Preparation of SXSMD.
SXSMD is composed of 13 medicinal materials, including: Radix panacis quinquefolii, Radix salvia miltiorrhiza, Ganoderma, Carthami flos, Radix notoginseng, Chuanxiong, Crataegi fructus, Ginkgo folium, Trionycis carapax, Dendrobii caulis, Schisandrae chinese fructus, Cassiae semen, and Nelumbinis folium. The batch numbers of the thirteen Chinese herbal formula granules were 20031493, 19096434, 19116164, 19106094, 19076054, 20026034, 19076224, 19010813, 19037364, 19126784, 19116754, 19066234, and 19037454, respectively (Supplementary Fig. 1). Most of the active ingredients of traditional Chinese medicine are poorly defined, thus, thin-layer chromatography (TLC) is used to qualitatively identify the dispensed granules of traditional Chinese medicine. The TLC identification of the formula granules is performed according to the standard of Chinese Pharmacopoeia (version 2015). All data on herbal materials, dispensed granules, and quality control of SXSMD were supplied by Tianjiang Pharmaceutical Co., Ltd (China).
Experimental animals.
All animal experiments were approved by the Experimental Animal Ethics Committee of Changchun University of Chinese Medicine (2020239). Male Sprague-Dawley (SD) rats (17–20 weeks of age) were purchased from Liaoning Changsheng biotechnology Co., Ltd. (China) and reared in a standard experimental animal laboratory with ad libitum access to food and water.
Animal grouping.
The Control, Sham, MIRI, MIRI + DSI, and MIRI + SXSMD groups were set up according to the two-factor model and treatment, each group included 8 animals.
Drug administration.
The daily dosage of rat treatment was calculated according to the conversion factor of the equivalent dose of the drug between rats and humans. The equivalent dose of SXSMD is 3.38 g/kg, which is equivalent to adult clinical medication. To determine whether the protective effect of SXSMD on MIRI is dose-dependent, various doses were used: 3.38 g/kg (L), 6.76 g/kg (M), and 13.52 g/kg (H). SXSMD was used as granules dissolved in distilled water. In the experiment, an equal volume of gavage (15 mL/kg) was used. Rats in each group were gavaged once a day with the corresponding doses for 15 days. The positive control group was treated with DSI purchased from Zhengda Qingchunbao Pharmaceutical Co., Ltd (China). According to calculations, the daily dose of DSI in rats was 1.8 mL/kg diluted with normal saline to 15 mL/kg and administered continuously for 15 days. The control group was treated with normal saline at a dose of 15 mL/kg for 15 days.
Electrocardiogram (ECG) and hemodynamic monitoring.
Before the operation, electrocardiogram of the rats was recorder, and animals with abnormal electrocardiogram were excluded. An 8 gauge needle was inserted subcutaneously in a rat limb and the electrodes were connected; NEG (white) was connected to the right upper limb, GROUND (green) was connected to the right lower limb, and POS (black) was connected to the left lower limb (Fig. 2a). Carotid artery was catheterized with a catheter prefilled with heparin; an incision along the midline of the neck was performed and the subcutaneous muscle and blood vessel layers were separated using a blunt tool; a catheter was inserted into the left ventricle and fixed in place. The Power Lab data acquisition module was used to analyze and monitor the changes in left ventricular hemodynamics throughout the process: rat electrocardiogram (ECG), left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), maximum rate of increase in left ventricular pressure (+ dp/dtmax), and maximum rate of decrease in left ventricular pressure (-dp/dtmax).
Rat models.
MIRI model induction: Rats were anesthetized by intraperitoneal injection of 0.9% pentobarbital sodium (45 mg/kg). Electrocardiogram was monitored, and rats were intubated with a ventilator. The chest skin was longitudinally cut, and tissue and muscle layers were separated using a blunt tool; the pleura was punctured and the heart was exposed; a retractor was used to gently and quickly squeeze the heart out, and the left atrial appendage and the pulmonary artery cone were located. The left anterior descending (LAD) coronary artery is located at the junction. The coronary arteries were ligated with a 6 − 0 suture by tying a long slip knot, and the heart was returned into the chest. The changes in ECG were observed. The ST segment level was significantly elevated and arched upwards. After 30 min, the suture was removed to restore blood flow at the ligation site and the heart was reperfused for 120 min. The coronary arteries of rats in the Sham group were threaded in a similar manner without ligation. In the Control group, only ECG and hemodynamic changes were monitored (Fig. 1). MI model induction: Rats were intragastrically administered for 15 days. Isoproterenol (1 mg/kg) was injected intraperitoneally on day 13, and the heart was acquired on day 15.
Hematoxylin-eosin staining (H&E).
The pathological changes in the heart structure and heart tissue were investigated. Formaldehyde-fixed heart tissue specimens were embedded in paraffin, and 5 µm sections were prepared according to the standard operating procedures and stained with H&E. The images were acquired by Leica ICC50 HD (Leica,Germany). Analysis of each specimen was performed using an average of 3 fields of view.
Mitochondrial ultrastructure detection.
The sample of left ventricular infarct boundary area of the heart from each group (1 mm*1 mm*2 mm) was selected, fixed in 4% glutaraldehyde for 2 h or longer, and fixed in 1% osmic acid for 1 ~ 2 h; the samples were washed with PBS for 5 min (3 times) and subjected to dehydration, infiltration, embedding, and ultrathin sectioning. Transmission electron microscope (TEM) (Hitachi, Japan) was used to detect the ultrastructural changes of the heart tissue. Analysis of each specimen was performed using an average of 3 fields of view.
Quantitative RT-PCR analysis.
Total RNA was extracted from frozen myocardial tissue samples (50 mg) using the Trizol method, and a Nanodrop 2000c spectrophotometer (Thermo, Germany) was used to detect the A260/280 values of the resulting RNA solution to determine the total RNA concentration.
A NovoScript Plus all-in-one first strand cDNA synthesis supermix kit (Novoprotein, China) was used to synthesize cDNA. The reaction system (20 µl) included template RNA, 2 × NovoScript Plus first strand cDNA synthesis supermix, gDNA eraser, and RNase-free water; the samples were gently mixed, centrifuged, and incubated.
A NovoStart SYBR qPCR supermix plus kit (Novoprotein, China) was used for the assay. The samples contained 2 × NovoStart SYBR qPCR supermix plus, upstream and downstream primers, template, and RNase-free water. The samples in PCR (total reaction volume of 20 µl) were processed by the PCR amplification program: 95ºC, 1 min; 95ºC 20 s, 60ºC 20 s, and 72ºC 30 s for 40 cycles. The program is based on the dissolution curve starting at 60ºC and ending at 95ºC with 1 min holding time. After the reaction, the calculations are performed based on the measured dissolution and amplification curves and the Cq values. The 2−ΔΔt method was used to compare the Cq values of the target gene and the internal reference gene GAPDH to calculate the relative expression levels of target mRNAs. The primer sequences were synthesized by Bioengineering Co., Ltd (China).
Serum IL-6/TNF-α ELISA.
Blood was collected from the abdominal aorta of the rats and incubated for 30 min at 4 °C. The separated serum was placed in a tube and tested using a rat IL-6/TNF-α ELISA kit (JYMBIO, China). OD at 450 nm was detected by a Multiskan GO microplate reader (Thermo, Germany). The standard curve was constructed based on the OD values of the standards and the standard curve equations (IL-6: y = 13.595x + 4.2974; TNF-α: y = 75.695x-14.778). The concentrations of IL-6 and TNF-α in each sample were calculated based on the corresponding equations.
Western blot analysis.
The total protein of the myocardial tissue samples was extracted, and a BCA protein assay kit (Biyuntian, China) was used to determine protein concentration according to a standard curve (y = 0.737x + 0.0989). Stacking and separating gels were prepared; the samples were resolved by electrophoresis and transferred onto a membrane. The PVDF membrane (Millipore, USA) was blocked using 5% nonfat milk and incubated on a circular shaker at room temperature. The following primary antibodies were used: HMGB1 (Cell Signaling Technology, USA), TLR4 (ProteinTech, USA), MyD88 (Cell Signaling Technology, USA), NF-κB p65 (Abcam, UK), and GAPDH (Bioss, China). Antibodies were diluted 1:1,000 and incubated at 4 °C overnight. After washing with TBS and TBST, secondary horseradish peroxidase-labeled goat anti-rabbit (IgG) antibodies at 1:1,000 dilution was incubated on a shaker at 4 °C, and the membrane was washed again. An ultrasensitive ECL chemiluminescence kit (Biyuntian, China) and a FUSION FX Spectra system (VILBER, China) were used to develop and detect the target signals and quantify the protein expression.
Statistical analysis.
All analyses were performed using the SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). All data are presented as the mean ± standard deviation (SD). One-way analysis of variance (one-way ANOVA) was used to analyze the normally distributed data. The data that were not normally distributed were analyzed by nonparametric tests, including Friedman or Kruskal-Wallis tests. Variance was considered homogeneous if the P value of the homogeneity of variance test (F) was > 0.1. The test result at P < 0.05 were considered different, and the results at P < 0.01 were considered significantly different corresponding to statistically significant differences.