SM extract was purchased from Xi'an Honson Biotechnology Co., Ltd. (Xi'an, China; batch no. 161025). The major components were hydrophilic salvanolic acid and hydrophobic tanshinones, as evaluated by HPLC analysis. DOO was purchased from Jishui Jinhai Natural Perfume Oil Technology Co., Ltd. (Jiangxi, China; batch no. XC20160918) and the major components were evaluated by GC-MS. Standards for danshensu, rosmarinic acid, salvianolic acid B, salvianolic acid A, protocatechuic acid, protocatechuic aldehyde, dihydrotanshinone I, cryptotanshinone, tanshinone II_A and tanshinon II_A silate sodium were purchased from Shanghai ANPEL Laboratory Technologies, Inc. (Shanghai, China; batch no. Q2880010). Acetonitrile was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). All other reagents were analysis grade.

Deionized water was prepared using a Milli-Q water purification system (EMD Millipore, Bedford, MA, USA). 2,3,5-triphenyltetrazolium chloride (TTC) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Pig creatine kinase-MB (CK-MB), cardiac troponin I (cTnI) and myoglobin (Myo) ELISA kits were bought from Xitang Biology Technology Company (Shanghai, China). Lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) test kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All primary antibodies, including Bcl-2 associated X (Bax; cat. no. 2772), Bcl-2 (cat. no. 2870), Akt (cat. no. 9272), phosphorylated (p)-Akt (cat. no. 9271), glycogen synthase kinase (GSK)-3β (cat. no. 9315), p-GSK-3β (cat. no. 9336) and β-actin (cat. no. 4970), were purchased from Cell Signaling Technologies, Inc. (Danvers, MA, USA). The secondary antibody (anti-rabbit IgG-B; cat. no. sc-53804) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

SM was dried at 50°C and ground into powder (<1 mm). SM (150 g) was soaked in 8-fold the volume of water for 30 min at room temperature and extracted for 3 h (1.5 h/reflux), followed by reflux extraction with 75% ethanol. The filtrates were combined, concentrated under reduced pressure and freeze-dried. DOO was isolated by steam distillation for 5 h, with a yield of 0.5% DOO, which was stored at 4°C.

Primary standard solutions of the 10 components were prepared by dissolving the solid standards in methanol. Working mixed standard solutions (40 μg/ml) were prepared by mixing and diluting the stock solution with methanol. The mixed standard solution was filtered through a 0.22-μm membrane before HPLC analysis.

SM was dried at a temperature of 50°C and ground into powder. Subsequently, 0.25 g SM was soaked in 8-fold the volume of water for 30 min at room temperature and extracted for 4 h, followed by ultrasound-assisted extraction for 30 min with 80% methanol. The suspension was filtered, concentrated and diluted to a 50-ml volume with methanol. The solution was filtered through a 0.22-μm membrane prior to HPLC analysis.

An aliquot of 200 μl DOO was diluted in 20 ml of ethyl acetate and dried over anhydrous sodium sulfate. The supernatant was filtered through a 0.22-μm membrane prior to GC-MS analysis.

HPLC analysis was performed using an Agilent 1260 HPLC system (Agilent Technologies, Santa Clara, CA, USA) and chromatographic separation was accomplished using an Agilent TC-C18 column (4.6×250 mm, 5 μm). The column temperature was set at 30°C with a 20-μl injection volume. The mobile phase consisted of 0.2% formic acid (v/v) in water (A) and acetonitrile (B). The gradient program used was as follows: 10% B to 30% B (0-15 min), 30% B to 32% B (15-40 min), 32% B to 55% B (40-55 min), 55% B to 67% B (55-65 min), 67% B to 72% B (65-75 min), 72% B to 85% B (75-85 min) and 100% B (85-100 min). The flow rate was set at 0.8 ml/min throughout the analysis, and UV detection was set at 270 nm.

The components of DOO were analyzed using an Agilent 7890A gas chromatograph coupled with an Agilent 5975C mass selective detector (Agilent Technologies). Chromatographic separation was performed using an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm film thickness, Agilent Technologies). The split ratio was 15:1, and the injection volume was set to 2 μl. The inlet and ion source temperatures were set to 260°C and 230°C, respectively. The helium flow rate was set to 1.0 ml/min. The oven temperature was initially 60°C, followed by a 1.5°C/min increase to 105°C for 10 min, then a 1°C/min increase to 115°C for 10 min, a 2.5°C/min increase to 160°C for 5 min and finally to 260°C at a heating rate of 20°C/min. MS was used in full scan mode with the acquisition range set to 30-800 m/z. GC-MS post-run analysis was performed, and the main components were identified using the library of the National Institute of Standards and Technology (NIST) (16). The peak area normalization method was used to establish the relative content of each substance in the sample.

A total of 18 male Yorkshire pigs (25-30 kg) were purchased from the Institute of Experiment Animals at the Fuwai Hospital Experimental Animal Center (Beijing, China) and kept in an animal room at 25±1°C with 55±5% humidity. The Animal Welfare and Ethics Committee of Fuwai Hospital, Chinese Academy of Medical Sciences approved all experiments [approval no. 0072-1-27-HX (F)]. After one week of acclimation, the pigs were randomized into 3 groups as follows: i) Sham group (n=6), oral administration of regular diet and sham-operated without ameroid constrictor implantation; ii) Model group (n=6), oral administration of regular diet; iii) SM-DOO group (n=6), oral administration of regular diet with 1 g/kg/day SM and 0.1 ml/kg/day DOO. The dosages of SM and DOO were determined based on our previous studies, and adjusted for pigs according to a previously published method (15,17). Pigs were provided free access to water and were fed twice a day. All animals were observed during feeding to ensure the entire portion of food was consumed.

At 4 weeks after the dietary modification, pentobarbital sodium (25 mg/kg) was administered intramuscularly (i.m.) to induce anesthesia. Animals were intubated, ventilated with oxygen at 1.5-2 l/min and anesthetized with 3% isoflurane. The pigs were right lateral decubitus, and a small thoracotomy was performed in intercostal segments 3 and 4 by sterile techniques. An ameroid constrictor (Research Instruments NW, Inc., Lebanon, OR, USA) with a 2.5-mm internal diameter was implanted around the left anterior descending artery (LAD). The ameroid constrictor was designed to achieve myocardial ischemia by inducing progressive coronary artery stenosis with a progression similar to that observed in humans. The pericardium was closed using a 6-0 polypropylene suture (Ethicon, Inc., Somerville, NJ, USA) and the chest was sutured closed. After the surgery, penicillin (6,400,000 U/day, i.m.) was administered to all pigs to prevent infection.

After 8 weeks, echocardiography, X-ray coronary angiography and hemodynamics analysis were performed. All surgical protocols were designed to minimize the suffering of the pigs. A schematic presentation of the study protocol is shown in Fig. 1.

Blood samples were collected in heparinized tubes, allowed to clot for 30 min and centrifuged at 2,285 × g at 4°C for 15 min. Myocardial injury markers, including CK-MB, LDH, ALT, AST, cTnI and Myo, were measured using assay kits or pig-specific ELISA kits according to the manufacturers' protocols.

At weeks 2, 6 and 8, pH, PO₂ and PCO₂ were measured in arterial blood samples taken from the femoral artery (500-700 μl) using pre-heparinized syringes (1,000 IU/ml) and analyzed using an i-STAT 1 blood gas system (Abbott Laboratories, Chicago, IL, USA).

Echocardiography was performed at the end of the study using a Vivid 9 machine (GE Vingmed Ultrasound AS, Horten, Norway). Left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVEDs), end-diastolic volume (EDV), end-systolic volume (ESV), ejection fraction (EF) and fractional shortening (FS) were recorded to assess cardiac function. The FS value was calculated based on the following equation: FS (%) = [(LVEDd-LVEDs)/LVEDd] × 100%. Images were stored and analyzed using Echopac software (GE Vingmed Ultrasound AS).

X-ray coronary angiography (GE Medical Systems, Milwaukee, WI, USA) with iopromide (Juste SAQF, Madrid, Spain) was performed on all experimental animals to verify the occlusion of the LAD and assess collateral vessel filling at the end of the experiment. The grade of collateral filling was scored using a standard 4-point scale as follows (18): 0 = no collateral filling; 1 = minimal collateral filling; 2 = moderate collateral filling, but less than the contralateral or ipsilateral non-infarct coronary artery; 3 = normal collateral filling compared with the contralateral or ipsilateral non-infarct coronary artery.

The heart rate (HR), mean arterial blood pressure (MAP), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), left ventricular maximum upstroke velocity (+dp/dt_max) and left ventricular maximum descent velocity (−dp/dt_max) were recorded using a pressure transducer (Millar Instruments, Houston, TX, USA) connected to an anesthesia monitor (Philips, Eindhoven, Netherlands).

Animals were sacrificed by intravenous injection with potassium chloride after the imaging study, and their hearts were explanted. Hearts were serially sliced and incubated with 2% TTC for 20 min at 37°C. An Image-Pro Plus image system (Media Cybernetics, Bethesda, MD, USA) was used to analyze the infarct (white) and non-infarct (red) areas. The percentage of myocardial infarct was calculated as infarct area divided by total area.

The left ventricle was harvested, fixed in 4% paraformaldehyde, embedded in paraffin and sectioned. Sections were stained with hematoxylin and eosin (H&E) for histological examination. The pathology of heart sections was investigated using a light microscope, and photomicrographs were captured.

Myocardial apoptosis was measured using an In-Situ Cell Death Detection kit (Wanlei Bio, Shenyang, China) according to the manufacturer's protocol. Myocardial slices were subjected to terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining combined with a DAPI (Sigma-Aldrich; Merck KGaA) counterstain. Images were obtained under a fluorescence microscope at ×400 magnification (Nikon Corporation, Tokyo, Japan), to confirm the successful labeling of apoptotic cells and nuclei. The percentage of TUNEL-positive cells was calculated as the number of apoptotic cells divided by the total number of nuclei × 100.

Myocardial tissues were homogenized in RIPA buffer containing 1% PMSF and 1% protease inhibitor cocktail (Nanjing Jiancheng Bioengineering Institute), and the supernatant was harvested by concentration (18,447 x g at 4°C for 10 min). The protein concentration was determined based on the BCA method using a Protein Quantitative Analysis kit (Nanjing Jiancheng Bioengineering Institute). The lysates were separated by 10% SDS-PAGE. Following separation, the proteins were transferred onto polyvinyl difluoride membranes, blocked for 1 h at 37°C with 5% non-fat dried milk, washed 3 times with Tween in Tris-buffered saline, and incubated with primary antibodies against Bax, Bcl-2, Akt, p-Akt, GSK-3β, p-GSK-3β and β-actin (dilution, 1:1,000) overnight at 4°C. Subsequent to washing 3 times as described above, the membranes were incubated with secondary antibodies (dilution, 1:5,000) at 37°C for 1 h. Immunoreactive bands were detected using the enhanced chemiluminescence method and semi-quantified using ImageJ Software version 1.47 (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis was conducted using GraphPad Prism version 6.02 (GraphPad Software, Inc., La Jolla, CA, USA). All data are presented as the mean ± standard deviation. One-way analysis of variance with Tukey's multiple comparison post-hoc test was performed to analyze the data. P<0.05 was considered to represent a statistically significant difference.

The ameroid constrictor was used to induce coronary closure. Coronary angiography indicated that the rate of coronary stenosis reached 98-100% 4 weeks after ameroid constrictor implantation, indicating that chronic myocardial ischemia was stably established. As shown in Fig. 2, coronary angiography revealed LAD stenosis with no evidence of collateral vessel filling in the Model group (0.20±0.11); however, collateral filling was observed in the SM-DOO group (1.49±0.31). Collateral scores were significantly increased by the administration of SM-DOO compared with the Model group (P<0.01).

As presented in Fig. 3, no differences were observed in CK-MB, LDH, ALT, AST, cTnI and Myo between the 3 groups at week 2. CK-MB, LDH, ALT, AST, cTnI and Myo levels in the Model group were higher compared with the Sham group at weeks 6 and 8. In the Model group, at week 8, the expression of myocardial injury markers was higher compared with at week 6. Conversely, CK-MB, LDH, ALT, AST, cTnI and Myo were significantly decreased in the SM-DOO group compared with the Model group (P<0.05 or P<0.01), demonstrating that SM-DOO ameliorated injury in chronic myocardial ischemia.

To investigate whether the cardioprotective effects of SM-DOO were mediated by affecting myocardial oxygen consumption, blood gas was quantified. As presented in Fig. 4, pH, PO₂ and PCO₂ were the same in all 3 groups prior to the induction of chronic myocardial ischemia. By weeks 6 and 8, the arterial pH and PO₂ were markedly lower, while the PCO₂ was higher in the Model group than that in the Sham group (47.13±3.91 vs. 40.57±3.29 mmHg at week 6; 49.05±5.56 vs. 39.46±3.10 mmHg at week 8). Thus, compared with the Model group, SM-DOO markedly ameliorated the effects on pH, PO₂ and PCO₂ induced by myocardial ischemia.

To assess the cardioprotective effects of SM-DOO, left ventricular function was examined. Compared with the Sham group, the mitral and apical levels in the Model group were reduced, the papillary muscle was thinned, and the left ventricular anterior wall moments were weakened (Table I). Additionally, in the Model group, the echocardiography parameters of LVEDd, LVEDs, EDV and ESV were significantly increased, while EF and FS were markedly reduced (P<0.01 or P<0.05). The results indicated that the left ventricular function in the Model group was markedly altered. Conversely, the effects on the left ventricular anterior wall thickness and moments were ameliorated in the SM-DOO group; the levels of LVEDd, LVEDs, EDV and ESV were significantly reduced, whereas EF and FS were increased following treatment with SM-DOO (62.5±4.848% for EF, P<0.05; 44.5±8.526% for FS; P<0.01; Fig. 5).

As shown in Fig. 6, the hemodynamic parameters indicated that the left ventricular function of the Model group was significantly decreased compared with the Sham group. In the SM-DOO group, HR, LVEDP and −dp/dt_max were markedly reduced (P<0.05 or 0.01), whereas MAP, LVSP and +dp/dt_max were significantly increased, compared with the Model group (102.176±22.203 vs. 67.079±16.378 mmHg for MAP; 115.522±21.528 vs. 83.206±9.189 mmHg for LVSP; and 2,017.917±548.228 vs. 1,244.587±284.314 mmHg/sec for +dp/dt_max; P<0.05).

After the pigs were sacrificed, hearts were collected for morphometric and histopathological analysis. Cardiac sections were stained with TTC to compare the infarct size between the 3 groups. As shown in Fig. 7A, the mean infarct size in the Model group was larger than that in the SM-DOO group. The infarct size was significantly decreased in the SM-DOO group (5.91±1.25%) compared with the Model group (12.93±1.68%; P<0.01; Fig. 7B). Thus, SM-DOO treatment reduced the myocardial infarct size after chronic myocardial ischemia.

As presented in Fig. 7C, the myocardium was H&E stained for histological examination. Myocardial tissues in the Sham group exhibited no abnormalities. As expected, chronic myocardial ischemia induced evident injury to the myocardial structure, including chronic inflammatory cells, edema in the myocardial fibers and ruptured myocardial fibers in the Model group. Myocardial alterations following chronic myocardial ischemia, in particular, disruption to the myocardial fibers, were significantly ameliorated by SM-DOO.

As shown in Fig. 8A, more apoptotic cells were observed in the Model group compared with the Sham group. The percentage of TUNEL-positive cells was significantly lower in the SM-DOO group (19.78±2.51%) compared with the Model group (28.20±5.33%), as shown in Fig. 8B (P<0.01). To further examine the potential mechanisms of the anti-apoptotic effect of SM-DOO, the expression of Bax and Bcl-2 was determined. As shown in Fig. 8C, D and E, the expression of Bax was significantly increased and Bcl-2 was significantly decreased in the Model group. In addition, the Bcl-2/Bax ratio was significantly decreased in the Model group (Fig. 8F; P<0.01). Thus, treatment with SM-DOO ameliorated the effects of chronic myocardial ischemia on apoptosis.

To investigate the signaling pathways involved in the cardioprotective effects of SM-DOO, the expression of Akt, p-Akt, GSK-3β and p-GSK-3β was determined. As shown in Fig. 9, compared with the Sham group, the expression of p-Akt and p-GSK-3β was slightly increased, while Akt and GSK-3β levels were not altered in the Model group. Compared with the Model group, the SM-DOO group had significantly higher levels of p-Akt/Akt and p-GSK-3β/ GSK-3β (P<0.01), indicating that SM-DOO administration markedly increased p-Akt and p-GSK-3β levels.

By comparing the retention times of mixed standard solutions with SM (Fig. 10A), 8 components were identified as danshensu, rosmarinic acid, salvianolic acid B, salvianolic acid A, tanshinone II_A silate sodium, dihydrotanshinone I, cryptotanshinone and tanshinone II_A by chromatography (Fig. 10B). The main components of SM were salvianolic acid B (18.67%), crypto-tanshinone (11.83%) and tanshinone II_A (10.55%). A total of 9 chemical components of DOO were detected by GC-MS and identified by NIST Database retrieval, as shown in Table II. The major components of DOO were nerolidol (22.52%), caryophyllene oxide (12.70%), α-santalol (9.01%) and β-farnesene (5.16%). The total ion current (TIC) of DOO is given in Fig. 10C.