Cardiovasc Toxicol DOI 10.1007/s12012-014-9243-5
Cardioprotective Effect of Rhizomes of Acorus gramineus Against Isoproterenol-Induced Cardiac Damage in Pigs Jong-Hoon Kim • Hwan-Suck Chung • P. Antonisamy • Seung Ryel Lee • Hyunsu Bae
Ó Springer Science+Business Media New York 2014
Abstract The present study was designed to evaluate the cardioprotective potential of water extract of rhizomes of Acorus gramineus (AGR) against isoproterenol (ISO)induced myocardial infarction. Male pigs were orally administered with 250 or 500 mg/kg of AGR or with vehicle for 9 days, with concurrent subcutaneous injections of ISO on the 8th and 9th day. Administration of AGR significantly ameliorated ISO-induced cardiac dysfunctions as evidenced by the ventricular ST-segment interval and R-amplitude as well as the left ventricle fractional shortening and ejection fraction. Additionally, administration of AGR significantly attenuated increased cardiac injury markers, such as cardiac troponin T, tumor necrosis factora, and myeloperoxidase activity, and cardiac marker enzymes, and prevented the depletion of antioxidant parameters. Malondialdehyde formation was also inhibited by AGR. Based on the results, it is concluded that AGR possesses significant cardioprotective potential and may serve as an adjunct in the treatment and prophylaxis of myocardial infarction.
Jong-Hoon Kim and Hwan-Suck Chung have contributed equally to this work. J.-H. Kim P. Antonisamy Department of Veterinary Physiology, Biosafety Research Institute, College of Veterinary Medicine, Chonbuk National University, 664-14, 1GA, Duckjin-Dong, Duckjin-Gu, Jeollabuk-Do, Chonju City 561-756, Republic of Korea H.-S. Chung S. R. Lee H. Bae (&) Department of Physiology, College of Korean Medicine, Kyung Hee University, #1 Hoeki-Dong, Dongdaemoon-gu, Seoul 130-701, Republic of Korea e-mail: [email protected]
Keywords Acorus gramineus Myocardial infarction Hemodynamics Antioxidant enzymes Cardioprotection
Introduction Myocardial infarction (MI) continues to be a health problem causing mortality and morbidity in developed countries, despite clinical care and public concern [1]. It is widely understood that MI is a common symptom of myocardial ischemia and occurs when cardiac injury surpasses a critical threshold, resulting in mortal cardiac damage [2]. In MI, reactive oxygen species (ROS) are the primary components of oxidative damage of cardiomyocyte [3]. Extensive research, including studies on myocardial ischemia–reperfusion injury, has focused on the roles of antioxidants in the prevention of MI [4, 5]. Catecholamines are responsible for cardiac necrosis [6], and oxidation of catecholamines is also known to result in the generation of toxic ROS [7]. Therefore, it is likely that ROS may play a key role in catecholamine-induced toxicity by membrane peroxidation in cardiomyocytes. Isoproterenol (ISO), a b-adrenergic agonist, has been known to produce cardiac ischemia through auto-oxidation and free radical production [4]. ISO-induced cardiac ischemia results in increased cardiac enzymes and oxidative stress, and abnormal electrocardiograph and cardiac functions [8]. It has also been reported that the pathophysiological and morphological abnormality induced by ISO was shown to be comparable with cardiac ischemia in humans [9]. Among the numerous mechanisms of cardiac ischemia induced by ISO, production of ROS by peroxidation of catecholamines has been shown to play a key role in inducing cardiac ischemia. Previous studies have shown that antioxidants may inhibit the progression of cardiac
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ischemia [10, 11]. Furthermore, many natural antioxidants are now considered to be potential herbal medicines for reducing the risk of heart disease [12, 13]. Rhizomes of Acorus gramineus Solander (Acoraceae, AGR) has been used as a traditional Korean medicine to treat various diseases including digestive problems, depression, epilepsy, and central nervous system (CNS) disorders [14]. It contains essential oil, such as b-asarone, a-asarone, caryophyllene, isosarone, methyl isoeugenol, safrole, and asaryl aldehyde as its main chemical constituents. AGR has anti-fungal, anti-bacterial, and neuroprotective activities [15, 16]. It is reported that AGR protects cultured rat neurons from glutamate-induced cytotoxicity [17], attenuated ischemia-induced neuronal death, inhibited methamphetamine-induced hyperactivity in mice [18], and improved cognitive deficits in rats [19]. Additionally, we have reported previously that AGR has distinct antiischemic effects on ischemia-induced isolated rat heart [20]. The present study was designed to test the effect of AGR pretreatment on ISO-induced myocardial damage in the pig model. This study also attempted to demonstrate the efficacy of AGR by studying electrocardiographic changes, hemodynamics, biochemical markers, and the antioxidant defense system.
Materials and Methods Animals Domestic Yorkshire–Landrace pigs (n = 25) ranging from 25 to 30 kg were divided into 5 groups. Pig of either sex was obtained from Hanil Experiment Animal Breeding (Yeumsung, South Korea) and was housed at an ambient temperature of 25 ± 2 °C with alternating 12-h cycles of light and dark. The pig had free access to standard food and water ad libitum for 3 days for acclimatization. The experimental protocol was approved by the Kyung Hee University Ethics Committee for the use of experimental animals and conformed to the Guide for the Care and Use of Laboratory Animals in accordance with the National Institutes of Health. Efforts were made to minimize the numbers of animals used and to reduce their suffering. Preparation of AGR In this study, we purchased the standardized AGR extract from Sun-Ten Pharmaceutical Company (Taipei, Taiwan). The spray-dried water extract of AGR was dissolved in pure water and filtered through a 0.2-lm syringe filter. Trans-asarone content analysis revealed a concentration of 0.19 ± 0.00 mg/g (0.02 ± 0.00 %) [20].
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Induction of Myocardial Infarction It was well known that ISO induced experimental myocardial infarction in rodents and pigs [21, 22]. Furthermore, Soltysinska et al. reported that daily intraperitoneal injection of ISO (final dose of 1 mg/kg) for 3 months induced heart failure as evidenced by cardiac hypertrophy (increased cardiac weights and left ventricular posterior wall thickness), basal systolic dysfunction (reduced LV fractional shortening (FS) determined by echocardiography), and reduced contractile reserve in guinea pigs [23]. In previous research, it was reported that subcutaneous ISO injections (100 mg/kg every 24 h) in Wistar rats resulted in elevated levels of serum cardiac troponin T after the 9th and 10th day. In a pilot study, we found that the level of serum cardiac troponin T (cTnT) was increased by subcutaneous injections of 85 and 150 mg/ kg of ISO twice at an interval of 24 h. Based on the pilot study preliminary results, an ISO dose of 150 mg/kg was selected because this dosage resulted in significant alteration in biochemical parameters such as cTnT, which to the best of our knowledge has not been previously reported. All animals were killed 24 h after the second injection of ISO. Experimental Protocols For the experiments, a total of 25 pigs, of both sexes, were equally divided into five groups (n = 5, each group). The normal control group was administered saline for 9 days. In the AGR group, pigs were administered oral 500 mg/kg doses of AGR once per day for 9 days as a sham control. In the ISO group, pigs were administered saline for 9 days and injected with ISO (150 mg/kg, injected subcutaneously twice at a 24-h interval) on the 8th and 9th days. In the 250 and 500 mg/kg AGR administered groups, pigs were administered AGR (250 and 500 mg/kg by gastric gavage, respectively) for 9 days. On the 8th and 9th days, two subcutaneous injections of 150 mg/kg of ISO at a 24-h interval were administered (Fig. 1). Electrocardiography (ECG) At the end of the above experiments, anesthesia was induced with a combination of atropine (0.04 mg/kg), xylazine (2 mg/kg), and zoletil (5 mg/kg), injected intramuscularly and maintained by 0.5–2.0 % isoflurane delivered in 100 % oxygen, followed by ECG recordings. Needle electrodes were briefly inserted under the skin, in lead II position, of the anesthetized pigs. ECG recordings were made using a computerized MP30 data acquisition system (BIOPAC, Santa Barbara, California, USA), and changes in ECG pattern were noted. In each group, elevation and decline of ST-segment intervals and R-amplitude were determined.
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Fig. 1 Experimental protocol. All experimental groups began with 3-day stabilization and divided into the normal control group (N/C), the AGR control, ISO control, 250 mg/kg AGR pretreatment, and 500 mg/kg AGR pretreatment group. In AGR-pretreated group, the
animals were pretreated with 250 and 500 mg/kg of AGR for 7 days. And, ISO was injected subcutaneously to porcine (150 mg/kg) daily for 2 consecutive days to induce experimental myocardial infarction
Echocardiography
Myeloperoxidase and Cardiac Troponin I Assay
Echocardiography was performed using the GE VingMed Vivid FiVe Ultrasound System (GE Healthcare, Denmark) equipped with a 10-MHz pediatric probe within 20 min of the anesthetic administration. Left ventricle systolic function was assessed by calculating the LV endocardial FS and ejection fraction (EF) [24] using a 16-channel PowerLab system (AD Instruments, Oxford, UK). Each echocardiographic variable was determined in at least four separate LV images taken from the same heart. The mean values were used for statistical analysis.
Serum Troponin T (cTnT) levels in were measured by ACS: 180 automated chemiluminescence system using commercial kits (Wuhan USCN sciences Co. Ltd.) by enzyme-linked immunosorbent assay [30]. To quantify neutrophil infiltration, the activity of myeloperoxidase (MPO), an abundant enzyme found in neutrophils, was evaluated using a modified method [31]. Briefly, myocardial tissue was homogenized in a 50 mM K2HPO4 buffer (pH 6), containing 0.5 % hexadecyltrimethylammonium bromide, using a Polytron tissue homogenizer. After freeze-thawing three times, the samples were centrifuged at 11,0009g for 30 min at 4 °C, and the resultant supernatants were assayed spectrophotometrically for MPO determination. In brief, 40 lL of sample was mixed with 960 lL of 50 mM phosphate buffer (pH 6) containing 0.167 mg/mL of O-dianisidine dihydrochloride and 0.0005 % H2O2. Changes in absorbance at 460 nm were studied using a spectrophotometer. One unit of enzyme activity was considered equal to the amount of MPO absorbance measured at 460 nm for 3 min. MPO activity data are presented as U/mg of tissue.
Cardiac Marker Enzymes in Serum The collected serum was used for the estimation of cardiac marker enzymes, including creatinine kinase-MB (CKMB), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate transaminase (AST), and alanine transaminase (ALT). Analysis was performed using commercially available standard enzymatic kits (Span Diagnostics Pvt. Ltd., India). Antioxidant Enzyme Activity in Cardiac Tissue
Determination of Tumor Necrosis Factor-a After electrocardiography and echocardiography, pigs were killed and the hearts were excised for biochemical experiments. The heart tissues were stored at -80 °C until further analysis in each group. A 10 % homogenate of heart tissue was prepared in phosphate buffer saline (pH 7.4, 50 mM). Aliquots of tissue homogenate were cold centrifuged at 5,000 rpm for 20 min, and the supernatant was used for estimation of protein [25], superoxide dismutase (SOD) [26], catalase (CAT) [27], and glutathione peroxidase (GPx) [28] content. The MDA, indicative of lipid peroxidation, was assessed according to its absorbencies by spectrophotometry [29].
Left ventricle samples were collected and immediately stored at -70 °C until the performance of assays as previously described [30]. In brief, the cardiac tissue was homogenized in RIPA (150 mmol/L NaCl, 0.1 % SDS, 0.5 % sodium deoxycholate, 1 % NP-40, 50 mmol/L Tris, pH 7.4, 20 mmol/L NaF, 2 mmol/L EGTA, 0.5 % levamisole, 1 mmol/L NaVO4, 1 lg/mL leupeptin) containing 1 mmol/L of PMSF. Supernatants were obtained by centrifugation (18,0009g, 15 min, 4 °C). Tumor necrosis factor-a (TNF-a) levels in the supernatants were determined using an ELISA kit (R&D Systems, Inc.,
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Cardiovasc Toxicol Fig. 2 The effect of 250 and 500 mg/kg AGR on ST-segment (a) and R-amplitude (b). STsegment and R-amplitude were estimated at 30-min intervals throughout the 120-min reperfusion period. Results were representative of five independent experiments. Values are expressed as mean ± SD. #P \ 0.01 compared with the control; *P \ 0.05, **P \ 0.01 compared with the isoproterenol
Minneapolis, MN, USA). The final results were expressed as pg/mg of protein. Statistical Analysis The results are expressed as the mean ± SD, whereby n = 5 for all data. Statistically significant differences between groups at baseline and at the end of the study were analyzed by one-way analysis of variance (ANOVA) with Tukey’s post hoc analysis using SPSS 9.0 software. Differences with P values \0.05 were considered to be statistically significant.
Results Effect of AGR on ECG Parameters ST-segment intervals and R-amplitude results on ECG parameters are shown in each group (Fig. 2). Normal controls and AGR controls showed normal ranges of STsegment intervals and R-amplitude, whereas ISO injection resulted in significantly elevated ST-segment intervals and decreased R-amplitude, which is indicative of myocardial infarction, compared to the controls [4]. However, the administration of AGR (250 and 500 mg/kg) significantly decreased ST-segment intervals and increased R-amplitude as shown in Fig. 2a, b. There was no difference between the control and AGR control group for ST-segment intervals and R-amplitude, suggesting that AGR (500 mg/kg) alone did not change ST-segment intervals and R-amplitude in this experiment.
Effect of AGR on Hemodynamic Functions As shown in Figs. 3 and 4, hemodynamic data such as fractional shortening (FS) and ejection fraction (EF) were
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determined in each group. Decreased values of FS and EF were observed in ISO controls as shown in Fig. 4. No differences in the FS and EF data between the control and AGR groups were observed. Left ventricle (LV) structural remodeling, such as LV dilatation and hypertrophy, was not observed; however, both FS and EF by ARG (250 and 500 mg/kg) exhibited significant percent increases compared to the ISO group. The ISO group has an average EF of 58.86 ± 3.72 %, and the EF values were 70.5 ± 4.27 and 80.28 ± 2.6 % for 250 and 500 mg/kg AGR, respectively. Furthermore, the ISO group has an average FS of 31.24 ± 3.14 %. The FS values were 38.58 ± 3.58 % for 250 mg/kg AGR and 45.8 ± 4.15 % for 500 mg/kg AGR, respectively, as seen in Fig. 4a, b. The hemodynamic data of AGR groups were significantly ameliorated than those seen in ISO group. These results consistently indicate that the pretreatment of AGR at 250 and 500 mg/kg doses is significantly effective in the preservation of hemodynamic function.
Effect of AGR on Myocardial Injury Biomarkers The cTnT activities, a sensitive and specific biomarker of myocardial injury [32], of all groups are shown in Fig. 5. The cTnT levels from the ISO controls were significantly higher than in the control groups. Significantly lower values of cTnT were determined in the 250 and 500 mg/kg AGR groups compared to the ISO group. Additionally, a significant increase of TNF-a (966.12 ± 23.12 pg/mg protein) was detected in the ISO-treated hearts. Administration of AGR at 250 and 500 mg/kg for 7 days before ISO treatment produced a significant decrease in the levels of TNF-a (749.76 ± 89.32 pg/mg protein for 250 mg/kg, and 628.4 ± 61.04 pg/mg protein for 500 mg/kg, ** P \ 0.01, Fig. 5). The activity of inflammatory markers, such as MPO, is shown in Fig. 5. The ISO control group showed a
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Fig. 3 The effect of AGR on echocardiographic evaluations. a echocardiography was performed on normal porcine’ hearts, showing the normal cardiac function such as fractional shortening (FS; rightwards white arrow) and ejection fraction (EF; Notched white rightwards arrow). FS was calculated by following formula such as [maximum cavity (vertical broken arrow)—minimum cavity (vertical nonbroken arrow)]/2 9 100. EF was autonomously calculated by a
program that calculates EF using FS values. b echocardiography was shown in porcine hearts pretreated with AGR only for 7 days, also showing the normal cardiac function. c ISO control exhibit worsening cardiac function such as FS and EF. d, e echocardiography was performed on pretreatment of 250 and 500 mg/kg AGR for 7 days before ISO injection, showing the recovery of FS and EF, respectively
Fig. 4 The effect of 250 and 500 mg/kg AGR on ejection fraction (EF) (a) and fractional shortening (b). FS and EF were shown with histogram by measurement of echocardiography. Data were presented as mean ± SD from total 5 porcines/group (#P \ 0.01 vs control,
**P \ 0.01 vs ISO control). Control; normal control, AGR, AGR control; ISO control; 250 AGR ? isoproterenol and 500 AGR ? isoproterenol groups which means pretreatment of 250 and 500 mg/kg AGR for 7 days before ISO injection
significant increase in the MPO activity when compared to control and AGR group. Administration of AGR led to a significant decrease in the myocardial MPO activity compared to ISO control. For the ISO controls, a significant increase of 14.4 ± 1.18 U/mg of tissue was
detected. Administration of AGR at 250 and 500 mg/kg doses for 7 days produced a significant decrease in elevated MPO activity (12.02 ± 0.98 and 10.48 ± 1.22 U/ mg of tissue for 250 and 500 mg/kg, respectively, Fig. 5).
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Cardiovasc Toxicol Fig. 5 The effect of 250 and 500 mg/kg AGR on the myocardial injury biomarkers in the heart. Control; normal control, AGR, AGR control; ISO control; 250 AGR ? isoproterenol and 500 AGR ? isoproterenol groups which means pretreatment of 250 and 500 mg/kg AGR for 7 days before ISO injection. # P \ 0.01 vs control; *P \ 0.05, **P \ 0.01 vs ISO control
The effects of AGR on pig serum enzymes such as CKMB and LDH levels are shown in Fig. 5. The activities of these enzymes increased significantly in ISO-treated pig
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compared to the normal controls. Conversely, the administration of AGR in ISO-treated pig significantly decreased these enzyme activities. Overall, AGR controls (500 mg/
Cardiovasc Toxicol Table 1 Effects of AGR on antioxidant parameters in heart of myocardial injury pigs (mean ± SD) Superoxide dismutase (U/mg protein)
Catalase (U/mg protein)
Glutathione peroxidase (U/mg protein)
Control
1,164.5 ± 126.8
17.63 ± 1.52
137.85 ± 10.52
AGR
1,232.7 ± 95.7
18.75 ± 1.76
141.53 ± 7.85
Malondialdehyde (nmol/mg protein) 8.35 ± 1.37 8.43 ± 1.28
Isoproterenol
696.4 ± 118.1a
8.43 ± 0.25a
73.27 ± 9.61a
13.76 ± 1.51a
Isoproterenol ? 250 AGR
953.8 ± 133.9c
12.37 ± 1.38c
89.43 ± 13.87b
11.13 ± 1.54b
Isoproterenol ? 500 AGR
c
c
1,062.3 ± 108.2
14.58 ± 2.42
115.75 ± 8.71
c
9.84 ± 1.12c
Compared with control: aP \ 0.01, Compared with isoproterenol: bP \ 0.05, cP \ 0.01
kg) did not show any significant changes in cTnT activity, TNF-a, MPO activity, CK-MB, and LDH compared to controls, indicating that AGR per se does not exert any adverse effects (Fig. 5). Effect of AGR on Antioxidant Enzymes Activity The antioxidant enzyme activities of SOD, CAT, GSH-Px, and MDA are shown in Table 1. SOD, CAT, and GSH-Px activities were significantly decreased in the cardiac tissues of ISO-treated group compared to the control group. Moreover, ISO injection produced significant increases in MDA levels in cardiac tissues. However, the administration of AGR for 9 days with ISO injections on the 8th and 9th days produced significant increases in SOD, CAT, and GPx levels (bP \ 0.05 for 250 mg/kg AGR and cP \ 0.01 for 500 mg/kg AGR). In addition, the administration of AGR (250 and 500 mg/kg) for 9 days with ISO injections on the 8th and 9th days significantly decreased the elevated MDA levels compared to the ISO group (bP \ 0.05 and c P \ 0.01). However, the 500 mg/kg AGR group did not show any significant changes in MDA levels or the enzyme activities of SOD, CAT, and GPx, indicating that AGR per se does not exert any additional effects.
Discussion A synthetic catecholamine, isoproterenol (ISO), is used to produce myocardial injury in animals and serves as an experimental model for the pharmacological evaluation of cardioprotective agents [33]. High doses of ISO treatment induce ischemia or hypoxia due to myocardial hyperactivity and coronary hypotension [34, 35] and are used to induce myocardial ischemia. In our study, we observed that high-dose ISO treatment resulted in myocardial infarction, increased activities of cardiac marker enzymes, decreased antioxidant parameters, and inhibition of hemodynamic function, which were consistent with previous reports [36]. ECG is the most widely used method of diagnosing myocardial infarction. ISO injection caused elevated ST-
segments and depressed R-amplitudes. The ST-segment increase reflects the potential difference in the boundary between the ischemic and non-ischemic zones and the consequent loss of cell membrane function, whereas the decreased R-amplitude may be due to the onset of myocardial edema following ISO administration [37]. AGR administration significantly attenuated pathologic alterations in the ECG, which was indicating its protective effects on cell membrane function. Echocardiogram results showed that left ventricular (LV) percent fractional shortening (FS) and LV ejection fraction (EF) were decreased in the ISO group. Treatment with AGR attenuated the effect of ISO on FS and EF. To examine the cardioprotective mechanism of AGR, we analyzed cTnT from sera, TNF-a from left ventricles, and MPO from myocardial tissues. AGR significantly inhibited the increases of TnT, TNF-a, and MPO by ISO treatment. cTnT is now regarded as the most specific biochemical marker of myocardial injury. These cardiac troponins appear in the blood as early as 3–4 h and remain elevated for 4–14 days [38]. TNF-a is highly expressed in the myocardium following ischemia and reperfusion; therefore, TNF-a may significantly influence the apoptosis of cells within the heart. This pro-inflammatory cytokine has been known to exert a cardiotoxic effect and depresses contractile function, diminishing b-adrenergic inotropic responsiveness, and activating pro-apoptotic pathways in the myocardium [39]. Jiang et al. reported the anti-inflammatory effect of AGR in the activated microglia [40], and our results suggest that AGR inhibited the increase of TNF-a induced by ISO. It has been reported that the neutrophils, a major source of free radicals, invade myocardial tissue during ischemia [41]. The results of this study show that AGR pretreatment blocked the elevation of MPO activity and indicated that AGR suppressed neutrophil infiltration into the injured myocardium. The inhibition of neutrophil infiltration and function results in reduced generation of oxygen free radicals during ischemia and may contribute to the protective action of AGR against myocardial ischemia. Although the o-dianisidine assay has been typically used for an MPO assay, it is rather a peroxidase assay than a
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MPO assay because other peroxidase enzymes, e.g., COX1 and COX-2, also metabolize o-dianisidine. So a highly specific and sensitive assay protocol using MPO antibody has been developed recently [42]. Even though o-dianisidine assay is not an exact way to assay MPO, we supposed that the increased values we assayed from the ISO-treated cardiac tissue were mainly caused from neutrophils infiltration rather than other peroxidase when we compared to others results. Trans-asarone, the major constituent of the essential oil from AGR, is regarded as carcinogenic in rodents and potentially genotoxic. Thus, the limit for the ingestion of this constituent from herbal medicinal products has been set at 0.115 mg beta-asarone/person/day. But, Chen et al. showed that the content of trans-asarone in dried herbal drug ranged from 15.22 to 25.34 mg/g decreased more than 85 % during a 1-h decoction. If this aqueous extract was heated for a further 2 h, the final content of trans-asarone was reduced to the equivalent of no more than 0.005 mg/g of herbal drug. This low level of beta-asarone should be acceptable for therapeutic use [43]. Moreover, in acute and chronic toxicity experiments, ethanolic extract and hydroalcoholic extract of Acorus calamus did not cause significant changes in rodents [44, 45]. The present study showed that there is a significant increase in oxidative stress after myocardial ischemia in pigs and that AGR produces a significant protective effect against this oxidative damage. Consistent with previous studies, there was a significant elevation in serum lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB), confirming the occurrence of acute myocardial infarction [46]. The cells are damaged with increased muscle contractility, which results in increased cell membrane permeability, which allows cardiac enzymes to leak into the bloodstream. CK-MB is localized, predominantly in the heart, which makes it a valuable diagnostic tool for MI because damage specific to the myocardium would result in elevated CK-MB levels [47, 48]. LDH have been considered as one of the important diagnostic markers of myocyte damage. LDH usually rises within 6–12 h of MI. Level of LDH peaks within 48 h, remains at that peak for 4–14 days. It is known that myocardial infarction increases oxidative stress and then causes oxidative damage to myocytes [49, 50]. Free radical scavenging enzymes such as SOD, catalase, and GPx [51] are the first-line cellular defense against oxidative stress, eliminating reactive oxygen radicals, and preventing the formation of more reactive radicals. Decreases in the values of SOD and CAT, following ISO administration, indicate overwhelming of free radicals, which ensues oxidative damage to the myocardium. MDA is a major lipid peroxidant end product. The increased levels of MDA indicate activation of the lipid peroxidative
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process, resulting in irreversible damage to the hearts of animals subjected to ISO stress [52]. Because tissue necrosis in oxidative stress increase oxidative stress by increasing lipid peroxidation products and decreasing antioxidants over again. At the moment, it is hard to tell which mechanism is involved on the myocardial protective effects of AGR. Further studies are needed to evaluate the mechanism of AGR’s effects.
Conclusion To conclude, present study findings show that AGR treatment maintained antioxidant levels, inhibited inflammatory responses, and improved cardiac performance following ISO administration. The results of this study suggest that AGR may serve as an adjunct in prophylaxis and treatment in delaying the progression of MI. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A4A01011658). Conflict of interest of interest.
The authors declare that there are no conflicts
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Cardioprotective Effect of Rhizomes of Acorus gramineus Against Isoproterenol-Induced Cardiac Damage in Pigs Jong-Hoon Kim • Hwan-Suck Chung • P. Antonisamy • Seung Ryel Lee • Hyunsu Bae
Ó Springer Science+Business Media New York 2014
Abstract The present study was designed to evaluate the cardioprotective potential of water extract of rhizomes of Acorus gramineus (AGR) against isoproterenol (ISO)induced myocardial infarction. Male pigs were orally administered with 250 or 500 mg/kg of AGR or with vehicle for 9 days, with concurrent subcutaneous injections of ISO on the 8th and 9th day. Administration of AGR significantly ameliorated ISO-induced cardiac dysfunctions as evidenced by the ventricular ST-segment interval and R-amplitude as well as the left ventricle fractional shortening and ejection fraction. Additionally, administration of AGR significantly attenuated increased cardiac injury markers, such as cardiac troponin T, tumor necrosis factora, and myeloperoxidase activity, and cardiac marker enzymes, and prevented the depletion of antioxidant parameters. Malondialdehyde formation was also inhibited by AGR. Based on the results, it is concluded that AGR possesses significant cardioprotective potential and may serve as an adjunct in the treatment and prophylaxis of myocardial infarction.
Jong-Hoon Kim and Hwan-Suck Chung have contributed equally to this work. J.-H. Kim P. Antonisamy Department of Veterinary Physiology, Biosafety Research Institute, College of Veterinary Medicine, Chonbuk National University, 664-14, 1GA, Duckjin-Dong, Duckjin-Gu, Jeollabuk-Do, Chonju City 561-756, Republic of Korea H.-S. Chung S. R. Lee H. Bae (&) Department of Physiology, College of Korean Medicine, Kyung Hee University, #1 Hoeki-Dong, Dongdaemoon-gu, Seoul 130-701, Republic of Korea e-mail: [email protected]
Keywords Acorus gramineus Myocardial infarction Hemodynamics Antioxidant enzymes Cardioprotection
Introduction Myocardial infarction (MI) continues to be a health problem causing mortality and morbidity in developed countries, despite clinical care and public concern [1]. It is widely understood that MI is a common symptom of myocardial ischemia and occurs when cardiac injury surpasses a critical threshold, resulting in mortal cardiac damage [2]. In MI, reactive oxygen species (ROS) are the primary components of oxidative damage of cardiomyocyte [3]. Extensive research, including studies on myocardial ischemia–reperfusion injury, has focused on the roles of antioxidants in the prevention of MI [4, 5]. Catecholamines are responsible for cardiac necrosis [6], and oxidation of catecholamines is also known to result in the generation of toxic ROS [7]. Therefore, it is likely that ROS may play a key role in catecholamine-induced toxicity by membrane peroxidation in cardiomyocytes. Isoproterenol (ISO), a b-adrenergic agonist, has been known to produce cardiac ischemia through auto-oxidation and free radical production [4]. ISO-induced cardiac ischemia results in increased cardiac enzymes and oxidative stress, and abnormal electrocardiograph and cardiac functions [8]. It has also been reported that the pathophysiological and morphological abnormality induced by ISO was shown to be comparable with cardiac ischemia in humans [9]. Among the numerous mechanisms of cardiac ischemia induced by ISO, production of ROS by peroxidation of catecholamines has been shown to play a key role in inducing cardiac ischemia. Previous studies have shown that antioxidants may inhibit the progression of cardiac
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ischemia [10, 11]. Furthermore, many natural antioxidants are now considered to be potential herbal medicines for reducing the risk of heart disease [12, 13]. Rhizomes of Acorus gramineus Solander (Acoraceae, AGR) has been used as a traditional Korean medicine to treat various diseases including digestive problems, depression, epilepsy, and central nervous system (CNS) disorders [14]. It contains essential oil, such as b-asarone, a-asarone, caryophyllene, isosarone, methyl isoeugenol, safrole, and asaryl aldehyde as its main chemical constituents. AGR has anti-fungal, anti-bacterial, and neuroprotective activities [15, 16]. It is reported that AGR protects cultured rat neurons from glutamate-induced cytotoxicity [17], attenuated ischemia-induced neuronal death, inhibited methamphetamine-induced hyperactivity in mice [18], and improved cognitive deficits in rats [19]. Additionally, we have reported previously that AGR has distinct antiischemic effects on ischemia-induced isolated rat heart [20]. The present study was designed to test the effect of AGR pretreatment on ISO-induced myocardial damage in the pig model. This study also attempted to demonstrate the efficacy of AGR by studying electrocardiographic changes, hemodynamics, biochemical markers, and the antioxidant defense system.
Materials and Methods Animals Domestic Yorkshire–Landrace pigs (n = 25) ranging from 25 to 30 kg were divided into 5 groups. Pig of either sex was obtained from Hanil Experiment Animal Breeding (Yeumsung, South Korea) and was housed at an ambient temperature of 25 ± 2 °C with alternating 12-h cycles of light and dark. The pig had free access to standard food and water ad libitum for 3 days for acclimatization. The experimental protocol was approved by the Kyung Hee University Ethics Committee for the use of experimental animals and conformed to the Guide for the Care and Use of Laboratory Animals in accordance with the National Institutes of Health. Efforts were made to minimize the numbers of animals used and to reduce their suffering. Preparation of AGR In this study, we purchased the standardized AGR extract from Sun-Ten Pharmaceutical Company (Taipei, Taiwan). The spray-dried water extract of AGR was dissolved in pure water and filtered through a 0.2-lm syringe filter. Trans-asarone content analysis revealed a concentration of 0.19 ± 0.00 mg/g (0.02 ± 0.00 %) [20].
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Induction of Myocardial Infarction It was well known that ISO induced experimental myocardial infarction in rodents and pigs [21, 22]. Furthermore, Soltysinska et al. reported that daily intraperitoneal injection of ISO (final dose of 1 mg/kg) for 3 months induced heart failure as evidenced by cardiac hypertrophy (increased cardiac weights and left ventricular posterior wall thickness), basal systolic dysfunction (reduced LV fractional shortening (FS) determined by echocardiography), and reduced contractile reserve in guinea pigs [23]. In previous research, it was reported that subcutaneous ISO injections (100 mg/kg every 24 h) in Wistar rats resulted in elevated levels of serum cardiac troponin T after the 9th and 10th day. In a pilot study, we found that the level of serum cardiac troponin T (cTnT) was increased by subcutaneous injections of 85 and 150 mg/ kg of ISO twice at an interval of 24 h. Based on the pilot study preliminary results, an ISO dose of 150 mg/kg was selected because this dosage resulted in significant alteration in biochemical parameters such as cTnT, which to the best of our knowledge has not been previously reported. All animals were killed 24 h after the second injection of ISO. Experimental Protocols For the experiments, a total of 25 pigs, of both sexes, were equally divided into five groups (n = 5, each group). The normal control group was administered saline for 9 days. In the AGR group, pigs were administered oral 500 mg/kg doses of AGR once per day for 9 days as a sham control. In the ISO group, pigs were administered saline for 9 days and injected with ISO (150 mg/kg, injected subcutaneously twice at a 24-h interval) on the 8th and 9th days. In the 250 and 500 mg/kg AGR administered groups, pigs were administered AGR (250 and 500 mg/kg by gastric gavage, respectively) for 9 days. On the 8th and 9th days, two subcutaneous injections of 150 mg/kg of ISO at a 24-h interval were administered (Fig. 1). Electrocardiography (ECG) At the end of the above experiments, anesthesia was induced with a combination of atropine (0.04 mg/kg), xylazine (2 mg/kg), and zoletil (5 mg/kg), injected intramuscularly and maintained by 0.5–2.0 % isoflurane delivered in 100 % oxygen, followed by ECG recordings. Needle electrodes were briefly inserted under the skin, in lead II position, of the anesthetized pigs. ECG recordings were made using a computerized MP30 data acquisition system (BIOPAC, Santa Barbara, California, USA), and changes in ECG pattern were noted. In each group, elevation and decline of ST-segment intervals and R-amplitude were determined.
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Fig. 1 Experimental protocol. All experimental groups began with 3-day stabilization and divided into the normal control group (N/C), the AGR control, ISO control, 250 mg/kg AGR pretreatment, and 500 mg/kg AGR pretreatment group. In AGR-pretreated group, the
animals were pretreated with 250 and 500 mg/kg of AGR for 7 days. And, ISO was injected subcutaneously to porcine (150 mg/kg) daily for 2 consecutive days to induce experimental myocardial infarction
Echocardiography
Myeloperoxidase and Cardiac Troponin I Assay
Echocardiography was performed using the GE VingMed Vivid FiVe Ultrasound System (GE Healthcare, Denmark) equipped with a 10-MHz pediatric probe within 20 min of the anesthetic administration. Left ventricle systolic function was assessed by calculating the LV endocardial FS and ejection fraction (EF) [24] using a 16-channel PowerLab system (AD Instruments, Oxford, UK). Each echocardiographic variable was determined in at least four separate LV images taken from the same heart. The mean values were used for statistical analysis.
Serum Troponin T (cTnT) levels in were measured by ACS: 180 automated chemiluminescence system using commercial kits (Wuhan USCN sciences Co. Ltd.) by enzyme-linked immunosorbent assay [30]. To quantify neutrophil infiltration, the activity of myeloperoxidase (MPO), an abundant enzyme found in neutrophils, was evaluated using a modified method [31]. Briefly, myocardial tissue was homogenized in a 50 mM K2HPO4 buffer (pH 6), containing 0.5 % hexadecyltrimethylammonium bromide, using a Polytron tissue homogenizer. After freeze-thawing three times, the samples were centrifuged at 11,0009g for 30 min at 4 °C, and the resultant supernatants were assayed spectrophotometrically for MPO determination. In brief, 40 lL of sample was mixed with 960 lL of 50 mM phosphate buffer (pH 6) containing 0.167 mg/mL of O-dianisidine dihydrochloride and 0.0005 % H2O2. Changes in absorbance at 460 nm were studied using a spectrophotometer. One unit of enzyme activity was considered equal to the amount of MPO absorbance measured at 460 nm for 3 min. MPO activity data are presented as U/mg of tissue.
Cardiac Marker Enzymes in Serum The collected serum was used for the estimation of cardiac marker enzymes, including creatinine kinase-MB (CKMB), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate transaminase (AST), and alanine transaminase (ALT). Analysis was performed using commercially available standard enzymatic kits (Span Diagnostics Pvt. Ltd., India). Antioxidant Enzyme Activity in Cardiac Tissue
Determination of Tumor Necrosis Factor-a After electrocardiography and echocardiography, pigs were killed and the hearts were excised for biochemical experiments. The heart tissues were stored at -80 °C until further analysis in each group. A 10 % homogenate of heart tissue was prepared in phosphate buffer saline (pH 7.4, 50 mM). Aliquots of tissue homogenate were cold centrifuged at 5,000 rpm for 20 min, and the supernatant was used for estimation of protein [25], superoxide dismutase (SOD) [26], catalase (CAT) [27], and glutathione peroxidase (GPx) [28] content. The MDA, indicative of lipid peroxidation, was assessed according to its absorbencies by spectrophotometry [29].
Left ventricle samples were collected and immediately stored at -70 °C until the performance of assays as previously described [30]. In brief, the cardiac tissue was homogenized in RIPA (150 mmol/L NaCl, 0.1 % SDS, 0.5 % sodium deoxycholate, 1 % NP-40, 50 mmol/L Tris, pH 7.4, 20 mmol/L NaF, 2 mmol/L EGTA, 0.5 % levamisole, 1 mmol/L NaVO4, 1 lg/mL leupeptin) containing 1 mmol/L of PMSF. Supernatants were obtained by centrifugation (18,0009g, 15 min, 4 °C). Tumor necrosis factor-a (TNF-a) levels in the supernatants were determined using an ELISA kit (R&D Systems, Inc.,
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Cardiovasc Toxicol Fig. 2 The effect of 250 and 500 mg/kg AGR on ST-segment (a) and R-amplitude (b). STsegment and R-amplitude were estimated at 30-min intervals throughout the 120-min reperfusion period. Results were representative of five independent experiments. Values are expressed as mean ± SD. #P \ 0.01 compared with the control; *P \ 0.05, **P \ 0.01 compared with the isoproterenol
Minneapolis, MN, USA). The final results were expressed as pg/mg of protein. Statistical Analysis The results are expressed as the mean ± SD, whereby n = 5 for all data. Statistically significant differences between groups at baseline and at the end of the study were analyzed by one-way analysis of variance (ANOVA) with Tukey’s post hoc analysis using SPSS 9.0 software. Differences with P values \0.05 were considered to be statistically significant.
Results Effect of AGR on ECG Parameters ST-segment intervals and R-amplitude results on ECG parameters are shown in each group (Fig. 2). Normal controls and AGR controls showed normal ranges of STsegment intervals and R-amplitude, whereas ISO injection resulted in significantly elevated ST-segment intervals and decreased R-amplitude, which is indicative of myocardial infarction, compared to the controls [4]. However, the administration of AGR (250 and 500 mg/kg) significantly decreased ST-segment intervals and increased R-amplitude as shown in Fig. 2a, b. There was no difference between the control and AGR control group for ST-segment intervals and R-amplitude, suggesting that AGR (500 mg/kg) alone did not change ST-segment intervals and R-amplitude in this experiment.
Effect of AGR on Hemodynamic Functions As shown in Figs. 3 and 4, hemodynamic data such as fractional shortening (FS) and ejection fraction (EF) were
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determined in each group. Decreased values of FS and EF were observed in ISO controls as shown in Fig. 4. No differences in the FS and EF data between the control and AGR groups were observed. Left ventricle (LV) structural remodeling, such as LV dilatation and hypertrophy, was not observed; however, both FS and EF by ARG (250 and 500 mg/kg) exhibited significant percent increases compared to the ISO group. The ISO group has an average EF of 58.86 ± 3.72 %, and the EF values were 70.5 ± 4.27 and 80.28 ± 2.6 % for 250 and 500 mg/kg AGR, respectively. Furthermore, the ISO group has an average FS of 31.24 ± 3.14 %. The FS values were 38.58 ± 3.58 % for 250 mg/kg AGR and 45.8 ± 4.15 % for 500 mg/kg AGR, respectively, as seen in Fig. 4a, b. The hemodynamic data of AGR groups were significantly ameliorated than those seen in ISO group. These results consistently indicate that the pretreatment of AGR at 250 and 500 mg/kg doses is significantly effective in the preservation of hemodynamic function.
Effect of AGR on Myocardial Injury Biomarkers The cTnT activities, a sensitive and specific biomarker of myocardial injury [32], of all groups are shown in Fig. 5. The cTnT levels from the ISO controls were significantly higher than in the control groups. Significantly lower values of cTnT were determined in the 250 and 500 mg/kg AGR groups compared to the ISO group. Additionally, a significant increase of TNF-a (966.12 ± 23.12 pg/mg protein) was detected in the ISO-treated hearts. Administration of AGR at 250 and 500 mg/kg for 7 days before ISO treatment produced a significant decrease in the levels of TNF-a (749.76 ± 89.32 pg/mg protein for 250 mg/kg, and 628.4 ± 61.04 pg/mg protein for 500 mg/kg, ** P \ 0.01, Fig. 5). The activity of inflammatory markers, such as MPO, is shown in Fig. 5. The ISO control group showed a
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Fig. 3 The effect of AGR on echocardiographic evaluations. a echocardiography was performed on normal porcine’ hearts, showing the normal cardiac function such as fractional shortening (FS; rightwards white arrow) and ejection fraction (EF; Notched white rightwards arrow). FS was calculated by following formula such as [maximum cavity (vertical broken arrow)—minimum cavity (vertical nonbroken arrow)]/2 9 100. EF was autonomously calculated by a
program that calculates EF using FS values. b echocardiography was shown in porcine hearts pretreated with AGR only for 7 days, also showing the normal cardiac function. c ISO control exhibit worsening cardiac function such as FS and EF. d, e echocardiography was performed on pretreatment of 250 and 500 mg/kg AGR for 7 days before ISO injection, showing the recovery of FS and EF, respectively
Fig. 4 The effect of 250 and 500 mg/kg AGR on ejection fraction (EF) (a) and fractional shortening (b). FS and EF were shown with histogram by measurement of echocardiography. Data were presented as mean ± SD from total 5 porcines/group (#P \ 0.01 vs control,
**P \ 0.01 vs ISO control). Control; normal control, AGR, AGR control; ISO control; 250 AGR ? isoproterenol and 500 AGR ? isoproterenol groups which means pretreatment of 250 and 500 mg/kg AGR for 7 days before ISO injection
significant increase in the MPO activity when compared to control and AGR group. Administration of AGR led to a significant decrease in the myocardial MPO activity compared to ISO control. For the ISO controls, a significant increase of 14.4 ± 1.18 U/mg of tissue was
detected. Administration of AGR at 250 and 500 mg/kg doses for 7 days produced a significant decrease in elevated MPO activity (12.02 ± 0.98 and 10.48 ± 1.22 U/ mg of tissue for 250 and 500 mg/kg, respectively, Fig. 5).
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Cardiovasc Toxicol Fig. 5 The effect of 250 and 500 mg/kg AGR on the myocardial injury biomarkers in the heart. Control; normal control, AGR, AGR control; ISO control; 250 AGR ? isoproterenol and 500 AGR ? isoproterenol groups which means pretreatment of 250 and 500 mg/kg AGR for 7 days before ISO injection. # P \ 0.01 vs control; *P \ 0.05, **P \ 0.01 vs ISO control
The effects of AGR on pig serum enzymes such as CKMB and LDH levels are shown in Fig. 5. The activities of these enzymes increased significantly in ISO-treated pig
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compared to the normal controls. Conversely, the administration of AGR in ISO-treated pig significantly decreased these enzyme activities. Overall, AGR controls (500 mg/
Cardiovasc Toxicol Table 1 Effects of AGR on antioxidant parameters in heart of myocardial injury pigs (mean ± SD) Superoxide dismutase (U/mg protein)
Catalase (U/mg protein)
Glutathione peroxidase (U/mg protein)
Control
1,164.5 ± 126.8
17.63 ± 1.52
137.85 ± 10.52
AGR
1,232.7 ± 95.7
18.75 ± 1.76
141.53 ± 7.85
Malondialdehyde (nmol/mg protein) 8.35 ± 1.37 8.43 ± 1.28
Isoproterenol
696.4 ± 118.1a
8.43 ± 0.25a
73.27 ± 9.61a
13.76 ± 1.51a
Isoproterenol ? 250 AGR
953.8 ± 133.9c
12.37 ± 1.38c
89.43 ± 13.87b
11.13 ± 1.54b
Isoproterenol ? 500 AGR
c
c
1,062.3 ± 108.2
14.58 ± 2.42
115.75 ± 8.71
c
9.84 ± 1.12c
Compared with control: aP \ 0.01, Compared with isoproterenol: bP \ 0.05, cP \ 0.01
kg) did not show any significant changes in cTnT activity, TNF-a, MPO activity, CK-MB, and LDH compared to controls, indicating that AGR per se does not exert any adverse effects (Fig. 5). Effect of AGR on Antioxidant Enzymes Activity The antioxidant enzyme activities of SOD, CAT, GSH-Px, and MDA are shown in Table 1. SOD, CAT, and GSH-Px activities were significantly decreased in the cardiac tissues of ISO-treated group compared to the control group. Moreover, ISO injection produced significant increases in MDA levels in cardiac tissues. However, the administration of AGR for 9 days with ISO injections on the 8th and 9th days produced significant increases in SOD, CAT, and GPx levels (bP \ 0.05 for 250 mg/kg AGR and cP \ 0.01 for 500 mg/kg AGR). In addition, the administration of AGR (250 and 500 mg/kg) for 9 days with ISO injections on the 8th and 9th days significantly decreased the elevated MDA levels compared to the ISO group (bP \ 0.05 and c P \ 0.01). However, the 500 mg/kg AGR group did not show any significant changes in MDA levels or the enzyme activities of SOD, CAT, and GPx, indicating that AGR per se does not exert any additional effects.
Discussion A synthetic catecholamine, isoproterenol (ISO), is used to produce myocardial injury in animals and serves as an experimental model for the pharmacological evaluation of cardioprotective agents [33]. High doses of ISO treatment induce ischemia or hypoxia due to myocardial hyperactivity and coronary hypotension [34, 35] and are used to induce myocardial ischemia. In our study, we observed that high-dose ISO treatment resulted in myocardial infarction, increased activities of cardiac marker enzymes, decreased antioxidant parameters, and inhibition of hemodynamic function, which were consistent with previous reports [36]. ECG is the most widely used method of diagnosing myocardial infarction. ISO injection caused elevated ST-
segments and depressed R-amplitudes. The ST-segment increase reflects the potential difference in the boundary between the ischemic and non-ischemic zones and the consequent loss of cell membrane function, whereas the decreased R-amplitude may be due to the onset of myocardial edema following ISO administration [37]. AGR administration significantly attenuated pathologic alterations in the ECG, which was indicating its protective effects on cell membrane function. Echocardiogram results showed that left ventricular (LV) percent fractional shortening (FS) and LV ejection fraction (EF) were decreased in the ISO group. Treatment with AGR attenuated the effect of ISO on FS and EF. To examine the cardioprotective mechanism of AGR, we analyzed cTnT from sera, TNF-a from left ventricles, and MPO from myocardial tissues. AGR significantly inhibited the increases of TnT, TNF-a, and MPO by ISO treatment. cTnT is now regarded as the most specific biochemical marker of myocardial injury. These cardiac troponins appear in the blood as early as 3–4 h and remain elevated for 4–14 days [38]. TNF-a is highly expressed in the myocardium following ischemia and reperfusion; therefore, TNF-a may significantly influence the apoptosis of cells within the heart. This pro-inflammatory cytokine has been known to exert a cardiotoxic effect and depresses contractile function, diminishing b-adrenergic inotropic responsiveness, and activating pro-apoptotic pathways in the myocardium [39]. Jiang et al. reported the anti-inflammatory effect of AGR in the activated microglia [40], and our results suggest that AGR inhibited the increase of TNF-a induced by ISO. It has been reported that the neutrophils, a major source of free radicals, invade myocardial tissue during ischemia [41]. The results of this study show that AGR pretreatment blocked the elevation of MPO activity and indicated that AGR suppressed neutrophil infiltration into the injured myocardium. The inhibition of neutrophil infiltration and function results in reduced generation of oxygen free radicals during ischemia and may contribute to the protective action of AGR against myocardial ischemia. Although the o-dianisidine assay has been typically used for an MPO assay, it is rather a peroxidase assay than a
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MPO assay because other peroxidase enzymes, e.g., COX1 and COX-2, also metabolize o-dianisidine. So a highly specific and sensitive assay protocol using MPO antibody has been developed recently [42]. Even though o-dianisidine assay is not an exact way to assay MPO, we supposed that the increased values we assayed from the ISO-treated cardiac tissue were mainly caused from neutrophils infiltration rather than other peroxidase when we compared to others results. Trans-asarone, the major constituent of the essential oil from AGR, is regarded as carcinogenic in rodents and potentially genotoxic. Thus, the limit for the ingestion of this constituent from herbal medicinal products has been set at 0.115 mg beta-asarone/person/day. But, Chen et al. showed that the content of trans-asarone in dried herbal drug ranged from 15.22 to 25.34 mg/g decreased more than 85 % during a 1-h decoction. If this aqueous extract was heated for a further 2 h, the final content of trans-asarone was reduced to the equivalent of no more than 0.005 mg/g of herbal drug. This low level of beta-asarone should be acceptable for therapeutic use [43]. Moreover, in acute and chronic toxicity experiments, ethanolic extract and hydroalcoholic extract of Acorus calamus did not cause significant changes in rodents [44, 45]. The present study showed that there is a significant increase in oxidative stress after myocardial ischemia in pigs and that AGR produces a significant protective effect against this oxidative damage. Consistent with previous studies, there was a significant elevation in serum lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB), confirming the occurrence of acute myocardial infarction [46]. The cells are damaged with increased muscle contractility, which results in increased cell membrane permeability, which allows cardiac enzymes to leak into the bloodstream. CK-MB is localized, predominantly in the heart, which makes it a valuable diagnostic tool for MI because damage specific to the myocardium would result in elevated CK-MB levels [47, 48]. LDH have been considered as one of the important diagnostic markers of myocyte damage. LDH usually rises within 6–12 h of MI. Level of LDH peaks within 48 h, remains at that peak for 4–14 days. It is known that myocardial infarction increases oxidative stress and then causes oxidative damage to myocytes [49, 50]. Free radical scavenging enzymes such as SOD, catalase, and GPx [51] are the first-line cellular defense against oxidative stress, eliminating reactive oxygen radicals, and preventing the formation of more reactive radicals. Decreases in the values of SOD and CAT, following ISO administration, indicate overwhelming of free radicals, which ensues oxidative damage to the myocardium. MDA is a major lipid peroxidant end product. The increased levels of MDA indicate activation of the lipid peroxidative
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process, resulting in irreversible damage to the hearts of animals subjected to ISO stress [52]. Because tissue necrosis in oxidative stress increase oxidative stress by increasing lipid peroxidation products and decreasing antioxidants over again. At the moment, it is hard to tell which mechanism is involved on the myocardial protective effects of AGR. Further studies are needed to evaluate the mechanism of AGR’s effects.
Conclusion To conclude, present study findings show that AGR treatment maintained antioxidant levels, inhibited inflammatory responses, and improved cardiac performance following ISO administration. The results of this study suggest that AGR may serve as an adjunct in prophylaxis and treatment in delaying the progression of MI. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A4A01011658). Conflict of interest of interest.
The authors declare that there are no conflicts
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