A total of 60 healthy adult male Sprague-Dawley (SD) rats, aged 6–8 weeks and weighing 220–250 g, were provided by the Experimental Animal Center of PLA Academy of Military Medical Sciences (Beijing, China) and acclimated for at least three days [license no. SCXK (Army) 2007–004]. All animals were housed in separated cages with laboratory chow and tap water ad libitum. All animal experiments were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, and experiments were approved by the University Laboratory Animal Research Committee of Tianjin Medical University (Tianjin, China).

SD rats were randomly allocated into five equal groups (n=12/group): Sham-operated control group (S), model group (M), and small (0.55 g/kg/day; GL), medium (1.1 g/kg/day; GM), and large (2.2 g/kg/day; GH) dosage GMT groups. GMT was provided by Tianjin Tongrentang Group Co., Ltd. (Tianjin, China). Control and model groups received an equal quantity of vehicle. After 14 days of treatment, animals underwent AMI surgery.

An AMI model was established in rats by ligation of the left anterior descending coronary artery for 24 h. The surgical procedure was performed according to a previous study (18), with minor modifications. Briefly, SD rats were anesthetized with 10% chloral hydrate (0.3 ml/100 g, intraperitoneally; Sigma-Aldrich, St. Louis, MO, USA), then a left thoracotomy was performed. The incised area was extended using forceps and the pericardium was opened. Following tracheal intubation, the rats were ventilated using a respirator (ALC-V8; Alcott Biotech Co., Ltd., Shanghai, China) with room air at a tidal volume of 25 ml/min and a respiratory rate of 70 breaths/min. The heart was exteriorized and ligated at the proximal left anterior descending coronary artery 2–3 mm from its origin between the pulmonary artery conus and the left atrium using a 5-0 Prolene suture (WEGO Inc., Shandong, China). The heart was returned to its normal position and the thorax was closed. Sham-operated rats underwent an identical surgical procedure as described above except that the suture was not tightened around the coronary artery.

A 2,3,5-triphenyltetrazolium chloride (TTC) assay was used to determine myocardial infarct size. TTC was provided by Amresco (Amresco LLC, Solon, OH, USA). In brief, the heart was transversely cut across the left ventricle, and sections 2–3 mm thick were incubated in 0.5% TTC solution prepared in phosphate buffer (pH 7.4; Sangon Biotech Co., Ltd., Shanghai, China) for 30 min at 37°C, following which they were fixed with 10% formalin (Sangon Biotech Co., Ltd.). Non-ischemic and viable ischemic myocardium were stained red, while the infarcted myocardium appeared pale grey or white. For histological analysis, 5-µm sections from the left ventricle were stained with hematoxylin and eosin (HE; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China). The pathological features were observed using a microscope (BX53; Olympus Corporation, Tokyo, Japan) at a magnification of ×400.

Abdominal aortic blood samples (4 ml) was separated by centrifugation at 840 × g for 10 min at 4°C. The activities of serum creatine kinase (CK; 812060103), creatine kinase-MB (CK-MB; 812060202), and lactate dehydrogenase (LDH; 812060403) were measured spectrophotometrically according to the specifications of commercial diagnostic kits (Shanghai Kehua Medical Instruments Co., Ltd., Shanghai, China).

Rats were sacrificed with 10% chloral hydrate (2 ml/100 g) and total RNA was extracted from the rat heart tissues using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. RNA yields and purity were assessed by spectrophotometric analysis (BioPhotometer Plus; Eppendorf, Shanghai, China) at 260 and 280 nm. Total RNA (1 µg) from each well was subjected to reverse transcription with oligo dT (19) primers, dNTPs (both Takara Bio, Inc., Otsu, Japan) and M-MLV reverse transcriptase (Promega Corporation, Madison, WI, USA) in a total reaction volume of 20 µl. Following DNase treatment (Sigma-Aldrich), the 20-µl RT-qPCR reaction system consisted of 10 µl SYBR Green Mix (Takara Bio, Inc.,), 0.5 µl of each primer (Table I) (Invitrogen), 1 µl cDNA and 8 µl double-distilled water. RT-qPCR was performed using an ABI 7300 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) and data were analyzed using the accompanying ABI 7300 software. Thermal cycling conditions were as follows: Initial denaturation at 95°C for 5 min; 40 cycles at 95°C for 30 sec and 58°C for 30 sec; elongation at 72°C for 30 sec; a final cycle at 95°C for 15 sec and 60°C for 15 sec; followed by dissociation at 95°C for 15 sec. All values obtained with the tumor necrosis factor-α (TNF-α), interleukin (IL-1) or ICAM-1 primers were normalized against the values obtained with the GAPDH primers, according to the 2^−∆∆Cq method (20). The results are expressed as the relative integrated intensity. Negative controls (no cDNA) and RT controls (no reverse transcriptase) were performed. All reactions were performed in triplicate.

ELISA measurements of IL-1 expression were performed in duplicate using a specific, commercially available IL-1 ELISA kit (Cusabio Biotech Co., Ltd., Wuhan, China) in accordance with the manufacturer's instructions, and analyzed using an ELISA reader (Tecan Trading AG, Männedorf, Switzerland) at 450 nm.

Myocardial tissue (100 mg) was grinded in liquid nitrogen and incubated with radioimmunoprecipitation assay lysis buffer, containing 20 mM HEPES, 0.5% NP-40 (both Sigma-Aldrich), 1% protease inhibitor cocktail (Promega Corporation), 200 mM KCl, 20% glycerol and 0.5 mM EDTA (all Sangon Biotech Co., Ltd.), to extract total protein. Subsequently, 30 µg protein/lane was subjected to 10% SDS-PAGE and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% bovine serum albumin and subsequently incubated with antibodies against B-cell lymphoma 2 (Bcl-2; 1:1,000; 2876), Bcl-2-associated X protein (Bax; 1:1,000; 2772), TNF-α (1:2,000; MAB510) ICAM-1 (1:1,000; ab124760) and GAPDH (1:2,000; AB-M-M001), as the internal control, at 4°C overnight. Following washing six times for 30 min with Tris-buffered saline with Tween 20 (TBST), the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (7074) or horse anti-mouse IgG (7076) secondary antibodies (both 1:3,000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature to detect the primary antibody. Following further washing with TBST for 30 min, the intensity of immunoreactive bands was estimated using an imaging densitometer (Gene Tools 3.06; Gene Company Ltd., Hong Kong). Rabbit polyclonal anti-Bax and anti-Bcl-2 antibodies were purchased from Cell Signaling Technology, Inc., monoclonal anti-TNF-α antibody from R&D Systems (Minneapolis, MN, USA), rabbit polyclonal anti-ICAM-1 antibody from Abcam (Cambridge, MA, USA) and anti-GAPDH antibody (1:2,000; AB-M-M001) from Hangzhou Xianzhi Biotechnology (Hangzhou, China).

Tissues were conventionally fixed with 10% formalin, then dehydrated with alcohol, embedded with paraffin wax and continuously sectioned at 5 µm. Sections were incubated overnight at 4°C with primary anti-Bcl-2 (BA0412) and anti-Bax (BA0315) antibodies (both 1:500; Wuhan Boster Biological Technology Ltd., Wuhan, China). Negative control were performed which involved the omission of primary antibody and use of phosphate-buffered saline (PBS). Sections were then rinsed with PBS and incubated for 1 h with HRP-conjugated secondary antibody (1:2,000; ZB2301; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.). The reaction was visualized using a solution of 3,3′-diaminobenzidine (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.). For quantification, the integral optical density of Bax and Bcl-2 staining were calculated using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

Data are reported as the mean ± standard deviation. Statistical significance was determined using one-way analysis of variance tests followed by Dunnett's test. Statistical analyses were performed using the software package StatView 5.0J (SAS Institute, Cary, NC, USA). P<0.05 was considered to indicate a statistically significant difference.

Representative illustrations of infarction tissue size (pale grey areas) as stained by TTC are shown in Fig. 1A. Sham-operated rats exhibited no evidence of infarcted tissue in the left ventricle. The model group presented with a large unstained area (30.02±4.32% vs. sham group; Table II). The infarction size significantly reduced in GMT groups compared with the model group, and the size was smallest in the high dosage group (15.71±3.32%) compared with the medium (17.83±2.37%) and small (23.16±4.01%) dosage groups. Regarding HE staining, the model group showed augmentation and loose arrangement of the myocardial fibers, staining asymmetry, deformity of the nucleus and marked edema and infiltration. By contrast, tissues from the SD rats treated with GMT exhibited markedly improved pathological changes compared with model group (Fig. 1B).

Fig. 2 shows that compared with the sham-operated group, the serum CK, CK-MB and LDH activities in the model group increased significantly (P<0.05). Treatment with GMT attenuated these elevations, and the various GMT dosages produced varying degrees of reduction (Fig. 2).

Adult cardiomyocytes are post-mitotic cells, and thus have a limited response capability to damage (21). Prior studies have suggested that apoptosis is increased in acute and chronic heart pathologies, were the process appears to be crucially involved (21). To clarify the possible mechanism underlying anti-apoptotic effect exerted by GMT, two key factors of intrinsic pathway, Bcl-2 and Bax, in the infarct heart tissues. Using western blot and immunohistochemical assays (Fig. 3), a significant reduction in Bcl-2 expression and an increase in Bax expression was observed in the cardiocytes of the model group, as compared with the sham-operated group. Treatment with GMT resulted in enhanced Bcl-2 expression, reduced Bax expression and attenuated the ratio of Bcl-2/Bax (P<0.05) (Table III). Furthermore, the higher dosage groups (1.1 and 2.2 g/kg/day) exhibited more marked differences compared with the low dosage group (0.55 g/kg/day).

At 24 h after AMI induction the mRNA expression levels of IL-1, TNF-α and ICAM-1 in the model group significantly increased (P<0.05 vs. sham group; Fig. 4), and a reversed trend was observed in GMT groups. In particular, the mRNA expression levels of TNF-α and ICAM-1 were significantly reduced in all three GMT-treated groups (P<0.05 vs. model group), and were dosage-correlated. The expression levels of IL-1 mRNA were significantly reduced in the medium and high dosage groups (P<0.05 vs. model group), whereas no significant difference was detected in the low dosage group.

The western blot analysis results indicated that ICAM-1 expression increased in the model group (vs. sham group) and decreased significantly in the medium and high dosage GMT groups (P<0.05 vs. model group; Fig. 5A). By contrast, a statistically significant elevation in TNF-α protein expression was detected in all three GMT groups (P<0.05 vs. model group; Fig. 5B). The results of the ELISA assay indicate that the protein expression levels of IL-1 increased significantly in the model group (P<0.05 vs. sham group), and that this elevation was significantly inhibited in all three GMT groups (P<0.05 vs. model group).