Comprehensive Myocardial Redox and Inflammatory Analysis

Minced myocardium (approximately 100 mg) was homogenized in nine-fold precooled normal saline using an Ultra-Turrax homogenizer at a low temperature. The homogenates were centrifuged at 3000 rpm for 15 min at 4 °C, and the resultant supernatants were collected for subsequent measurements. Redox-related parameters were measured using commercial kits from the Nanjing Jiancheng Bioengineering Institute of China. GSH concentration (A006-2-1), H₂O₂ concentration (A064-1-1), total antioxidant capacity (A015-1-1), and Thioredoxin Reductase activity (A119-1-1) were measured by spectrophotometric method; MDA concentration (A003-1-1) was measured by the thiobarbituric acid method; superoxide dismutase activity (A001-1-1) was measured by the hydroxylamine method; catalase activity (A007-1-1) was measured by the ammonium molybdate method. Inflammatory cytokines (including IL-1β (JM-00567B1), IL-6 (JM-00561B1), IL-8 (JM-08328B1), IL-10 (JM-00568B1), and IL-12 (JM-08322B1)) were measured with commercial enzyme-linked immunosorbent assay kits from Jiangsu Jingmei biotechnology Institute of China. The OD₄₅₀ was detected using a microplate reader (BioTek Epoch, Winooski, VT, USA). The R² of the standard curve was more than 0.9900, and the intra- and inter-plate coefficients of variation for cytokines were less than 15%. The detailed steps were performed according to the manufacturer’s instructions. All redox and inflammation-related parameters were normalized to protein concentrations, which were measured using the BCA method (Meilunbio, Dalian, China, cat. No. MA0082-2).