Female Wistar rats (age, 130 days; weight, 250-300 g) were obtained from Shandong University and acclimatized for 1 week prior to any experimentation. Rats were allowed free access to water and standard chow diet. All animals were housed under the following conditions: Standard lighting at 12:12 h light-dark cycle, temperature, 22±0.5˚C and humidity, 60±10%. The present study was approved by the Ethics Committees of the Second Hospital of Shandong University, and all procedures were performed in accordance with the Institutional Animal Care and Use Committee and National Institutes of Health Guidelines.

A total of 60 rats were randomly and equally allocated into 5 groups (n=12/group): i) Sham group; ii) I/R group; iii) I/R + atorvastatin group; iv) I/R + fasudil group; and v) I/R + atorvastatin + fasudil group. The sham group was used as a control and received a standard diet and water prior to the sham operation. The I/R group was used as a control and received a standard diet and water before I/R. The I/R + atorvastatin group was orally administered atorvastatin (10 mg/kg body weight daily) for 2 weeks prior to I/R. The I/R + fasudil group was administered intravenously fasudil (10 mg/kg body weight daily) for 2 weeks prior to I/R. The I/R + atorvastatin + fasudil group was administered the same doses of atorvastatin and fasudil prior to I/R.

Rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally) and subjected to 30 min left anterior descending coronary artery (LAD) ligation using a 4-0 silk suture, followed by a 180 min reperfusion according to our previous published protocol (13). The sham control animals were subjected to the entire surgical procedure and silk suture was passed beneath the coronary artery, but the LAD coronary artery was not ligated. At the end of reperfusion, rats were exposed to overdoses of isoflurane for ~10 min and euthanatized by exsanguination.

Following reperfusion, arterial blood samples were collected and placed in heparinized centrifuge tubes, and were centrifuged at 1,500 x g for 20 min at 4˚C. The supernatant was collected and stored at -80˚C. Serum IL-6 and TNF-α activities were measured using Rat IL-6 and TNF-α ELISA kits (Bio-Swamp, Inc.; cat. no. RA20607; cat. no. RA20035, respectively), according to the manufacturer's protocols. Total nitric oxide (NO) production was determined by measuring the concentration of nitrate and nitrite, a stable metabolite of NO, by the modified Griess reaction method; the procedure involved the use of the Total NO kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocols. Each sample supernatant (100 µl) was reacted with Nitrate Reductase for 30 min and Griess reagent for 10 min at room temperature in the dark. Nitrite levels were determined by measuring absorbance at 540 nm using an OPTImax multiplate reader. The levels of nitrite were normalized to standard values.

At the end of reperfusion, the coronary artery was re-occluded and Evans blue dye solution (3 ml, 2% wt/vol) was injected into the left ventricle to determine between ischemic [area at risk (AAR), unstained] and non-ischemic myocardium (area not at risk, stained blue). The hearts were subsequently harvested, rinsed in normal saline and the atria, right ventricle and great vessels were removed. The heart was excised and cut into transverse slices (thickness, 1 mm). The AAR was separated from the area not at risk and subsequently incubated with nitro-blue tetrazolium (NBT, 1% wt/vol, 15 min at 37˚C) to distinguish between ischemic (stained blue) and infarcted tissue (unstained). Then, the AAR and infarct size were calculated after weighing the respective tissue samples, and infarct size was expressed as the percentage of the AAR.

TUNEL was used to evaluate apoptotic activity following I/R injury. Heart sections were fixed in 4% paraformaldehyde for 24 h at room temperature and embedded in paraffin. Each section (thickness, 6 µm) was deparaffinized with xylene and rehydrated with serial changes ethanol (100, 95, 90, 80, 70, 60 and 50%). TUNEL staining was performed using an in situ Cell Death Detection kit (Roche Diagnostics) according to the manufacturer's protocol. The TdT reaction was carried out for 1 h at 37^˚C in a humidified chamber, and then DAB chromogen (OriGene Technologies, Inc; cat. no. ZLI-9019) was applied. Hematoxylin was used as a counterstain. The results were viewed using a confocal FV 1000 SPD Laser Scanning microscope (Olympus Corporation; magnification, x400). TUNEL-positive cells were determined by randomly selecting 10 fields of view and were expressed as a percentage of normal nuclei.

A total of 20 mg myocardial tissue was minced and homogenized in ice-cold RIPA buffer (Sigma-Aldrich; Merck KGaA). Homogenates were subject to centrifugation at 13,000 x g for 15 min at 4˚C to obtain the supernatant as sample tissue for total protein preparation. The protein concentration was determined by bicinchoninic acid (BCA) assay kit (Beyotime Institute of Biotechnology). Myocardial tissue SOD activity and MDA content were assayed, according to the manufacturer's protocols (Jiancheng Biotech Ltd.; cat.no. A001-3-2; cat. no. A003-1-2). The results of SOD and MDA assays were expressed as units per mg of protein.

Rho-kinase activity was assessed by examining the phosphorylation state of myosin phosphatase targeting subunit 1 (MYPT-1), a well-established Rho-kinase specific substrate (26). Western blotting was performed on heart tissue obtained from the AAR. Tissue proteins were extracted using the Membrane and Cytosol Protein Extraction kit (Beyotime Institute of Biotechnology; cat. no. P0033) and protein concentration was determined using the BCA assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. After protein quantification, equal amounts of protein (50 µg) were separated on 10% Tris-glycine SDS gel by electrophoresis and subsequently transferred to PVDF membranes. After blocking with 5% BSA (Beyotime Institute of Biotechnology) in Tris-buffer for 1 h at room temperature, membranes were incubated overnight at 4^˚C with the primary antibodies: β-actin (1:1,000; Santa Cruz Biotechnology, Inc.; cat. no. sc-69879), rabbit polyclonal MYPT-1 antibody (1:500; Bioworld Technology, Inc.; cat. no. BS8367), rabbit polyclonal phosphorylated (p)-MYPT-1 antibody (1:500; Bioworld Technology, Inc.; cat. no. BS64148) and rabbit monoclonal cleaved caspase-3 antibody (1:1,000; Cell Signaling Technology, Inc.; cat. no. 9664). Then, membranes were incubated at room temperature for 2 h with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:10,000; Santa Cruz Biotechnology, Inc.; cat. no. sc-2004). Immunoreactive bands were visualized with the SuperSignal West Pico enhanced chemoluminescence kit (Piece; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Band intensities were quantified using a densitometer analysis system Quantity One 4.6.6 (Bio-Rad Laboratories, Inc.).

Data are presented as the mean ± standard deviation. Statistical analysis of the results was carried out via one-way ANOVA followed by the Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using SPSS 19.0 (SPSS, Inc.).

It was identified that the average infarct size of the heart was 57.68±3.95% in the I/R group. Furthermore, administration of atorvastatin or fasudil caused a significant reduction in infarct size; the infarct size of the heart was 37.87±5.19 and 36.21±5.01% in the I/R + atorvastatin group and I/R + fasudil group, respectively. Moreover, it was found that there were no significant differences between the I/R + atorvastatin group, I/R + fasudil group and I/R + atorvastatin + fasudil group (Fig. 1). Therefore, the present results suggested that atorvastatin and fasudil reduced myocardial infarct size in rat heart I/R injury.

Following examination of microscopy images, it was indicated that apoptotic cell nuclei were stained dark brown, while healthy myocardial cell nuclei were stained blue. The apoptotic rate was 2.04±1.34% in the sham group, while the number of TUNEL-positive cells was significantly increased in I/R group (36.83±6.39%). In addition, it was demonstrated that the apoptotic rates were significantly reduced in the I/R + atorvastatin, I/R + fasudil and I/R + atorvastatin + fasudil groups (P<0.05 vs. I/R group); however, there were no significant differences among these three groups (Fig. 2).

Activation of caspase-3 is a hallmark of apoptotic cell death, and caspase-3 cleavage is indicative of its activation (27). The western blotting results identified that I/R caused a significant increase in the protein expression of cleaved capsase-3. Moreover, it was found that capsase-3 activity was attenuated in the I/R + atorvastatin, I/R + fasudil and I/R + atorvastatin + fasudil groups (Fig. 3). Thus, the results suggested that atorvastatin and fasudil reduced cell apoptosis in rat heart I/R injury.

Compared with the sham group, the serum concentrations of IL-6 and TNF-α were significantly elevated in the I/R group (P<0.05). Furthermore, these increases in IL-6 and TNF-α levels were significantly suppressed in the I/R + atorvastatin, I/R + fasudil and I/R + atorvastatin + fasudil groups (P<0.05 vs. I/R group); however, there were no significant differences among these three groups (Fig. 4A and B; Table I). It was also found that NO production in the I/R group was significantly decreased compared with the sham group (P<0.05). Moreover, when treated with atorvastatin, fasudil or their combination, NO production increased compared with the I/R group (P<0.05), but there were no significant differences among these three groups (Fig. 4C; Table I).

It was indicated that MDA level was increased following I/R injury, while SOD decreased significantly (P<0.05 vs. sham group). In addition, when treated with atorvastatin, fasudil or their combination, the level of MDA was significantly suppressed, while SOD activity significantly increased (P<0.05 vs. I/R group; Fig. 5A and B; Table II). Therefore, the results suggested that atorvastatin and fasudil antagonized oxidative stress induced by I/R injury.

Rho-kinase activity was assessed by examining the phosphorylation of MYPT-1 using western blot analysis and it was found that the phosphorylation of MYPT-1 was significantly increased during I/R protocol (P<0.05 vs. the sham group). Furthermore, atorvastatin, fasudil or their combination therapy resulted in significant reduction in p-MYPT-1/MYPT-1 ratio (P<0.05 vs. I/R group; Fig. 6). Collectively, the results indicated that atorvastatin, similar to fasudil, inhibited Rho-kinase activity in heart I/R injury.