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Protective effects of arjunolic acid against cardiac toxicity induced by oral sodium nitrite: Effects on cytokine balance and apoptosis
Life Sciences 111 (2014) 18–26
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Protective effects of arjunolic acid against cardiac toxicity induced by oral sodium nitrite: Effects on cytokine balance and apoptosis Mohammed M.H. Al-Gayyar a,b,⁎, Abdullah Al Youssef c, Iman O. Sherif a, Mohamed E.E. Shams d, Ahmed Abbas e a
Dept. of Clinical Biochemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt Faculty of Pharmacy, University of Tabuk, Tabuk 71491, Saudi Arabia Dept. of Internal Medicine, Faculty of Medicine, University of Tabuk, Tabuk 71471, Saudi Arabia d Dept. of Pharmaceutics, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt e Dept. of Pharmacognosy, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt b c
a r t i c l e
i n f o
Article history: Received 24 March 2014 Accepted 7 July 2014 Available online 23 July 2014 Keywords: Caspase-3/8/9 Cytochrome C oxidase Interleukin (IL)-1β/4/10 JNK MAPK Tumor necrosis factor (TNF)-α
a b s t r a c t Aims: Sodium nitrite, a preservative used in meat products, helps in the production of free radicals, leading to increased lipid peroxidation, which plays a vital role in posing toxic effects in different body organs. On the other hand, arjunolic acid possesses antioxidant properties and plays protective roles against chemically induced organ pathophysiology. We investigated the effect of sodium nitrite on cardiac tissue in rats on the inflammatory cytokine balance and the type of induced apoptosis, and we analyzed the protective role of arjunolic acid. Main methods: Sixty adult male Sprague-Dawley rats were injected with 80 mg/kg sodium nitrite in the presence/absence of arjunolic acid (100 and 200 mg/kg). Cardiac pro-inflammatory cytokines (TNF-α and IL-1β), c-reactive protein (CRP) and anti-inflammatory cytokines (IL-4 and IL-10) were measured by ELISA. Cardiac mitochondrial activity (cytochrome-C-oxidase), JNK activation and apoptosis (caspase-3, caspase-8 and caspase-9) were assessed. Key findings: Sodium nitrite resulted in increased TNF-α (1.6-fold), IL-1β (3.7-fold) and CRP (2.4-fold) levels accompanied by 52%, 59% and 40% reductions in IL-10, IL-4 and cytochrome-C-oxidase, respectively, as well as enhanced JNK, caspase-3, caspase-8 and caspase-9 activities. Arjunolic acid markedly ameliorated these effects. Significance: Arjunolic acid attenuated sodium nitrite-induced cardiac damage in rats and restored the normal balance between pro- and anti-inflammatory cytokines. Moreover, arjunolic acid protected cardiac tissues from both extrinsic and intrinsic cell death pathways. © 2014 Elsevier Inc. All rights reserved.
Introduction Food additives are common and play an important role in our lives. Sodium nitrite is routinely used as a color fixative and preservative in meats and fish. The wide use of these food additives in food technology elevates the importance of studying their effects on mammals (Ezz ElArab et al., 2006). The hazardous effect of sodium nitrite is derived from the reaction of nitrite with amines and amides in food to produce nitrosamines and nitrosamides, respectively, which are considered to play vital roles in posing toxic effects in different organs (Tong et al., 2010; El-Sheikh and Khalil, 2011). Sodium nitrite was reported to cause cancer, hepatotoxicity, nephrotoxicity, dysregulation of inflammation and tissue injury (El-Sheikh and Khalil, 2011; Salama et al., 2013).
⁎ Corresponding author at: Faculty of Pharmacy, University of Tabuk, Saudi Arabia. Tel.: +966 50 8114998. E-mail address: [email protected] (M.M.H. Al-Gayyar).
http://dx.doi.org/10.1016/j.lfs.2014.07.002 0024-3205/© 2014 Elsevier Inc. All rights reserved.
Recent studies have shown that many compounds of herbal origin are able to reduce plasma triacylglycerol and total cholesterol levels and elevate the high-density lipoprotein concentration, thereby reducing the risk of cardiovascular diseases. The plant Terminalia arjuna (Combretaceae) has had a long history of medicinal uses, primarily in the preparation of ayurvedic formulations, for more than three centuries (Nammi et al., 2003). Arjunolic acid, a natural pentacyclic triterpenoid saponin, is one of the major constituents present in arjuna barks. Arjunolic acid has been shown to possess protective activities in various organ pathophysiologies (Manna et al., 2008; Manna and Sil, 2012; Ghosh and Sil, 2013; Rashid et al., 2013). The potent antioxidant property of arjunolic acid combined with its metal-chelating ability protects organs from metal- and drug-induced toxicity (Hemalatha et al., 2010; Ghosh and Sil, 2013). In addition, arjunolic acid acts as a prophylactic agent against oxidative cardiac damage induced by arsenic (Manna et al., 2008), isoproterenol (Sumitra et al., 2001) and doxorubicin (Ghosh et al., 2011). We previously published two studies illustrating the effect of sodium nitrite on hepatic dysfunction and focused on oxidative stress,
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inflammation, fibrosis and apoptosis (Salama et al., 2013; Sherif and Al-Gayyar, 2013). Less attention has been paid to measuring the cardiac toxicity effects of sodium nitrite. Therefore, this study is designed to investigate the impact of sodium nitrite on cardiac tissue in rats by measuring its effect on the balance between pro- and anti-inflammatory cytokines and the type of induced apoptosis. In addition, we aimed to investigate the cardiac protective effects of arjunolic acid.
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Measuring heart function The serum aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels were measured by standard methodologies using commercially available kits provided by Diamond Company (Egypt). Assessment of oxidative stress Oxidative stress was estimated through the following parameters:
Materials & methods Animals and their treatment outlines The animal protocol was approved by the ethical committee of the Faculty of Pharmacy, University of Mansoura. Male Sprague-Dawley rats weighing 180–200 g were used. All animals were maintained under standard conditions, and all treatments were given via oral gavage. Rats were classified into the following groups with 10 rats in each group: Control group. Rats received the standard diet without any treatment. Arjunolic acid-treated control group (100 mg/kg). Rats received a daily standard diet that was supplemented orally with 100 mg/kg arjunolic acid (Hangzhou Dayangchem, Zhejiang, China) daily for 12 weeks. Arjunolic acid-treated control group (200 mg/kg). Rats received a daily standard diet that was supplemented orally with 200 mg/kg arjunolic acid daily for 12 weeks. Sodium nitrite group. Rats were orally given 80 mg/kg sodium nitrite (Sigma-Aldrich, St. Louis, MO, USA) daily for 12 weeks. Arjunolic acid (100 mg/kg)-treated group. Rats supplemented orally with 100 mg/kg arjunolic acid followed by 80 mg/kg sodium nitrite daily for 12 weeks. Arjunolic acid (200 mg/kg)-treated group. Rats supplemented orally with 200 mg/kg arjunolic acid followed by 80 mg/kg sodium nitrite daily for 12 weeks. The doses and time courses of the experiments that were used for sodium nitrite and arjunolic acid were in the range of those used in other studies and applied for the same animal species (Yin et al., 2012; Salama et al., 2013; Sherif and Al-Gayyar, 2013).
Serum and cardiac malondialdhyde (MDA) concentrations were measured as described previously (Al-Gayyar et al., 2007; Shams et al., 2011). Briefly, after precipitation of the proteins by trichloroacetic acid, thiobarbituric acid was reacted with MDA to form thiobarbituric acid-reactive substances, which were measured at 532 nm. Blood and cardiac reduced glutathione (GSH) concentrations were measured as described previously (Beutler et al., 1963). This assay depended on the reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) by glutathione, which formed a highly colored, yellow anion that could be measured at 412 nm. Assessment of lipid contents Serum and cardiac homogenates were used for cholesterol, triglyceride and phospholipid measurements by standard methodologies using commercially available kits provided by Biodiagnostic Company (Egypt). ELISA determination The levels of biochemical parameters in the cardiac homogenates were measured by ELISA assays using commercially available tumor necrosis factor (TNF)-α, c-reactive protein (CRP), interleukin (IL)-1β, IL-4 and IL-10 ELISA kits (eBioscience Inc., San Diego, CA, USA) in accordance with the manufacturer's instructions. Western blot analysis
The animals were anesthetized by intra-peritoneal injection of (30 mg/kg) thiopental sodium. Needle electrodes were inserted under the skin of the four limbs, and electrocardiograms were recorded from standard lead II limb leads using a single-channel ECG (Fukuda ME Kogyo Co. Ltd., Model: 501-B ΙΙΙ, Tokyo, Japan). The electrocardiograph was standardized before each tracing to obtain sensitivity (2 mV pulse produces 20 mm height) and chart (paper) speed (50 mm/s).
Cardiac tissues were homogenized in RIPA buffer (Millipore, Temecula, CA, USA), and the total amount of protein was determined by protein assays (Bio-Rad Laboratories Inc., Hercules, CA, USA). Samples (30 μg protein) were separated by SDS-PAGE and electroblotted onto nitrocellulose membranes, which were blocked in PBS, 0.02% Tween 20 (PBST) containing 5% non-fat milk. Antibodies for cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 (Cell Signaling Technology, Inc., Danvers, MA) were used in 1:500 dilutions in PBST containing 5% non-fat milk and applied overnight at 4 °C. The membranes were reprobed with 1:2000 β-actin (Sigma-Aldrich) in PBST containing 5% non-fat milk for 2 h to confirm equal loading. Primary antibody was detected using 1:5000 dilutions of horseradish peroxidase-conjugated sheep anti-mouse antibodies in PBST and enhanced chemiluminescence (GE Healthcare, Piscataway, NJ, USA).
Animal sacrifice and collection of samples
Measurement of cell activation of the pro-apoptotic pJNK/JNK MAPK pathway
Electrocardiogram (ECG)
The animals were sacrificed by decapitation. Rat trunk blood was collected and centrifuged at 3000 rpm for 5 min, and serum samples were separated and stored at − 80 °C. Rat hearts were removed, cleaned with ice-cold saline, weighed and chilled on crushed ice. A piece of the heart was homogenized in a 10-fold volume of ice-cold sodium potassium phosphate buffer (0.01 M, pH 7.4) containing 1.15% KCl. The homogenates were centrifuged at 600 g at 4 °C for 10 min, and the supernatant was stored at −80 °C until use.
Renal tissue activation of JNK MAPK, as measured as the ratio of pJNK/JNK, was determined using a commercially available ELISA kit from RayBio® Company. Determination of cardiac mitochondrial function Cardiac mitochondrial function was measured via determining the cytochrome C oxidase activity using a commercially available kit
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(Sigma-Aldrich) and was based on observing the decrease in absorbance at 550 nm of ferrocytochrome C, which is caused by its oxidation to ferricytochrome C by cytochrome C oxidase. Estimation of the activities of caspase-3, caspase-8 and caspase-9 Caspase-3, caspase-8 and caspase-9 enzyme activities were measured colorimetrically using commercially available kits (GenScript, Piscataway, NJ, USA) following the manufacturer's procedure. Statistical analysis For descriptive statistics of quantitative variables, the mean ± standard error was used. Normality of the sample distribution of each continuous variable was tested with the Kolmogorov–Smirnov (K–S) test. One-way analysis of variance (ANOVA) was used to compare the means between groups. Once differences between the means were found, post hoc Bonferroni correction tests were calculated. Statistical computations were performed on a personal computer using Microsoft Excel 2007. Statistical significance was predefined as P ≤ 0.05. Results Arjunolic acid reversed the cardiac biomarker changes induced by sodium nitrite Administration of sodium nitrite resulted in marked cellular, molecular and biochemical changes that included fibrosis, mitochondrial function impairment, inflammation and DNA degradation (Hassan et al., 2010; Salama et al., 2013; Sherif and Al-Gayyar, 2013). The serum levels of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) were used as indicators for cardiac function. As shown in Table 1, sodium nitrite caused significant increases in the serum LDH and AST levels compared with the control groups (p b 0.05). Treatment with arjunolic acid showed a significant, dose-dependent reduction in the LDH and AST levels compared with the sodium nitrite group (p b 0.05) and did not affect the control rats. Arjunolic acid improved sodium nitrite-induced changes in ECG variables As shown in Table 2, examination of ECG from sodium nitriteadministered rats revealed significant and serious changes. There was a significant increase in the heart rate, ST segment elevation, QT interval, T-wave amplitude and QRS amplitude in comparison to the normal control group. Abnormal T-waves and QRS complexes were also observed. Based on the aforementioned changes, it appeared that sodium nitrite significantly impaired myocardial contractility with severe conducting tissue impairment in comparison to the normal control group. The treatment of rats with arjunolic acid reduced the heart rate and ameliorated the deleterious effects observed due to sodium nitrite on the heart. Therefore, according to the heart function and ECG results, arjunolic acid exerts protective effects against sodium nitrite-induced cardiac damage.
Arjunolic acid ameliorated the bad lipid profile induced by sodium nitrite The correlation between the lipid profile and changes in heart markers is well established (Gokkusu and Mostafazadeh, 2003). As shown in Table 3, sodium nitrite caused significant increases in the serum and cardiac cholesterol and triglyceride levels as well as significant decreases in phospholipids when compared with the control group (p b 0.05). Treatment with arjunolic acid daily for 12 weeks showed a significant, dose-dependent reduction in serum and cardiac cholesterol and triglycerides as well as a significant elevation of phospholipids when compared with the sodium nitrite group (p b 0.05). In addition, arjunolic acid showed no significant effects on the levels of lipids in the control rats. Arjunolic acid blocked sodium nitrite-induced enhancement in oxidative stress Oxidative stress has been associated with diverse pathophysiological events, including cancer, renal disease and cardiac damage (Hassan et al., 2010). We therefore measured the cardiac levels of MDA and reduced glutathione. Sodium nitrite showed significant increases in the serum and cardiac levels of MDA as well as significant decreases in the reduced glutathione concentrations compared with the control groups (p b 0.05). Treatment of rats with arjunolic acid showed a significant, dose-dependent reduction in the MDA levels and significant increases in the concentrations of reduced glutathione compared with the sodium nitrite group, but the control group was not affected (Table 4). Therefore, the cardio-protective effects of arjunolic acid can be partially attributed to its antioxidant activity. Arjunolic acid reversed sodium nitrite-induced deactivation of cardiac mitochondrial activity In the failing heart, the capacity of the mitochondria for oxygen consumption and oxidative phosphorylation is significantly reduced compared to the normal heart (Horn and Barrientos, 2008). As shown in Fig. 1, we found an approximately 40% reduction in the cardiac activity of cytochrome C oxidase in rats that received sodium nitrite when compared with control rats (p b 0.05). Treatment with 100 or 200 mg/kg arjunolic acid restored cytochrome C oxidase activity in the sodium nitrite group and did not affect the control group, illustrating the ability of arjunolic acid to restore the sodium nitrite-induced reduction in mitochondrial activity. Arjunolic acid reversed the sodium nitrite-induced elevation in cardiac inflammatory cytokines It appears that the heart failure syndrome is in large part due to an imbalance of pro- and anti-inflammatory mediators (Van Tassell et al., 2013). Regarding proinflammatory cytokines, as demonstrated in Fig. 2, we found a significant elevation in the cardiac concentrations of TNF-α (315.7 ± 27.4 pg/mg) and IL-1β (95.3 ± 8.3 pg/mg) in the sodium nitrite group compared with the control group (193.4 ± 18.4 and
Table 1 Effects of sodium nitrite (80 mg/kg/day) alone and in combination with arjunolic acid (100 and 200 mg/kg/day) for 12 weeks on heart function tests (mean ± SE).
AST (U/l) LDH (U/l) a b c
Control
Control + arjunolic acid (100 mg/kg)
Control + arjunolic acid (200 mg/kg)
Sodium nitrite (80 mg/kg)
Sodium nitrite + arjunolic acid (100 mg/kg)
Sodium nitrite + arjunolic acid (200 mg/kg)
29.43 ± 3.13 17.29 ± 1.39
30.11 ± 2.59 16.57 ± 1.61
29.11 ± 1.97 17.42 ± 2.11
51.3 ± 5.12a 38.8 ± 3.54a
42.18 ± 3.46a,b 25.17 ± 2.11a,b
36.47 ± 3.37b 18.7 ± 1.91b,c
Significant difference as compared with the control groups at p b 0.05. Significant difference as compared with sodium nitrite groups at p b 0.05. Significant difference as compared with arjunolic acid (100 mg/kg) group at p b 0.05.
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Table 2 Effects of sodium nitrite (80 mg/kg/day) alone and in combination with arjunolic acid (100 and 200 mg/kg/day) for 12 weeks on electrocardiogram (ECG) variables (mean ± SE). Control Heart rate/min ST segment elevation (mV) QT interval (msecond) T wave amplitude (mV) QRS complex amplitude (mV) a b c
343 0.091 72 0.34 1.6
± ± ± ± ±
11.3 0.0092 4.3 0.024 0.09
Control + arjunolic acid (100 mg/kg)
Control + arjunolic acid (200 mg/kg)
Sodium nitrite (80 mg/kg)
352 0.094 71.6 0.33 1.7
341 0.093 71.1 0.33 1.6
436 0.38 89.1 0.69 2.4
± ± ± ± ±
10.5 0.0087 3.1 0.03 0.1
± ± ± ± ±
11.4 0.0063 4.1 0.02 0.08
± ± ± ± ±
14.6a 0.021a 4.2a 0.039a 0.09a
Sodium nitrite + arjunolic acid (100 mg/kg) 412 0.29 79.8 0.56 2.02
± ± ± ± ±
12.1a 0.018a,b 2.7a,b 0.031a 0.12a,b
Sodium nitrite + arjunolic acid (200 mg/kg) 377 0.16 71 0.37 1.83
± ± ± ± ±
9.6a,b,c 0.0098a,b,c 3.4b,c 0.028b,c 0.06a,b
Significant difference as compared with the control groups at p b 0.05. Significant difference as compared with sodium nitrite groups at p b 0.05. Significant difference as compared with arjunolic acid (100 mg/kg) group at p b 0.05.
25.8 ± 2.3 pg/mg, respectively). The treatment of sodium nitrite rats with arjunolic acid showed a significant, dose-dependent reduction in the cardiac levels of both TNF-α and IL-1β compared with the sodium nitrite group, and it did not affect the control group. Arjunolic acid ameliorated the sodium nitrite-induced activation of cardiac acute inflammation markers We found a significant increase in the cardiac CRP level in the sodium nitrite group (18.6 ± 1.53 ng/mg) compared with the control group (7.9 ± 0.72 ng/mg). Rats in the sodium nitrite group treated with 100 or 200 mg/kg arjunolic acid showed a significant, dosedependent reduction in the cardiac levels of CRP (13.2 ± 1.29 and 8.2 ± 0.71 ng/mg) compared with the sodium nitrite group. Treatment with arjunolic acid did not affect the control group (7.7 ± 0.69 ng/mg, Fig. 3). Arjunolic acid elevated the sodium nitrite-induced inhibition of cardiac anti-inflammatory cytokines We found a significant decrease in the cardiac levels of IL-4 and IL-10 (6.3 ± 0.53 and 4.8 ± 0.45 pg/mg, respectively) in the sodium nitrite group compared with the control group (15.4 ± 1.43 and 9.94 ± 0.85 pg/mg, respectively). The treatment of sodium nitrite-treated rats with 100 of 200 mg/kg arjunolic acid showed a significant, dosedependent increase in the cardiac levels of IL-4 (7.9 ± 0.68 and 12.6 ± 1.17 pg/mg, respectively) and IL-10 (7.4 ± 0.69 and 8.6 ± 0.72 pg/g, respectively) compared with the sodium nitrite group, and it did not affect the control group (Fig. 4). Therefore, arjunolic acid has the ability to restore the normal balance between pro- and anti-inflammatory cytokines. Arjunolic acid inhibited the sodium nitrite-induced activation of the JNK MAPK pathway There has been accumulating evidence in both human and animal models suggesting that apoptosis may be an important mode of cell
death during heart failure (Kaminska, 2005). Therefore, we investigated the activation of the JNK MAPK apoptotic pathway in cardiac tissue. Statistical analysis of rat cardiac tissue showed a 3.2-fold elevation in the activation of JNK MAPK in the sodium nitrite group when compared with the control group. Treatment with arjunolic acid showed a dosedependent reduction in JNK MAPK in sodium nitrite-treated rats, but it did not affect control rats (Fig. 5). Arjunolic acid blocked sodium nitrite-induced expression of enzymes involved in cardiac apoptosis Next, we investigated the effect of both sodium nitrite and arjunolic acid on cardiac caspases. Sodium nitrite caused a significant increase in the cardiac activities of caspase-3, caspase-8 and caspase-9 (28.4 ± 2.1, 34.67 ± 2.91 and 47.71 ± 3.31 U/mg, respectively) when compared with the control group (11.2 ± 1.03, 20.42 ± 1.6 and 34.23 ± 2.91 U/mg, respectively). Treatment with 100 and 200 mg/kg arjunolic acid showed a significant, dose-dependent reduction in the cardiac activities of caspase-3 (22.7 ± 1.9 and 16.9 ± 1.5 U/mg, respectively), caspase-8 (30.12 ± 2.61 and 23.43 ± 2.1 U/mg, respectively) and caspase-9 (41.84 ± 2.79 and 35.18 ± 2.27 U/mg, respectively), but it did not affect the control group (Figs. 6a–c). In parallel, Western blot analysis revealed significant increases the in cardiac levels of cleaved caspase-3, -8 and -9 in the sodium nitrite group. The treatment of rats with arjunolic acid showed a significant reduction in the cardiac level of cleaved caspases, indicating the ability of arjunolic acid to ameliorate both the intrinsic and extrinsic apoptotic pathways (Fig. 6d). Discussion Natural and synthetic food additives are used to maintain or improve the safety, nutrient value, taste and texture of food. Sodium nitrite and other additives may react with amines of food in the stomach and produce nitrosamines and free radicals. Such products can be harmful to different organs (Choi et al., 2002). In addition, dietary intake of naturally occurring antioxidants may be the most sensible means to prevent biochemical alterations and disease risk factors associated with
Table 3 Effects of sodium nitrite (80 mg/kg/day) alone and in combination with arjunolic acid (100 and 200 mg/kg/day) for 12 weeks on serum and cardiac lipids (mean ± SE). Control
Serum cholesterol (mg/dl) Serum triglycerides (mg/dl) Serum phospholipids (mg/dl) Cardiac cholesterol (mg/g protein) Cardiac triglycerides (mg/g protein) Cardiac phospholipids (mg/g protein) a b c
148.1 98.3 114.3 72.15 44.79 17.84
± ± ± ± ± ±
11.3 7.4 9.4 7.59 4.19 1.75
Control + arjunolic acid (100 mg/kg)
Control + arjunolic acid (200 mg/kg)
Sodium nitrite (80 mg/kg)
146.8 99.1 117.8 70.11 45.84 18.33
145.1 97.2 115.6 70.43 46.32 18.93
239.1 148.1 84.1 103.65 83.42 10.87
± ± ± ± ± ±
12.9 8.7 10.7 6.95 3.96 1.72
Significant difference as compared with the control groups at p b 0.05. Significant difference as compared with sodium nitrite groups at p b 0.05. Significant difference as compared with arjunolic acid (100 mg/kg) group at p b 0.05
± ± ± ± ± ±
9.4 7.3 9.9 6.76 4.47 1.26
± ± ± ± ± ±
19.4a 11.7a 7.5a 6.76a 7.94a 0.94a
Sodium nitrite + arjunolic acid (100 mg/kg) 201.6 123.9 97.6 99.65 74.37 12.29
± ± ± ± ± ±
16.1a,b 11.6a,b 6.1a 6.13a 5.93a 1.17a
Sodium nitrite + arjunolic acid (200 mg/kg) 168.6 106.8 104.5 89.34 56.78 16.74
± ± ± ± ± ±
13.4b,c 8.3b,c 8.9a 7.92a,b 5.61a,b,c 1.56b,c
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Table 4 Effects of sodium nitrite (80 mg/kg/day) alone and in combination with arjunolic acid (100 and 200 mg/kg/day) for 12 weeks on levels of malondialdehyde (MDA) and reduced glutathione (mean ± SE). Control Serum MDA (nM) Blood glutathione (mM) Cardiac MDA (nmol/g protein) Cardiac glutathione (mmol/g protein) b c
± ± ± ±
11.9 0.52 12.9 1.17
Control + arjunolic acid (200 mg/kg)
Sodium nitrite (80 mg/kg)
183.9 5.91 130.8 12.13
181.1 5.82 136.9 11.98
384.4 3.81 346.73 7.12
± ± ± ±
9.8 0.68 12.3 1.13
± ± ± ±
11.2 0.51 11.6 1.01
± ± ± ±
24.1a 0.29a 28.5a 0.68a
Sodium nitrite + arjunolic acid (100 mg/kg) 263.1 5.11 218.7 9.98
± ± ± ±
18.1a,b 0.41b 18.2a,b 0.84b
Sodium nitrite + arjunolic acid (200 mg/kg) 201.8 5.98 151.4 11.73
± ± ± ±
19.4b,c 0.57b 14.9b,c 1.09b
Significant difference as compared with the control groups at p b 0.05. Significant difference as compared with sodium nitrite groups at p b 0.05. Significant difference as compared with arjunolic acid (100 mg/kg) group at p b 0.05.
free radical formation. Therefore, we aimed to determine whether arjunolic acid could inhibit sodium nitrite-induced cardiac inflammation. Our results clearly showed that administration of sodium nitrite alone for 12 weeks resulted in cardiac dysfunction, as indicated by a significant elevation in serum LDH and AST as well as of negative ECG variables compared to the control group. However, the free radical species produced by the exposure to nitrite is considered one of the most important causes of tissue destruction. Our finding is consistent with that of a previous study (Hassan et al., 2010). Our results revealed that the administration of arjunolic acid improved cardiac function markers. Arjunolic acid supplementation was previously reported to reduce the leakage of membrane-bound enzymes in diabetic-induced cardiac dysfunction rats (Trofimiuk and Braszko, 2011; Manna and Sil, 2012). Moreover, arjunolic acid protects cells and tissues from several toxicities, such as arsenic (Manna et al., 2007) and acetaminophen (Ghosh et al., 2010b). Lipids play an important role in cardiovascular diseases, not only by way of hyperlipidemia and the development of atherosclerosis but also by modifying the composition, structure and stability of the myocardium. We found that sodium nitrite worsened the lipid profile in rats when compared with the control groups. High levels of circulating cholesterol along with triglycerides and their accumulation in cardiac tissues is usually accompanied by cardiovascular damage (Gokkusu and Mostafazadeh, 2003). Sodium nitrite-induced oxidative stress primarily attacks polyunsaturated fatty acids, which are initiators of lipid peroxide formation that lead to impaired membrane function and structural integrity (Karthika et al., 2011). In addition, the reduction in cardiac phospholipids is associated with increased lipid peroxidation, which may occur due to the increased peroxidative degradation of phospholipids in the brains of sodium nitrite-intoxicated rats (Axelrad et al., 2002; Lau et al., 2006). However, we found that arjunolic acid
ameliorated the bad lipid profile in sodium nitrite-treated rats. Arjunolic acid has protective roles against cardiovascular complications in Type 1 diabetes mellitus (Manna and Sil, 2012), and the cardioprotective effect of arjunolic acid through the prevention of myocardial necrosis, platelet aggregation, coagulation, and lowering of the blood pressure and heart rate as well as the reduction of serum cholesterol levels was proven in experimental studies (Hemalatha et al., 2010). Regarding oxidative stress, we found significant increases in serum and cardiac MDA and a significant decrease in reduced glutathione in the sodium nitrite group. Our results agreed with those of a previous study (El-Sheikh and Khalil, 2011). The high MDA level may reflect the oxidative stress generated from sodium nitrite, which depleted the reduced glutathione, in different tissues (Shahjahan et al., 2005). The antioxidant effects of arjunolic acid are thought to be mainly attributed to its ability to scavenge oxygen-derived free radicals and to chelate metal ions (Hemalatha et al., 2010). Arjunolic acid protects cardiac
a Cardiac TNF-α (pg/mg protein)
a
179.3 6.18 137.4 11.63
Control + arjunolic acid (100 mg/kg)
400
*
350 300
#
250
150 100 50 0
1.2 1
*
0.8 0.6
*
0.4 0.2
b Cardiac IL-1β (pg/mg protein)
Relative cardiac cytochrome C oxidase level
# $
#
200
C
1.4
*
C+AA (100mg/kg)
C+AA (200mg/kg)
120
SN
*
100
SN+AA (100mg/kg)
* #
SN+AA (200mg/kg)
* # $
80 60 40 20 0 C
0 C
C+AA C+AA (100mg/kg) (200mg/kg)
SN
SN+AA SN+AA (100mg/kg) (200mg/kg)
Fig. 1. The effect of sodium nitrite (SN, 80 mg/kg/day) alone and in combination with arjunolic acid (AA, 100 and 200 mg/kg/day) for 12 weeks on cardiac cytochrome C oxidase activity. *Significant difference compared with the control groups at p b 0.05. # Significant difference compared with the sodium nitrite groups at p b 0.05. $Significant difference compared with the arjunolic acid (100 mg/kg) group at p b 0.05.
C+AA (100mg/kg)
C+AA (200mg/kg)
SN
SN+AA (100mg/kg)
SN+AA (200mg/kg)
Fig. 2. Effect of sodium nitrite (SN, 80 mg/kg/day) alone and in combination with arjunolic acid (AA, 100 and 200 mg/kg/day) for 12 weeks on cardiac tumor necrosis factor (TNF)-α (a) and interleukin (IL)-1β (b) levels. *Significant difference compared with the control groups at p b 0.05. #Significant difference compared with the sodium nitrite groups at p b 0.05. $Significant difference compared with the arjunolic acid (100 mg/kg) group at p b 0.05.
M.M.H. Al-Gayyar et al. / Life Sciences 111 (2014) 18–26 4
*
20
* #
15
10
# $
5
C+AA C+AA (100mg/kg) (200mg/kg)
SN
tissues from arsenic-induced organ dysfunction by maintaining the levels of reduced glutathione, MDA and other markers as those of the control group (Manna et al., 2007). Mitochondrial cytochrome C oxidase, a copper-containing metalloenzyme, is required for aerobic ATP production (Horn and Barrientos, 2008). We found a significant decrease in cardiac cytochrome C oxidase activity in rats that received sodium nitrite orally. However, we previously reported a significant decrease in hepatic cytochrome C oxidase activity in rats treated with oral sodium nitrite (Sherif and Al-Gayyar, 2013). In addition, the decrease in cytochrome C oxidase activity was previously linked with oxidative stress and apoptosis (Papadopoulou and Tsiftsoglou, 1996; You et al., 2002). Moreover, we
Cardiac IL-4 (pg/mg protein)
2.5
*
2
* # $
1.5 1 0.5
*
20
# $
18 16 14 12 10
*
8 6
*
4 2 0 C
C+AA C+AA (100mg/kg) (200mg/kg)
SN
12 10
SN+AA SN+AA (100mg/kg) (200mg/kg)
*
#
SN+AA (100mg/kg)
SN+AA (200mg/kg)
#
8
*
6 4 2 0 C
C+AA (100mg/kg)
C+AA (200mg/kg)
SN
C+AA (100mg/kg)
C+AA (200mg/kg)
SN
SN+AA (100mg/kg)
SN+AA (200mg/kg)
SN+AA SN+AA (100mg/kg) (200mg/kg)
Fig. 3. Effect of sodium nitrite (SN, 80 mg/kg/day) alone and in combination with arjunolic acid (AA, 100 and 200 mg/kg/day) for 12 weeks on the cardiac C reactive protein (CRP) level. *Significant difference compared with the control groups at p b 0.05. #Significant difference compared with the sodium nitrite groups at p b 0.05. $Significant difference compared with the arjunolic acid (100 mg/kg) group at p b 0.05.
Cardiac IL-10 (pg/mg protein)
3
C
C
b
*
3.5
0 0
a
Relative cardiac pJNK/JNK
Cardiac CRP (ng/mg protein)
25
23
Fig. 4. Effect of sodium nitrite (SN, 80 mg/kg/day) alone and in combination with arjunolic acid (AA, 100 and 200 mg/kg/day) for 12 weeks on cardiac IL-4 (a) and IL-10 (b) levels. *Significant difference compared with the control groups at p b 0.05. #Significant difference compared with the sodium nitrite groups at p b 0.05. $Significant difference compared with the arjunolic acid (100 mg/kg) group at p b 0.05.
Fig. 5. Effect of sodium nitrite (SN, 80 mg/kg/day) alone and in combination with arjunolic acid (AA, 100 and 200 mg/kg/day) for 12 weeks on cardiac pJNK/JNK MAPK. *Significant difference compared with the control groups at p b 0.05. #Significant difference compared with sodium nitrite groups at p b 0.05. $Significant difference compared with the arjunolic acid (100 mg/kg) group at p b 0.05.
found that treatment of rats with arjunolic acid restored the activity of cytochrome C oxidase. No previous study had reported the significance of using arjunolic acid to restore cytochrome C oxidase activity. We examined the effect of sodium nitrite on cardiac proinflammatory and anti-inflammatory cytokines. We observed significant elevations in the levels of cardiac inflammatory cytokines TNF-α and IL-1β and significant reductions in those of the cardiac anti-inflammatory cytokines IL-4 and IL-10 in the sodium nitrite group. This result may be attributed to the ability of sodium nitrite to initiate peroxidative damage to the cell as well as to activate pro-inflammatory cytokines (Mittal et al., 2006). Exposure to nitrosamines has been shown to increase lipid peroxidation and to alter the antioxidant status of experimental animals (Mittal et al., 2006, 2007). Therefore, pro-inflammatory cytokines may play a major role in the risk of cardiovascular events (Guan et al., 2012; Yu et al., 2012; Van Tassell et al., 2013). However, administration of arjunolic acid reversed the effects of sodium nitrite on pro- and anti-inflammatory cytokines. Similarly, arjunolic acid was reported to affect arachidonic acid metabolism by inhibiting cyclooxygenase (Hemalatha et al., 2010). Furthermore, treatment with arjunolic acid reduced vascular inflammation and prevented the activation of oxidative stress-induced signaling cascades that lead to cell death (Manna et al., 2010; Manna and Sil, 2012). MAPK cascades comprise one of the major signaling systems by which cells transduce and integrate diverse intracellular signals. Extracellular signal-regulated kinases (ERKs) are activated primarily in response to proliferation stimuli (Roux and Blenis, 2004). Although oxidative stress is the most notable JNK activator, JNK MAPK can also be activated by cytokines and adhesion molecules (Kaminska, 2005). We observed a significant increase in cardiac JNK activation that was associated with significant increases in inflammatory cytokines in the sodium nitrite group. However, treatment with arjunolic acid significantly reduced cardiac JNK activation. Arjunolic acid was reported to reduce JNK activation in doxorubicin-induced cardiovascular burden (Ghosh et al., 2011) and acetaminophen-induced hepatotoxicity (Ghosh et al., 2010b). We observed significant elevations in the cardiac activities of caspase-3, -8 and -9 that were associated with increased protein expression of the cleaved forms of these caspases after administration of sodium nitrite, and this effect was blocked by arjunolic acid. Caspase-8 plays a central role in the extrinsic cell death pathways involving transmembrane receptor-mediated interactions, whereas caspase-9 is an important component in the intrinsic cell death pathway (Shi et al., 2012). Extrinsic apoptosis is initiated by the binding of specific ligands such as TNF-α (Lee et al., 2012; Al-Gayyar and Elsherbiny, 2013). Once activated, caspase-8 is released into the cytoplasm and initiates downstream cleavage of caspase-3 (Lee et al., 2012; Broughton et al., 2009). Of note, we found a significant increase in cardiac TNF-α, which is
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Cardiac caspase-3 (U/mg protein)
a
Cardiac caspase-9 (U/mg protein)
c
*
35 30 25 20 15 10 5 0
*
60 50
* *
* #
# $
b Cardiac caspase-8 (U/mg protein)
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* *
40 35 30 25 20 15 10 5 0
# $
# $
40 30 20 10 0
d
C
C+AA 100
C+AA 200
1
0.9±0.1
0.9±.2
1
1.1±0.1
0.9±.1 3.6±0.5* 2.3±0.2*# 0.9±0.1#$
1
1.1±0.1
1.2±.1
Cleaved caspase-3 ROD
Cleaved caspase-8 ROD
Cleaved caspase-9 ROD
SN
SN+AA SN+AA 100 200
2.7±0.3* 1.9±0.3*# 1.1±0.1#$
2.1±0.2* 1.7±0.2* 0.9±0.1#$
Acn Fig. 6. Effect of sodium nitrite (SN, 80 mg/kg/day) alone and in combination with arjunolic acid (AA, 100 and 200 mg/kg/day) for 12 weeks on the cardiac activity of caspase-3 (a), caspase8 (b) and caspase-9 (c) as well as Western blot analysis of the cardiac levels of the cleaved caspases (d). *Significant difference compared with the control groups at p b 0.05. #Significant difference compared with the sodium nitrite groups at p b 0.05. $Significant difference compared with the arjunolic acid (100 mg/kg) group at p b 0.05.
associated with elevated caspase-8, in rats that received sodium nitrite. On the other hand, in response to mitochondrial stress, caspase-9 is activated, leading to cytochrome C release and caspase-3 activation, which are involved in various pathological conditions (Chwa et al., 2008). However, we observed an elevated MDA level associated with reduced cytochrome C oxidase that led to mitochondrial activation, enhancing the intrinsic cell death pathway. These findings strongly suggest that sodium nitrite mediates oxidative stress and contributes to oxidantinduced apoptotic cell death in cardiac cells. Many studies reported the anti-apoptotic effect of arjunolic acid through the reduction of cadmium-induced oxidative stress, attenuation of NFκB activation and inhibition of the apoptotic signaling pathways (Pal et al., 2011). In addition, arjunolic acid supplementation resulted in anti-apoptotic action in
hyperglycemia-induced cardiac pathophysiology through the suppression of a caspase-dependent pathway (Ghosh and Sil, 2013). Arjunolic acid was previously reported to block both extrinsic and intrinsic cell death in cadmium-induced hepatic damage (Pal et al., 2011) and acetaminophen-induced renal injury (Ghosh et al., 2010a). However, no previous study has reported the protective effects of arjunolic acid against cell death in sodium nitrite induced-cardiac dysfunction. Conclusion Arjunolic acid ameliorated the impairment of cardiac function in rats that ingested sodium nitrite via multiple mechanisms, such as the following: (1) reducing sodium nitrite-induced oxidative stress, as
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Sodium nitrite
Increase Cyto C oxidase
Increase oxidative stress Decrease IL-4 - IL10
Increase pJNK/JNK
Increase Caspase-9
Increase DNA frag
Arjunolic acid
Recruitment of inflammatory cells Increase TNF-α IL-1β Increase CRP
Increase Caspase-8 Cardiac apoptosis
Cardiac inflam
Fig. 7. Schematic representation of the mechanism of the cardiac protective action of arjunolic acid against sodium nitrite-induced cardiac damage.
indicated by a reduction in the serum and cardiac MDA levels and restoration of the levels of reduced glutathione; (2) blocking sodium nitriteinduced inhibition of cardiac cytochrome C oxidase; (3) blocking sodium nitrite-induced elevation of cardiac pro-inflammatory cytokines such as TNF-α and IL-1β; (4) blocking sodium nitrite-induced inhibition of the anti-inflammatory cytokines IL-4 and IL-10; (5) reducing sodium nitrite-induced activation of the JNK MAPK pathway; and (6) reducing sodium nitrite-induced activation of both extrinsic and intrinsic cell death pathways. The mechanism is summarized in Fig. 7. Although, both doses of arjunolic acid produced protective effects, the use of 200 mg/kg arjunolic acid produced better results. All changes in the tested parameters indicate that sodium nitrite induces cardiac injury that is similar to atherosclerosis. Conflicts of interest All authors declare no potential conflicts of interest, including any financial, personal or other relationships with other people or organizations within that could inappropriately influence, or be perceived to influence, this work.
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Protective effects of arjunolic acid against cardiac toxicity induced by oral sodium nitrite: Effects on cytokine balance and apoptosis Mohammed M.H. Al-Gayyar a,b,⁎, Abdullah Al Youssef c, Iman O. Sherif a, Mohamed E.E. Shams d, Ahmed Abbas e a
Dept. of Clinical Biochemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt Faculty of Pharmacy, University of Tabuk, Tabuk 71491, Saudi Arabia Dept. of Internal Medicine, Faculty of Medicine, University of Tabuk, Tabuk 71471, Saudi Arabia d Dept. of Pharmaceutics, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt e Dept. of Pharmacognosy, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt b c
a r t i c l e
i n f o
Article history: Received 24 March 2014 Accepted 7 July 2014 Available online 23 July 2014 Keywords: Caspase-3/8/9 Cytochrome C oxidase Interleukin (IL)-1β/4/10 JNK MAPK Tumor necrosis factor (TNF)-α
a b s t r a c t Aims: Sodium nitrite, a preservative used in meat products, helps in the production of free radicals, leading to increased lipid peroxidation, which plays a vital role in posing toxic effects in different body organs. On the other hand, arjunolic acid possesses antioxidant properties and plays protective roles against chemically induced organ pathophysiology. We investigated the effect of sodium nitrite on cardiac tissue in rats on the inflammatory cytokine balance and the type of induced apoptosis, and we analyzed the protective role of arjunolic acid. Main methods: Sixty adult male Sprague-Dawley rats were injected with 80 mg/kg sodium nitrite in the presence/absence of arjunolic acid (100 and 200 mg/kg). Cardiac pro-inflammatory cytokines (TNF-α and IL-1β), c-reactive protein (CRP) and anti-inflammatory cytokines (IL-4 and IL-10) were measured by ELISA. Cardiac mitochondrial activity (cytochrome-C-oxidase), JNK activation and apoptosis (caspase-3, caspase-8 and caspase-9) were assessed. Key findings: Sodium nitrite resulted in increased TNF-α (1.6-fold), IL-1β (3.7-fold) and CRP (2.4-fold) levels accompanied by 52%, 59% and 40% reductions in IL-10, IL-4 and cytochrome-C-oxidase, respectively, as well as enhanced JNK, caspase-3, caspase-8 and caspase-9 activities. Arjunolic acid markedly ameliorated these effects. Significance: Arjunolic acid attenuated sodium nitrite-induced cardiac damage in rats and restored the normal balance between pro- and anti-inflammatory cytokines. Moreover, arjunolic acid protected cardiac tissues from both extrinsic and intrinsic cell death pathways. © 2014 Elsevier Inc. All rights reserved.
Introduction Food additives are common and play an important role in our lives. Sodium nitrite is routinely used as a color fixative and preservative in meats and fish. The wide use of these food additives in food technology elevates the importance of studying their effects on mammals (Ezz ElArab et al., 2006). The hazardous effect of sodium nitrite is derived from the reaction of nitrite with amines and amides in food to produce nitrosamines and nitrosamides, respectively, which are considered to play vital roles in posing toxic effects in different organs (Tong et al., 2010; El-Sheikh and Khalil, 2011). Sodium nitrite was reported to cause cancer, hepatotoxicity, nephrotoxicity, dysregulation of inflammation and tissue injury (El-Sheikh and Khalil, 2011; Salama et al., 2013).
⁎ Corresponding author at: Faculty of Pharmacy, University of Tabuk, Saudi Arabia. Tel.: +966 50 8114998. E-mail address: [email protected] (M.M.H. Al-Gayyar).
http://dx.doi.org/10.1016/j.lfs.2014.07.002 0024-3205/© 2014 Elsevier Inc. All rights reserved.
Recent studies have shown that many compounds of herbal origin are able to reduce plasma triacylglycerol and total cholesterol levels and elevate the high-density lipoprotein concentration, thereby reducing the risk of cardiovascular diseases. The plant Terminalia arjuna (Combretaceae) has had a long history of medicinal uses, primarily in the preparation of ayurvedic formulations, for more than three centuries (Nammi et al., 2003). Arjunolic acid, a natural pentacyclic triterpenoid saponin, is one of the major constituents present in arjuna barks. Arjunolic acid has been shown to possess protective activities in various organ pathophysiologies (Manna et al., 2008; Manna and Sil, 2012; Ghosh and Sil, 2013; Rashid et al., 2013). The potent antioxidant property of arjunolic acid combined with its metal-chelating ability protects organs from metal- and drug-induced toxicity (Hemalatha et al., 2010; Ghosh and Sil, 2013). In addition, arjunolic acid acts as a prophylactic agent against oxidative cardiac damage induced by arsenic (Manna et al., 2008), isoproterenol (Sumitra et al., 2001) and doxorubicin (Ghosh et al., 2011). We previously published two studies illustrating the effect of sodium nitrite on hepatic dysfunction and focused on oxidative stress,
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inflammation, fibrosis and apoptosis (Salama et al., 2013; Sherif and Al-Gayyar, 2013). Less attention has been paid to measuring the cardiac toxicity effects of sodium nitrite. Therefore, this study is designed to investigate the impact of sodium nitrite on cardiac tissue in rats by measuring its effect on the balance between pro- and anti-inflammatory cytokines and the type of induced apoptosis. In addition, we aimed to investigate the cardiac protective effects of arjunolic acid.
19
Measuring heart function The serum aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels were measured by standard methodologies using commercially available kits provided by Diamond Company (Egypt). Assessment of oxidative stress Oxidative stress was estimated through the following parameters:
Materials & methods Animals and their treatment outlines The animal protocol was approved by the ethical committee of the Faculty of Pharmacy, University of Mansoura. Male Sprague-Dawley rats weighing 180–200 g were used. All animals were maintained under standard conditions, and all treatments were given via oral gavage. Rats were classified into the following groups with 10 rats in each group: Control group. Rats received the standard diet without any treatment. Arjunolic acid-treated control group (100 mg/kg). Rats received a daily standard diet that was supplemented orally with 100 mg/kg arjunolic acid (Hangzhou Dayangchem, Zhejiang, China) daily for 12 weeks. Arjunolic acid-treated control group (200 mg/kg). Rats received a daily standard diet that was supplemented orally with 200 mg/kg arjunolic acid daily for 12 weeks. Sodium nitrite group. Rats were orally given 80 mg/kg sodium nitrite (Sigma-Aldrich, St. Louis, MO, USA) daily for 12 weeks. Arjunolic acid (100 mg/kg)-treated group. Rats supplemented orally with 100 mg/kg arjunolic acid followed by 80 mg/kg sodium nitrite daily for 12 weeks. Arjunolic acid (200 mg/kg)-treated group. Rats supplemented orally with 200 mg/kg arjunolic acid followed by 80 mg/kg sodium nitrite daily for 12 weeks. The doses and time courses of the experiments that were used for sodium nitrite and arjunolic acid were in the range of those used in other studies and applied for the same animal species (Yin et al., 2012; Salama et al., 2013; Sherif and Al-Gayyar, 2013).
Serum and cardiac malondialdhyde (MDA) concentrations were measured as described previously (Al-Gayyar et al., 2007; Shams et al., 2011). Briefly, after precipitation of the proteins by trichloroacetic acid, thiobarbituric acid was reacted with MDA to form thiobarbituric acid-reactive substances, which were measured at 532 nm. Blood and cardiac reduced glutathione (GSH) concentrations were measured as described previously (Beutler et al., 1963). This assay depended on the reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) by glutathione, which formed a highly colored, yellow anion that could be measured at 412 nm. Assessment of lipid contents Serum and cardiac homogenates were used for cholesterol, triglyceride and phospholipid measurements by standard methodologies using commercially available kits provided by Biodiagnostic Company (Egypt). ELISA determination The levels of biochemical parameters in the cardiac homogenates were measured by ELISA assays using commercially available tumor necrosis factor (TNF)-α, c-reactive protein (CRP), interleukin (IL)-1β, IL-4 and IL-10 ELISA kits (eBioscience Inc., San Diego, CA, USA) in accordance with the manufacturer's instructions. Western blot analysis
The animals were anesthetized by intra-peritoneal injection of (30 mg/kg) thiopental sodium. Needle electrodes were inserted under the skin of the four limbs, and electrocardiograms were recorded from standard lead II limb leads using a single-channel ECG (Fukuda ME Kogyo Co. Ltd., Model: 501-B ΙΙΙ, Tokyo, Japan). The electrocardiograph was standardized before each tracing to obtain sensitivity (2 mV pulse produces 20 mm height) and chart (paper) speed (50 mm/s).
Cardiac tissues were homogenized in RIPA buffer (Millipore, Temecula, CA, USA), and the total amount of protein was determined by protein assays (Bio-Rad Laboratories Inc., Hercules, CA, USA). Samples (30 μg protein) were separated by SDS-PAGE and electroblotted onto nitrocellulose membranes, which were blocked in PBS, 0.02% Tween 20 (PBST) containing 5% non-fat milk. Antibodies for cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 (Cell Signaling Technology, Inc., Danvers, MA) were used in 1:500 dilutions in PBST containing 5% non-fat milk and applied overnight at 4 °C. The membranes were reprobed with 1:2000 β-actin (Sigma-Aldrich) in PBST containing 5% non-fat milk for 2 h to confirm equal loading. Primary antibody was detected using 1:5000 dilutions of horseradish peroxidase-conjugated sheep anti-mouse antibodies in PBST and enhanced chemiluminescence (GE Healthcare, Piscataway, NJ, USA).
Animal sacrifice and collection of samples
Measurement of cell activation of the pro-apoptotic pJNK/JNK MAPK pathway
Electrocardiogram (ECG)
The animals were sacrificed by decapitation. Rat trunk blood was collected and centrifuged at 3000 rpm for 5 min, and serum samples were separated and stored at − 80 °C. Rat hearts were removed, cleaned with ice-cold saline, weighed and chilled on crushed ice. A piece of the heart was homogenized in a 10-fold volume of ice-cold sodium potassium phosphate buffer (0.01 M, pH 7.4) containing 1.15% KCl. The homogenates were centrifuged at 600 g at 4 °C for 10 min, and the supernatant was stored at −80 °C until use.
Renal tissue activation of JNK MAPK, as measured as the ratio of pJNK/JNK, was determined using a commercially available ELISA kit from RayBio® Company. Determination of cardiac mitochondrial function Cardiac mitochondrial function was measured via determining the cytochrome C oxidase activity using a commercially available kit
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(Sigma-Aldrich) and was based on observing the decrease in absorbance at 550 nm of ferrocytochrome C, which is caused by its oxidation to ferricytochrome C by cytochrome C oxidase. Estimation of the activities of caspase-3, caspase-8 and caspase-9 Caspase-3, caspase-8 and caspase-9 enzyme activities were measured colorimetrically using commercially available kits (GenScript, Piscataway, NJ, USA) following the manufacturer's procedure. Statistical analysis For descriptive statistics of quantitative variables, the mean ± standard error was used. Normality of the sample distribution of each continuous variable was tested with the Kolmogorov–Smirnov (K–S) test. One-way analysis of variance (ANOVA) was used to compare the means between groups. Once differences between the means were found, post hoc Bonferroni correction tests were calculated. Statistical computations were performed on a personal computer using Microsoft Excel 2007. Statistical significance was predefined as P ≤ 0.05. Results Arjunolic acid reversed the cardiac biomarker changes induced by sodium nitrite Administration of sodium nitrite resulted in marked cellular, molecular and biochemical changes that included fibrosis, mitochondrial function impairment, inflammation and DNA degradation (Hassan et al., 2010; Salama et al., 2013; Sherif and Al-Gayyar, 2013). The serum levels of lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) were used as indicators for cardiac function. As shown in Table 1, sodium nitrite caused significant increases in the serum LDH and AST levels compared with the control groups (p b 0.05). Treatment with arjunolic acid showed a significant, dose-dependent reduction in the LDH and AST levels compared with the sodium nitrite group (p b 0.05) and did not affect the control rats. Arjunolic acid improved sodium nitrite-induced changes in ECG variables As shown in Table 2, examination of ECG from sodium nitriteadministered rats revealed significant and serious changes. There was a significant increase in the heart rate, ST segment elevation, QT interval, T-wave amplitude and QRS amplitude in comparison to the normal control group. Abnormal T-waves and QRS complexes were also observed. Based on the aforementioned changes, it appeared that sodium nitrite significantly impaired myocardial contractility with severe conducting tissue impairment in comparison to the normal control group. The treatment of rats with arjunolic acid reduced the heart rate and ameliorated the deleterious effects observed due to sodium nitrite on the heart. Therefore, according to the heart function and ECG results, arjunolic acid exerts protective effects against sodium nitrite-induced cardiac damage.
Arjunolic acid ameliorated the bad lipid profile induced by sodium nitrite The correlation between the lipid profile and changes in heart markers is well established (Gokkusu and Mostafazadeh, 2003). As shown in Table 3, sodium nitrite caused significant increases in the serum and cardiac cholesterol and triglyceride levels as well as significant decreases in phospholipids when compared with the control group (p b 0.05). Treatment with arjunolic acid daily for 12 weeks showed a significant, dose-dependent reduction in serum and cardiac cholesterol and triglycerides as well as a significant elevation of phospholipids when compared with the sodium nitrite group (p b 0.05). In addition, arjunolic acid showed no significant effects on the levels of lipids in the control rats. Arjunolic acid blocked sodium nitrite-induced enhancement in oxidative stress Oxidative stress has been associated with diverse pathophysiological events, including cancer, renal disease and cardiac damage (Hassan et al., 2010). We therefore measured the cardiac levels of MDA and reduced glutathione. Sodium nitrite showed significant increases in the serum and cardiac levels of MDA as well as significant decreases in the reduced glutathione concentrations compared with the control groups (p b 0.05). Treatment of rats with arjunolic acid showed a significant, dose-dependent reduction in the MDA levels and significant increases in the concentrations of reduced glutathione compared with the sodium nitrite group, but the control group was not affected (Table 4). Therefore, the cardio-protective effects of arjunolic acid can be partially attributed to its antioxidant activity. Arjunolic acid reversed sodium nitrite-induced deactivation of cardiac mitochondrial activity In the failing heart, the capacity of the mitochondria for oxygen consumption and oxidative phosphorylation is significantly reduced compared to the normal heart (Horn and Barrientos, 2008). As shown in Fig. 1, we found an approximately 40% reduction in the cardiac activity of cytochrome C oxidase in rats that received sodium nitrite when compared with control rats (p b 0.05). Treatment with 100 or 200 mg/kg arjunolic acid restored cytochrome C oxidase activity in the sodium nitrite group and did not affect the control group, illustrating the ability of arjunolic acid to restore the sodium nitrite-induced reduction in mitochondrial activity. Arjunolic acid reversed the sodium nitrite-induced elevation in cardiac inflammatory cytokines It appears that the heart failure syndrome is in large part due to an imbalance of pro- and anti-inflammatory mediators (Van Tassell et al., 2013). Regarding proinflammatory cytokines, as demonstrated in Fig. 2, we found a significant elevation in the cardiac concentrations of TNF-α (315.7 ± 27.4 pg/mg) and IL-1β (95.3 ± 8.3 pg/mg) in the sodium nitrite group compared with the control group (193.4 ± 18.4 and
Table 1 Effects of sodium nitrite (80 mg/kg/day) alone and in combination with arjunolic acid (100 and 200 mg/kg/day) for 12 weeks on heart function tests (mean ± SE).
AST (U/l) LDH (U/l) a b c
Control
Control + arjunolic acid (100 mg/kg)
Control + arjunolic acid (200 mg/kg)
Sodium nitrite (80 mg/kg)
Sodium nitrite + arjunolic acid (100 mg/kg)
Sodium nitrite + arjunolic acid (200 mg/kg)
29.43 ± 3.13 17.29 ± 1.39
30.11 ± 2.59 16.57 ± 1.61
29.11 ± 1.97 17.42 ± 2.11
51.3 ± 5.12a 38.8 ± 3.54a
42.18 ± 3.46a,b 25.17 ± 2.11a,b
36.47 ± 3.37b 18.7 ± 1.91b,c
Significant difference as compared with the control groups at p b 0.05. Significant difference as compared with sodium nitrite groups at p b 0.05. Significant difference as compared with arjunolic acid (100 mg/kg) group at p b 0.05.
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Table 2 Effects of sodium nitrite (80 mg/kg/day) alone and in combination with arjunolic acid (100 and 200 mg/kg/day) for 12 weeks on electrocardiogram (ECG) variables (mean ± SE). Control Heart rate/min ST segment elevation (mV) QT interval (msecond) T wave amplitude (mV) QRS complex amplitude (mV) a b c
343 0.091 72 0.34 1.6
± ± ± ± ±
11.3 0.0092 4.3 0.024 0.09
Control + arjunolic acid (100 mg/kg)
Control + arjunolic acid (200 mg/kg)
Sodium nitrite (80 mg/kg)
352 0.094 71.6 0.33 1.7
341 0.093 71.1 0.33 1.6
436 0.38 89.1 0.69 2.4
± ± ± ± ±
10.5 0.0087 3.1 0.03 0.1
± ± ± ± ±
11.4 0.0063 4.1 0.02 0.08
± ± ± ± ±
14.6a 0.021a 4.2a 0.039a 0.09a
Sodium nitrite + arjunolic acid (100 mg/kg) 412 0.29 79.8 0.56 2.02
± ± ± ± ±
12.1a 0.018a,b 2.7a,b 0.031a 0.12a,b
Sodium nitrite + arjunolic acid (200 mg/kg) 377 0.16 71 0.37 1.83
± ± ± ± ±
9.6a,b,c 0.0098a,b,c 3.4b,c 0.028b,c 0.06a,b
Significant difference as compared with the control groups at p b 0.05. Significant difference as compared with sodium nitrite groups at p b 0.05. Significant difference as compared with arjunolic acid (100 mg/kg) group at p b 0.05.
25.8 ± 2.3 pg/mg, respectively). The treatment of sodium nitrite rats with arjunolic acid showed a significant, dose-dependent reduction in the cardiac levels of both TNF-α and IL-1β compared with the sodium nitrite group, and it did not affect the control group. Arjunolic acid ameliorated the sodium nitrite-induced activation of cardiac acute inflammation markers We found a significant increase in the cardiac CRP level in the sodium nitrite group (18.6 ± 1.53 ng/mg) compared with the control group (7.9 ± 0.72 ng/mg). Rats in the sodium nitrite group treated with 100 or 200 mg/kg arjunolic acid showed a significant, dosedependent reduction in the cardiac levels of CRP (13.2 ± 1.29 and 8.2 ± 0.71 ng/mg) compared with the sodium nitrite group. Treatment with arjunolic acid did not affect the control group (7.7 ± 0.69 ng/mg, Fig. 3). Arjunolic acid elevated the sodium nitrite-induced inhibition of cardiac anti-inflammatory cytokines We found a significant decrease in the cardiac levels of IL-4 and IL-10 (6.3 ± 0.53 and 4.8 ± 0.45 pg/mg, respectively) in the sodium nitrite group compared with the control group (15.4 ± 1.43 and 9.94 ± 0.85 pg/mg, respectively). The treatment of sodium nitrite-treated rats with 100 of 200 mg/kg arjunolic acid showed a significant, dosedependent increase in the cardiac levels of IL-4 (7.9 ± 0.68 and 12.6 ± 1.17 pg/mg, respectively) and IL-10 (7.4 ± 0.69 and 8.6 ± 0.72 pg/g, respectively) compared with the sodium nitrite group, and it did not affect the control group (Fig. 4). Therefore, arjunolic acid has the ability to restore the normal balance between pro- and anti-inflammatory cytokines. Arjunolic acid inhibited the sodium nitrite-induced activation of the JNK MAPK pathway There has been accumulating evidence in both human and animal models suggesting that apoptosis may be an important mode of cell
death during heart failure (Kaminska, 2005). Therefore, we investigated the activation of the JNK MAPK apoptotic pathway in cardiac tissue. Statistical analysis of rat cardiac tissue showed a 3.2-fold elevation in the activation of JNK MAPK in the sodium nitrite group when compared with the control group. Treatment with arjunolic acid showed a dosedependent reduction in JNK MAPK in sodium nitrite-treated rats, but it did not affect control rats (Fig. 5). Arjunolic acid blocked sodium nitrite-induced expression of enzymes involved in cardiac apoptosis Next, we investigated the effect of both sodium nitrite and arjunolic acid on cardiac caspases. Sodium nitrite caused a significant increase in the cardiac activities of caspase-3, caspase-8 and caspase-9 (28.4 ± 2.1, 34.67 ± 2.91 and 47.71 ± 3.31 U/mg, respectively) when compared with the control group (11.2 ± 1.03, 20.42 ± 1.6 and 34.23 ± 2.91 U/mg, respectively). Treatment with 100 and 200 mg/kg arjunolic acid showed a significant, dose-dependent reduction in the cardiac activities of caspase-3 (22.7 ± 1.9 and 16.9 ± 1.5 U/mg, respectively), caspase-8 (30.12 ± 2.61 and 23.43 ± 2.1 U/mg, respectively) and caspase-9 (41.84 ± 2.79 and 35.18 ± 2.27 U/mg, respectively), but it did not affect the control group (Figs. 6a–c). In parallel, Western blot analysis revealed significant increases the in cardiac levels of cleaved caspase-3, -8 and -9 in the sodium nitrite group. The treatment of rats with arjunolic acid showed a significant reduction in the cardiac level of cleaved caspases, indicating the ability of arjunolic acid to ameliorate both the intrinsic and extrinsic apoptotic pathways (Fig. 6d). Discussion Natural and synthetic food additives are used to maintain or improve the safety, nutrient value, taste and texture of food. Sodium nitrite and other additives may react with amines of food in the stomach and produce nitrosamines and free radicals. Such products can be harmful to different organs (Choi et al., 2002). In addition, dietary intake of naturally occurring antioxidants may be the most sensible means to prevent biochemical alterations and disease risk factors associated with
Table 3 Effects of sodium nitrite (80 mg/kg/day) alone and in combination with arjunolic acid (100 and 200 mg/kg/day) for 12 weeks on serum and cardiac lipids (mean ± SE). Control
Serum cholesterol (mg/dl) Serum triglycerides (mg/dl) Serum phospholipids (mg/dl) Cardiac cholesterol (mg/g protein) Cardiac triglycerides (mg/g protein) Cardiac phospholipids (mg/g protein) a b c
148.1 98.3 114.3 72.15 44.79 17.84
± ± ± ± ± ±
11.3 7.4 9.4 7.59 4.19 1.75
Control + arjunolic acid (100 mg/kg)
Control + arjunolic acid (200 mg/kg)
Sodium nitrite (80 mg/kg)
146.8 99.1 117.8 70.11 45.84 18.33
145.1 97.2 115.6 70.43 46.32 18.93
239.1 148.1 84.1 103.65 83.42 10.87
± ± ± ± ± ±
12.9 8.7 10.7 6.95 3.96 1.72
Significant difference as compared with the control groups at p b 0.05. Significant difference as compared with sodium nitrite groups at p b 0.05. Significant difference as compared with arjunolic acid (100 mg/kg) group at p b 0.05
± ± ± ± ± ±
9.4 7.3 9.9 6.76 4.47 1.26
± ± ± ± ± ±
19.4a 11.7a 7.5a 6.76a 7.94a 0.94a
Sodium nitrite + arjunolic acid (100 mg/kg) 201.6 123.9 97.6 99.65 74.37 12.29
± ± ± ± ± ±
16.1a,b 11.6a,b 6.1a 6.13a 5.93a 1.17a
Sodium nitrite + arjunolic acid (200 mg/kg) 168.6 106.8 104.5 89.34 56.78 16.74
± ± ± ± ± ±
13.4b,c 8.3b,c 8.9a 7.92a,b 5.61a,b,c 1.56b,c
22
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Table 4 Effects of sodium nitrite (80 mg/kg/day) alone and in combination with arjunolic acid (100 and 200 mg/kg/day) for 12 weeks on levels of malondialdehyde (MDA) and reduced glutathione (mean ± SE). Control Serum MDA (nM) Blood glutathione (mM) Cardiac MDA (nmol/g protein) Cardiac glutathione (mmol/g protein) b c
± ± ± ±
11.9 0.52 12.9 1.17
Control + arjunolic acid (200 mg/kg)
Sodium nitrite (80 mg/kg)
183.9 5.91 130.8 12.13
181.1 5.82 136.9 11.98
384.4 3.81 346.73 7.12
± ± ± ±
9.8 0.68 12.3 1.13
± ± ± ±
11.2 0.51 11.6 1.01
± ± ± ±
24.1a 0.29a 28.5a 0.68a
Sodium nitrite + arjunolic acid (100 mg/kg) 263.1 5.11 218.7 9.98
± ± ± ±
18.1a,b 0.41b 18.2a,b 0.84b
Sodium nitrite + arjunolic acid (200 mg/kg) 201.8 5.98 151.4 11.73
± ± ± ±
19.4b,c 0.57b 14.9b,c 1.09b
Significant difference as compared with the control groups at p b 0.05. Significant difference as compared with sodium nitrite groups at p b 0.05. Significant difference as compared with arjunolic acid (100 mg/kg) group at p b 0.05.
free radical formation. Therefore, we aimed to determine whether arjunolic acid could inhibit sodium nitrite-induced cardiac inflammation. Our results clearly showed that administration of sodium nitrite alone for 12 weeks resulted in cardiac dysfunction, as indicated by a significant elevation in serum LDH and AST as well as of negative ECG variables compared to the control group. However, the free radical species produced by the exposure to nitrite is considered one of the most important causes of tissue destruction. Our finding is consistent with that of a previous study (Hassan et al., 2010). Our results revealed that the administration of arjunolic acid improved cardiac function markers. Arjunolic acid supplementation was previously reported to reduce the leakage of membrane-bound enzymes in diabetic-induced cardiac dysfunction rats (Trofimiuk and Braszko, 2011; Manna and Sil, 2012). Moreover, arjunolic acid protects cells and tissues from several toxicities, such as arsenic (Manna et al., 2007) and acetaminophen (Ghosh et al., 2010b). Lipids play an important role in cardiovascular diseases, not only by way of hyperlipidemia and the development of atherosclerosis but also by modifying the composition, structure and stability of the myocardium. We found that sodium nitrite worsened the lipid profile in rats when compared with the control groups. High levels of circulating cholesterol along with triglycerides and their accumulation in cardiac tissues is usually accompanied by cardiovascular damage (Gokkusu and Mostafazadeh, 2003). Sodium nitrite-induced oxidative stress primarily attacks polyunsaturated fatty acids, which are initiators of lipid peroxide formation that lead to impaired membrane function and structural integrity (Karthika et al., 2011). In addition, the reduction in cardiac phospholipids is associated with increased lipid peroxidation, which may occur due to the increased peroxidative degradation of phospholipids in the brains of sodium nitrite-intoxicated rats (Axelrad et al., 2002; Lau et al., 2006). However, we found that arjunolic acid
ameliorated the bad lipid profile in sodium nitrite-treated rats. Arjunolic acid has protective roles against cardiovascular complications in Type 1 diabetes mellitus (Manna and Sil, 2012), and the cardioprotective effect of arjunolic acid through the prevention of myocardial necrosis, platelet aggregation, coagulation, and lowering of the blood pressure and heart rate as well as the reduction of serum cholesterol levels was proven in experimental studies (Hemalatha et al., 2010). Regarding oxidative stress, we found significant increases in serum and cardiac MDA and a significant decrease in reduced glutathione in the sodium nitrite group. Our results agreed with those of a previous study (El-Sheikh and Khalil, 2011). The high MDA level may reflect the oxidative stress generated from sodium nitrite, which depleted the reduced glutathione, in different tissues (Shahjahan et al., 2005). The antioxidant effects of arjunolic acid are thought to be mainly attributed to its ability to scavenge oxygen-derived free radicals and to chelate metal ions (Hemalatha et al., 2010). Arjunolic acid protects cardiac
a Cardiac TNF-α (pg/mg protein)
a
179.3 6.18 137.4 11.63
Control + arjunolic acid (100 mg/kg)
400
*
350 300
#
250
150 100 50 0
1.2 1
*
0.8 0.6
*
0.4 0.2
b Cardiac IL-1β (pg/mg protein)
Relative cardiac cytochrome C oxidase level
# $
#
200
C
1.4
*
C+AA (100mg/kg)
C+AA (200mg/kg)
120
SN
*
100
SN+AA (100mg/kg)
* #
SN+AA (200mg/kg)
* # $
80 60 40 20 0 C
0 C
C+AA C+AA (100mg/kg) (200mg/kg)
SN
SN+AA SN+AA (100mg/kg) (200mg/kg)
Fig. 1. The effect of sodium nitrite (SN, 80 mg/kg/day) alone and in combination with arjunolic acid (AA, 100 and 200 mg/kg/day) for 12 weeks on cardiac cytochrome C oxidase activity. *Significant difference compared with the control groups at p b 0.05. # Significant difference compared with the sodium nitrite groups at p b 0.05. $Significant difference compared with the arjunolic acid (100 mg/kg) group at p b 0.05.
C+AA (100mg/kg)
C+AA (200mg/kg)
SN
SN+AA (100mg/kg)
SN+AA (200mg/kg)
Fig. 2. Effect of sodium nitrite (SN, 80 mg/kg/day) alone and in combination with arjunolic acid (AA, 100 and 200 mg/kg/day) for 12 weeks on cardiac tumor necrosis factor (TNF)-α (a) and interleukin (IL)-1β (b) levels. *Significant difference compared with the control groups at p b 0.05. #Significant difference compared with the sodium nitrite groups at p b 0.05. $Significant difference compared with the arjunolic acid (100 mg/kg) group at p b 0.05.
M.M.H. Al-Gayyar et al. / Life Sciences 111 (2014) 18–26 4
*
20
* #
15
10
# $
5
C+AA C+AA (100mg/kg) (200mg/kg)
SN
tissues from arsenic-induced organ dysfunction by maintaining the levels of reduced glutathione, MDA and other markers as those of the control group (Manna et al., 2007). Mitochondrial cytochrome C oxidase, a copper-containing metalloenzyme, is required for aerobic ATP production (Horn and Barrientos, 2008). We found a significant decrease in cardiac cytochrome C oxidase activity in rats that received sodium nitrite orally. However, we previously reported a significant decrease in hepatic cytochrome C oxidase activity in rats treated with oral sodium nitrite (Sherif and Al-Gayyar, 2013). In addition, the decrease in cytochrome C oxidase activity was previously linked with oxidative stress and apoptosis (Papadopoulou and Tsiftsoglou, 1996; You et al., 2002). Moreover, we
Cardiac IL-4 (pg/mg protein)
2.5
*
2
* # $
1.5 1 0.5
*
20
# $
18 16 14 12 10
*
8 6
*
4 2 0 C
C+AA C+AA (100mg/kg) (200mg/kg)
SN
12 10
SN+AA SN+AA (100mg/kg) (200mg/kg)
*
#
SN+AA (100mg/kg)
SN+AA (200mg/kg)
#
8
*
6 4 2 0 C
C+AA (100mg/kg)
C+AA (200mg/kg)
SN
C+AA (100mg/kg)
C+AA (200mg/kg)
SN
SN+AA (100mg/kg)
SN+AA (200mg/kg)
SN+AA SN+AA (100mg/kg) (200mg/kg)
Fig. 3. Effect of sodium nitrite (SN, 80 mg/kg/day) alone and in combination with arjunolic acid (AA, 100 and 200 mg/kg/day) for 12 weeks on the cardiac C reactive protein (CRP) level. *Significant difference compared with the control groups at p b 0.05. #Significant difference compared with the sodium nitrite groups at p b 0.05. $Significant difference compared with the arjunolic acid (100 mg/kg) group at p b 0.05.
Cardiac IL-10 (pg/mg protein)
3
C
C
b
*
3.5
0 0
a
Relative cardiac pJNK/JNK
Cardiac CRP (ng/mg protein)
25
23
Fig. 4. Effect of sodium nitrite (SN, 80 mg/kg/day) alone and in combination with arjunolic acid (AA, 100 and 200 mg/kg/day) for 12 weeks on cardiac IL-4 (a) and IL-10 (b) levels. *Significant difference compared with the control groups at p b 0.05. #Significant difference compared with the sodium nitrite groups at p b 0.05. $Significant difference compared with the arjunolic acid (100 mg/kg) group at p b 0.05.
Fig. 5. Effect of sodium nitrite (SN, 80 mg/kg/day) alone and in combination with arjunolic acid (AA, 100 and 200 mg/kg/day) for 12 weeks on cardiac pJNK/JNK MAPK. *Significant difference compared with the control groups at p b 0.05. #Significant difference compared with sodium nitrite groups at p b 0.05. $Significant difference compared with the arjunolic acid (100 mg/kg) group at p b 0.05.
found that treatment of rats with arjunolic acid restored the activity of cytochrome C oxidase. No previous study had reported the significance of using arjunolic acid to restore cytochrome C oxidase activity. We examined the effect of sodium nitrite on cardiac proinflammatory and anti-inflammatory cytokines. We observed significant elevations in the levels of cardiac inflammatory cytokines TNF-α and IL-1β and significant reductions in those of the cardiac anti-inflammatory cytokines IL-4 and IL-10 in the sodium nitrite group. This result may be attributed to the ability of sodium nitrite to initiate peroxidative damage to the cell as well as to activate pro-inflammatory cytokines (Mittal et al., 2006). Exposure to nitrosamines has been shown to increase lipid peroxidation and to alter the antioxidant status of experimental animals (Mittal et al., 2006, 2007). Therefore, pro-inflammatory cytokines may play a major role in the risk of cardiovascular events (Guan et al., 2012; Yu et al., 2012; Van Tassell et al., 2013). However, administration of arjunolic acid reversed the effects of sodium nitrite on pro- and anti-inflammatory cytokines. Similarly, arjunolic acid was reported to affect arachidonic acid metabolism by inhibiting cyclooxygenase (Hemalatha et al., 2010). Furthermore, treatment with arjunolic acid reduced vascular inflammation and prevented the activation of oxidative stress-induced signaling cascades that lead to cell death (Manna et al., 2010; Manna and Sil, 2012). MAPK cascades comprise one of the major signaling systems by which cells transduce and integrate diverse intracellular signals. Extracellular signal-regulated kinases (ERKs) are activated primarily in response to proliferation stimuli (Roux and Blenis, 2004). Although oxidative stress is the most notable JNK activator, JNK MAPK can also be activated by cytokines and adhesion molecules (Kaminska, 2005). We observed a significant increase in cardiac JNK activation that was associated with significant increases in inflammatory cytokines in the sodium nitrite group. However, treatment with arjunolic acid significantly reduced cardiac JNK activation. Arjunolic acid was reported to reduce JNK activation in doxorubicin-induced cardiovascular burden (Ghosh et al., 2011) and acetaminophen-induced hepatotoxicity (Ghosh et al., 2010b). We observed significant elevations in the cardiac activities of caspase-3, -8 and -9 that were associated with increased protein expression of the cleaved forms of these caspases after administration of sodium nitrite, and this effect was blocked by arjunolic acid. Caspase-8 plays a central role in the extrinsic cell death pathways involving transmembrane receptor-mediated interactions, whereas caspase-9 is an important component in the intrinsic cell death pathway (Shi et al., 2012). Extrinsic apoptosis is initiated by the binding of specific ligands such as TNF-α (Lee et al., 2012; Al-Gayyar and Elsherbiny, 2013). Once activated, caspase-8 is released into the cytoplasm and initiates downstream cleavage of caspase-3 (Lee et al., 2012; Broughton et al., 2009). Of note, we found a significant increase in cardiac TNF-α, which is
M.M.H. Al-Gayyar et al. / Life Sciences 111 (2014) 18–26
Cardiac caspase-3 (U/mg protein)
a
Cardiac caspase-9 (U/mg protein)
c
*
35 30 25 20 15 10 5 0
*
60 50
* *
* #
# $
b Cardiac caspase-8 (U/mg protein)
24
* *
40 35 30 25 20 15 10 5 0
# $
# $
40 30 20 10 0
d
C
C+AA 100
C+AA 200
1
0.9±0.1
0.9±.2
1
1.1±0.1
0.9±.1 3.6±0.5* 2.3±0.2*# 0.9±0.1#$
1
1.1±0.1
1.2±.1
Cleaved caspase-3 ROD
Cleaved caspase-8 ROD
Cleaved caspase-9 ROD
SN
SN+AA SN+AA 100 200
2.7±0.3* 1.9±0.3*# 1.1±0.1#$
2.1±0.2* 1.7±0.2* 0.9±0.1#$
Acn Fig. 6. Effect of sodium nitrite (SN, 80 mg/kg/day) alone and in combination with arjunolic acid (AA, 100 and 200 mg/kg/day) for 12 weeks on the cardiac activity of caspase-3 (a), caspase8 (b) and caspase-9 (c) as well as Western blot analysis of the cardiac levels of the cleaved caspases (d). *Significant difference compared with the control groups at p b 0.05. #Significant difference compared with the sodium nitrite groups at p b 0.05. $Significant difference compared with the arjunolic acid (100 mg/kg) group at p b 0.05.
associated with elevated caspase-8, in rats that received sodium nitrite. On the other hand, in response to mitochondrial stress, caspase-9 is activated, leading to cytochrome C release and caspase-3 activation, which are involved in various pathological conditions (Chwa et al., 2008). However, we observed an elevated MDA level associated with reduced cytochrome C oxidase that led to mitochondrial activation, enhancing the intrinsic cell death pathway. These findings strongly suggest that sodium nitrite mediates oxidative stress and contributes to oxidantinduced apoptotic cell death in cardiac cells. Many studies reported the anti-apoptotic effect of arjunolic acid through the reduction of cadmium-induced oxidative stress, attenuation of NFκB activation and inhibition of the apoptotic signaling pathways (Pal et al., 2011). In addition, arjunolic acid supplementation resulted in anti-apoptotic action in
hyperglycemia-induced cardiac pathophysiology through the suppression of a caspase-dependent pathway (Ghosh and Sil, 2013). Arjunolic acid was previously reported to block both extrinsic and intrinsic cell death in cadmium-induced hepatic damage (Pal et al., 2011) and acetaminophen-induced renal injury (Ghosh et al., 2010a). However, no previous study has reported the protective effects of arjunolic acid against cell death in sodium nitrite induced-cardiac dysfunction. Conclusion Arjunolic acid ameliorated the impairment of cardiac function in rats that ingested sodium nitrite via multiple mechanisms, such as the following: (1) reducing sodium nitrite-induced oxidative stress, as
M.M.H. Al-Gayyar et al. / Life Sciences 111 (2014) 18–26
Sodium nitrite
Increase Cyto C oxidase
Increase oxidative stress Decrease IL-4 - IL10
Increase pJNK/JNK
Increase Caspase-9
Increase DNA frag
Arjunolic acid
Recruitment of inflammatory cells Increase TNF-α IL-1β Increase CRP
Increase Caspase-8 Cardiac apoptosis
Cardiac inflam
Fig. 7. Schematic representation of the mechanism of the cardiac protective action of arjunolic acid against sodium nitrite-induced cardiac damage.
indicated by a reduction in the serum and cardiac MDA levels and restoration of the levels of reduced glutathione; (2) blocking sodium nitriteinduced inhibition of cardiac cytochrome C oxidase; (3) blocking sodium nitrite-induced elevation of cardiac pro-inflammatory cytokines such as TNF-α and IL-1β; (4) blocking sodium nitrite-induced inhibition of the anti-inflammatory cytokines IL-4 and IL-10; (5) reducing sodium nitrite-induced activation of the JNK MAPK pathway; and (6) reducing sodium nitrite-induced activation of both extrinsic and intrinsic cell death pathways. The mechanism is summarized in Fig. 7. Although, both doses of arjunolic acid produced protective effects, the use of 200 mg/kg arjunolic acid produced better results. All changes in the tested parameters indicate that sodium nitrite induces cardiac injury that is similar to atherosclerosis. Conflicts of interest All authors declare no potential conflicts of interest, including any financial, personal or other relationships with other people or organizations within that could inappropriately influence, or be perceived to influence, this work.
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