Thirty-five healthy male SD rats (200 ± 20 g) were obtained from the Experiment Animal Center of Shanghai University of Traditional Chinese Medicine. The animals were received humane care and kept on a 12-hours light/dark cycle with free access to water and food. Temperature and relative humidity in the animal breeding room ranged between 23~25℃ and 50~70%, respectively. The protocol was approved by the animal ethics committee of Shanghai University of TCM and the Animal Research Committee of Shanghai. Every effort was made to minimize animal suffering and the number of rats used during experimental manipulation.
Production of MI model
The rats were fixed on the surgical plate in a supine position after being anesthetized by 1% sodium pentobarbital (40 mg/kg, i.p.). The rats were orally intubated and mechanically ventilated with a rodent ventilator (Harvard Apparatus, USA). Under sterile conditions, the heart was exposed through a lateral thoracotomy. After that, the pericardium was gently removed and the left anterior descending coronary artery was permanently occluded at 2 to 3 mm below the starting point which is located between the pulmonary arterial cone and the left atrial appendage. Acute MI was confirmed by electrocardiography with ST elevation appearance and visual inspection for a rapid whitish discoloration of anterior wall of the LV. The muscle layer and skin were sutured, and the rats were allowed to recover on a warm heating pad (World Precision Instruments Inc., FL, USA).
Intragastric administration of MTHSWD
There were 5 rats were died during and after the surgery. The survival MI rats were then divided into three groups randomly at the second day after modeling, including MI group, THSWD group and MTHSWD group with 10 rats in each group. The rats in THSWD group or MTHSWD group were intragastrically administered THSWD or MTHSWD respectively twice a day for 2 weeks (1 ml each time). The rats in MI group were intragastrically administered an equivalent amount of physiological saline for the same time. The composition and dosage of THSWD were the same as that used in our published article . MTHSWD for each rat contained 10 components including Semen Persicae (0.16 g), Flos Carthami (0.16 g), Angelica Sinensis (0.22 g), Radix Paeoniae Alba (0.18 g), Rhizoma Chuanxiong (0.14 g), Radix Rehmanniae Praeparata (0.27 g), Salvia miltiorrhiza (0.54 g), Radix Astragali (0.4 g),Epimedium (0.27 g) and Notopterygii Rhizoma et Radix (0.27 g), which were provided by Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine. The doses of MTHSWD for rat was converted from a human equivalent dose commonly used in the clinical practice based on the body surface area. THSWD and MTHSWD were prepared according to our reported method . In brief, the crude herbal drugs were mixed and extracted with boiling water. The decoction was concentrated through rotary evaporation under vacuum to an equivalent 2.61 g/mL of the crude herbal drugs.
Echocardiography was performed on isoflurane anesthetized rats with a Vevo 2100 system (Visualsonics, Toronto, Canada) by an operator in a blinded manner at 1 and 2 weeks after treatment. M-mode images of the left ventricle were captured in the parasternal long-axis view and the left ventricle internal diameter at end-diastole (LVIDd) and end-systole (LVIDs) were measured for three consecutive cardiac cycles. The left-ventricular end-systolic (LVESV) and end-diastolic volume (LVEDV) were calculated according to LVIDs and LVIDd, and the ejection fraction (EF) and fractional shortening (FS) of LV were calculated as (LVEDV-LVESV)/LVEDV (%) and (LVIDd-LVIDs)/LVIDd (%), respectively.
Terminal dUTP nick-end labeling assay
The heart of rat was rapidly excised under anesthesia after the echocardiographic assessment and was cut into two parts transversally along the center of the infarct zone. The tissue near cardiac base was fixed with 4% formaldehyde solution after removing the atrium. Then the sample was embedded in Tissue-Tek OCT (Sakura, USA) and cut into 10-μm sections through a freezing microtome (HM525, Thermo Fisher Scientific, USA). To investigate the role of MTHSWD administration on cardioprotection after MI, the cell apoptosis was detected by the terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling (TUNEL) staining kit (Roche, Mannheim, Germany) according to the instruction recommended by the manufacturer. For each section, at least 5 high-power fields (HPFs) were photographed and the number of TUNEL-positive cells were counted in each field.
The tissue near cardiac apex was homogenized in RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Roche, Mannheim, Germany) to extract protein. After being incubated on ice for 30 min, the sample was centrifuged at 13,000g at 4°C for 15 min. The supernatant was harvested and the concentration of total protein was measured by BCA protein assay (Pierce Biotechnology, USA). The samples were subsequently boiled for 8 min to denature the protein. For the detection, 30 μg of each protein was loaded into each well and separated on a 10% SDS-polyacrylamide gel electrophoresis. The proteins were then transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, USA) using a semidry transfer system. After that, the unoccupied sites were blocked in 5% skim milk for 1 h and immunoblotted with primary antibodies of p-Akt (1:1000, CST), cleaved caspase 3 (1:200, Sigma), Akt (1:2000, CST), matrix metallopeptidase-2 (MMP-2, 1:2000, Abcam), tissue inhibitor of metalloproteinases-2 (TIMP-2, 1:500, Abcam) and VEGF (1:1000, Beyotime Biotechnology, China) overnight at 4°C. After the incubation with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature, the protein bands were visualized by enhanced chemi-luminescence (Pierce Biotechnology, USA) and photographed. The optical densities of the reactive bands normalized to GAPDH were quantified by the image analysis software ImageJ (NIH, Bethesda, MD, USA).
The infarcted myocardium tissues were weighed and homogenized in PBS by tissue grinder on the ice. The samples were then centrifugated at 3,000 rpm for 20 min at 4°C and the supernatants were harvested for the evaluation of cytokine levels through the specific enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Enzyme-linked Biotechnology, Shanghai, China) according to the manufacturer’s instruction.
Masson’s trichrome staining
Masson’s trichrome staining was performed to detect the infarct size and collagen content in the infarcted area through the routine procedures. The blue area indicated fibrotic tissue and the viable myocardium was stained red. The infarction size and collagen content were measured by Image-Pro Plus software (Bethesda, USA), and respectively defined as the percentage of blue area in the cross-sectional area of whole myocardium in the LV wall and the percentage of fibrotic tissue area in each HPF covering the infarcted and surrounding area.
The frozen sections of hearts were incubated with 5% normal goat serum for 1 h at room temperature to block non-specific protein-protein interactions followed by primary antibodies Cx43 (1:200; CST, USA), cTnT (1:100; Abcam, USA), CD31 (1:100; Abcam, USA) and α-SMA (1:200; Abcam, USA) overnight at 4°C. The sections were then washed with PBS for 3 times and probed with appropriate secondary antibodies (1:200; Invitrogen, USA) for 2 h at room temperature. The nuclei were counter-stained with 4’,6-diamidino-2-phenylindole (DAPI) in the detection of cTnT and Cx43 expression. At least 5 HPFs in each section were randomly selected and the number of CD31 or α-SMA positive blood vessels per HPF was counted to determine the angiogenesis. The fluorescence intensity of 5 HPFs in each section was analyzed using Image-Pro Plus software for the semi-quantitative analysis of cTnT and Cx43 expression.
All data were represented as means ± standard deviation. Multiple groups were compared using one-way analysis of variance (ANOVA) followed by a Student-Newman-Keuls (SNK) post hoc test through a statistical software (IBM SPSS Statistics for Windows, Version 23.0, IBM Corp., NY, USA). Differences were considered to be statistically significant when P < 0.05.