Myocardial ischaemia‒reperfusion model

The rats were weighed, fasted, and dehydrated for 12 h before surgery. Sodium pentobarbital (50 mg/kg) was injected intraperitoneally (Item No. P3761, Sigma, USA). Each rat was fixed in the supine position and connected to a Powerlab data acquisition and analysis system. A standard II-lead ECG was recorded, and any abnormalities were excluded. The cervical trachea was incised, and a small animal ventilator (Rivard, USA) was connected for assisted breathing (tidal volume, 5 mL/100 g, frequency 60–80 breaths/min, respiratory ratio 2:1, continuous positive end-expiratory pressure). The skin was cut longitudinally 0.5 cm on the left side of the sternum, using the 3rd and 4th ribs as the upper and lower borders. The subcutaneous tissue, pectoralis major muscle, and intercostal muscle were bluntly separated with forceps and a scalpel. Then, 6–0 ophthalmic sutures were passed under the left atrium and 2–3 mm below the intersection of the cone of the pulmonary artery. The left anterior descending branch of the coronary artery was ligated for 30 min, after which the sutures were cut. After successful ligation, the anterior wall of the left ventricle was bruised or pale, the pulsation was reduced, and the ECG showed ST-segment elevation (≥ 0.25 mV), which is a sign of myocardial ischemia. Thirty minutes after ligation, the ligature was cut with scissors to form a reperfusion, and the ECG showed a gradual decrease in the ST segment by approximately 50% and the pale or cyanotic myocardium gradually turned red when blood flow was restored. Reperfusion was allowed to occur for 2.5 h [17]. After the end of reperfusion, rats were euthanized by a single intraperitoneal injection of an overdose (150 mg/kg) of sodium pentobarbital (Item No. P3761, Sigma, USA).