kits were used in the assessment of the hearts’ content of malondialdehyde (MDA), reduced glutathione (GSH), enzymatic activities of superoxide dismutase (SOD), catalase (CAT) (Cat. No. MD 2529, GR 2511, SD 2521, and CA 2517, Biodiagnostic, Giza, Egypt, respectively), and protein carbonyl, interlukin-6 (IL-6), and tumor necrosis factor α (TNF-α) (Cat. No. ab238536, ab234570, and ab100785, Abcam, Cambridge, UK, respectively).
Nuclear fractions of tissue homogenates were obtained using NE-PER nuclear and cytoplasmic extraction kit (Cat. No. 78833, Thermo Fisher Scientific, Waltham, MA, USA). Protein content of the nuclear extracts was determined, and a volume equivalent to 80 μg was employed in the assessment of the DNA-binding activity of NF-kB p65 using NF-κB p65 ELISA Kit (Cat. No. ab133112, Abcam, Cambridge, UK). Results are expressed as fold change of control.

Each liver sample (1 g) was homogenized with 5 ml phosphate buffer, pH 7.4, on ice using an electrical homogenizer. Tissue homogenate was centrifuged at 1,200 × g for 20 min at 4°C for the separation of supernatants. The supernatant was used for determining the activities of antioxidant enzymes (CAT, SOD, and GPx) and MDA concentration.
Briefly, CAT activity was assessed according to the method described by Aebi (21 (link)) and expressed as microns per gram of tissue. Determination of SOD was done following the instructions of the kits (Biodiagnostic, # SD 2521, Egypt). The increase of the absorbance was monitored at 560 nm over 5 min and expressed as microns per gram (22 (link)). GPx was determined according to the instructions of the Biodiagnostic kit (Biodiagnostic, #GP 2524, Egypt). The decrease of absorbance was recorded at 340 nm over a period of 3 min and expressed as microns per gram (23 (link)). For determination of MDA, the manufacturer's protocol of Biodiagnostic kit was followed (Biodiagnostic, #MD 2529, Egypt), measured spectrophotometrically at 534 nm, and expressed as nanomole per gram of tissue (24 (link)).

The renal homogenate was used to determine the SOD activity using a commercially available kit (SD 25 21, Biodiagnostic, Egypt).