Male Sprague-Dawley rats (Sam Tako Inc., Osan, Korea) weighing between 220 and 250 g were used for this study. All animals were maintained in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health in 1996. Animal preparation and surgery were done according to previous report.13 Animals received general anesthesia with an intramuscular injection of 80 mg/kg ketamine (ketamine HCl®, Huons, Seoul, Korea) and 8 mg/kg zylazine (Rompun®, Bayer Korea, Seoul, Korea). Once the rat tail moved, we judged the rat as awakening from the anesthesia. We additionally injected 40 mg/kg ketamine and 4 mg/kg zylazine to maintain the level of anesthesia. The trachea was intubated, and the rats were mechanically ventilated (tidal volume, 8 mL/kg; respiratory rate, 80 bpm) with ambient air using a volume-controlled rodent ventilator (Type 7025; Ugo Basile, Comerio, Italy). Arterial blood pH and gases were maintained within normal physiological limits (pH 7.35-7.45, PaCO₂ 35-40 mmHg, PaO₂ 70-100 mmHg).

A paramedian sternotomy was performed, and the pericardium was opened to expose the heart. For the I/R experiments, a 4-0 black silk, nonabsorbable suture (Mersilk; Ethicon Inc., Somerville, NJ, USA) was passed around the left coronary artery (LCA) territory to induce regional myocardial ischemia. Coronary artery occlusion was achieved by tightening the snare through a small vinyl chloride tube and clamping it with a mosquito hemostat. Reperfusion was achieved by releasing the clamp. Myocardial ischemia was verified by the appearance of regional epicardial cyanosis over the myocardial surface and arrhythmia. After 30 min of ischemia, the myocardium was reperfused by loosening the snare, which was maintained for 2 or 24 hours of reperfusion. Successful reperfusion was confirmed by the disappearance of epicardial cyanosis and production of epicardial hyperemia and arrhythmia after releasing the snare. The body temperature was monitored with a rectal thermometer (Sirecust 1260; Siemens Medical Electronics, MA, USA) and maintained at 36-38℃ with an electrical heating pad.

Each drug was administered intraperitoneally 1 hour before the ischemic insult. We grasped the abdominal wall with two fingers to protect the bowel organs from the drug injection. We injected the drugs into the intraperitoneal space with a 23 G needle and syringe. All animals underwent a 30 min LCA occlusion followed by 2 or 24 hours of reperfusion. The animals were randomly allocated into two sets of experiments, with each set consisting of four groups. The randomization was allocated by one author with a numbered container. The sample size calculation is based on our preliminary study. The appropriate sample size was 7 by using PASS 2008 (Statistical & Power Analysis Software; NCSS, Utah, USA).

In the first set of experiments, 48 rats were divided into four groups as follows. 1) Sham + 4 mL lactated Ringer's solution (LR) group, sham-operated rats in which the LCA was not tightened (n = 12); 2) sham + EP50 group, sham-operated rats in which 50 mg/kg EP dissolved in 4 mL Ringer's solution was administered intraperitoneally 1 hour before the sham operation (n = 12); 3) I/R + LR group, rats in which 4 mL LR was administered intraperitoneally 1 hour before ischemic insult (n = 12); and 4) I/R + EP50 group, rats in which 50 mg/kg EP dissolved in 4 mL Ringer's solution was administered intraperitoneally 1 hour before ischemic insult (n = 12). Vehicle (LR 4 mL) or EP (50 mg/kg) dissolved in 4 mL Ringer's solution was administered by intraperitoneal injection 1 hour before ischemia. The animals were exposed to 30 min of ischemia followed by 2 hours of reperfusion, and tissue samples (n = 6) were used to detect NF-κB translocation and myeloperoxidase (MPO) activity. Blood samples (1 mL in each group, n = 6) were collected at baseline and after 2 hours reperfusion in tubes containing heparin to measure the tumor necrosis factor (TNF)-α and interleukin (IL)-β concentrations. The entire sample was immediately centrifuged. Plasma was collected and frozen at -70℃ until analyzed.

In the second set of experiments, animals were divided into four groups as follows. 1) Sham + 4 mL lactated Ringer's solution (LR) group, sham-operated rats in which the LCA was not tightened (n = 15); 2) I/R + LR group, rats in which 4 mL LR was administered intraperitoneally 1 hour before ischemic insult (n = 15); 3) I/R + EP25 group dissolved in 4 mL Ringer's solution, rats in which 25 mg/kg EP was administrated intraperitoneally 1 hour before ischemic insult (n = 15); and 4) I/R + EP50 group, rats in which 50 mg/kg EP dissolved in 4 mL Ringer's solution was administered intraperitoneally 1 hour before ischemia insult (n = 15). The animals were exposed to 30 min of ischemia followed by a 24 hours reperfusion, and infarct size and heart function was estimated.

The isolation of nuclear and cytoplasmic protein was done according to previous report.14 The heart sample area at risks (AARs) were suspended in a buffer [10 mM Tris (pH 7.5), 1.5 mM MgCl2, 10 mM KCl, and 0.1% Triton X-100], and lysed by homogenisation. The lysed sample was isolated by microcentrifugation at 7,500 rpm for 5 min. The supernatant, which contained cytoplasmic protein, was collected and stored at -80℃ prior to Western blot analysis. Nuclear proteins were extracted at 4℃ by resuspending the nuclear pellets in a buffer [20 mM Tris (pH 7.5), 20% glycerol, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM ethylenediaminetetraacetic acid (EDTA), and 0.1% Triton X-100]. The nuclear pellets were left at 4℃ with occasional vortex during 1 hour incubation. The pellets were centrifuged at 13,000 rpm for 15 min, the supernatant specimen that contained nuclear protein was collected. The protein concentrations of the samples were equal in all experiments.

Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with monoclonal antibodies to nuclear factor (NF)-κB p65 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4℃ overnight. Proteins were detected with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) [1 : 5,000 in Tris-buffered saline and Tween 20 (TBS-T) containing 5% skimmed milk powder for 1 hour at room temperature] and were visualised by enhanced chemiluminescence.

ELISA kits (R&D Systems, Minneapolis, MN, USA) were used for detection of TNF-α and IL-1β according to the manufacturer's instructions as earlier described.14 The plasma samples reacted with the assay reagents in the TNF-α and IL-1β kits, respectively, and analyzed spectrophotometrically (Infinite® F200; Tecan Group, Mannedorf, Switzerland) at an absorbance of 450 nm.

MPO activity, a marker for neutrophil accumulation, was evaluated according to the method of Bradley, et al. Myocardial tissue (ca. 500 mg) was excised from the AAR after 2 hours reperfusion and homogenized in 50 mM/L potassium phosphate buffer at pH 6.0. The samples were centrifuged for 30 min at 20,000 g and 4℃ before extraction. The resulting pellets were used to measure MPO activity after suspension in 50 mM/L potassium phosphate buffer containing 0.5% hexadecyltrimethyl ammonium bromide. The pellets were centrifuged at 20,000 g for 15 min; a 0.2 mL supernatant specimen was added to 0.8 mL of 50 mM/L potassium phosphate buffer (pH 6.0) containing 0.167 mg/mL 3,3'-dimethoxybenzidine dihydrochloride and 0.0005% hydrogen peroxide. Absorbance was measured spectrophotometrically at 460 nm using a spectrophotometer (Lambda EZ 210; PerkinElmer, Waltham, MA, USA) for 2 min. One unit of MPO was defined as the activity that degraded 1 µM/min peroxide at 25℃.

After the 24 hours reperfusion, rats were anesthetised with 30 mg/kg ketamine and 3 mg/kg xylazine. The right common carotid artery was cannulated with a 2-Fr Millar catheter (Model SPR-407; Millar Instruments, Houston, TX, USA) and the catheter was advanced into the left ventricle (LV) to measure heart rate (HR), left ventricular end diastolic pressure (LVEDP), and maximum rate of LV pressure increase (+dP/dt_max) and decrease (-dP/dt_max).

After the cardiac function analysis, the LCA was occluded again and 2 mL of 1% Evans blue dye was injected into the pulmonary artery to facilitate AAR determination. The Evans blue dye stained the perfused myocardium, whereas the occluded vascular bed remained unstained. The atria, right ventricles, and major vessels were removed from the heart, and the AAR was separated from the non-ischemic area. The AAR was cut into small pieces and incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC) at 37℃ for 20 min. The TTC stained the non-infarcted myocardium a deep red color, and the infarcted area (IA) remained pale yellow. The sections were fixed in 2% formalin solution for 24 hours. The LV, AAR, and IA weights were calculated. The AAR was expressed as a percentage of the LV weight, and the IA as a percentage of the AAR weight.

All values are expressed as the mean ± SD. All statistical analyses were performed using statistics software (ver. 10.0 for Windows; SPSS Inc., Chicago, IL, USA). Differences in the relationship amongst groups were evaluated by the Friedman one-way ANOVA and post hoc Duncan's multiple comparison test. Statistical significance was determined as p < 0.05.

The changes in the cytosolic and nuclear extractions are shown in Fig. 1. NF-κB expression in the cytosolic fraction was attenuated significantly in the I/R + LR group compared to the sham + LR group. In contrast, expression in the nuclear fraction was enhanced in the I/R + LR group; however, this change was inhibited by EP pretreatment, which suggests that I/R-induced NF-κB translocation into the nucleus was inhibited by EP.

I/R induced a significant increase in the plasma levels of inflammatory cytokines after the 2 hours reperfusion (Fig. 2). However, in the I/R + EP50 group, the elevation in plasma TNF-α concentration was inhibited significantly compared to that in the I/R + LR group (75 ± 36.7 vs. 152 ± 48.9 pg/mL plasma, p = 0.010). The IL-1β level was also reduced in the I/R + EP50 group compared to the I/R + LR group (105 ± 56 vs. 216 ± 85 pg/mL plasma, p = 0.040). The TNF-α and IL-1β levels were not significantly different between the Sham + LR and Sham + EP50. MPO activity in the ischemic area of the myocardium was reduced significantly in the I/R + EP50 group compared to the I/R + LR group (64 ± 7.4 vs. 86 ± 12.1 U/g tissue, p = 0.030) after regional I/R injury (Fig. 3).

Hemodynamic measurements recorded at 24 hours after reperfusion revealed substantial LV dysfunction as demonstrated by an increased LVEDP (11.9 ± 2.2 vs. 5.7 ± 1.3 mmHg, p < 0.001) and decreased contractility. The +dP/dt_max shows the decreased contractility in the I/R + LR group compared to the sham + LR group (+3,202 ± 260 vs. +3,958 ± 189 mmHg/sec, p < 0.001). The -dP/dt_max shows the decreased contractivity in the I/R + LR group compared to the sham + LR group (- 2,605 ± 247 vs. - 3,236 ± 321 mmHg/sec, p < 0.001) (Table 1). The +dP/dt_max shows an inhibited decrease of contractility in the I/R + EP50 compared to the I/R + LR group (+3,510 ± 247 vs. +3,202 ± 260 mmHg/sec, p = 0.002). Also, the -dP/dt_max shows an inhibited decrease of contractility in the I/R + EP50 compared to the I/R + LR group (- 2,979 ± 202 vs. - 2,605 ± 247 mmHg/sec, p < 0.001). Moreover, the elevated LVEDP was significantly attenuated in the I/R + EP50 group (8.2 ± 2.0 vs. 11.9 ± 2.2 mmHg, p < 0.001). Consequently, EP significantly recovered heart function after myocardial I/R injury (Table 1).

No significant differences were found in the AAR/LV induced by LCA occlusion among the groups. The IA/AAR was 51 ± 4% in the I/R + LR group and 44 ± 3% in the I/R + EP25 group (p = 0.040 vs. I/R + LR group). However, in the I/R + EP50 group, IA/AAR was reduced significantly to 36 ± 2% (p = 0.014 vs. I/R + LR group) (Fig. 4).