Assessment of nuclear cytoplasmic ratio of Nrf2, myocardial H2S and HIF-1α levels

After ischemia-reperfusion injury, the hearts were isolated for biochemical estimations. One half of the heart portion was homogenized in phosphate buffer saline (PBS, pH: 7.4). It was followed by centrifugation at 5000g for 15 min to obtain clear supernatant solution. The levels of H₂S and HIF-1α were determined in clear supernatant solution. The levels of H₂S were measured in the heart homogenates following ischemia-reperfusion injury using reverse phase high-performance liquid chromatography (HPLC) method and data were represented as μM/mg of protein. The levels of proteins in the heart homogenate were measured using Folin–Lowry method. The levels of HIF-1α were also determined in supernatant solution using ELISA kit. The other half portion of the heart was used to quantify nuclear cytoplasmic ratio of Nrf2. The nuclear and cytoplasmic fractions were separated using an extraction kit (BioVision, USA). The levels of Nrf2 were assessed in the supernatant using commercially available ELISA kits.