CK-MB (cat. no. H197), LDH (cat. no. A020-1), total superoxide dismutase (SOD; cat. no. A001-1) and malondialdehyde (MDA; cat. no. A003-1) diagnostic agents were obtained from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). Meis1 antibody (cat. no. BS3488) was obtained from Bioworld Technology, Co., Ltd. (Nanjing, China). GAPDH polyclonal primary antibody (cat. no. 10494-1-AP) and goat anti-rabbit horseradish peroxidase-conjugated immunoglobulin G secondary antibody (cat. no. SA00001-2) were purchased from Wuhan Sanying Biotechnology (Wuhan, China). All other reagents used were of commercial analytical grade.

The dry stems of Dendrobium officinale were purchased from Xi'an Xiaocao Botanical Development Co., Ltd. (Xi'an, China). The voucher specimens were deposited at Southwest University (Chongqing, China). Briefly, the dry stems were ground into a fine powder through a 100-mesh filter. The powdered materials (100 g) were pre-extracted using petroleum ether and the residues were then extracted three times using hot distilled water. The crude extracts were filtered through a 0.22 µm microporous membrane, concentrated via the alcohol precipitation method and centrifuged at 625 × g for 10 min. The extracts were then dried by lyophilization, and 23.5 g powder extracts were produced. The DOE predominantly consisted of polysaccharide, and the concentration of polysaccharide was determined by the phenol-sulfuric acid method to be 97.4%.

A total of 60 Kunming male mice (age, 4 weeks; weight, 22±2 g) were purchased from Chongqing Tengxin Bio-technology Co., Ltd. (Chongqing, China). The present study was carried out according to recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (2015). In addition, the experiments were approved by the Ethical Committee for Animals of Southwest University. The mice were housed in standard conditions: Humidity, 50%; temperature, 25±2°C, under a 12:12 h light/dark cycle, with ad libitum access to normal food and tap water. Mice were allowed to acclimate to the environment for 1 week prior to experimentation.

Mice were randomly assigned to five groups (n=10/group): Sham group, model group, and LAD + DOE pretreatment groups (75, 150 and 300 mg/kg DOE; oral administration). DOE was dissolved in distilled water. Mice were administered normal saline (10 ml/kg) or DOE (75, 150 or 300 mg/kg) intragastrically once daily for 2 weeks. Subsequently, the mice were subjected to coronary artery ligation; LAD occlusion was performed as previously reported (20,21). Briefly, the left chest cavity of the mice was opened and the heart was exposed following anesthetization with sodium pentobarbital (40 mg/kg, i.p.). Subsequently, in the model and LAD + DOE pretreatment groups, 7–0 silk was used to ligate the LAD for 24 h, after which the heart was returned to its normal position and the thoracic cavity was closed. In the sham group, 7–0 silk was passed through the LAD, without ligation. After 24 h, mice were sacrificed by an overdose of pentobarbital sodium (100 mg/kg), and blood and heart samples were collected.

The lead II ECG was recorded using the BL-420F biological function experiment system (Chengdu Taimeng Technology Co. Ltd., Chengdu, China). Significant ECG changes, including elevation of the ST segment and widening of the QRS complex, indicated successful coronary occlusion.

A total of 24 h after ischemia, the hearts were collected and dissected at 1.2 mm cross-section. Subsequently, the heart sections were stained with 1% triphenyl tetrazolium chloride (TTC) for 15 min at 37°C in 0.2 M phosphate-buffered saline (22). The viable myocardium was stained red, whereas the infarcted tissue remained pale. Images were captured and the infarct size was analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

Blood samples were collected and were centrifuged at 625 × g for 10 min at 4°C to obtain serum samples. The CK-MB, LDH, SOD and MDA activity levels in the serum samples were determined using commercially available kits according to the manufacturer's protocols.

Heart tissues harvested from the mice were washed immediately with ice-cold saline. The biopsies were then fixed in 4% neutral paraformaldehyde and were embedded in paraffin. Heart tissues were cut into 5 µm sections, which were stained with hematoxylin and eosin (H&E) for 90 min at room temperature. Morphological evaluation and observation were performed under a light microscope.

TUNEL assays were performed as previously reported (23). The total number of TUNEL-positive cardiomyocyte nuclei in the infarcted zone was counted. Individual nuclei were visualized at a magnification of 400x (Olympus CKX41; Olympus Corporation, Tokyo, Japan), and the percentage of apoptotic nuclei (apoptotic nuclei/total nuclei) was calculated in six randomly selected fields per section and averaged for statistical analysis.

Heart tissues were lysed in radioimmunoprecipitation assay buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1% SDS, 1 mM phenylmethylsulfonyl fluoride], and concentration was determined using the bicinchoninic acid method. Heart tissue lysates (100 µg) were separated by 12% SDS-PAGE, and the proteins were electrophoretically transferred to polyvinylidene difluoride membranes. After blocking non-specific binding sites for 2 h with 5% dried skim milk at room temperature, the membranes were individually exposed to primary antibodies (anti-Meis1, 1:6,000; anti-GAPDH, 1:10,000) at 4°C overnight. The membranes were subsequently incubated for 2 h at room temperature with HRP-conjugated secondary anti-rabbit antibodies. The immunoreactive proteins were detected using an enhanced chemiluminescence reagent (Advansta Inc., Menlo Park, CA, USA). The proteins were visualized and representative images were obtained. Densitometric analysis was performed using Quantity One 4.6.2 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and GAPDH expression was used as an internal standard.

All statistical analyses were performed using SPSS statistical software (version 16.0 for Windows; SPSS Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation. Statistical comparisons were performed using one-way analysis of variance followed by Tukey's post-hoc test for multiple group comparisons. P<0.05 was considered to indicate a statistically significant difference.

The Lead II ECG is presented in Fig. 1. Marked elevation of the ST segment was detected in the myocardial ischemia model group, whereas a normal ECG was observed in the sham group. Ischemia-induced alterations to the ECG were significantly ameliorated by pretreatment with DOE, as compared with in the model group.

To evaluate infarct size, TTC staining was performed. The cross-section of the heart stained with TTC is presented in Fig. 2A. As shown in Fig. 2B, DOE markedly decreased infarct size compared with the model group. A modest reduction in infarct size was detected in mice treated with 75 mg/kg DOE (5.6% decrease). Notably, pretreatment with 150 mg/kg DOE more significantly reduced infarct size (10.0% decrease).

As shown in Fig. 3, CK-MB and LDH activities, which are indicators of myocardial ischemia, were significantly increased in the myocardial ischemia model group compared with the sham group. Conversely, pretreatment with DOE for 2 weeks decreased myocardial CK-MB and LDH release. Furthermore, the most obvious reduction was exhibited in mice pretreated with the middle dose of DOE (150 mg/kg). However, there was no marked difference in the alterations in CK-MB and LDH levels between the middle dose group (150 mg/kg) and the high dose group (300 mg/kg).

Myocardial ischemia is associated with oxidative stress (24); therefore, MDA and SOD levels were measured in the present study. As shown in Fig. 4, MDA activity was markedly increased in the model group, whereas pretreatment with DOE for 2 weeks decreased serum MDA levels. Furthermore, the most obvious decrease was observed in the middle dose group (150 mg/kg). Conversely, SOD activity was reduced in the model group compared with the sham group, whereas SOD levels were increased by pretreatment with DOE, as compared with in the model group. However, no significant change was detected in serum SOD levels between the low dose DOE group (75 mg/kg) and the model group.

Histopathological evaluation of cardiac tissue was conducted using H&E staining, as presented in Fig. 5. The cardiac tissue in the sham group exhibited a clear structure without infiltration of inflammatory cardiomyocytes (Fig. 5A). However, myocardial structural abnormalities, including cytoplasmic vacuolization, cardiomyocyte necrosis and inflammatory infiltration, were detected in the model group (Fig. 5B). Conversely, the presence of necrotic cardiomyocytes was rare, and vacuolization and myofibrillar loss were almost undetectable following pretreatment with DOE (Fig. 5C-E).

Apoptotic alterations in cardiac tissue and the percentage of TUNEL-positive cells are presented in Fig. 6. As shown in Fig. 6A, TUNEL-positive cells were rarely detected in the sham group. However, the number of TUNEL-positive cells was markedly increased in the myocardial ischemia model group (Fig. 6B). The number of TUNEL-positive cells was significantly decreased (42.5±12.4, 37.3±1.6 and 18.2±4.5%) by pretreatment with DOE (75, 150 and 300 mg/kg) compared with the model group (54.2±7.5%).

To determine the underlying mechanism of DOE, the protein expression levels of Meis1 were assessed by western blotting. The results presented in Fig. 7 indicate that the expression levels of Meis1 were significantly decreased in the model group compared with the sham group. Conversely, pretreatment with DOE (75 and 150 mg/kg) markedly upregulated the expression of Meis1.