Scoparone was purchased from Dalian Meilun Biology Technology Co., Ltd., (Dalian, China).

Cardiac myocytes were prepared from 20 neonatal Sprague-Dawley rats (Male, 1–3 days-old, 10 g) as previously described (13). Rats were obtained from Hebei Medical University. Rats were housed under the same standard environmental conditions of light (a 12-h light/dark cycle), temperature (22±2°C), and ambient humidity of 50±10% with free access to food and water. Briefly, the obtained ventricles were cut into sections, which were digested with trypsin at 37°C for 8 min. Subsequently, the supernatant was added to Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) containing 10% fetal bovine serum (FBS, Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The digestion step was repeated 6–8 times, until the tissue sections were digested. Cell suspensions were centrifuged at 800 × g for 5 min at 37°C, and the cells were cultured with DMEM containing 10% FBS at 37°C in an atmosphere containing 5% CO₂ for 2 h. The non-adherent cell suspension was collected to separate fibroblast cells and cardiac myocytes based on the varying durations of adherence. The fibroblast cells and cardiac myocytes were separated by its adherent at different time. The cardiac myocytes (1×10⁵ cells/ml) were cultured in DMEM containing 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 g/ml vitamin B12. After 48 h, dead cells were removed, and the medium was replaced with fresh medium containing 0.1 mM bromodeoxyuridine. The cardiac myocytes were assessed by immunofluorescence staining with α-actin (cat. no. sc-58670; Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) as previously described (13); myocardial cell purity was confirmed at >95%.

OGD/R was performed on primary cultured neonatal rat cardiac myocytes according to previously described methods (14). Briefly, primary cultured neonatal rat cardiac myocytes were randomly divided into five groups: Control group, OGD/R group, scoparone low-dose group (S-L), scoparone mid-dose group (S-M) and scoparone high-dose group (S-H). The control group was incubated without any treatment. As for the OGD/R group, the cell medium was replaced with DMEM (glucose-free), which was preincubated with a mixture of 5% CO₂ and 95% N₂ for 20 min to remove O₂. Subsequently, cells were cultured in an incubator containing 5% CO₂ and 95% N₂ at 37°C. After 3 h, the medium was replaced with DMEM containing glucose, and cardiac myocytes were transferred into an incubator containing 5% CO₂ at 37°C for 1 h. The cells in the S-L, S-M and S-H groups were pretreated with scoparone at 100, 500 and 1,000 mg/ml, respectively, for 1 h prior to OGD/R, which was conducted as described for the OGD/R group.

Male Wistar rats with body weight ranging from 240–260 g, 7-weeks old were used in the present study. Rats were obtained from Hebei Medical University and were housed under the same standard environmental conditions of light (12-h light/dark cycle), temperature (22±2°C), and ambient humidity of 50±10% with free access to food and water. A total of 120 Wistar rats were randomly divided into five groups: Sham-operated group (sham), I/R group, scoparone low-dose group (S-L), scoparone mid-dose group (S-M) and scoparone high-dose group (S-H) (n=24 rats/group). Briefly, the rats were anesthetized with ether and an incision was made in the chest to expose the heart. The left anterior descending branch of the coronary artery was then isolated. With the exception of the sham group, in the other groups, the coronary artery was immediately ligated with line 0 (15); the groups underwent ischemia for 30 min, followed by 120 min of reperfusion. Scoparone was administered 1 h prior to ligation. The rats in the sham and I/R groups were intravenously injected with 2 ml/kg normal saline, whereas the rats in the S-L, S-M and S-H groups were intravenously injected with 25, 50, 100 mg/kg scoparone, respectively. The rats were sacrificed after reperfusion and myocardial tissues were collected. The present study was approved by the Ethics Committee of Hebei Medical University (Shijiazhaung, China).

Following OGD/R in vitro, cell viability was determined using MTT reagent. Cells (1×10⁵/ml) were cultured with MTT reagent (final concentration, 0.5 mg/ml) for 4 h at 37°C. The medium was then removed and dimethyl sulfoxide (150 µl) was added to each well for 15 min at 37°C, in order to solubilize formazan. The absorbance of formazan was measured at 492 nm, which is directly proportional to cell viability.

The apoptotic rates of cardiac myocytes subjected to OGD/R injury in vitro and I/R injury in vivo were measured by terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) assay using a TUNEL kit (In situ Cell Death Detection kit, fluorescein; Roche Diagnostics, Indianapolis, IN, USA) according to manufacturer's protocols. The cell nucleus was stained with DAPI (Roche Diagnostics) according to manufacturer's protocols. Positive TUNEL staining was observed under a fluorescence microscope (TE2000U; Nikon Corporation, Tokyo, Japan) using a B-2A filter (450–490 nm excitation filter, 505 nm dichroic mirror, 520 nm bandpass filter). The ratio was determined by calculating the number of TUNEL-positive cells to the total number of cells in each of the 10 fields of view.

Following OGD/R in vitro, cell culture medium was collected. The levels of lactate dehydrogenase (LDH) and creatine kinase (CK) were measured using commercial ELISA kits (cat. nos. JL13677 and 0-025486; Shanghai Jiang Lai Biological Technology Co., Ltd., Shanghai, China), according to the manufacturer's protocols. In addition, the cells were collected by centrifugation (1,000 × g, 5 min, 37°C), and the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were measured using commercial ELISA kits (cat. no. JL13297 and JL11065; Shanghai Jiang Lai Biological Technology Co., Ltd.) according to the protocols recommended by the manufacturer.

Following I/R in vivo, serum samples were collected by centrifugation (3,000 × g, 10 min, 4°C) from myocardial tissues. The concentrations of LDH and CK in the serum were detected using ELISA kits (cat. nos. JL13677 and 0-025486; Shanghai Jiang Lai Biological Technology Co., Ltd.) according to manufacturer's protocols. In addition, myocardial tissues were collected from all rats to detect MDA and SOD levels. Ice physiological saline is added to the myocardial tissues. A total of 10% myocardial tissue homogenate was made by using a high-speed homogenizer and centrifuged 3,000 × g at 4°C. The supernatant was collected. The levels of MDA and SOD in the supernatant were determined using commercial kits (cat. nos. JL13297 and JL11065; Shanghai Jiang Lai Biological Technology Co., Ltd.) according to the manufacturer's protocols.

Intracellular ROS levels were measured using a fluorescent carboxy-H₂DCFDA probe, as previously described (16). Carboxy-H₂DCFDA is hydrolyzed to H₂DCF in cells; H₂DCF emits no fluorescence and cannot leave the cell through the cell membrane. However, ROS can oxidize H₂DCF to DCF, which emits a green fluorescence; therefore, detection of the fluorescence intensity of DCF can reflect intracellular ROS levels; the fluorescence intensity is proportional to the concentration of ROS. Following I/R injury, the myocardium was homogenized in Hank's buffered salt solution and the supernatant was collected. The samples were cultured with 10 µM carboxy-H₂DCFDA for 20 min at 37°C in the dark. The fluorescence signal intensity of DCF was detected using a flow cytometer with 488 nm excitation wavelength and 600 nm detection wavelength. Expo 32 ADC (Beckman Coulter, Inc., Brea, CA, USA) was used to analyze the fluorescence data.

Myocardial IA was determined using the triphenyl tetrazolium chloride (TTC) staining method (17). Briefly, the heart samples were frozen and sliced into 1 mm sections along the vertical axis. The sections were incubated for 15 min at 37°C in 1% TTC, and were then immersed in 4% formaldehyde for 12 h at 37°C. IA were determined using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Following OGD/R in vitro, cardiac myocytes were collected and lysed at 4°C with lysis buffer (Tiangen Biotech, Co., Ltd., Beijing, China) containing 1 µM phenylmethylsulfonyl fluoride. Protein concentration was determined according to the bicinchoninic acid method. Equal amounts of protein (100 µg) were separated by 10% SDS-PAGE and were electrotransferred onto a polyvinylidene fluoride membrane. The membrane was blocked with Tris-buffered saline (TBS) containing 5% skimmed milk for 1 h at 37°C, and was then incubated with primary antibodies against B-cell lymphoma 2 (Bcl-2; cat. no. ab692), Bcl-2-associated X protein (Bax; cat. no. ab77566), caspase-3 (cat. no. ab13585) and cytochrome c (Cyt C; cat. no. ab110325; Abcam, Cambridge, MA USA), all 1:100 dilution, at 4°C overnight. Subsequently, the membrane was washed with TBST [TBS, (pH 7.5) containing 0.1% Tween-20] and was incubated with an anti-Mouse immunoglobulin G (IgG) H&L secondary antibody (1:10,000; cat. no. ab6785; Abcam) for 1 h at 37°C. Signals were then developed using the Enhanced Chemiluminescence Plus Western Blotting Detection system (Amersham; GE Healthcare Life Sciences, Little Chalfont, UK). Signal intensities were semi-quantified by densitometry using ImageJ v1.45 software (National Institutes of Health, Bethesda, MD, USA) and were normalized against the corresponding β-actin (cat. no. ab8226; Abcam) signals. The antibody was diluted by 1:1,000 and incubated for 1 h at 37°C.

Harvested myocardial tissues were fixed in formalin overnight at 37°C, paraffin embedded; serial sections (3–5 µm) obtained. The myocardial sections were blocked with 3% hydrogen peroxide for 15 min at room temperature. Subsequently, the sections were microwaved in 10 mmol/l (pH 8.0) EDTA (Sangon Biotech Co., Ltd., Shanghai, China) for 2 min, incubated with 5% goat serum (Shanghai Haoran Bio, Shanghai, China) for 1 h at 37°C, and incubated overnight at 4°C with various antibodies (Cyt c, caspase-3, Bcl-2 and Bax) as aforementioned. Anti-Mouse IgG H&L was used as aforementioned. To analyze staining, the following systems were used: PicTure PV6000 and Elivision Plus (Fuzhou Maixin Biotech Development Co., Ltd., Fuzhou, China). Finally, the sections were counterstained with hematoxylin (3 min, 37°C). An Olympus BX41 brightfield microscope (Olympus Corporation, Tokyo, Japan) was used to observe sections.

Data are presented as the means ± standard deviation. Experiments were repeated in triplicate. All data were analyzed using one-way analysis of variance followed by Bonferroni post hoc test (SPSS software package version 19.0; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Following OGD/R injury in cardiac myocytes, cell viability and SOD activity were significantly reduced, whereas LDH release, MDA levels, CK activity and cell apoptosis were markedly increased. Scoparone, at concentrations of 100, 500 and 1,000 mg/ml, attenuated these alterations in a dose-dependent manner (Figs. 1–3). In cardiac myocytes treated with 100, 500 and 1,000 mg/ml scoparone prior to OGD/R injury, cell viability was increased by 17.9, 28.7 and 65.7%, respectively; cell apoptosis was decreased by 27.0, 41.7 and 62.2%, respectively; LDH levels were decreased by 12.8, 32.1 and 52.8%, respectively; MDA production was decreased by 13.7, 28.6 and 47.4%, respectively; SOD activity was increased by 24.4, 56 and 82.4%, respectively; and CK activity was decreased by 24.2, 47.2 and 62.7%, respectively.

Following I/R injury in rats, the levels of LDH and MDA, and SOD and CK activities were detected. I/R significantly increased the LDH levels (4,152.51±487.31 U/l), MDA production (10.547±0.92 mmol/ml), and CK activity (1,137.18±106.35 U/ml), whereas SOD activity was significantly decreased (1,684.68±143.56 U/l) compared with in the sham group (Fig. 4; P<0.05). Conversely, compared with the I/R group, scoparone was revealed to significantly decrease the LDH levels (4,034.15±428.23, 3,502.53±412.04 U/l and 3,425.14±482.47 U/l, respectively; P<0.05), MDA production (8.677±0.495, 7.621±0.587 and 6.805±0.647 mmol/ml, respectively; P<0.05), and CK activity (924.478±38.841, 766.758±43.812 and 708.158±83.958 U/l, respectively; P<0.05), whereas SOD activity was increased (1,977.341±176.902, 2,173.923±185.483 and 2,365.837±166.384 U/l, respectively; P<0.05) in a dose-dependent manner, compared with the I/R group (Fig. 4).

The apoptotic rate of cardiac myocytes was detected by TUNEL staining. The results indicated that the percentage of apoptotic cardiac myocytes was significantly increased in the I/R group (34.95±1.53%), compared with in the sham group (Fig. 5; P<0.05). Treatment with scoparone, at doses of 25, 50 and 100 mg/kg, resulted in a dose-dependent reduction in the apoptotic rate of cardiac myocytes following I/R injury, to 26.14±1.71, 22.11±1.98 and 12.13±1.54%, respectively.

TTC staining was conducted to determine the effects of scoparone on myocardial infarct size in I/R rats (Fig. 6). In the I/R group, myocardial IA was 85.09±2.53%, which was a significantly increased compared with the sham group (P<0.05). Treatment of scoparone, at a dose of 25, 50 and 100 mg/kg, significantly reduced myocardial IA (43.98±1.96, 25.64±2.36 and 9.05±1.87%, respectively) compared with in the I/R group (P<0.05).

ROS concentration was determined following I/R using a carboxy-H₂DCFDA probe. The fluorescence intensity of DCF represents the concentration of ROS. As shown in Fig. 7, I/R injury significantly increased fluorescence intensity compared with in the sham group (677.93±37.44 % of control). However, scoparone pretreatment, at a dose of 25, 50 and 100 mg/kg, significantly decreased fluorescence intensity (454.29±32.32, 346.64±30.90, 224.45±27.44% of control, respectively) induced by I/R in a dose-dependent manner.

As shown in Fig. 8, the results of western blot analysis indicated that caspase-3 and Cyt C expression were increased following OGD/R injury. Conversely, scoparone, at concentrations of 100, 500, 1,000 mg/ml, significantly and dose-dependently decreased caspase-3 and Cyt C expression following OGD/R injury of cardiac myocytes.

The effects of scoparone on caspase-3 and Cyt C expression in I/R rats were detected by immunohistochemical staining. As shown in Fig. 9, I/R administration markedly increased caspase-3 and Cyt C expression compared with in the sham group; however, scoparone inhibited I/R-induced caspase-3 and Cyt C expression in a dose-dependent manner.

As shown in Figs. 8 and 9, I/R injury induced an increase in Bax expression and a decrease in Bcl-2 expression in a cell model of OGD/R model and a rat model of I/R. Conversely, treatment with scoparone attenuated these alterations in a dose-dependent manner.