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Annals of Clinical & Laboratory Science 35:440-448 (2005)
© 2005 Association of Clinical Scientists

In Vivo Effects of Caffeic Acid Phenethyl Ester on Myocardial Ischemia-Reperfusion Injury and Apoptotic Changes in Rats

Kerim Cagli1, Cahit Bagci2, Mukaddes Gulec3, Beyhan Cengiz2, Omer Akyol4, Ibrahim Sari5, Sevgi Cavdar2, Sadrettin Pence2 and Hakan Dinckan6
1 Division of Cardiovascular Surgery, Yuksek Ihtisas Hospital, Ankara, Turkey; 2 Departments of Physiology, 5 Pathology and 6 Cardiology, Gaziantep University Medical Faculty, Gaziantep, Turkey; 3 Division of Biochemistry, Akyurt State Hospital, Ankara, Turkey; and 4 Department of Biochemistry, Hacettepe University Medical Faculty, Ankara, Turkey

Omer Akyol, M.D., Ph.D., Hacettepe University Medical Faculty, Department of Biochemistry, 06100 Sihhiye, Ankara, Turkey; tel 90 312 3051652 (x116); fax 90 312 3100580; e-mail oakyol{at}hacettepe.edu.tr.


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 References
 
Ischemia/reperfusion (I/R) has been reported to induce apoptotic cellular death in myocardium. This study tested the hypothesis that caffeic acid phenethyl ester (CAPE), one of the active components of propolis, may ameliorate myocardial apoptosis and oxidative myocardial injury. Wistar rats were divided into 4 groups: (i) sham operated, (ii) I/R, (iii) I/R+CAPE, and (iv) I/R+glutathione (GSH). CAPE (10 µmol/kg) was infused iv 10 min before occlusion of the left anterior descending coronary artery (30 min) followed by reperfusion (120 min). GSH (5 mg/kg) was infused iv after the occlusion and immediately before reperfusion. The TdT-mediated in situ nick end-labeling (TUNEL) method was used to evaluate apoptotic activity. I/R resulted in myocardial apoptosis, alterations of antioxidant status, elevation of serum creatine kinase (CK) and aspartate aminotransferase (AST) activities, evidence of lipid peroxidation, and elevated nitric oxide levels, compared to the sham-operation group. No apoptotic cells were found in the myocardial tissue of sham-operated rats. The TUNEL-positive myocardial cells averaged 60%, 30%, and 40% in the I/R, I/R+CAPE, and I/R+GSH groups, respectively. This study demonstrates that pretreatment with CAPE provides cardio-protection from I/R injury. The I/R+CAPE group showed reduced apoptosis, attenuated NO production, elevated myocardial superoxide dismutase (SOD) activity, and diminished serum CK and AST activities, compared to the I/R group.

Keywords: caffeic acid phenethyl ester, myocardial ischemia/reperfusion injury, apoptosis


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 References
 
Ischemia-reperfusion (I/R) injury of myocardium is a condition that results from coronary angioplasty, heart transplantation, myocardial infarction, etc. The reperfusion period of the injury is believed to be associated with increased generation of reactive oxygen species (ROS) [1]. The ROS include superoxide radicals (O2•–), hydrogen peroxide (H2O2), peroxyl radicals, singlet oxygen (1O2), and hydroxyl radicals (•OH). Among them, •OH is a particularly reactive molecule that can be formed from H2O2 in a nonenzymatic reaction catalyzed by ferric iron (Fe3+) [2]. It can react with cellular and subcellular structures, such as proteins, nucleic acids, lipids, and other molecules, altering their functions and producing tissue damage. On the other hand, nitric oxide (NO) may react with O2•– to produce peroxynitrite (ONOO–), which is capable of oxidizing or nitrating various biological substrates. Again, ONOO– is protonated to form •OH radical. The end product of peroxidation of membrane polyunsaturated fatty acids (PUFA) is malondialdehyde (MDA). Enormous production of ROS may result in arrhythmias, membrane permeability changes, and abnormal contractile function of the myocardium [3]. ROS may also depress sarcolemmal calcium transport and cause cardiac dysfunction [4].

Proinflammatory cytokines and tumor necrosis factor-alpha are released after reperfusion of the myocardium [5]. Apoptosis is a major contributor to I/R injury of the myocardium in experimental models [6,7]. Heart injury after open heart surgery may be related to apoptosis [8]. Attempts have been made to attenuate apoptotic myocardial injury after I/R [9,10].

Caffeic acid phenethyl ester (CAPE) is an active component of honeybee propolis extracts. It has long been used in folk medicine in various regions of the world. At a concentration of 10 µM, it blocks the production of ROS in human neutrophils by the xanthine/xanthine oxidase (XO) system [11]. Although CAPE has antioxidant [12–14] and anti-inflammatory [15] effects, its effects on myocardial I/R injury by oxidant/antioxidant pathways have not been investigated in detail. The present study tested whether CAPE pretreatment ameliorates I/R injury and apoptotic changes in the myocardium.

Materials and Methods  Animals:  Forty-two male Wistar rats weighing 250–350 g were used in the study. Rats were housed in a vivarium with 12-hr light/dark cycle (8 am; 8 pm). Rats were allowed access to water and food ad libitum. This study was in compliance with the Guide for the Care and Use of Laboratory Animals.

Experimental design:  Rats were divided into 4 groups: Group 1 consisted of sham-operated rats; Group 2 consisted of rats undergoing I/R and treated with iv vehicle (ethanol diluted in saline); Group 3 consisted of I/R+CAPE-treated rats; Group 4 consisted of I/R+GSH-treated rats. After completion of the surgical procedures, all hearts were allowed to stabilize for 20 min prior to the experimental protocol. The left anterior descending (LAD) coronary artery was occluded for 30 min for ischemia and then reperfused for 120 min.

Drug administration:  CAPE (10 µmol/kg) (Sigma, >97% pure by HPLC and TLC) and GSH (5 mg/kg) (Sigma, 99% pure) were each administered in 0.1 ml of vehicle solution (ethanol 10% (v/v) in saline solution (NaCl, 9 g/L)). Control rats received an equal volume of the ethanol/saline vehicle solution. The CAPE infusion was given iv 10 min before occlusion, and the GSH infusion was given iv before reperfusion (see below).

Surgical preparation of animals:  The rats were anesthetized with ip thiopental sodium (50 mg/kg, Pental Sodyum, I.E. Ulagay, Istanbul, Turkey). The jugular vein was cannulated for drug administration. A tracheotomy was performed, and the trachea was intubated with a cannula connected to a rodent ventilator (SAR-830 IITC Life Science, Woodland Hills, CA, USA) for artificial respiration. The rats were ventilated with room air supplemented with O2 at 60–65 breaths/min. A blood pressure monitor (PowerLab, AD Instrument Co., Castle Hill, Australia) was used to measure blood pressure by cannulating the transducer through the carotid artery. Body temperature was maintained at 37 ± 1°C using a heating pad.

A left thoracotomy was performed 10 mm from the sternum to expose the heart at the fifth intercostal space. The pericardium was opened, and the left atrial appendage was moved to reveal the LAD coronary artery. A ligature (6–0 silk) was passed below the left vein and coronary artery from the area immediately below the left atrial appendage to the right portion of the left ventricle. The ends of the suture were threaded through a propylene tube to form a snare. Following a stabilization period of 20 min, the snare around the LAD coronary artery was sutured and held in place with a small clip to induce transient regional myocardial ischemia for 30 min. Pulling the ends of the suture taut and clamping the snare onto the epicardial surface with a hemostat elicited occlusion of the coronary artery and resulted in regional left ventricular ischemia. Successful occlusion was confirmed by a 20–30% reduction in the arterial blood pressure relative to the pre-ischemic values. Any animal in which this procedure produced an arrhythmia or a sustained fall in the mean arterial pressure to <70 mm Hg was removed from the study at this point. Reperfusion of the ischemic myocardium, initiated by unclamping the hemostat and loosening the snare, was confirmed an epicardial hyperemic response.

Tissue preparation:  Histological studies and measurements of enzyme activities and analyte levels were performed on samples of the left anterior ventricular wall obtained from hearts of the rats in the 4 experimental groups. The rats were anesthetized and sacrificed, and the hearts rapidly excised, rinsed in cold PBS (pH 7.4) containing 0.16 mg/ml heparin to remove red blood cells and clots, and divided into 2 parts for the histological and biochemical studies. For biochemical analyses, the ventricular wall sample was frozen in liquid nitrogen and stored at –25°C. The remainder of the heart was fixed in 10% formalin for 24 hr. Myocardial ischemic foci were identified by visual inspection of pale areas, and were cut at 6–7 nm thickness. The 2 center slices were embedded in paraffin and histological sections were prepared.

Apoptotic activity was evaluated by the TdT-mediated in situ nick end-labeling (TUNEL) method. Each section was deparaffinized and rehydrated with serial changes of xylene and ethanol. Proteinase K (20 ng/ml) was applied to the section for 15 min to produce proteolysis. The endogenous peroxidase was inhibited with 3% hydrogen peroxide for 5 min. An apoptosis detection kit (Apoptag, Oncor, Gaithers-burg, MD, USA) was used. The TdT reaction was applied for 1 hr at 37 °C. Anti-digoxigenin-peroxidase was applied for 30 min at room temperature. Biochemical controls were made with positive slides treated with DNase-I (1 mg/ml) instead of proteinase K, and with negative slides treated with phosphate-buffered saline instead of TdT. Methyl green was used as the counter stain. Nonspecific cytoplasmic staining without nuclear involvement was considered to be negative. By microscopy at x400 magnification, 3 fields were searched; the number of positive cells, divided by the total number of cells, was defined as the TUNEL index.

For biochemical analyses, cardiac tissues were washed twice with cold saline solution, placed in glass vials, labeled, and stored in a freezer (–30°C) until processing. Tissue samples were minced with scissors and homogenized in 4 volumes of ice-cold Tris-HCl buffer (50 mM, pH 7.4) using a glass Teflon homogenizer (Ultra Turrax IKA T18 Basic) for 2 min at 5,000x g. Malondialdehyde (MDA), xanthine oxidase (XO), nitric oxide (NO), and protein levels were measured.

The homogenate was then centrifuged at 5,000x g for 60 min. The supernatant was collected; glutathione peroxidase (GSH-Px) and catalase (CAT) activities and protein level were measured at this stage. The supernatant solution was extracted with an equal volume of ethanol/chloroform (5/3, v/v). After centrifugation at 5000 x G for 30 min, the clear upper layer (the ethanol phase) was collected and assayed for superoxide dismutase (SOD) activity and protein level. All of these biochemical procedures were performed at 4°C.

Blood samples were collected at the end of the experiment. The samples were centrifuged at 5,000 rpm, 4°C, for 15 min; the plasma was removed and stored at –30°C. Total creatine kinase (CK) and aspartate transaminase (AST) activities were analyzed by an automated analyzer (Olympus AU-600, Shizuoka-ken, Japan) using commercial kits (Olympus).

Superoxide dismutase (SOD) activity:  Total (Cu-Zn, extra-cellular and Mn) SOD (E.C. 1.15.1.1 [EC] ) activity was determined in heart tissue according to Sun et al [16]. The method is based on inhibition of nitroblue tetrazolium (NBT) reduction by the xanthine-xanthine oxidase system as a superoxide generator. SOD activity was assessed in the ethanol phase of the myocardial homogenates after 1 ml ethanol/chloroform mixture (5/3, v/v) was added to the same volume of sample that was centrifuged. One unit of SOD was defined as the enzyme amount causing 50% inhibition of the NBT reduction rate. SOD activity was expressed as U/mg protein.

Glutathione peroxidase (GSH-Px) activity:  Glutathione peroxidase (E.C. 1.6.4.2 [EC] ) activity was measured in heart tissue by the method of Paglia et al [17]. The enzymatic reaction in the tube, which contained NADPH, reduced glutathione (GSH), sodium azide, and glutathione reductase, was initiated by adding H2O2, and the change in absorbance at 340 nm was measured by a spectrophotometer. The activity was expressed as U/g protein.

Catalase (CAT) activity:  CAT activity was assayed in heart tissue by Aebi’s method [18]. This method is based on determination of the rate constant (s–1, k) for H2O2 decomposition at 240 nm. Results were expressed as k/g protein.

Malondialdehyde (MDA):  The MDA level was determined by reaction with thiobarbituric acid (TBA) at 90–100°C [19]. MDA or MDA-like substances, and TBA react to produce a pink pigment with absorption maximum at 532 nm. The sample was mixed with 2 volumes of cold 10% (w/v) trichloro-acetic acid to precipitate protein. The precipitate was pelleted by centrifugation and an aliquot of the supernatant was reacted with an equal volume of 0.67% (w/v) TBA at 90°C for 15 min. After cooling, the absorbance was read at 532 nm (UV-1601 spectrophotometer, Shimadzu, Kyoto, Japan). The results were expressed as nmol/g wet tissue, based on a graph prepared with 1,1,3,3-tetramethoxypropane standards.

Nitric oxide (NO) level:  Tissue nitrite (NO2–) and nitrate (NO3–) were estimated as an index of NO production, based on the Griess reaction [20]. Tissue samples were deproteinized with Somogyi’s reagent and total nitrite (nitrite+nitrate) was measured by spectrophotometry at 545 nm after reduction of nitrate to nitrite with copperized cadmium granules. The assay was calibrated with standard solutions (10–8 to 10–3 mol/L) of sodium nitrite. Linear regression was performed and the resulting equation was used to calculate the unknown sample concentrations. Results were expressed as µmol/g wet tissue.

Xanthine oxidase (XO) activity:  Xanthine oxidase activity was assayed spectrophotometrically at 293 mn by the formation of uric acid from xanthine according to Prajda and Weber [21]. A calibration curve was constructed by using standard XO solutions (10–50 mU/ml, Sigma X-1875, Sigma Cemical Co, St Louis, MO, USA). One unit of activity was defined as 1 µmol of uric acid formed/min at 37 °C, pH 7.5; results were expressed as U/g of tissue.

Protein concentration:  Protein was assayed by the method of Lowry et al [22].

Statistics:  Data were analyzed by the SPSS for Windows computing program. Significant differences between the groups were determined using one-way ANOVA followed by post-hoc LSD. A p value <0.05 was accepted as significant. Results were expressed as mean ± SD or mean ± SE.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 References
 
I/R caused a significant increase in the serum level of CK and AST. The increases of CK and AST induced by I/R were ameliorated by administration of CAPE (10 µmol/kg) or GSH (5 mg/kg) (Figs. 1AGo and 1BGo). The apparent reduction of CK level induced by GSH was not statistically significant.



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  		Fig. 1A. Effects of myocardial ischemia and reperfusion (I/R) on plasma total creatine kinase (CK) activity. * p <0.0001 vs sham group; ** p <0.001 vs I/R group.

 


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  		Fig. 1B. Effects of myocardial ischemia and reperfusion (I/R) on plasma aspartate aminotransferase (AST) activity. *p <0.0001 vs I/R group; ** p <0.0001 vs sham group {dagger} p <0.002 vs sham group; # p <0.004 vs sham group.

 
TUNEL staining of myocardial samples was performed in the possible ischemic areas in the 4 groups. No apoptotic cells were found in the myocardial tissues of sham-operated rats. The mean proportions of TUNEL positive cells were 60%, 30%, and 40% in the I/R group, I/R+CAPE group, and I/R+GSH group, respectively (Figs. 2Go–5GoGoGo).



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  		Fig 2. TdT-mediated in situ nick end-labeling (TUNEL) staining (x400) of myocardium from the sham group.

 


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  		Fig 3. TdT-mediated in situ nick end-labeling (TUNEL) staining (x400) of myocardium from the I/R group.

 


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  		Fig 4. TdT-mediated in situ nick end-labeling (TUNEL) staining (x400) of myocardium from the I/R+CAPE group.

 


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  		Fig 5. TdT-mediated in situ nick end-labeling (TUNEL) staining (x400) of myocardium from the sham group.

 
Statistical analyses of results for the various physiological variables did not show any significant differences among the 4 groups. The heart rate was unaffected by the I/R procedure; administration of CAPE and GSH did not affect the heart rate after I/R (Table 1Go). Left ventricular pressure was unchanged upon I/R and was unaffected by administration of CAPE or GSH (Table 2Go).


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  		Table 1. The effects of caffeic acid phenethyl ester (CAPE) and glutathione (GSH) on heart rate (beat/minute) after 30 min of myocardial ischemia (AI) and after 30, 60, and 120 min of myocardial reperfusion (AR). Data are means ± SE.
 

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  		Table 2. The effects of caffeic acid phenethyl ester (CAPE) and glutathione (GSH) on left ventricular pressure (mm Hg) after 30 min of myocardial ischemia (AI) and after 30, 60, and 120 min of myocardial reperfusion (AR). Data are means ± SE.
 
The results for biochemical parameters are summarized in Tables 3Go and 4Go. There was significant decrease in SOD activity in the I/R group compared to the sham group (p <0.034). SOD activity in the I/R+CAPE group was higher than in those of sham, I/R, or I/R+GSH groups. GSH caused reduced SOD activity compared to the sham and I/R groups. There was a significant increase in GSH-Px activity in the I/R group compared to the sham operation group (p <0.0001). CAPE and GSH had no effects on GSH-Px activity induced by the I/R procedure. Tissue CAT activity was increased in the I/R group compared to the sham group. GSH, but not CAPE, caused a significant decrease in CAT activity after I/R (p <0.025).


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  		Table 3. Cardiac antioxidant enzyme actvities: superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) after myocardial I/R injury. Results are expressed as mean ± SD; n.s. = not significant.
 

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  		Table 4. Cardiac malondialdehyde (MDA) and nitric oxide (NO) levels as well as xanthine oxidase (XO) activity after myocardial I/R injury. Results were expressed as mean ± SE.; n.s. = not significant.
 
There were no significant changes in tissue XO activity in the I/R group compared to the sham, I/R+CAPE, and I/R+GSH groups. Tissue MDA levels in myocardium were increased in the I/R group compared to the sham group. Neither CAPE nor GSH caused reduced MDA levels after I/R. In contrast to MDA levels, tissue NO levels were decreased by CAPE (p <0.004) after a significant increase in the I/R group compared to the sham operation group. GSH had no attenuating effect on the increased NO levels after I/R.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
References
 
Coronary artery disease is one of the leading causes of death in Turkey, as in all Western societies [23]. Therefore, the search for new preventive agents for reperfusion injury of myocardium is one of the main goals of scientists studying in this area. The present study confirmed our hypothesis that administration of CAPE at a dose of 10 µmol/kg would provide some cardio-protection from I/R injury. Evidence for this conclusion can be summarized as follows; (i) reduced apoptosis in myocardium, (ii) diminished plasma activities of CK and AST, and (iii) attenuation of myocardial NO production in the I/R+CAPE group vs the I/R group.

One of the common disorders associated with cell death is myocardial infarction. This disease arises primarily as a result of an acute cessation of blood flow. In myocardial ischemia, cells within the central area of ischemia appear to die rapidly as a result of necrosis. However, outside the central ischemic zone, cells die over a more protracted period and morphologically appear to die by apoptosis [24]. Ischemia of cardiac myocytes in culture results in the induction of apoptosis [25]. Agents known to be effective in inhibition of apoptosis might limit myocardial infarct size.

Further tissue injury frequently occurs during establishment of reperfusion after ischemia in the heart. The reperfusion process is associated with increases in ROS production and intracellular calcium ions (Ca2+); both are known as inducers of apoptosis. It has been suggested that reperfusion injury can be prevented by using agents that alter apoptotic threshold and inhibit apoptosis [9]. Our findings show that CAPE decreases apoptotic changes in myocardial tissue after I/R in rats. On the other hand, CAPE has been suggested to induce apoptosis in transformed cells as well as cytotoxicity in oral cancer cells [26]. This inducing effect of CAPE on apoptosis may be a unique effect that pertains to neoplastic changes in cells and tissues. Cells from a variety of human malignancies have decreased ability to undergo apoptosis in response to some physiologic stimuli [27]. Chen et al [28] suggested that CAPE induces apoptosis by targeting intracellular GSH and mitochondria, whereas the antioxidant activity of CAPE probably relates to a scavenging effect for H2O2.

I/R is known to impair the intracellular anti-oxidant defense system [29]. This impairment may be one of the main causes of myocardial I/R injury. In this study, we investigated the effects of CAPE, which exerts antioxidant properties in vivo. We showed in the present I/R model that CAPE protects myocardium to some extent against reperfusion injury. Previously, we reported that CAPE was a good agent against several organ I/R injuries [30–35]. Recently, Ozer et al [36] investigated the effects of CAPE on I/R-induced infarct size and hemodynamic parameters in rat hearts [36]. Our findings on the hemodynamic changes of rats were in accordance with their results as hemodynamic parameters did not change in I/R group and study groups (CAPE and GSH) compared to control group. They found that CAPE administration (50 µmol/kg) iv reduced infarct size/risk area in rat heart after I/R. They noted that CAPE provides protection against I/R injury in rat heart. The modulation of lipid peroxidation, antioxidant enzymes, NO, apoptosis, infarct size, and cardiac enzymes such as CK, AST, and ALT that was induced by CAPE in our study and the other investigation can probably be attributed to its antioxidant and tissue stabilizing effects.

NO donors were shown to enhance the recovery of myocardial functions and coronary flow in isolated rat hearts subjected to global I/R, and the effect was abolished by NOS inhibitors [37,38]. NO production has been shown to have both detrimental and beneficial effects after I/R. NO is known to react with O2•– to produce a potentially damaging oxidant, peroxynitrite (ONOO–) [35]. CAPE may inhibit iNOS gene expression and iNOS enzyme activity, resulting in reduction in NO production [39]. On the other hand, NO could inactivate pro-inflammatory inducible enzymes [40] that may induce some forms of heart injury. Tissue NO level was found to be increased after I/R in this study; CAPE diminished the NO levels, protecting myocardial tissue from the detrimental effect of ONOO–. NO may directly stimulate or inhibit apoptosis. The potential direct relationship has been reported between increased NO and increased apoptosis [41]. Apoptosis induced by increased NO production in I/R of myocardium might be reversed by reduced NO levels in case of CAPE administration.

It must be emphasized that the indirect estimation of total myocardial NO using the Griess reaction (nitrate plus nitrite) is inferior to the direct measurement of NO using a porphyrinic microelectrode impaled in individual cells [42]. Moreover, the Griess reaction is insensitive to local fluctuations in NO concentrations [43].

Enzymatic elimination of ROS by antioxidant enzymes such as SOD and CAT lacked in vivo efficacy in animal models [44] although myocyte protection was achieved in vitro [45]. Our findings with antioxidant enzymes in the present study are consistent with our previous reports. Intracellular GSH level appears to be decreased after I/R injury. Seiler and Starnes found that, in isolated perfused rat hearts, GSH supplementation protected the myocardium against I/R injury [46]. We observed a significant increase in GSH-Px activity after I/R. In the I/R+GSH group, there was a small but insignificant increase in GSH-Px activity.

In conclusion, studies in humans and animals indicate that myocardial I/R induces oxidative stress, especially as a mechanism of reperfusion injury. In the present study, the beneficial effects of CAPE against I/R injury and oxidative stress were confirmed by an in vivo experiment. Pretreatment with CAPE decreased the apoptosis in rat myocardium that was induced by I/R injury. Pretreatment with CAPE also influenced some oxidative/antioxidative mechanisms. This report thus provides valuable information about the effects of caffeic acid substances derived from propolis on myocardial I/R injury.


   Acknowledgment
 
This study was supported by Project #TF-01-07 from the Gaziantep University Scientific Research Projects Fund.


   References
Top
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 Introduction
 Results
 Discussion
 References
 

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