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Caffeic acid phenethyl ester (CAPE) protects rat skeletal muscle against ischemia–reperfusion-induced oxidative stress
Vascular Pharmacology 47 (2007) 108 – 112 www.elsevier.com/locate/vph
Caffeic acid phenethyl ester (CAPE) protects rat skeletal muscle against ischemia–reperfusion-induced oxidative stress Huseyin Ozyurt a,⁎, Birsen Ozyurt b , Kenan Koca c , Salih Ozgocmen d a
d
Gaziosmanpasa University, Faculty of Medicine, Department of Biochemistry, Tokat, Turkey b Gaziosmanpasa University, Faculty of Medicine, Department of Anatomy, Tokat, Turkey c Sarikamis Military Hospital, Department of Orthopaedics and Traumatology, Kars, Turkey Firat University, Faculty of Medicine Department of Physical Medicine and Rehabilitation, Elazig, Turkey Received 17 November 2006; accepted 27 April 2007
Abstract Oxygen-derived free radicals have been implicated in the pathogenesis of skeletal muscle injury after ischemia–reperfusion. Caffeic acid phenethyl ester, an active component of propolis extract, exhibits antioxidant properties. The aim of this study was to assess the effects of caffeic acid phenethyl ester (CAPE) and α-tocopherol (vit E) on ischemia/reperfusion (I/R) injury in a rat hind limb ischemia/reperfusion model. For this purpose, ischemia was induced in anesthetized rats by unilateral (right) femoral artery clipping for 2 h followed by 2 h of reperfusion. Four groups were studied: sham, I/R, I/R + CAPE and I/R + vit E. Drugs were administered intraperitoneally after 1 h of ischemia and I/R rats received saline vehicle. After 2 h of reperfusion, venous blood was sampled and the right gastrocnemius muscle was harvested. Plasma and tissue were assayed for malondialdehyde (MDA), superoxide dismutase (SOD) and nitric oxide (NO) metabolites. Tissue was also assayed for catalase (CAT) activity. Both tissue and plasma NO levels, MDA levels, SOD activities was significantly increased in I/R groups compared to control groups. The two treated groups showed decreased MDA and NO in both muscle and plasma compared to the I/R group. No differences were noted in muscle tissue SOD in three I/R groups, but SOD activity were increased in the plasma of I/R + CAPE and I/R + vit E groups compared with I/R group. Whereas tissue CAT activity was not changed among groups. Our results indicate that CAPE has antioxidant properties similar to those of vit E in this model and may attenuate the harmful effects of hind limb I/R in skeletal muscle. © 2007 Elsevier Inc. All rights reserved. Keywords: Reperfusion injury; Skeletal muscle; Caffeic acid phenethyl ester (CAPE); α-Tocopherol; Oxidative stress
1. Introduction Ischemia–reperfusion (I/R) injury in the skeletal muscle is often occurred as a consequence of a range of vascular events. These vascular events may include thrombolytic therapy, organ transplantation, limb trauma, and cardiovascular surgery. The molecular interactions that occur in reperfusion injury are known to involve the formation of reactive oxygen species (ROS), lipid peroxidation, eicosanoid generation, neutrophil activation, infiltration, complement activation and cytokine generation (Appell et al., 1999). ROS including hydroxyl radical (UOH), superoxide anion radical (O2U−), singlet oxygen (1O2), hydrogen ⁎ Corresponding author. Tel.: +90 356 212 9500/1042; fax: +90 356 212 9417. E-mail address: [email protected] (H. Ozyurt). 1537-1891/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.vph.2007.04.008
peroxide (H2O2) and nitric oxide (NOU) can cause cellular injury when they are generated excessively and hazardous to lipids, proteins, carbohydrates and nucleic acids (Clanton et al., 1999; Cheeseman, 1993; Marx and Chevion, 1986). Oxidative stress means an alteration in the delicate balance between free radicals and the scavenging capacity of antioxidant enzymes in favor of free radicals in the body systems (Frei, 1994). The ROS attack the polyunsaturated fatty acids (PUFAs) in the membrane lipids, thereby lipid peroxidation resulting in loss of fluidity of the membranes and ruptures leading to release of cell. Therefore, assessment of thiobarbituric acid-reactive substances (TBARS) is probably the most commonly applied method for the measurement of lipid peroxidation (Esterbauer, 1993). The role of NO in ischemia/reperfusion injury remains controversial that NO has biphasic actions as cytoprotection and cytotoxicity (Khanna
H. Ozyurt et al. / Vascular Pharmacology 47 (2007) 108–112
et al., 2005). SOD is a potent protective enzyme that can selectively scavenge O2U− by catalyzing its dismutation to H2O2 and oxygen (O2). The other antioxidant enzyme, CAT, catalyzes the conversion of H2O2 to water and oxygen. Assessment of the activities of particular free radical scavenging enzymes and lipid peroxidation end-product levels in plasma and/or muscle tissue would enhance our understanding of the mechanism in muscle ischemia–reperfusion injury. Caffeic acid phenethyl ester (CAPE) is an active component of honeybee propolis extract and has been used as a traditional medicine for many years. It has anti-inflammatory, anti-mitotic, antiviral and immunomodulatory properties, and has been shown to inhibit the growth of different types of transformed cells, and to inhibit pulmonary fibrosis (Chen et al., 1996; Ozyurt et al., 2001; Gurel et al., 2004). In a previous study, it was demonstrated that at a concentration of 10 μmol, CAPE completely blocked production of ROS in human neutrophils and xanthine/xanthine oxidase system (Sudʻina et al., 1993). It has been reported that CAPE suppresses lipid peroxidation, displays antioxidant activity and inhibits lipoxygenase activities. CAPE has been used in some I/R models and its protective effect in reperfusion injury has been demonstrated (Ozyurt et al., 2001; Koltuksuz et al., 1999). Although in previous studies only the effects of CAPE on the lung as a remote organ after performing hind limb I/R were investigated (Calikoglu et al., 2003; Akyol et al., 2006), its antioxidant potential effect has not yet been investigated in skeletal muscle tissue in a hind limb ischemia–reperfusion model. Vitamin E is known as lipid soluble antioxidant and widely used as anti-oxidant. A previous study showed that vitamin E is effective in reducing the oxidative muscle damage in humans lower limb skeletal muscle through biochemical and morphological analyses (Novelli et al., 1996). We aimed to control the potential antioxidant effects of CAPE in comparison to vit E. The aims of this study were to investigate whether endogenous indices of oxidative stress change with treatment with CAPE or vitamin E in a rat skeletal muscle I/R injury model. For this purpose, we assayed various oxidative stress markers in muscle tissue such as CAT, SOD enzyme activities and NO and MDA levels as well as MDA, NO levels and SOD activity in plasma in a rat hind limb I/R injury model. 2. Materials and methods Twenty-four healthy adult male Wistar Albino rats (230– 270 g) were divided into four groups: group I (n = 6), sham; group II (n = 6), I/R; group III (n = 6), I/R+ CAPE; and group IV (n = 6), I/R + α-tocopherol (vit E). The principles of laboratory animal care were followed (NIH publication No: 86-23, revised 1984). The rats were kept at room temperature (20–22 °C), humidity level of 40–50% and photoperiod of 12-h day/12-h night. All surgical procedures were performed while the rats were under anesthesia with intraperitoneally administered 60 mg kg− 1 ketamine and 10 mg kg− 1 xylazine cocktail. A skin incision was made over the anteromedial surface of the right hind limb, starting at the level of femoral artery, extending upward to the inguinal ligament. The right femoral artery was isolated by clamping with an
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atraumatic microvascular clamp. After the animals were anesthetized, an ischemic insult was created in the right femoral artery for 2 h, followed by 2 h of reperfusion. Animals with I/R injury received either saline, CAPE (10 μmol kg− 1, from 25 μmol ml− 1 solution in 10% ethanol) or α-tocopherol (Evigen®-AKSU) (10 mg kg− 1) with intraperitoneal injection 1 h before the reperfusion. The dose of CAPE was based on previous studies (Ozyurt et al., 2001; Koltuksuz et al., 1999; Calikoglu et al., 2003). After 2 h of ischemia, the microvascular clamp was removed. A heat lamp was used to maintain the body temperature at 37 ± 0.5 °C. At the end of the reperfusion period for biochemical assays blood were drawn from the inferior vena cava with heparinized syringes while still anesthetized. Blood samples were collected into polyethylene tubes and centrifuged (3500×g for 30 min at 4 °C) to separate platelet-poor plasma. We measured MDA, NO levels and SOD activities in the plasma. Moreover, the right gastrocnemius muscle was harvested and immediately stored at −30 °C for the assessment of SOD, CAT activities and measurement of NO and MDA levels. Animals in the sham operation group underwent a surgical procedure similar to the other groups but the artery was not occluded. At the end of each experimental procedure, the harvested right gastrocnemius muscle was weighed, then tissues were homogenized in four volumes of ice-cold Tris–HCl buffer (50 mM, pH 7.4) containing 0.50 ml l− 1 Triton X-100 with a homogenizer (IKA Ultra Turrax T 25 Basic, Germany) for 3 min at 22,000 rpm. MDA levels were determined in the homogenate. The homogenate was then centrifuged at 5000×g for 20 min to remove debris. Clear supernatant was taken and used in CAT activity determination. For a further extraction procedure, the supernatant was extracted in ethanol/chloroform mixture (5/3, v/v). After second centrifugation at 5000×g for 20 min, the clear upper layer (the ethanol phase) was taken and used in SOD activity determination. All procedures were performed at + 4 °C. 2.1. Superoxide dismutase (SOD) activity measurement The principle of the total (Cu–Zn and Mn) superoxide dismutase (t-SOD) (EC 1.1.15.1.1) enzyme activity method is based, briefly, on the inhibition of nitroblue tetrazolium (NBT) reduction by O2U− generated by the xanthine/XO system (Sun et al., 1988; Durak et al., 1993). Activity was assessed in the ethanol phase of the plasma or supernatant from muscle tissue after 1.0 ml ethanol/chloroform mixture (5/3, v/v) was added to the same volume plasma or supernatant and centrifuged. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate. Plasma SOD activity was expressed as units per milliliter plasma (U ml− 1). Muscle tissue SOD activity was also expressed as units per gram protein (U g− 1 protein). 2.2. Catalase (CAT) activity measurement Muscle tissue CAT (EC 1.11.1.6) activity was measurement according to Aebi's method (Aebi, 1974). The essential of the method was based on the determination of the rate
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constant k (s− 1 , k) of the H2O2 decomposition rate at 240 nm. Results were expressed as k (rate constant per gram protein; k g− 1 protein). 2.3. Thiobarbituric acid-reactive substances (TBARS) determination
sodium nitrite. Linear regression was done by using the peak area from nitrite standards. The resulting equation was used to calculate the unknown sample concentrations. Results were expressed as micromoles per liter plasma (μmol l− 1) and μmol g− 1 wet tissue. 2.6. Statistical analysis
Plasma and muscle tissue TBARS level was determined by reaction with thiobarbituric acid (TBA) at 90–100 °C MDA or MDA-like substances and TBA react together for tee production of a pink pigment having an absorption maximum at 532 nm. The reaction was performed at pH 2–3 at 90 °C for 15 min. The sample was mixed with 2 volumes of cold 10% (w/v) trichloroacetic 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 in a boiling water bath for 10 min. After cooling, the absorbance was read at 532 nm (Ultraspec Plus, Pharmacia LKB Biocrom, England). A standard curve was prepared using serial dilutions of 1,1,3,3tetramethoxypropane. Tissue TBARS levels were expressed as nanomoles per gram wet tissue (nmol g− 1 wet tissue). Plasma TBARS levels was expressed as micromoles per liter (μmol l− 1) (Wasowicz et al., 1993). 2.4. Protein determination Protein assays were made by the method of Lowry et al. (1951). 2.5. Nitric oxide analyses NO has a half-life of only a few seconds because it is readily oxidized to nitrite (NO2−) and subsequently to nitrate (NO3−) which serve as index parameters of NO production. The method for plasma nitrite and nitrate levels was based on the Griess reaction (Cortas and Wakid, 1990). Samples were initially deproteinized with Somogyi reagent. Total nitrite (nitrite + nitrate) was measured by spectrophotometry at 545 nm after conversion of nitrate to nitrite by copperized cadmium granules. A standard curve was established with a set of serial dilutions (10− 8 – 10− 3 mol l− 1) of
Data were analyzed by using Statistical Package for Social Sciences (SPSS Inc. Chicago, IL) for Windows software. Distribution of the groups was analyzed with one sample Kolmogorov– Smirnov test. As all groups showed normal distribution, group differences were analyzed using parametric statistical methods, independent samples t test following one-way ANOVA. Bivariate comparisons were examined using Pearson's correlation coefficients (r). Results were presented as mean ± standard deviation. A p value less than 0.05 was considered statistically significant. 3. Results Results of oxidative stress markers in each group were shown in Tables 1 and 2. We measured the amount of the MDA as a biomarker of lipid peroxidation. We found higher skeletal muscle tissue and plasma TBARS levels in I/R groups compared to sham group (p b 0.003, and p b 0.0001, respectively). CAPE administration effectively reduced muscle tissue and plasma MDA production nearly to the levels of sham-operated control ( p b 0.001 and p b 0.0001, respectively) (Table 1). On the other hand, I/R led significantly elevated tissue SOD activity ( p b 0.005) and reduced plasma SOD activity ( p b 0.004) compared to sham group. CAPE and α-tocopherol administration augmented plasma SOD activity that reached to the levels of sham group ( p b 0.009 and p b 0.0001, respectively) (Table 2). There was no statistically significant difference in the CAT enzyme activity between treatment groups and sham group. In the I/R group, both tissue and plasma NO levels were significantly higher than those of the sham group ( p b 0.0001 for tissue and p b 0.007 for plasma, respectively). In I/R + CAPE group NO levels in plasma and tissue were significantly reduced nearly to the levels of sham group (Tables 1 and 2). Although
Table 1 Superoxide dismutase (SOD), catalase (CAT) enzyme activities and malondialdehyde (MDA) levels in muscle tissue of rats with muscle ischemia-reperfusion (I/R) injury 1– 2– 3– 4–
Sham (n = 6) I/R (n = 6) I/R + CAPE (n = 6) I/R + vit E (n = 6)
P values 1–2 1–3 1–4 2–3 2–4 3–4
SOD (U g− 1 protein)
CAT (k g− 1 protein)
MDA (nmol g− 1 protein)
NO (μmol g− 1 protein)
32.17 ± 10.36 78.33 ± 18.98 58.33 ± 32.52 55.46 ± 28.76
0.530 ± 0.203 0.693 ± 0.123 0.781 ± 0.342 0.751 ± 0.129
10.54 ± 1.79 16.46 ± 3.24 9.33 ± 2.85 11.23 ± 4.25
0.077 ± 0.034 0.376 ± 0.139 0.153 ± 0.075 0.155 ± 0.063
0.005 ns ns ns ns ns
ns ns ns ns ns ns
0.003 ns ns 0.001 0.0001 ns
0.0001 ns ns 0.0001 0.0001 ns
Results were expressed as mean ± standard deviation. One-way ANOVA test was performed intergroup for meaningfulness comparisons. n.s.: non-significant.
H. Ozyurt et al. / Vascular Pharmacology 47 (2007) 108–112 Table 2 Plasma SOD activity and MDA levels of rats with I/R injury 1– 2– 3– 4–
Sham (n = 6) I/R (n = 6) I/R + CAPE (n = 6) I/R + vit E (n = 6)
P values 1–2 1–3 1–4 2–3 2–4 3–4
MDA (μmol l− 1)
SOD (U ml− 1)
NO (μmol l− 1)
2.04 ± 0.22 3.30 ± 0.37 1.73 ± 0.62 2.25 ± 0.68
5.18 ± 3.65 3.64 ± 0.76 5.02 ± 0.91 5.87 ± 0.62
31.12 ± 8.07 54.39 ± 18.01 33.44 ± 6.69 44.83 ± 17.16
0.0001 ns ns 0.0001 0.002 ns
0.004 ns ns 0.009 0.0001 ns
0.007 ns ns 0.014 ns ns
Results were expressed as mean ± standard deviation values. One-way ANOVA test was performed intergroup for meaningfulness comparisons. n.s.: nonsignificant.
tissue NO levels in I/R + vit E group significantly decreased compared to I/R group, reduction in plasma NO levels did not reach statistical significance compared to I/R group (Tables 1 and 2). There was no significant difference in both plasma and tissue NO levels in I/R + CAPE compared with I/R + vit E groups. 4. Discussion Oxidative stress has been described as a disturbance in the equilibrium status of pro-oxidant/antioxidant systems in intact cells. Thus, ROS have a number of features which include impaired muscle contractions, muscle necrosis, endothelial cell swelling, release of cellular proteins and increased microvascular permeability to proteins (Belkin et al., 1989; Petrasek et al., 1994). Since ROS during ischemia and reperfusion is supposed to play a major role, several antioxidants like N-acetylcysteine, vasoactive intestinal peptide, SOD, iloprost, and vitamin E have been used to reverse skeletal muscle I/R damage (Koksal et al., 2003; Tuncel et al., 1997). Although skeletal muscle reperfusion injury is characterized by an increase in ROS, the real mechanism is still somehow controversial since paradoxical results have been reported in different animal models. For example, vitamin E has been shown to have no protective effect on maximum contraction force or histologic damage, but serum creatine kinase (CK) and pyruvate kinase enzyme activities were decreased in treatment with vit E in another study (Clanton et al., 1999). In contrast, another study showed that administration of vitamin E prevent the muscles from oxidative stress, endothelial damage, intramuscular edema, and major muscle fiber damage (Appell et al., 1999). Additionally, Novelli et al. demonstrated that vitamin E had a protective effect on oxidative muscle damage after a period of I/R in human. They observed that MDA level and neutrophil infiltration in muscle fibers decreased in vitamin E-treated patients undergoing aortic aneurysm resection (Novelli et al., 1996). CAPE has antioxidant activity because of having two hydroxyl groups in its structure. Recently, two studies demonstrated the protective effects of CAPE on the lungs as remote organ by biochemical and histopathological analyses in a hind limb model
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of I/R injury (Calikoglu et al., 2003; Akyol et al., 2006). Furthermore, in our previous study, we showed that administration of CAPE significantly decreased tissue protein carbonyl (PC) levels as a marker of protein oxidation, myeloperoxidase (MPO) as a marker of neutrophil infiltration, and xanthine oxidase (XO) activities in skeletal muscle compared to I/R group (Ozyurt et al., 2006). Accordingly, in this study, we also demonstrated the effect of CAPE on tissue MDA, NO levels and SOD, CAT activity and plasma MDA, NO, SOD activity in a hind limb model of I/R injury. Moreover, in the present study, we compared the effects of CAPE with a potent antioxidant, i.e. vitamin E. Our results revealed that I/R injury led increases in MDA and NO levels in both skeletal muscle tissue and plasma. In contrast, CAPE led a decrease in MDA and NO levels in both plasma and muscle tissue. Reactive oxygen species attack the polyunsaturated fatty acids in the membrane lipids and result in peroxidation, which may lead to disorganization of cell structure and function. This process results in an excess of free radicals, which can react with cellular lipids, proteins and nucleic acids, leading to local injury and eventually organ dysfunction. It has been suggested that the protective effects of CAPE depend mainly on its antioxidant properties. The production of NOU from L-arginine is catalysed by the dioxygenase, NOS, which has three isoforms, i.e., neuronal (nNOS), inducible (iNOS), and endothelial (eNOS). Endothelium-derived NO, which causes vasodilatation, probably exerts a protective action on ischemia induced tissue damage. On the other hand, NO derived from inducible nitric oxide synthase (iNOS) has been considered to enhance the tissue damage observed in I/R injury partly via the formation of peroxynitrite anion, a NOderived oxidant formed from the interaction of NO with superoxide. Ikebe and colleagues reported that contractile function of the gastrocnemius muscle in the L-NMMA as a NOS inhibitor and SOD-treated groups but not L-NMMA + S-nitrosoglutathionetreated group was better than saline-treated controls (Ikebe et al., 2002). In another study, during the first 10 min of ischemia, there was doubling of the basal level in NO which was considered predominantly eNOS-mediated and, at a later time, iNOS was expressed through the action of pro-inflammatory cytokines and started to produce large amounts of NO (Khanna et al., 2005). It is well known that peroxynitrite (ONOOU−) is rapidly protonated and decays, generating the highly toxic hydroxyl radicals that lead eventually tissue destruction. Increased NO level was determined in both tissue and plasma in I/R group compared with the other groups in our study. Moreover, CAPE was more effective in reducing increased NO levels than vit E. Song et al. suggested that CAPE may exert its anti-inflammatory effect by inhibiting the iNOS gene expression at the transcriptional level and by directly inhibiting the catalytic activity of iNOS (Song et al., 2002). Therefore, reduction in NO levels by CAPE is causally important. Seyama (1993) reported that MDA level in the gastrocnemius muscle after 2 h of reperfusion increased significantly compared to 1 h of ischemia. This effect was suppressed by the administration of free radical scavengers (CAT, SOD). The antioxidant enzymes SOD, CAT, and GSH-Px have complementary activities in the anti-oxidative defense system. We assayed these two enzymes to investigate the enzymatic antioxidant status in skeletal
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muscle I/R injury. We found increased tissue SOD activity but decreased plasma SOD activity in I/R group compared to sham group. However, we could not observe a significant increase or decrease in the activity of CAT. Increased SOD enzyme activity in skeletal muscle tissue may reflect a preceding cellular oxidative stress or may be involved in compensatory mechanisms. On the other hand, the fact that SOD is the first enzymatic antioxidant defense system, reduction of plasma SOD activity in I/R injury may lead or trigger lipid peroxidation in the cellular membranes. The decreased plasma SOD enzyme activity returned to nearly normal levels I/R + CAPE and I/R + vit E groups. Although the real mechanism of CAPE on enzyme activity is not known, we can speculate that CAPE prevents the induction of SOD enzyme by inhibiting the formation of toxic oxidative products. It is known that SOD can inhibit the expression of free radicals and the adhesion molecules, and reasonably prevents skeletal muscle I/R injury. Our present findings are generally in agreement with the reports of previous studies on CAPE. In addition, it may be meaningful to investigate the effect of skeletal muscle ischemia/ reperfusion injury as apoptotic histological changes. In conclusion, this study demonstrated that prophylactic administration of CAPE might protect skeletal muscle from reperfusion injury to the extent that vitamin E did. Our data also revealed that this protective effect of CAPE is probably ascribed to its free radical scavenging activity. We underscore the necessity of human studies with CAPE that would be hypothetically beneficial preventing skeletal muscle I/R injury particularly during surgical interventions. References Aebi, H., 1974. Catalase. In: Bergmeyer, U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York, pp. 673–677. Akyol, A., Ulusoy, H., Imamoglu, M., Cay, A., Yulug, E., Alver, A., Erturk, E., Kosucu, M., Besir, A., Akyol, A., Ozen, I., 2006. Does propofol or caffeic acid phenethyl ester prevent lung injury after hindlimb ischemia– reperfusion in ventilated rats? Injury 37 (5), 380–387. Appell, H.J., Glöser, S., Soares, J.M.C., Duarte, J.A., 1999. Structural alterations of skeletal muscle induced by ischemia and reperfusion. Basic Appl. Myol. 9 (5), 263–268. Belkin, M., LaMorte, W.L., Wright, J.G., Hobson, R.W., 1989. The role of leukocytes in the pathophysiology of skeletal muscle ischemic injury. J. Vasc. Surg. 10, 14–19. Calikoglu, M., Tamer, L., Sucu, N., Coskun, B., Ercan, B., Gul, A., Calikoglu, I., Kanik, A., 2003. The effects of caffeic acid phenethyl ester on tissue damage in lung after hindlimb ischemia–reperfusion. Pharmacol. Res. 48 (4), 397–403. Cheeseman, K.H., 1993. Mechanisms and effects of lipid peroxidation. Mol. Aspects Med. 14 (3), 191–197. Chen, J.H., Shao, Y., Huang, M.T., Chin, C.K., Ho, C.T., 1996. Inhibitory effect of caffeic acid phenethyl ester on human leukemia HL 60 cells. Cancer Lett 108, 211–214. Clanton, T.L., Zuo, L., Klawitter, P., 1999. Oxidants and skeletal muscle function: physiologic and pathophysiologic implications. Proc. Soc. Exp. Biol. Med. 222 (3), 253–262.
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Caffeic acid phenethyl ester (CAPE) protects rat skeletal muscle against ischemia–reperfusion-induced oxidative stress Huseyin Ozyurt a,⁎, Birsen Ozyurt b , Kenan Koca c , Salih Ozgocmen d a
d
Gaziosmanpasa University, Faculty of Medicine, Department of Biochemistry, Tokat, Turkey b Gaziosmanpasa University, Faculty of Medicine, Department of Anatomy, Tokat, Turkey c Sarikamis Military Hospital, Department of Orthopaedics and Traumatology, Kars, Turkey Firat University, Faculty of Medicine Department of Physical Medicine and Rehabilitation, Elazig, Turkey Received 17 November 2006; accepted 27 April 2007
Abstract Oxygen-derived free radicals have been implicated in the pathogenesis of skeletal muscle injury after ischemia–reperfusion. Caffeic acid phenethyl ester, an active component of propolis extract, exhibits antioxidant properties. The aim of this study was to assess the effects of caffeic acid phenethyl ester (CAPE) and α-tocopherol (vit E) on ischemia/reperfusion (I/R) injury in a rat hind limb ischemia/reperfusion model. For this purpose, ischemia was induced in anesthetized rats by unilateral (right) femoral artery clipping for 2 h followed by 2 h of reperfusion. Four groups were studied: sham, I/R, I/R + CAPE and I/R + vit E. Drugs were administered intraperitoneally after 1 h of ischemia and I/R rats received saline vehicle. After 2 h of reperfusion, venous blood was sampled and the right gastrocnemius muscle was harvested. Plasma and tissue were assayed for malondialdehyde (MDA), superoxide dismutase (SOD) and nitric oxide (NO) metabolites. Tissue was also assayed for catalase (CAT) activity. Both tissue and plasma NO levels, MDA levels, SOD activities was significantly increased in I/R groups compared to control groups. The two treated groups showed decreased MDA and NO in both muscle and plasma compared to the I/R group. No differences were noted in muscle tissue SOD in three I/R groups, but SOD activity were increased in the plasma of I/R + CAPE and I/R + vit E groups compared with I/R group. Whereas tissue CAT activity was not changed among groups. Our results indicate that CAPE has antioxidant properties similar to those of vit E in this model and may attenuate the harmful effects of hind limb I/R in skeletal muscle. © 2007 Elsevier Inc. All rights reserved. Keywords: Reperfusion injury; Skeletal muscle; Caffeic acid phenethyl ester (CAPE); α-Tocopherol; Oxidative stress
1. Introduction Ischemia–reperfusion (I/R) injury in the skeletal muscle is often occurred as a consequence of a range of vascular events. These vascular events may include thrombolytic therapy, organ transplantation, limb trauma, and cardiovascular surgery. The molecular interactions that occur in reperfusion injury are known to involve the formation of reactive oxygen species (ROS), lipid peroxidation, eicosanoid generation, neutrophil activation, infiltration, complement activation and cytokine generation (Appell et al., 1999). ROS including hydroxyl radical (UOH), superoxide anion radical (O2U−), singlet oxygen (1O2), hydrogen ⁎ Corresponding author. Tel.: +90 356 212 9500/1042; fax: +90 356 212 9417. E-mail address: [email protected] (H. Ozyurt). 1537-1891/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.vph.2007.04.008
peroxide (H2O2) and nitric oxide (NOU) can cause cellular injury when they are generated excessively and hazardous to lipids, proteins, carbohydrates and nucleic acids (Clanton et al., 1999; Cheeseman, 1993; Marx and Chevion, 1986). Oxidative stress means an alteration in the delicate balance between free radicals and the scavenging capacity of antioxidant enzymes in favor of free radicals in the body systems (Frei, 1994). The ROS attack the polyunsaturated fatty acids (PUFAs) in the membrane lipids, thereby lipid peroxidation resulting in loss of fluidity of the membranes and ruptures leading to release of cell. Therefore, assessment of thiobarbituric acid-reactive substances (TBARS) is probably the most commonly applied method for the measurement of lipid peroxidation (Esterbauer, 1993). The role of NO in ischemia/reperfusion injury remains controversial that NO has biphasic actions as cytoprotection and cytotoxicity (Khanna
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et al., 2005). SOD is a potent protective enzyme that can selectively scavenge O2U− by catalyzing its dismutation to H2O2 and oxygen (O2). The other antioxidant enzyme, CAT, catalyzes the conversion of H2O2 to water and oxygen. Assessment of the activities of particular free radical scavenging enzymes and lipid peroxidation end-product levels in plasma and/or muscle tissue would enhance our understanding of the mechanism in muscle ischemia–reperfusion injury. Caffeic acid phenethyl ester (CAPE) is an active component of honeybee propolis extract and has been used as a traditional medicine for many years. It has anti-inflammatory, anti-mitotic, antiviral and immunomodulatory properties, and has been shown to inhibit the growth of different types of transformed cells, and to inhibit pulmonary fibrosis (Chen et al., 1996; Ozyurt et al., 2001; Gurel et al., 2004). In a previous study, it was demonstrated that at a concentration of 10 μmol, CAPE completely blocked production of ROS in human neutrophils and xanthine/xanthine oxidase system (Sudʻina et al., 1993). It has been reported that CAPE suppresses lipid peroxidation, displays antioxidant activity and inhibits lipoxygenase activities. CAPE has been used in some I/R models and its protective effect in reperfusion injury has been demonstrated (Ozyurt et al., 2001; Koltuksuz et al., 1999). Although in previous studies only the effects of CAPE on the lung as a remote organ after performing hind limb I/R were investigated (Calikoglu et al., 2003; Akyol et al., 2006), its antioxidant potential effect has not yet been investigated in skeletal muscle tissue in a hind limb ischemia–reperfusion model. Vitamin E is known as lipid soluble antioxidant and widely used as anti-oxidant. A previous study showed that vitamin E is effective in reducing the oxidative muscle damage in humans lower limb skeletal muscle through biochemical and morphological analyses (Novelli et al., 1996). We aimed to control the potential antioxidant effects of CAPE in comparison to vit E. The aims of this study were to investigate whether endogenous indices of oxidative stress change with treatment with CAPE or vitamin E in a rat skeletal muscle I/R injury model. For this purpose, we assayed various oxidative stress markers in muscle tissue such as CAT, SOD enzyme activities and NO and MDA levels as well as MDA, NO levels and SOD activity in plasma in a rat hind limb I/R injury model. 2. Materials and methods Twenty-four healthy adult male Wistar Albino rats (230– 270 g) were divided into four groups: group I (n = 6), sham; group II (n = 6), I/R; group III (n = 6), I/R+ CAPE; and group IV (n = 6), I/R + α-tocopherol (vit E). The principles of laboratory animal care were followed (NIH publication No: 86-23, revised 1984). The rats were kept at room temperature (20–22 °C), humidity level of 40–50% and photoperiod of 12-h day/12-h night. All surgical procedures were performed while the rats were under anesthesia with intraperitoneally administered 60 mg kg− 1 ketamine and 10 mg kg− 1 xylazine cocktail. A skin incision was made over the anteromedial surface of the right hind limb, starting at the level of femoral artery, extending upward to the inguinal ligament. The right femoral artery was isolated by clamping with an
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atraumatic microvascular clamp. After the animals were anesthetized, an ischemic insult was created in the right femoral artery for 2 h, followed by 2 h of reperfusion. Animals with I/R injury received either saline, CAPE (10 μmol kg− 1, from 25 μmol ml− 1 solution in 10% ethanol) or α-tocopherol (Evigen®-AKSU) (10 mg kg− 1) with intraperitoneal injection 1 h before the reperfusion. The dose of CAPE was based on previous studies (Ozyurt et al., 2001; Koltuksuz et al., 1999; Calikoglu et al., 2003). After 2 h of ischemia, the microvascular clamp was removed. A heat lamp was used to maintain the body temperature at 37 ± 0.5 °C. At the end of the reperfusion period for biochemical assays blood were drawn from the inferior vena cava with heparinized syringes while still anesthetized. Blood samples were collected into polyethylene tubes and centrifuged (3500×g for 30 min at 4 °C) to separate platelet-poor plasma. We measured MDA, NO levels and SOD activities in the plasma. Moreover, the right gastrocnemius muscle was harvested and immediately stored at −30 °C for the assessment of SOD, CAT activities and measurement of NO and MDA levels. Animals in the sham operation group underwent a surgical procedure similar to the other groups but the artery was not occluded. At the end of each experimental procedure, the harvested right gastrocnemius muscle was weighed, then tissues were homogenized in four volumes of ice-cold Tris–HCl buffer (50 mM, pH 7.4) containing 0.50 ml l− 1 Triton X-100 with a homogenizer (IKA Ultra Turrax T 25 Basic, Germany) for 3 min at 22,000 rpm. MDA levels were determined in the homogenate. The homogenate was then centrifuged at 5000×g for 20 min to remove debris. Clear supernatant was taken and used in CAT activity determination. For a further extraction procedure, the supernatant was extracted in ethanol/chloroform mixture (5/3, v/v). After second centrifugation at 5000×g for 20 min, the clear upper layer (the ethanol phase) was taken and used in SOD activity determination. All procedures were performed at + 4 °C. 2.1. Superoxide dismutase (SOD) activity measurement The principle of the total (Cu–Zn and Mn) superoxide dismutase (t-SOD) (EC 1.1.15.1.1) enzyme activity method is based, briefly, on the inhibition of nitroblue tetrazolium (NBT) reduction by O2U− generated by the xanthine/XO system (Sun et al., 1988; Durak et al., 1993). Activity was assessed in the ethanol phase of the plasma or supernatant from muscle tissue after 1.0 ml ethanol/chloroform mixture (5/3, v/v) was added to the same volume plasma or supernatant and centrifuged. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate. Plasma SOD activity was expressed as units per milliliter plasma (U ml− 1). Muscle tissue SOD activity was also expressed as units per gram protein (U g− 1 protein). 2.2. Catalase (CAT) activity measurement Muscle tissue CAT (EC 1.11.1.6) activity was measurement according to Aebi's method (Aebi, 1974). The essential of the method was based on the determination of the rate
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constant k (s− 1 , k) of the H2O2 decomposition rate at 240 nm. Results were expressed as k (rate constant per gram protein; k g− 1 protein). 2.3. Thiobarbituric acid-reactive substances (TBARS) determination
sodium nitrite. Linear regression was done by using the peak area from nitrite standards. The resulting equation was used to calculate the unknown sample concentrations. Results were expressed as micromoles per liter plasma (μmol l− 1) and μmol g− 1 wet tissue. 2.6. Statistical analysis
Plasma and muscle tissue TBARS level was determined by reaction with thiobarbituric acid (TBA) at 90–100 °C MDA or MDA-like substances and TBA react together for tee production of a pink pigment having an absorption maximum at 532 nm. The reaction was performed at pH 2–3 at 90 °C for 15 min. The sample was mixed with 2 volumes of cold 10% (w/v) trichloroacetic 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 in a boiling water bath for 10 min. After cooling, the absorbance was read at 532 nm (Ultraspec Plus, Pharmacia LKB Biocrom, England). A standard curve was prepared using serial dilutions of 1,1,3,3tetramethoxypropane. Tissue TBARS levels were expressed as nanomoles per gram wet tissue (nmol g− 1 wet tissue). Plasma TBARS levels was expressed as micromoles per liter (μmol l− 1) (Wasowicz et al., 1993). 2.4. Protein determination Protein assays were made by the method of Lowry et al. (1951). 2.5. Nitric oxide analyses NO has a half-life of only a few seconds because it is readily oxidized to nitrite (NO2−) and subsequently to nitrate (NO3−) which serve as index parameters of NO production. The method for plasma nitrite and nitrate levels was based on the Griess reaction (Cortas and Wakid, 1990). Samples were initially deproteinized with Somogyi reagent. Total nitrite (nitrite + nitrate) was measured by spectrophotometry at 545 nm after conversion of nitrate to nitrite by copperized cadmium granules. A standard curve was established with a set of serial dilutions (10− 8 – 10− 3 mol l− 1) of
Data were analyzed by using Statistical Package for Social Sciences (SPSS Inc. Chicago, IL) for Windows software. Distribution of the groups was analyzed with one sample Kolmogorov– Smirnov test. As all groups showed normal distribution, group differences were analyzed using parametric statistical methods, independent samples t test following one-way ANOVA. Bivariate comparisons were examined using Pearson's correlation coefficients (r). Results were presented as mean ± standard deviation. A p value less than 0.05 was considered statistically significant. 3. Results Results of oxidative stress markers in each group were shown in Tables 1 and 2. We measured the amount of the MDA as a biomarker of lipid peroxidation. We found higher skeletal muscle tissue and plasma TBARS levels in I/R groups compared to sham group (p b 0.003, and p b 0.0001, respectively). CAPE administration effectively reduced muscle tissue and plasma MDA production nearly to the levels of sham-operated control ( p b 0.001 and p b 0.0001, respectively) (Table 1). On the other hand, I/R led significantly elevated tissue SOD activity ( p b 0.005) and reduced plasma SOD activity ( p b 0.004) compared to sham group. CAPE and α-tocopherol administration augmented plasma SOD activity that reached to the levels of sham group ( p b 0.009 and p b 0.0001, respectively) (Table 2). There was no statistically significant difference in the CAT enzyme activity between treatment groups and sham group. In the I/R group, both tissue and plasma NO levels were significantly higher than those of the sham group ( p b 0.0001 for tissue and p b 0.007 for plasma, respectively). In I/R + CAPE group NO levels in plasma and tissue were significantly reduced nearly to the levels of sham group (Tables 1 and 2). Although
Table 1 Superoxide dismutase (SOD), catalase (CAT) enzyme activities and malondialdehyde (MDA) levels in muscle tissue of rats with muscle ischemia-reperfusion (I/R) injury 1– 2– 3– 4–
Sham (n = 6) I/R (n = 6) I/R + CAPE (n = 6) I/R + vit E (n = 6)
P values 1–2 1–3 1–4 2–3 2–4 3–4
SOD (U g− 1 protein)
CAT (k g− 1 protein)
MDA (nmol g− 1 protein)
NO (μmol g− 1 protein)
32.17 ± 10.36 78.33 ± 18.98 58.33 ± 32.52 55.46 ± 28.76
0.530 ± 0.203 0.693 ± 0.123 0.781 ± 0.342 0.751 ± 0.129
10.54 ± 1.79 16.46 ± 3.24 9.33 ± 2.85 11.23 ± 4.25
0.077 ± 0.034 0.376 ± 0.139 0.153 ± 0.075 0.155 ± 0.063
0.005 ns ns ns ns ns
ns ns ns ns ns ns
0.003 ns ns 0.001 0.0001 ns
0.0001 ns ns 0.0001 0.0001 ns
Results were expressed as mean ± standard deviation. One-way ANOVA test was performed intergroup for meaningfulness comparisons. n.s.: non-significant.
H. Ozyurt et al. / Vascular Pharmacology 47 (2007) 108–112 Table 2 Plasma SOD activity and MDA levels of rats with I/R injury 1– 2– 3– 4–
Sham (n = 6) I/R (n = 6) I/R + CAPE (n = 6) I/R + vit E (n = 6)
P values 1–2 1–3 1–4 2–3 2–4 3–4
MDA (μmol l− 1)
SOD (U ml− 1)
NO (μmol l− 1)
2.04 ± 0.22 3.30 ± 0.37 1.73 ± 0.62 2.25 ± 0.68
5.18 ± 3.65 3.64 ± 0.76 5.02 ± 0.91 5.87 ± 0.62
31.12 ± 8.07 54.39 ± 18.01 33.44 ± 6.69 44.83 ± 17.16
0.0001 ns ns 0.0001 0.002 ns
0.004 ns ns 0.009 0.0001 ns
0.007 ns ns 0.014 ns ns
Results were expressed as mean ± standard deviation values. One-way ANOVA test was performed intergroup for meaningfulness comparisons. n.s.: nonsignificant.
tissue NO levels in I/R + vit E group significantly decreased compared to I/R group, reduction in plasma NO levels did not reach statistical significance compared to I/R group (Tables 1 and 2). There was no significant difference in both plasma and tissue NO levels in I/R + CAPE compared with I/R + vit E groups. 4. Discussion Oxidative stress has been described as a disturbance in the equilibrium status of pro-oxidant/antioxidant systems in intact cells. Thus, ROS have a number of features which include impaired muscle contractions, muscle necrosis, endothelial cell swelling, release of cellular proteins and increased microvascular permeability to proteins (Belkin et al., 1989; Petrasek et al., 1994). Since ROS during ischemia and reperfusion is supposed to play a major role, several antioxidants like N-acetylcysteine, vasoactive intestinal peptide, SOD, iloprost, and vitamin E have been used to reverse skeletal muscle I/R damage (Koksal et al., 2003; Tuncel et al., 1997). Although skeletal muscle reperfusion injury is characterized by an increase in ROS, the real mechanism is still somehow controversial since paradoxical results have been reported in different animal models. For example, vitamin E has been shown to have no protective effect on maximum contraction force or histologic damage, but serum creatine kinase (CK) and pyruvate kinase enzyme activities were decreased in treatment with vit E in another study (Clanton et al., 1999). In contrast, another study showed that administration of vitamin E prevent the muscles from oxidative stress, endothelial damage, intramuscular edema, and major muscle fiber damage (Appell et al., 1999). Additionally, Novelli et al. demonstrated that vitamin E had a protective effect on oxidative muscle damage after a period of I/R in human. They observed that MDA level and neutrophil infiltration in muscle fibers decreased in vitamin E-treated patients undergoing aortic aneurysm resection (Novelli et al., 1996). CAPE has antioxidant activity because of having two hydroxyl groups in its structure. Recently, two studies demonstrated the protective effects of CAPE on the lungs as remote organ by biochemical and histopathological analyses in a hind limb model
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of I/R injury (Calikoglu et al., 2003; Akyol et al., 2006). Furthermore, in our previous study, we showed that administration of CAPE significantly decreased tissue protein carbonyl (PC) levels as a marker of protein oxidation, myeloperoxidase (MPO) as a marker of neutrophil infiltration, and xanthine oxidase (XO) activities in skeletal muscle compared to I/R group (Ozyurt et al., 2006). Accordingly, in this study, we also demonstrated the effect of CAPE on tissue MDA, NO levels and SOD, CAT activity and plasma MDA, NO, SOD activity in a hind limb model of I/R injury. Moreover, in the present study, we compared the effects of CAPE with a potent antioxidant, i.e. vitamin E. Our results revealed that I/R injury led increases in MDA and NO levels in both skeletal muscle tissue and plasma. In contrast, CAPE led a decrease in MDA and NO levels in both plasma and muscle tissue. Reactive oxygen species attack the polyunsaturated fatty acids in the membrane lipids and result in peroxidation, which may lead to disorganization of cell structure and function. This process results in an excess of free radicals, which can react with cellular lipids, proteins and nucleic acids, leading to local injury and eventually organ dysfunction. It has been suggested that the protective effects of CAPE depend mainly on its antioxidant properties. The production of NOU from L-arginine is catalysed by the dioxygenase, NOS, which has three isoforms, i.e., neuronal (nNOS), inducible (iNOS), and endothelial (eNOS). Endothelium-derived NO, which causes vasodilatation, probably exerts a protective action on ischemia induced tissue damage. On the other hand, NO derived from inducible nitric oxide synthase (iNOS) has been considered to enhance the tissue damage observed in I/R injury partly via the formation of peroxynitrite anion, a NOderived oxidant formed from the interaction of NO with superoxide. Ikebe and colleagues reported that contractile function of the gastrocnemius muscle in the L-NMMA as a NOS inhibitor and SOD-treated groups but not L-NMMA + S-nitrosoglutathionetreated group was better than saline-treated controls (Ikebe et al., 2002). In another study, during the first 10 min of ischemia, there was doubling of the basal level in NO which was considered predominantly eNOS-mediated and, at a later time, iNOS was expressed through the action of pro-inflammatory cytokines and started to produce large amounts of NO (Khanna et al., 2005). It is well known that peroxynitrite (ONOOU−) is rapidly protonated and decays, generating the highly toxic hydroxyl radicals that lead eventually tissue destruction. Increased NO level was determined in both tissue and plasma in I/R group compared with the other groups in our study. Moreover, CAPE was more effective in reducing increased NO levels than vit E. Song et al. suggested that CAPE may exert its anti-inflammatory effect by inhibiting the iNOS gene expression at the transcriptional level and by directly inhibiting the catalytic activity of iNOS (Song et al., 2002). Therefore, reduction in NO levels by CAPE is causally important. Seyama (1993) reported that MDA level in the gastrocnemius muscle after 2 h of reperfusion increased significantly compared to 1 h of ischemia. This effect was suppressed by the administration of free radical scavengers (CAT, SOD). The antioxidant enzymes SOD, CAT, and GSH-Px have complementary activities in the anti-oxidative defense system. We assayed these two enzymes to investigate the enzymatic antioxidant status in skeletal
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muscle I/R injury. We found increased tissue SOD activity but decreased plasma SOD activity in I/R group compared to sham group. However, we could not observe a significant increase or decrease in the activity of CAT. Increased SOD enzyme activity in skeletal muscle tissue may reflect a preceding cellular oxidative stress or may be involved in compensatory mechanisms. On the other hand, the fact that SOD is the first enzymatic antioxidant defense system, reduction of plasma SOD activity in I/R injury may lead or trigger lipid peroxidation in the cellular membranes. The decreased plasma SOD enzyme activity returned to nearly normal levels I/R + CAPE and I/R + vit E groups. Although the real mechanism of CAPE on enzyme activity is not known, we can speculate that CAPE prevents the induction of SOD enzyme by inhibiting the formation of toxic oxidative products. It is known that SOD can inhibit the expression of free radicals and the adhesion molecules, and reasonably prevents skeletal muscle I/R injury. Our present findings are generally in agreement with the reports of previous studies on CAPE. In addition, it may be meaningful to investigate the effect of skeletal muscle ischemia/ reperfusion injury as apoptotic histological changes. In conclusion, this study demonstrated that prophylactic administration of CAPE might protect skeletal muscle from reperfusion injury to the extent that vitamin E did. Our data also revealed that this protective effect of CAPE is probably ascribed to its free radical scavenging activity. We underscore the necessity of human studies with CAPE that would be hypothetically beneficial preventing skeletal muscle I/R injury particularly during surgical interventions. References Aebi, H., 1974. Catalase. In: Bergmeyer, U. (Ed.), Methods of Enzymatic Analysis. Academic Press, New York, pp. 673–677. Akyol, A., Ulusoy, H., Imamoglu, M., Cay, A., Yulug, E., Alver, A., Erturk, E., Kosucu, M., Besir, A., Akyol, A., Ozen, I., 2006. Does propofol or caffeic acid phenethyl ester prevent lung injury after hindlimb ischemia– reperfusion in ventilated rats? Injury 37 (5), 380–387. Appell, H.J., Glöser, S., Soares, J.M.C., Duarte, J.A., 1999. Structural alterations of skeletal muscle induced by ischemia and reperfusion. Basic Appl. Myol. 9 (5), 263–268. Belkin, M., LaMorte, W.L., Wright, J.G., Hobson, R.W., 1989. The role of leukocytes in the pathophysiology of skeletal muscle ischemic injury. J. Vasc. Surg. 10, 14–19. Calikoglu, M., Tamer, L., Sucu, N., Coskun, B., Ercan, B., Gul, A., Calikoglu, I., Kanik, A., 2003. The effects of caffeic acid phenethyl ester on tissue damage in lung after hindlimb ischemia–reperfusion. Pharmacol. Res. 48 (4), 397–403. Cheeseman, K.H., 1993. Mechanisms and effects of lipid peroxidation. Mol. Aspects Med. 14 (3), 191–197. Chen, J.H., Shao, Y., Huang, M.T., Chin, C.K., Ho, C.T., 1996. Inhibitory effect of caffeic acid phenethyl ester on human leukemia HL 60 cells. Cancer Lett 108, 211–214. Clanton, T.L., Zuo, L., Klawitter, P., 1999. Oxidants and skeletal muscle function: physiologic and pathophysiologic implications. Proc. Soc. Exp. Biol. Med. 222 (3), 253–262.
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