Materials
The seeds of A. fistulosum was collected in Zigong, Sichuan Province, China, and authenticated by Prof. Han-Ming Zhang of the Department of Pharmacognosy, School of Pharmacy, Naval Medical University, Shanghai, China; a voucher specimen (No. 20040947) was deposited in the Department of Pharmacognosy, School of Pharmacy, Naval Medical University. SAFE was prepared by the following method: the air-dried seeds of A. fistulosum were refluxed three times with water at 100°C (2 h, each), the extract was passed through an AB-8 resin column, the column was washed with 5 volumes of water, eluted with 5 volumes of 50% ethanol, and the eluent was recovered after drying at 60°C. Afterward, the extract was freshly prepared, when needed, by dissolving it in normal saline. Xingling granule (XLKL, Shanghai Xingling Technology Pharmaceutical Co., Ltd., Shanghai, China; batch No. 20030201) was employed as a positive control.
Animal groupings
Healthy hybrid dogs were randomly divided into five groups: the model group (normal saline, 2 mL·kg− 1), SAFE low dose group (SAFE-L, 15 mg·kg− 1), SAFE medium dose group (SAFE-M, 45 mg·kg− 1), SAFE high dose group (SAFE-H, 90 mg·kg− 1), and XLKL group (300 mg·kg− 1) in the experimental model of hemodynamics and myocardial oxygen consumption. All methods are reported in accordance with ARRIVE guidelines.
Dog hemodynamics model
The dogs were anesthetized intravenously with 3% sodium pentobarbital (30 mg·kg− 1) and fixed to the operating table in the supine position. Then, the trachea was separated and intubated. The right femoral artery was separated, and a cardiac catheter was inserted retrogradely. The arterial blood pressure was recorded by inserting an arterial catheter, and the right common carotid artery was isolated. Two catheters were connected to the pressure sensor. The pressure signal was amplified by a carrier wave and connected to the Powerlab System 8S Physiological Recorder (ML785/8S, AD Instruments, Sydney, NSW, Australia). Needle electrodes were inserted subcutaneously into the limbs and connected to an AC Amplifier (AB -621 G) for monitoring a standard two-lead ECG. The data were monitored and stored in real time by computer chart 4.12 software (ML785/8S, AD Instruments, Sydney, NSW, Australia). The dogs were stable for 30 minutes after the operation for recording base values before administration. The negative control group was given normal saline (2 mL·kg− 1), the positive control group was given XLKL (300 mg·kg− 1), and the treatment group was given SAFE. HR, SBP, DBP, MAP, LVSP, LVEDP, +dp/dt max and -dp/dt max were observed. The observation time was before and 15, 30, 60, 90, 120, and 180 min after administration. After the test, the related indices were analyzed by chart 4.12 software [31, 32].
Dog myocardial oxygen consumption model
The dogs were anesthetized with 30 mg·kg− 1 iv sodium pentobarbital. Their bodies were kept at a constant temperature using a thermostatic operating table, and tracheal intubation was performed. An SC-M5 anesthesia ventilator was connected after exposing the chest to provide mechanical ventilation with a breathing rate of 16–18 times·min− 1 and ventilation with room air (tidal volume, 350–550 mL·kg− 1·min− 1). The left femoral artery was separated, and a catheter was inserted centripetally to monitor and record the peripheral blood pressure. The right femoral artery was separated for arterial blood gas analysis. The chest was opened along the left fourth intercostal space to expose the heart, and the cardiac package was cut to make a heart hammock. The root of the ascending aorta and the upper part of the left anterior descending coronary artery were freed, and the aortic flow (cardiac output) and coronary flow were measured by a magnetic flow meter with a proper internal diameter. The right jugular vein was separated, a cardiac catheter was inserted, and the opening of the coronary sinus was detected with the finger along the junction of the right atrium and right ventricle inferior vena cava. A cardiac catheter was inserted into the coronary sinus, and the cardiac catheter was fixed. Blood samples of the coronary sinus and femoral artery (0.5% heparin anticoagulation) were collected at the same time. PO2, pH, and arterial and venous oxygen content were measured by a blood gas analyzer. The arterial and venous blood gases were analyzed before and 15, 30, 60, 90, 120, and 180 min after administration of saline, SAFE and XLKL, and mean blood pressure, cardiac output and coronary flow were recorded at 15, 30, 60, 90, 120, and 180 min at the same time. At the end of the experiment, the heart was weighed and counter current perfused with 10–15 ml 10% Shanghai advanced carbon ink into the area below the measurement site of the electromagnetic flowmeter. Black areas were excised and weighed to calculate myocardial oxygen consumption. Myocardial oxygen consumption was calculated before and 15, 30, 60, 90, 120, and 180 min after administration. The formula is:
(1). Myocardial oxygen consumption (ml/min/100 g) = coronary blood flow (ml/min/100 g)× (arterial oxygen content -coronary sinus oxygen content).
(2). Myocardial oxygen uptake rate (%) = (arterial oxygen content-coronary sinus oxygen content)/arterial oxygen×100%.
(3). Vascular resistance (TPR, dyn·s·cm− 5) = mean arterial pressure [MAP (KPa)] × 80 /cardiac output [CO (L/min)].
(4). Coronary resistance (KPa/mL/min) = mean blood pressure [MAP(KPa)]/coronary blood flow [CBF(mL/min/100 g)] [33–36].
Statistical Analysis
All experimental data were expressed as ± S. Statistical analysis was performed with one-way ANOVA, using SPSS 10.0 statistical software (SPSS Inc., Chicago, Illinois, USA), p < 0.05 was considered significant.