Cardiovasc Toxicol DOI 10.1007/s12012-013-9238-7
Preventive Effect of Total Flavones of Choerospondias axillaries on Ischemia/Reperfusion-Induced Myocardial Infarction-Related MAPK Signaling Pathway Chunmei Li • Jie He • Yonglin Gao • Yanli Xing • Jian Hou • Jingwei Tian
Ó Springer Science+Business Media New York 2013
Abstract Choerospondias axillaris (Guangzao) is a medicinal plant used in Mongolia, and its fruit is commonly used for the treatment of cardiovascular diseases in clinic. The constituents responsible for this effect are always conceded to be the total flavonoids of C. axillaries (TFC). The present study was to evaluate the preventive effect of TFC on acute myocardial infarction induced by ischemia/reperfusion (I/R) in rats and the possible signaling pathway involved. The model of myocardial I/R was caused by occlusion of the left anterior descending coronary artery for 30 min followed by reperfusion for 2 h. Cardiac dysfunction, infarct size, pathologic histology and apoptosis were examined. Heart tissues were homogenized for biochemistry assays and Western blots analysis. The results indicated that pretreatment with TFC strongly improved cardiac function, obviously reduced heart pathologic lesion in I/R rat hearts. TFC also could protect the heart from I/R injury by increasing the levels of catalase, glutathione peroxidase and superoxide dismutase in heart homogenate, and decreasing that of malondialdehyde level. These beneficial effects were associated with the decrease in TUNEL-positive nuclear staining, Bax and caspase-3 levels, and the increase in Bcl-2 expression. Moreover,
C. Li J. Tian (&) School of Pharmacy, Yantai University, Yantai 264005, Shandong, People’s Republic of China e-mail: [email protected]; [email protected] J. He Y. Xing J. Hou Shandong Luye Research and Development for Natural Drugs Co. Ltd., Yantai 264003, Shandong, People’s Republic of China Y. Gao School of Life Sciences, Yantai University, Yantai 264005, Shandong, People’s Republic of China
TFC counteracted the I/R-induced decreased activation of p38 mitogen-activated protein kinase (MAPK) and Jun N-terminal kinase. These data suggested that TFC improved I/R-induced myocardium impairment via antioxidative and anti-apoptotic activities, and this beneficial effects were intervened by MAPK signaling pathway. Keywords Total flavones of Choerospondias axillaries Ischemia/reperfusion Anti-oxidative Anti-apoptotic MAPK pathways
Introduction Acute myocardial infarction (AMI) is the sharp decline in coronary artery or the interruption of blood supply to a part of the heart, resulting heart cells to die. The resulting ischemia (restriction in blood supply) and ensuing oxygen shortage induce myocardium infarction or heart damage, if left untreated for a sufficient period of time. Reperfusion therapy must be treated as soon as possible after AMI in order to attenuate the ischemic injury. However, reperfusion is responsible for additional myocardial damage [1]. Reactive oxygen species (ROS) play a vital role in ischemia development as well as during the reperfusion phase after AMI [2]. One hypothesis is that antioxidants are severely consumed during infarction, and O2 abruptly increases metabolism after reperfusion in the damage of normal defenses [3]. Moreover, it is now implied that the readmission of oxygenated blood into previously ischemic myocardium can induce a cascade of events that will paradoxically produce additional myocardial cell dysfunction and cell apoptosis [4]. Based on these observations, it has been promised that inhibiting cardiomyocyte apoptosis and
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oxidative stress could serve as the basis for the potential development of drugs for ischemic heart diseases. Choerospondias axillaris (Guangzao) is a medicinal plant used in Mongolia, and its fruit is widely used for the treatment of cardiovascular diseases in clinic [5, 6]. For example, Guang-Zao-Qi-Wei-Wan, Compound Guangzao capsule and San-Wei-Guang-Zao capsule have been wildly used for many years in china [7–9]. The constituents responsible for this effect are generally conceded to be the total flavonoids of C. axillaries (TFC), because these kinds of compounds are known to be antioxidants [10]. Blood flow arrest and reperfusion during AMI cause myocytes and endothelium injury through oxidative stress and inflammatory response, both of which involve ROS and peroxides that consume antioxidant defenses. Of note, researchers have found that TFC could inhibit dexamethasone-induced thymocyte apoptosis [11]. Recently, Ao et al. have reported that TFC could attenuate the serum levels of CK, CK-MB and LDH in isoproterenol-induced MI injury in rats [12]. In light of the previous findings, we hypothesized that TFC can inhibit oxidative damage and cardiomyocyte apoptosis associated with AMI. To support our hypothesis, we used a rat model of ischemia/reperfusion (I/R) to investigate the cardioprotective properties of TFC and assessed its relationship to oxidative stress markers, apoptosis-inducing factors, hemodynamics and histopathological lesions. Moreover, the present study also aimed to investigate whether the cardioprotective effects of TFC are mediated by p38 mitogen-activated protein kinase (MAPK) pathways.
Materials and Methods Chemicals All the chemicals and reagents were analytical grade. The plant material used was the dried fruit of C. axillaries. The test compound, TFC, was isolated as described previously [13]. Briefly, the fruit was powdered and extracted in water at 100 °C for 40 min. The sample was filtered and extracted 3 times under the same conditions with new solvent. The crude extract was collected and refluxed for 1.5 h with 60 % alcohol (1:12) 4 times in succession; then, the test substance was obtained. I/R Procedure to Induce Myocardial Injury In the current study, male Sprague–Dawley rats, 250 ± 20 g, were divided into five groups: Group 1, sham; Group 2, I/R model; Group 3, I/R ? TFC 75 mg/kg; Group 4, I/R ? TFC 150 mg/kg; Group 5, I/R ? TFC 300 mg/kg.
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TFC 75, 150 and 300 mg/kg group animals were administered orally once a day for continues 30 days. Group 1 and 2 were treated with the same volume of distilled water. On day 29 and day 30 of treatment, the cardial I/R operation was carried out according to previous procedure [14]. Briefly, after rats were anesthetized by injection of urethane, coronary artery ligation was achieved with a gab occluder fixed onto the left anterior descending (LAD) coronary artery. A 7-0 silk suture was passed underneath the LAD (2–3 mm inferior to the left auricle) and tied. AMI injury was induced by 30 min of ischemia followed by 2 h of reperfusion. The respirator weaned when the animal recovered spontaneous breathing. All procedures involved in the use of laboratory animals were in accordance with the Guidelines of the Animal Care and Use of Laboratory Animals set by the Association of Laboratory Animal Science and the Center for Laboratory Animal Science. Hemodynamic Assessment Following 2 h after I/R, hemodynamic measurements, including left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and ±dp/dtmax, were performed and programmed by using a biotic signal collection and processing system (BIOPIC, American). A millar vessel was inserted into the left ventricular cavity of animals via the right common carotid artery. Analysis of Myocardial Infarction The rat hearts were removed, and the left ventricle area was divided into thick slices from the apex and incubated in 2 % triphenyltetrazolium chloride (TTC) in pH 7.4 buffer for 15 min at 37 °C. TTC stained viable tissue dark red, while the infarct portion remained grayish-white. The infarcted area (border zone) was traced by hand on image system (NIH image software) and measured by computerized planimetry. Histopathological Examination of Cardiac Tissues Hearts were fixed in 10 % formalin and embedded in paraffin. The paraffin-embedded tissues were sectioned and stained with hematoxylin–eosin (H&E). The histological sections were examined by an observer blinded to the treatment regimen. TUNEL Assays Hearts were rapidly removed from anaesthetized rats and the sections were obtained [15] using a microtome (Leica,
Cardiovasc Toxicol
RM 2135, Germany). Sections from LV were incubated for 1 h at 56 °C, deparaffinized in xylene and rehydrated through an ethanol series. TUNEL staining was done according to the manufacturer’s protocol (Sheng Ke Bo Yuan Biological Technology Co., Ltd., Beijing, China). Photographs were taken with the use of a microscope. TUNEL-positive cardiomyocytes were carefully evaluated under double-blind conditions. In each visual field, the number of positive cell nuclei was counted among 200 cell nuclei. The rate of apoptotic cell nuclei was expressed as apoptotic positive cell nuclei/total cell nuclei in the field. Biochemistry Examination The hearts were homogenated for assays of biochemistry parameters as previous study [16] including catalase, superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) levels, according to the manufacturer’s instructions (Jiancheng, Nanjing, China) and our previous study [17]. In brief, catalase activity was assayed by the absorbance of the remaining H2O2 using a colorimetric method, and the activity was expressed as U/mg protein. For the determination of GSH-Px activity, 5,50-dithiobis (2-nitrobenzoic acid) (DTNB) was used as substrate and also expressed as U/mg protein. SOD activity was detected by luciferase chemiluminescence elicited by xanthine oxidase system, and the activity was expressed as U/mg protein. MDA level was determined by the conjugation ability of thiobarbituric acid with MDA and expressed as nmol/mg protein. Western Blot Analysis Western blot analysis was processed on the heart samples of each animal. In brief, heart tissues were homogenized in an ice-cold buffer containing a protease inhibitor cocktail. Then, the supernatant was collected and total protein level was detected using a standard bicinchoninic acid method (Beyotime Institute of Biotechnology, China).
Fig. 1 Histopathological changes in rat cardiac tissue (H&E 9 400): a Sham-operated rats, b Model rats and c Rats treated with TFC 300 mg/kg
Table 1 Effects of TFC on hemodynamic changes and myocardial infarction Treatment
LVSP(mmHg)
Sham
131.43 ± 18.61
Model
92.28 ± 17.19**
LVEDP(mmHg)
?dp/dtmax(mmHg/s)
-dp/dtmax(mmHg/s)
4.45 ± 1.13
11,981.74 ± 2,496.16
5,150.67 ± 1,003.28
Infarcted area (%) –
8.60 ± 2.17**
6,994.92 ± 1,527.20**
3,065.83 ± 762.14**
32.67 ± 8.33
TFC (75 mg/kg)
95.39 ± 14.62
8.50 ± 2.27
6,226.53 ± 1,195.31
3,142.56 ± 1,390.85
35.17 ± 7.09
TFC (150 mg/kg)
114.21 ± 15.11#
6.66 ± 1.90#
8,396.69 ± 963.96#
4,082.01 ± 965.91#
29.81 ± 9.44
TFC (300 mg/kg)
120.11 ± 14.42##
6.03 ± 1.70##
8,995.47 ± 1,432.65#
4,171.59 ± 796.08#
33.67 ± 8.03
Values are expressed as the mean ± SD. Significance was determined by ANOVA followed by Dunnett’s test. P \ 0.05 and ##P \ 0.01 versus Model respectively
**
P \ 0.01 versus sham group;
#
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Sham Model TFC (75 mg/kg) TFC (150 mg/kg) TFC (300 mg/kg)
Catalase(U/mg protein)
12.0
##
9.0
6.0
#
**
3.0
C 150
GSH-Px(U/mg protein)
A
0.0
B
120 ## ##
90
** 60
30
0
D
6.0
18
#
4.0
**
2.0
MDA(nmol/mg protein)
SOD(U/mg protein)
** #
15 ##
12
##
9 6 3
0.0
0
Fig. 2 Effects of TFC on the levels of catalase a, glutathione peroxidase (GSH-Px) b, superoxide dismutase (SOD) c and malondialdehyde (MDA) d. Values are expressed as the mean ± S.D.
Significance was determined by ANOVA followed by Dunnett’s test. ** P \ 0.01 versus sham group; # P \ 0.05, ## P \ 0.01 versus model group
Statistical Analysis
Analysis of Histopathological Examination
All data were reported as mean ± S.D. Statistical analysis was performed using the SPSS 11.5 software for Windows. Significance was determined by ANOVA followed by Dunnett’s test. A value of P \ 0.05 was considered statistically.
TFC markedly attenuated reperfusion injury as demonstrated by significant amelioration of histopathological changes. Figure 1 illustrates myocardial tissue in shamand I/R-treated rats, respectively. Heart tissues from sham group rats did not have any obvious histopathological changes (Fig. 1a). However, heart tissues from I/R rats showed widespread myocardial structure disorder, the myocardial swelling, interstitial hemorrhage, leukocyte infiltration and intercellular space widening with myofibril focal degeneration (Fig. 1b) as compared with sham group. Rats treated with TFC showed mild degenerative changes in the cardiac muscle fiber (Fig. 1c).
Results Assessment of Hemodynamic Changes and Myocardial Infarction Table 1 depicted the hemodynamic values found in anesthetized animals. After I/R, damaged LVEDP, LVSP and ±dp/dtmax of left ventricle became worse 2 h after I/R, when compared with sham-operated rats (P \ 0.01). On the contrary, pretreatment with TFC (150 and 300 mg/kg) significantly improved these parameters to near normal levels in a dose-dependent manner (P \ 0.05, P \ 0.01). However, the mean infarcted area after I/R was alike between the model and the TFC-treated groups.
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Biochemistry Assays Figure 2 depicted the activities of catalase, SOD, GSH-Px and MDA in the heart of control and I/R rats. Rats with I/R showed significant reductions in catalase, SOD and GSHPx versus sham-operated rats (P \ 0.01). However, when pretreating with TFC at doses of 150 and 300 mg/kg,
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obvious elevations of catalase, SOD and GSH-Px were observed than that in model group (P \ 0.05, P \ 0.01). Furthermore, rats subjected to I/R significantly exhibited the increase in MDA, an index of lipid peroxidation, compared to sham-operated rats (P \ 0.01). Administration of TFC to I/R rats at doses of 150 and 300 mg/kg diminished ischemia-mediated lipid peroxidation with a dose-dependent manner (P \ 0.01).
TUNEL and Western Blot Analysis To confirm the protective effect of TFC on the functional improvement of myocardial apoptosis induced by I/R, we used TUNEL staining to locate apoptotic nuclei, and a significant number of apoptotic cell nuclei were found in I/R rat hearts (P \ 0.01, Fig. 3). In contrast, few apoptotic cell nuclei were confirmed in TFC-treated groups (150 and 300 mg/kg, all P \ 0.01). These results demonstrated that the apoptosis in I/R was attenuated by TFC in a dosedependent fashion. To corroborate the result that TFC inhibited cardiomyocyte apoptosis following I/R in rats, Western blot analysis was processed to detect the expressions of the apoptosisregulatory proteins including caspase-3, Bcl-2 and Bax in the rat heart tissue. Figure 4 demonstrated that caspase-3 expression in model group was abundantly increased when compared with the sham group (P \ 0.01). Nevertheless, when treated with TFC at doses of 150 and 300 mg/kg, a remarkable reduction in this protein expression was noted in I/R rats, as shown in Fig. 4a, b (P \ 0.05, P \ 0.01). Additionally, the Bcl-2 family proteins, Bcl-2 and Bax, were also considered to play vital role in the process of apoptotic cascades. The results showed that pretreatment with TFC at doses of 150 and 300 mg/kg strikingly elevated Bcl-2 level and decreased Bax expression in the heart tissue of infarcted rats, when compared with model group, as indicated in Fig. 4a, c, d (P \ 0.05, P \ 0.01). The results indicated that TFC inhibited the apoptosis, causing a dose-dependent effect in I/R-induced myocardial infarction.
The Signaling Pathway Evaluation
Fig. 3 Effects of TFC on cardiomyocyte apoptosis. Cardiomyocyte apoptosis was detected by the TUNEL assay in hearts obtained from a Sham-operated rats, b Model rats, c Rats treated with TFC 300 mg/ kg, d Effects of TFC on cardiomyocyte apoptotic index
To evaluate the signaling pathway of TFC-mediated antiapoptotic effects, we measured the MAPK cascades, the upstream signaling molecules in apoptotic reactions by Western blot, and the results are shown in Fig. 5. The activations of p38 and c-Jun N-terminal kinase (JNK) was increased in model group due to I/R when compared with sham group. However, this change was obviously blocked by pretreatment with TFC (Fig. 5b, c). Moreover, TFC did not change the phosphorylation of ERK1/2 induced by I/R
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A
C
3.0
Caspase-3 Bcl-2 Bax
Bcl-2 (% Sham)
##
##
2.0
1.0
GAPDH 0.0 Sham
B
D
Model
75
150
300
4.0
Bax (% Sham)
** ##
3.0 2.0
1.0 0.0 Sham
Model
75
150
300
Fig. 4 Effects of TFC on the protein expressions of Caspase-3, Bcl-2 and Bax. a indicated the representative images of Western blots with antibodies against caspase-3, Bcl-2 and Bax, respectively, in rat hearts from different groups. b c and d were the quantitative analysis of the protein levels of caspase-3, Bcl-2 and Bax in rats hearts from different
groups, respectively. Values are expressed as the mean ± S.D. Significance was determined by ANOVA followed by Dunnett’s test. ** P \ 0.01 versus sham group; # P \ 0.05, ## P \ 0.01 versus model group
(data not shown), suggesting the beneficial effects of TFC were mediated at least partly by MAPK pathways.
was investigated. Firstly, myocardial I/R injury was caused by the occlusion of LAD coronary artery for 30 min followed by reperfusion for 2 h. Following 2-h reperfusion, the developments of infarct size (IS), cardiovascular dysfunction and histopathological changes were found in these animals. However, TFC administration dose dependently improved cardiac dysfunction. These beneficial effects were along with amelioration of histopathological damage. On the basis of these obtained results, further investigation was performed to clarify a critical molecule involved in the mechanisms of TFC on protecting cardiomyocyte against I/R-induced myocardium damage. It is well known that the excessive generation of oxygen-free radicals is conceived of as an important factor that exacerbates cellular damage during ischemic insult. GSHPx, catalase and SOD constitute a mutually supportive enzyme system of the first line cellular defense against oxidative injury, decomposing H2O2 and O2 before their interaction to form the more harmful hydroxyl radical [18]. MDA, the degradation product of lipid oxidation and oxygen-derived free radicals, reflects ROS-induced damage [19] and is used as a pivotal index for evaluating
Discussion The imbalance between ROS production and antioxidant defenses leads to the condition known as oxidative stress. AMI is a complex event, which is accompanied with increasing production of ROS. Epidemiological studies also have shown that an increase in oxidative stress in AMI patients occurred as a result of an imbalance between antioxidants and oxidants. In addition, ROS-induced apoptosis plays a vital role in the development of I/R. The inhibition of apoptosis and ROS is regarded as important as in treatment of these heart diseases. TFC, the most active ingredient of C. axillaris has obvious anti-oxidative and anti-apoptotic bioactivities. In the present study, AMI rat model was used to assess the cardioprotective effects of TFC on hemodynamics and histopathological changes, and focused on the correlate to oxidative damage and cell apoptosis. Importantly, the possible signaling pathway also
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A p-P38 P38
p-p38 (% Sham group)
300
**
250 200
##
150 100 50 0 Sham
Model
TFC(300 mg/kg)
B p- JNK
p-JNK (% Sham group)
JNK 500
** 400 300
##
200 100 0 Sham
Model
TFC (300mg/kg)
Fig. 5 Effects of TFC on the activation of p38 MAPK and JNK. Phosphorylated MAPKs were detected with Western blot. Values are expressed as the mean ± S.D. Significance was determined by ANOVA followed by Dunnett’s test. ** P \ 0.01 versus. sham group; ##P \ 0.01 versus model group
structural oxidative injury of cell membrane. In the present study, TFC treatment to the infarcted rats resulted in the evident reduction in MDA and the significant elevation of SOD, catalase as well as GSH-Px, which indicated that the cardioprotection of TFC against AMI in rats was at least related to its antioxidant property. Cardiac damage after ischemic insults occured via oxidative stress can contribute to consequently initiate an apoptotic cascade. Many genes, such as Bcl-2 and Bax, are suggested to play pivotal roles in determining cell’s death or survival after apoptotic stimuli [20, 21]. Bcl-2 is regards as the repressor of apoptosis, while another member of the family, Bax, functions as the promoter of cell death [22]. It has been previously reported that myocardial infarction
dramatically increased the expression of the apoptotic promoting molecule Bax [23, 24]. Moreover, Bcl-2 expression in I/R rats was also increased slightly, indicating the initiation of the tissue homeostatic response [25]. Our findings depicted that an evident up-regulation of Bax and Bcl-2 (slightly) was found in rats following AMI, which was in accordance with previous reports. However, TFC could significantly decrease Bax level and strikingly elevated Bcl-2 expression. In addition, caspase-3 is involved in the common pathway of necrotic and apoptotic death in the myocytes [26], activation of caspase-3 results in cleavage of nuclear proteins and cytoskeletal and DNA nucleosomal fragmentation [27]. Cumulative investigations have revealed that up-regulation of caspase-3 expression was observed after AMI [23]. Results from the current study implicated that TFC treatment significantly caused the down-regulation of caspase-3 in AMI rats. Taken together, our data showed that TFC exerted the cardioprotective effects partially via its anti-apoptotic profile in AMI of rat model. Importantly, TUNEL tests, another important methods for detecting apoptotic programmed cell death, also indicated the same protective effects of TFC. A great number of studies have suggested that MAPK signaling cascades were regarded as an important pathway in oxidative-stress-induced apoptotic cell death [28, 29], and three MAPK subfamily members, ERK1/2 (beneficial), p38 MAPK and JNK (deleterious), are activated following myocardial I/R [30, 31]. Namely, activation of ERK contributes to cell differentiation, proliferation and survival, whereas JNK and p38 are activated by environmental stresses, promote apoptosis and pro-inflammatory cytokines [32]. To clarify the possible singling pathway of TFC on I/R-induced heart damage, we next tested the potential effects of TFC on different MAPK cascade activation induced by I/R. The results indicated that activations of ERK1/2, p38 and JNK were observed in rat hearts subjected to I/R. However, pretreatment with TFC significantly suppressed the activation of p38 and JNK, but not influenced ERK1/2 activity, suggesting clearly the involvement of p38 and JNK proteins in TFC preconditioning. In summary, the present investigations indicated that TFC dose dependently improved I/R-induced myocardium impairment via anti-apoptotic and anti-oxidative activities, and these beneficial effects were intervened by MAPK signaling pathway. Practically, these results also supported the fact that TFC might hold promise as a therapeutic intervention for the treatment of myocardial ischemia, but more detailed studies should be carried out. Conflict of interest
None.
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Preventive Effect of Total Flavones of Choerospondias axillaries on Ischemia/Reperfusion-Induced Myocardial Infarction-Related MAPK Signaling Pathway Chunmei Li • Jie He • Yonglin Gao • Yanli Xing • Jian Hou • Jingwei Tian
Ó Springer Science+Business Media New York 2013
Abstract Choerospondias axillaris (Guangzao) is a medicinal plant used in Mongolia, and its fruit is commonly used for the treatment of cardiovascular diseases in clinic. The constituents responsible for this effect are always conceded to be the total flavonoids of C. axillaries (TFC). The present study was to evaluate the preventive effect of TFC on acute myocardial infarction induced by ischemia/reperfusion (I/R) in rats and the possible signaling pathway involved. The model of myocardial I/R was caused by occlusion of the left anterior descending coronary artery for 30 min followed by reperfusion for 2 h. Cardiac dysfunction, infarct size, pathologic histology and apoptosis were examined. Heart tissues were homogenized for biochemistry assays and Western blots analysis. The results indicated that pretreatment with TFC strongly improved cardiac function, obviously reduced heart pathologic lesion in I/R rat hearts. TFC also could protect the heart from I/R injury by increasing the levels of catalase, glutathione peroxidase and superoxide dismutase in heart homogenate, and decreasing that of malondialdehyde level. These beneficial effects were associated with the decrease in TUNEL-positive nuclear staining, Bax and caspase-3 levels, and the increase in Bcl-2 expression. Moreover,
C. Li J. Tian (&) School of Pharmacy, Yantai University, Yantai 264005, Shandong, People’s Republic of China e-mail: [email protected]; [email protected] J. He Y. Xing J. Hou Shandong Luye Research and Development for Natural Drugs Co. Ltd., Yantai 264003, Shandong, People’s Republic of China Y. Gao School of Life Sciences, Yantai University, Yantai 264005, Shandong, People’s Republic of China
TFC counteracted the I/R-induced decreased activation of p38 mitogen-activated protein kinase (MAPK) and Jun N-terminal kinase. These data suggested that TFC improved I/R-induced myocardium impairment via antioxidative and anti-apoptotic activities, and this beneficial effects were intervened by MAPK signaling pathway. Keywords Total flavones of Choerospondias axillaries Ischemia/reperfusion Anti-oxidative Anti-apoptotic MAPK pathways
Introduction Acute myocardial infarction (AMI) is the sharp decline in coronary artery or the interruption of blood supply to a part of the heart, resulting heart cells to die. The resulting ischemia (restriction in blood supply) and ensuing oxygen shortage induce myocardium infarction or heart damage, if left untreated for a sufficient period of time. Reperfusion therapy must be treated as soon as possible after AMI in order to attenuate the ischemic injury. However, reperfusion is responsible for additional myocardial damage [1]. Reactive oxygen species (ROS) play a vital role in ischemia development as well as during the reperfusion phase after AMI [2]. One hypothesis is that antioxidants are severely consumed during infarction, and O2 abruptly increases metabolism after reperfusion in the damage of normal defenses [3]. Moreover, it is now implied that the readmission of oxygenated blood into previously ischemic myocardium can induce a cascade of events that will paradoxically produce additional myocardial cell dysfunction and cell apoptosis [4]. Based on these observations, it has been promised that inhibiting cardiomyocyte apoptosis and
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oxidative stress could serve as the basis for the potential development of drugs for ischemic heart diseases. Choerospondias axillaris (Guangzao) is a medicinal plant used in Mongolia, and its fruit is widely used for the treatment of cardiovascular diseases in clinic [5, 6]. For example, Guang-Zao-Qi-Wei-Wan, Compound Guangzao capsule and San-Wei-Guang-Zao capsule have been wildly used for many years in china [7–9]. The constituents responsible for this effect are generally conceded to be the total flavonoids of C. axillaries (TFC), because these kinds of compounds are known to be antioxidants [10]. Blood flow arrest and reperfusion during AMI cause myocytes and endothelium injury through oxidative stress and inflammatory response, both of which involve ROS and peroxides that consume antioxidant defenses. Of note, researchers have found that TFC could inhibit dexamethasone-induced thymocyte apoptosis [11]. Recently, Ao et al. have reported that TFC could attenuate the serum levels of CK, CK-MB and LDH in isoproterenol-induced MI injury in rats [12]. In light of the previous findings, we hypothesized that TFC can inhibit oxidative damage and cardiomyocyte apoptosis associated with AMI. To support our hypothesis, we used a rat model of ischemia/reperfusion (I/R) to investigate the cardioprotective properties of TFC and assessed its relationship to oxidative stress markers, apoptosis-inducing factors, hemodynamics and histopathological lesions. Moreover, the present study also aimed to investigate whether the cardioprotective effects of TFC are mediated by p38 mitogen-activated protein kinase (MAPK) pathways.
Materials and Methods Chemicals All the chemicals and reagents were analytical grade. The plant material used was the dried fruit of C. axillaries. The test compound, TFC, was isolated as described previously [13]. Briefly, the fruit was powdered and extracted in water at 100 °C for 40 min. The sample was filtered and extracted 3 times under the same conditions with new solvent. The crude extract was collected and refluxed for 1.5 h with 60 % alcohol (1:12) 4 times in succession; then, the test substance was obtained. I/R Procedure to Induce Myocardial Injury In the current study, male Sprague–Dawley rats, 250 ± 20 g, were divided into five groups: Group 1, sham; Group 2, I/R model; Group 3, I/R ? TFC 75 mg/kg; Group 4, I/R ? TFC 150 mg/kg; Group 5, I/R ? TFC 300 mg/kg.
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TFC 75, 150 and 300 mg/kg group animals were administered orally once a day for continues 30 days. Group 1 and 2 were treated with the same volume of distilled water. On day 29 and day 30 of treatment, the cardial I/R operation was carried out according to previous procedure [14]. Briefly, after rats were anesthetized by injection of urethane, coronary artery ligation was achieved with a gab occluder fixed onto the left anterior descending (LAD) coronary artery. A 7-0 silk suture was passed underneath the LAD (2–3 mm inferior to the left auricle) and tied. AMI injury was induced by 30 min of ischemia followed by 2 h of reperfusion. The respirator weaned when the animal recovered spontaneous breathing. All procedures involved in the use of laboratory animals were in accordance with the Guidelines of the Animal Care and Use of Laboratory Animals set by the Association of Laboratory Animal Science and the Center for Laboratory Animal Science. Hemodynamic Assessment Following 2 h after I/R, hemodynamic measurements, including left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and ±dp/dtmax, were performed and programmed by using a biotic signal collection and processing system (BIOPIC, American). A millar vessel was inserted into the left ventricular cavity of animals via the right common carotid artery. Analysis of Myocardial Infarction The rat hearts were removed, and the left ventricle area was divided into thick slices from the apex and incubated in 2 % triphenyltetrazolium chloride (TTC) in pH 7.4 buffer for 15 min at 37 °C. TTC stained viable tissue dark red, while the infarct portion remained grayish-white. The infarcted area (border zone) was traced by hand on image system (NIH image software) and measured by computerized planimetry. Histopathological Examination of Cardiac Tissues Hearts were fixed in 10 % formalin and embedded in paraffin. The paraffin-embedded tissues were sectioned and stained with hematoxylin–eosin (H&E). The histological sections were examined by an observer blinded to the treatment regimen. TUNEL Assays Hearts were rapidly removed from anaesthetized rats and the sections were obtained [15] using a microtome (Leica,
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RM 2135, Germany). Sections from LV were incubated for 1 h at 56 °C, deparaffinized in xylene and rehydrated through an ethanol series. TUNEL staining was done according to the manufacturer’s protocol (Sheng Ke Bo Yuan Biological Technology Co., Ltd., Beijing, China). Photographs were taken with the use of a microscope. TUNEL-positive cardiomyocytes were carefully evaluated under double-blind conditions. In each visual field, the number of positive cell nuclei was counted among 200 cell nuclei. The rate of apoptotic cell nuclei was expressed as apoptotic positive cell nuclei/total cell nuclei in the field. Biochemistry Examination The hearts were homogenated for assays of biochemistry parameters as previous study [16] including catalase, superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) levels, according to the manufacturer’s instructions (Jiancheng, Nanjing, China) and our previous study [17]. In brief, catalase activity was assayed by the absorbance of the remaining H2O2 using a colorimetric method, and the activity was expressed as U/mg protein. For the determination of GSH-Px activity, 5,50-dithiobis (2-nitrobenzoic acid) (DTNB) was used as substrate and also expressed as U/mg protein. SOD activity was detected by luciferase chemiluminescence elicited by xanthine oxidase system, and the activity was expressed as U/mg protein. MDA level was determined by the conjugation ability of thiobarbituric acid with MDA and expressed as nmol/mg protein. Western Blot Analysis Western blot analysis was processed on the heart samples of each animal. In brief, heart tissues were homogenized in an ice-cold buffer containing a protease inhibitor cocktail. Then, the supernatant was collected and total protein level was detected using a standard bicinchoninic acid method (Beyotime Institute of Biotechnology, China).
Fig. 1 Histopathological changes in rat cardiac tissue (H&E 9 400): a Sham-operated rats, b Model rats and c Rats treated with TFC 300 mg/kg
Table 1 Effects of TFC on hemodynamic changes and myocardial infarction Treatment
LVSP(mmHg)
Sham
131.43 ± 18.61
Model
92.28 ± 17.19**
LVEDP(mmHg)
?dp/dtmax(mmHg/s)
-dp/dtmax(mmHg/s)
4.45 ± 1.13
11,981.74 ± 2,496.16
5,150.67 ± 1,003.28
Infarcted area (%) –
8.60 ± 2.17**
6,994.92 ± 1,527.20**
3,065.83 ± 762.14**
32.67 ± 8.33
TFC (75 mg/kg)
95.39 ± 14.62
8.50 ± 2.27
6,226.53 ± 1,195.31
3,142.56 ± 1,390.85
35.17 ± 7.09
TFC (150 mg/kg)
114.21 ± 15.11#
6.66 ± 1.90#
8,396.69 ± 963.96#
4,082.01 ± 965.91#
29.81 ± 9.44
TFC (300 mg/kg)
120.11 ± 14.42##
6.03 ± 1.70##
8,995.47 ± 1,432.65#
4,171.59 ± 796.08#
33.67 ± 8.03
Values are expressed as the mean ± SD. Significance was determined by ANOVA followed by Dunnett’s test. P \ 0.05 and ##P \ 0.01 versus Model respectively
**
P \ 0.01 versus sham group;
#
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Sham Model TFC (75 mg/kg) TFC (150 mg/kg) TFC (300 mg/kg)
Catalase(U/mg protein)
12.0
##
9.0
6.0
#
**
3.0
C 150
GSH-Px(U/mg protein)
A
0.0
B
120 ## ##
90
** 60
30
0
D
6.0
18
#
4.0
**
2.0
MDA(nmol/mg protein)
SOD(U/mg protein)
** #
15 ##
12
##
9 6 3
0.0
0
Fig. 2 Effects of TFC on the levels of catalase a, glutathione peroxidase (GSH-Px) b, superoxide dismutase (SOD) c and malondialdehyde (MDA) d. Values are expressed as the mean ± S.D.
Significance was determined by ANOVA followed by Dunnett’s test. ** P \ 0.01 versus sham group; # P \ 0.05, ## P \ 0.01 versus model group
Statistical Analysis
Analysis of Histopathological Examination
All data were reported as mean ± S.D. Statistical analysis was performed using the SPSS 11.5 software for Windows. Significance was determined by ANOVA followed by Dunnett’s test. A value of P \ 0.05 was considered statistically.
TFC markedly attenuated reperfusion injury as demonstrated by significant amelioration of histopathological changes. Figure 1 illustrates myocardial tissue in shamand I/R-treated rats, respectively. Heart tissues from sham group rats did not have any obvious histopathological changes (Fig. 1a). However, heart tissues from I/R rats showed widespread myocardial structure disorder, the myocardial swelling, interstitial hemorrhage, leukocyte infiltration and intercellular space widening with myofibril focal degeneration (Fig. 1b) as compared with sham group. Rats treated with TFC showed mild degenerative changes in the cardiac muscle fiber (Fig. 1c).
Results Assessment of Hemodynamic Changes and Myocardial Infarction Table 1 depicted the hemodynamic values found in anesthetized animals. After I/R, damaged LVEDP, LVSP and ±dp/dtmax of left ventricle became worse 2 h after I/R, when compared with sham-operated rats (P \ 0.01). On the contrary, pretreatment with TFC (150 and 300 mg/kg) significantly improved these parameters to near normal levels in a dose-dependent manner (P \ 0.05, P \ 0.01). However, the mean infarcted area after I/R was alike between the model and the TFC-treated groups.
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Biochemistry Assays Figure 2 depicted the activities of catalase, SOD, GSH-Px and MDA in the heart of control and I/R rats. Rats with I/R showed significant reductions in catalase, SOD and GSHPx versus sham-operated rats (P \ 0.01). However, when pretreating with TFC at doses of 150 and 300 mg/kg,
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obvious elevations of catalase, SOD and GSH-Px were observed than that in model group (P \ 0.05, P \ 0.01). Furthermore, rats subjected to I/R significantly exhibited the increase in MDA, an index of lipid peroxidation, compared to sham-operated rats (P \ 0.01). Administration of TFC to I/R rats at doses of 150 and 300 mg/kg diminished ischemia-mediated lipid peroxidation with a dose-dependent manner (P \ 0.01).
TUNEL and Western Blot Analysis To confirm the protective effect of TFC on the functional improvement of myocardial apoptosis induced by I/R, we used TUNEL staining to locate apoptotic nuclei, and a significant number of apoptotic cell nuclei were found in I/R rat hearts (P \ 0.01, Fig. 3). In contrast, few apoptotic cell nuclei were confirmed in TFC-treated groups (150 and 300 mg/kg, all P \ 0.01). These results demonstrated that the apoptosis in I/R was attenuated by TFC in a dosedependent fashion. To corroborate the result that TFC inhibited cardiomyocyte apoptosis following I/R in rats, Western blot analysis was processed to detect the expressions of the apoptosisregulatory proteins including caspase-3, Bcl-2 and Bax in the rat heart tissue. Figure 4 demonstrated that caspase-3 expression in model group was abundantly increased when compared with the sham group (P \ 0.01). Nevertheless, when treated with TFC at doses of 150 and 300 mg/kg, a remarkable reduction in this protein expression was noted in I/R rats, as shown in Fig. 4a, b (P \ 0.05, P \ 0.01). Additionally, the Bcl-2 family proteins, Bcl-2 and Bax, were also considered to play vital role in the process of apoptotic cascades. The results showed that pretreatment with TFC at doses of 150 and 300 mg/kg strikingly elevated Bcl-2 level and decreased Bax expression in the heart tissue of infarcted rats, when compared with model group, as indicated in Fig. 4a, c, d (P \ 0.05, P \ 0.01). The results indicated that TFC inhibited the apoptosis, causing a dose-dependent effect in I/R-induced myocardial infarction.
The Signaling Pathway Evaluation
Fig. 3 Effects of TFC on cardiomyocyte apoptosis. Cardiomyocyte apoptosis was detected by the TUNEL assay in hearts obtained from a Sham-operated rats, b Model rats, c Rats treated with TFC 300 mg/ kg, d Effects of TFC on cardiomyocyte apoptotic index
To evaluate the signaling pathway of TFC-mediated antiapoptotic effects, we measured the MAPK cascades, the upstream signaling molecules in apoptotic reactions by Western blot, and the results are shown in Fig. 5. The activations of p38 and c-Jun N-terminal kinase (JNK) was increased in model group due to I/R when compared with sham group. However, this change was obviously blocked by pretreatment with TFC (Fig. 5b, c). Moreover, TFC did not change the phosphorylation of ERK1/2 induced by I/R
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A
C
3.0
Caspase-3 Bcl-2 Bax
Bcl-2 (% Sham)
##
##
2.0
1.0
GAPDH 0.0 Sham
B
D
Model
75
150
300
4.0
Bax (% Sham)
** ##
3.0 2.0
1.0 0.0 Sham
Model
75
150
300
Fig. 4 Effects of TFC on the protein expressions of Caspase-3, Bcl-2 and Bax. a indicated the representative images of Western blots with antibodies against caspase-3, Bcl-2 and Bax, respectively, in rat hearts from different groups. b c and d were the quantitative analysis of the protein levels of caspase-3, Bcl-2 and Bax in rats hearts from different
groups, respectively. Values are expressed as the mean ± S.D. Significance was determined by ANOVA followed by Dunnett’s test. ** P \ 0.01 versus sham group; # P \ 0.05, ## P \ 0.01 versus model group
(data not shown), suggesting the beneficial effects of TFC were mediated at least partly by MAPK pathways.
was investigated. Firstly, myocardial I/R injury was caused by the occlusion of LAD coronary artery for 30 min followed by reperfusion for 2 h. Following 2-h reperfusion, the developments of infarct size (IS), cardiovascular dysfunction and histopathological changes were found in these animals. However, TFC administration dose dependently improved cardiac dysfunction. These beneficial effects were along with amelioration of histopathological damage. On the basis of these obtained results, further investigation was performed to clarify a critical molecule involved in the mechanisms of TFC on protecting cardiomyocyte against I/R-induced myocardium damage. It is well known that the excessive generation of oxygen-free radicals is conceived of as an important factor that exacerbates cellular damage during ischemic insult. GSHPx, catalase and SOD constitute a mutually supportive enzyme system of the first line cellular defense against oxidative injury, decomposing H2O2 and O2 before their interaction to form the more harmful hydroxyl radical [18]. MDA, the degradation product of lipid oxidation and oxygen-derived free radicals, reflects ROS-induced damage [19] and is used as a pivotal index for evaluating
Discussion The imbalance between ROS production and antioxidant defenses leads to the condition known as oxidative stress. AMI is a complex event, which is accompanied with increasing production of ROS. Epidemiological studies also have shown that an increase in oxidative stress in AMI patients occurred as a result of an imbalance between antioxidants and oxidants. In addition, ROS-induced apoptosis plays a vital role in the development of I/R. The inhibition of apoptosis and ROS is regarded as important as in treatment of these heart diseases. TFC, the most active ingredient of C. axillaris has obvious anti-oxidative and anti-apoptotic bioactivities. In the present study, AMI rat model was used to assess the cardioprotective effects of TFC on hemodynamics and histopathological changes, and focused on the correlate to oxidative damage and cell apoptosis. Importantly, the possible signaling pathway also
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A p-P38 P38
p-p38 (% Sham group)
300
**
250 200
##
150 100 50 0 Sham
Model
TFC(300 mg/kg)
B p- JNK
p-JNK (% Sham group)
JNK 500
** 400 300
##
200 100 0 Sham
Model
TFC (300mg/kg)
Fig. 5 Effects of TFC on the activation of p38 MAPK and JNK. Phosphorylated MAPKs were detected with Western blot. Values are expressed as the mean ± S.D. Significance was determined by ANOVA followed by Dunnett’s test. ** P \ 0.01 versus. sham group; ##P \ 0.01 versus model group
structural oxidative injury of cell membrane. In the present study, TFC treatment to the infarcted rats resulted in the evident reduction in MDA and the significant elevation of SOD, catalase as well as GSH-Px, which indicated that the cardioprotection of TFC against AMI in rats was at least related to its antioxidant property. Cardiac damage after ischemic insults occured via oxidative stress can contribute to consequently initiate an apoptotic cascade. Many genes, such as Bcl-2 and Bax, are suggested to play pivotal roles in determining cell’s death or survival after apoptotic stimuli [20, 21]. Bcl-2 is regards as the repressor of apoptosis, while another member of the family, Bax, functions as the promoter of cell death [22]. It has been previously reported that myocardial infarction
dramatically increased the expression of the apoptotic promoting molecule Bax [23, 24]. Moreover, Bcl-2 expression in I/R rats was also increased slightly, indicating the initiation of the tissue homeostatic response [25]. Our findings depicted that an evident up-regulation of Bax and Bcl-2 (slightly) was found in rats following AMI, which was in accordance with previous reports. However, TFC could significantly decrease Bax level and strikingly elevated Bcl-2 expression. In addition, caspase-3 is involved in the common pathway of necrotic and apoptotic death in the myocytes [26], activation of caspase-3 results in cleavage of nuclear proteins and cytoskeletal and DNA nucleosomal fragmentation [27]. Cumulative investigations have revealed that up-regulation of caspase-3 expression was observed after AMI [23]. Results from the current study implicated that TFC treatment significantly caused the down-regulation of caspase-3 in AMI rats. Taken together, our data showed that TFC exerted the cardioprotective effects partially via its anti-apoptotic profile in AMI of rat model. Importantly, TUNEL tests, another important methods for detecting apoptotic programmed cell death, also indicated the same protective effects of TFC. A great number of studies have suggested that MAPK signaling cascades were regarded as an important pathway in oxidative-stress-induced apoptotic cell death [28, 29], and three MAPK subfamily members, ERK1/2 (beneficial), p38 MAPK and JNK (deleterious), are activated following myocardial I/R [30, 31]. Namely, activation of ERK contributes to cell differentiation, proliferation and survival, whereas JNK and p38 are activated by environmental stresses, promote apoptosis and pro-inflammatory cytokines [32]. To clarify the possible singling pathway of TFC on I/R-induced heart damage, we next tested the potential effects of TFC on different MAPK cascade activation induced by I/R. The results indicated that activations of ERK1/2, p38 and JNK were observed in rat hearts subjected to I/R. However, pretreatment with TFC significantly suppressed the activation of p38 and JNK, but not influenced ERK1/2 activity, suggesting clearly the involvement of p38 and JNK proteins in TFC preconditioning. In summary, the present investigations indicated that TFC dose dependently improved I/R-induced myocardium impairment via anti-apoptotic and anti-oxidative activities, and these beneficial effects were intervened by MAPK signaling pathway. Practically, these results also supported the fact that TFC might hold promise as a therapeutic intervention for the treatment of myocardial ischemia, but more detailed studies should be carried out. Conflict of interest
None.
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