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ORIGINAL ARTICLE
Heart, Lung and Circulation (2014) xx, 1–9 1443-9506/04/$36.00 http://dx.doi.org/10.1016/j.hlc.2014.11.018
Cardioprotective Effect of Hydrogen-rich Saline on Isoproterenol-induced Myocardial Infarction in Rats Lei Jing a,b,d,1, Yun Wang a,1, Xiao-Min Zhao a,b*, Bing Zhao a,b,e, Ji-Ju Han a, Shu-Cun Qin a, Xue-Jun Sun c a
Key Laboratory of Atherosclerosis in Universities of Shandong (Taishan Medical University), Taian, 271000, China Department of Pharmocology, Taishan Medical University, Taian, 271000, China Department of Diving Medicine, the Second Military Medical University, Shanghai, 200433, China d Department of Pharmacy, Dongping County People’s Hospital, Dongping, 271500, China e Department of Pharmacy, Boshan District Hospital of Zibo, Boshan, 255200, China b c
Received 30 July 2014; received in revised form 18 November 2014; accepted 24 November 2014; online published-ahead-of-print xxx
Background
Infusion with hydrogen gas-saturated saline has recently been reported to exert antioxidant and antiinflammatory activity that may protect against organ damage induced by oxidative stress. Therefore because oxidative stress plays a significant role in the pathophysiology of myocardial infarction (MI), the aim of our study was to investigate whether hydrogen-rich saline has cardioprotective effects against isoproterenol-induced MI in rats.
Methods
An acute MI model was induced in male Wistar rats by subcutaneous injection of isoproterenol. Different doses of hydrogen-rich saline (5, 7.5, and 10 mL/kg body weight i.p.) or Vitamin C (250 mg/kg body weight i.g.) were administered to the rats. Oxidative stress indices including levels of myocardial marker enzymes, inflammatory cytokines, membrane-bound myocardial enzymes and histopathological changes were measured.
Results
Compared with those in isoproterenol-MI group, hydrogen-rich saline decreased malondialdehyde and 8-hydroxy-desoxyguanosine concentrations, enhanced superoxide dismutase and Na+-K+-ATPase activity, lowered Ca2+-ATPase activity and decreased interleukin-6 and tumour necrosis factor-a levels in the serum and/or cardiac tissue of rats. Hydrogen-rich saline pretreatment also diminished infarct size, improved left heart function, and ameliorated pathological changes of the left heart.
Conclusion
From these results, hydrogen-rich saline exerts cardiovascular protective effects against isoproterenolinduced MI at least in part via interactions which evoke antioxidant and anti-inflammatory activities.
Keywords
Hydrogen Inflammation Isoproterenol Myocardial infarction Oxidative stress
Introduction As an acute condition, myocardial infarction (MI) occurs as the result of unbalanced coronary artery supply and myocardial demand [1]. It is still the main cause of death worldwide, although therapeutic modalities and clinical care have
improved [2]. Extensive previous studies have shown that the production of toxic, reactive oxygen species (ROS), such as hydrogen peroxide, hydroxyl radicals and superoxide radicals in ischaemic tissue, induce damage to myocardial cells, leading to oxidative damage to membrane lipids, proteins and DNA [3]. To date the major challenge remains in
*Corresponding author at: Key Laboratory of Atherosclerosis in Universities of Shandong, Taishan Medical University, Taian, 271000, China. Tel.: +86 538 6225275; fax: +86 538 6225275, Email: [email protected] 1
These two authors contributed equally to the study.
© 2014 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ). Published by Elsevier Inc. All rights reserved.
Please cite this article in press as: Jing L, et al. Cardioprotective Effect of Hydrogen-rich Saline on Isoproterenol-induced Myocardial Infarction in Rats. Heart, Lung and Circulation (2014), http://dx.doi.org/10.1016/j.hlc.2014.11.018
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finding a clinically practical and efficacious agent to limit this post-MI related injury. Isoproterenol (ISO) is a potent mixed beta-adrenergic agonist that at high acute dose has been reported to cause severe myocardial stress and induce infarct-like necrosis [4]. Isoproterenol-induced MI in a rat model replicates the acute myocardial necrosis, which is followed by increased release of cardiac enzymes, accumulation of lipid peroxidases, and impaired cardiac function [5]. The pathophysiological and morphologic alterations in this model mimic those of human MI [6]. Thus, to evaluate MI originating from oxidative stress, we considered the ISO-induced MI to be the most suitable. Hydrogen gas (H2) has been used to prevent decompression sickness [7]. In 2007, Ohsawa et al. found that hydrogen could be used as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals [8]. Several other studies have shown that H2 had therapeutic antioxidant activity and fought against organ damage, such as neonatal cerebral hypoxia-ischaemia, pulmonary hypertension, lung, hepatic or myocardial injury induced by ischaemia/reperfusion [9–13]. Taking convenience and safety into account, we used hydrogen-rich saline instead of hydrogen gas. This study was therefore designed to explore possible pharmacological effects, as well as the mechanism of hydrogen-rich saline, on ISO-induced MI in rats.
Materials and Methods Animals Male Wistar rats, weighing 220 20 g, were supplied by the Experimental Animal Center of Shandong University of Traditional Chinese Medicine (Shandong, China). Rats were acclimated for five days before any operation. All rats received humane care according to the Chinese Academy of Science’s Guide for the Care and Use of Laboratory Animals.
Drugs and Materials The method for the preparation of hydrogen-rich saline was described previously [14]. Briefly, hydrogen was dissolved in physiological saline for six hours under high pressure of 0.4 megapascal (MPa) to the supersaturated level. The saturated hydrogen saline was stored in an aluminum bag with no dead volume at 4 8C under atmospheric pressure. To ensure the hydrogen concentration of more than 0.6 mM, hydrogen-rich saline was made every week. Isoproterenol (ISO) was obtained from Sigma-Aldrich (Sigma, St. Louis, MO, USA). Malondialdehyde (MDA), superoxide dismutase (SOD), aspartate aminotransferase (AST), Na+-K+ATPase and Ca2+-ATPase assay reagents were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Interleukin-6 (IL-6), 8-hydroxy-desoxyguanosine (8-OHdG) and tumour necrosis factor-a (TNF-a) enzymelinked immunosorbent assay (ELISA) kits were obtained from Shanghai Bluegene Biotech Co., Ltd. (Shanghai, China). Creatine kinase isoenzyme (CK-MB) ELISA kits
were from R&D systems (Beijing, China). All the other chemicals used were of analytical grade.
Experimental Design Rats were divided randomly into six groups: Group 1 (n = 14): normal control rats pretreated with physiological saline intraperitoneally [i.p.] before physiological saline was given subcutaneously; Group 2 (n = 22): MI group in which rats pretreated with physiological saline i.p. before ISO was administered; Groups 3 to 5 (n = 22 in each group): rats pretreated with hydrogen-rich saline (5, 7.5, and 10 mL/kg body weight i.p.) before ISO was administered. Group 6 (n = 22): rats pretreated with Vitamin C (250 mg/kg body weight intragastrically [i.g.]) [15] before ISO was administered. ISO was given twice subcutaneously at an interval of 24 hours at a dose of 200 mg/kg body weight in 1 mL 0.9% saline [16,17], and physiological saline was given in the same way in group 1. Haemodynamic studies were performed before animals were sacrificed and cardiac tissues were collected from eight rats in each group for measurements of infarct area. Hearts from an additional six animals in each group were used for histopathological examination, and eight animals were used for preparation of homogenate. Infarct area was not measured in the normal control group, thus all 14 control animals were assessed for other measures. All animal experiments were approved by the Animal Ethics Committee of the Taishan Medical University.
Haemodynamic Studies At the end of the experiment (12 h after the second injection of ISO) [17], rats were anaesthetised i.p. with 1% pentobarbital sodium (40 mg/kg), and placed in the supine position. A PE-50 polyethylene tube was advanced into the left ventricle through the right carotid artery. Haemodynamic parameters, including left ventricular systolic pressure (LVSP), the maximal rate of pressure rise (+dP/dt max) and pressure fall (-dP/dt max) of the left ventricular, and left ventricle end-diastolic pressure (LVEDP) were recorded on an MP150 system (BIOPAC, USA).
Measurement of Infarct Area A triphenyl tetrazolium chloride (TTC) test was applied to evaluate infarct size [18]. After haemodynamic studies, the heart was removed, washed in cold saline to eliminate excess blood, trimmed of excess epicardial fat, sliced transversely into 2 mm thick sections, stained with 1% TTC solution, and scanned; the percentage of infarct size was calculated by dividing the infarct (white) area by the total (white and red) area using the Image-Pro1 Plus Version 6.0.
Determination of Biochemical Parameters, Enzymes, and Cytokines Blood was taken from the abdominal aorta for preparation of serum. Heart homogenate was prepared using cardiac apex
Please cite this article in press as: Jing L, et al. Cardioprotective Effect of Hydrogen-rich Saline on Isoproterenol-induced Myocardial Infarction in Rats. Heart, Lung and Circulation (2014), http://dx.doi.org/10.1016/j.hlc.2014.11.018
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tissues and was centrifuged. Protein concentration in the supernatant was determined using the Bradford method. Levels of CK-MB, AST, IL-6 and TNF-a in serum, MDA, SOD and 8-OHdG in serum and cardiac tissues, and Na+-K+-ATPase and Ca2+-ATPase activities in heart tissues, were measured according to the manufacturers’ instructions.
Histopathological Examination The cardiac apex tissues obtained from different groups were washed with cold saline, fixed in 10% buffered formalin solution, embedded in paraffin, sectioned and stained with haematoxylin and eosin (H & E). The sections were viewed under a high power microscope (200 ) and photomicrographs were taken.
Statistical Analysis Statistical analysis was carried out by a one-way analysis of variance (ANOVA) using SPSS software package version 11.5. Results were expressed as mean S.D. A P-value of less than 0.05 was considered statistically significant.
Results Hydrogen-rich Saline Diminished Infarct Area and Lowered Serum CK-MB and AST Levels Photographs (Fig. 1A) showed a large infarct area in the ISOtreated (MI) group. Groups pretreated with hydrogen-rich saline (5, 7.5, or 10 mL/kg) had reduced infarct size compared to the MI group. The percentage of infarct size is shown in Figure 1B. Figures 1C and 1D show the activity of CK-MB and AST in the serum of normal and experimental rats. The activity of these enzymes was enhanced significantly in ISO-treated rats. Pretreatment with hydrogen-rich saline at doses of 5, 7.5, or 10 mL/kg showed decreased CK-MB and AST activity.
Hydrogen-rich Saline Improved Cardiac Function Compared to the control group, ISO treatment led to left ventricular dysfunction, which presented an increase in
Figure 1 Hydrogen-rich saline reduced the infarct area and CK-MB and AST levels in serum. (A) Representative photographs of heart tissue in different groups stained with triphenyl tetrazolium chloride (TTC). The heart was sliced transversely into sequential sections and stained with TTC. White and red areas represent infarct and noninfarct areas respectively. (B) Infarct size (%). (C) and (D) Changes in serum CK-MB and AST levels. Control = normal control group; MI = ISO-treated group; 5, 7.5, and 10 = hydrogen-rich saline (5, 7.5, and 10 mL/kg body weight i.p.) pretreated group; Vc = Vitamin C pretreated group. AST = aspartate aminotransferase; CK-MB = creatine kinase isoenzyme. ~~P < 0.01 versus control; *P < 0.05, **P < 0.01 versus MI; ## P < 0.01 versus Vc.
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LVEDP, a decrease in LVSP, and a steep decline in +dP/dt max and -dP/dt max (Fig. 2). The elevation in LVEDP and decreases in LVSP, +dP/dt max, and -dP/dt max were partially or fully reversed by pretreatment with hydrogen-rich saline (Fig. 2).
Effect of Hydrogen-rich Saline on SOD Activity, 8-OHdG and MDA Concentrations in Serum and Cardiac Tissue ISO-treated rats showed higher concentrations of 8-OHdG and MDA and lower SOD activity in the serum and cardiac tissue when compared to the control group. After hydrogen-rich saline pretreatment, 8-OHdG and MDA levels decreased and SOD activity increased compared to the MI group (Fig. 3).
Hydrogen-rich Saline Reduced IL-6 and TNF-a Levels in Serum Results showed that the serum levels of IL-6 and TNF-a in the MI group significantly increased compared to the control
group (Fig. 4). Hydrogen-rich saline pretreatment reduced the increases in IL-6 and TNF-a.
Effects of Hydrogen-rich Saline on Ca2+-ATPase and Na+-K+-ATPase Activity in Cardiac Tissue The activity of two kinds of membrane-bound enzymes, including Ca2+-ATPase and Na+-K+-ATPase in the heart, are shown in Fig. 5. The activity of Na+-K+-ATPase decreased, and Ca2+-ATPase increased markedly in the heart of ISO-administered rats. Hydrogen-rich saline pretreatment at different doses significantly increased Na+-K+-ATPase activity, and decreased Ca2+-ATPase activity.
Histopathological Examination Histopathological findings of control myocardium showed normal cardiac fibres and no inflammatory cell infiltration (Fig. 6A). ISO caused the formation of infarction areas, characterised by disordered cardiac muscle fibres and infiltration of inflammatory cells (Fig. 6B). However, after pretreatment
Figure 2 Effect of hydrogen-rich saline on cardiac function. (A) LVSP. (B) LVEDP. (C) +dP/dt max. (D) -dP/dt max. Control = normal control group; MI = ISO-treated group; 5, 7.5, and 10 = hydrogen-rich saline (5, 7.5, and 10 mL/kg body weight i.p.) pretreated group; Vc = Vitamin C pretreated group. LVSP = left ventricular systolic pressure; LVEDP = left ventricular end diastolic pressure; +dP/dt max = maximal rate of pressure rise; -dP/dt max = maximal rate of pressure fall. ~~ P < 0.01 versus control; *P < 0.05, **P < 0.01 versus MI; ## P < 0.01 versus Vc.
Please cite this article in press as: Jing L, et al. Cardioprotective Effect of Hydrogen-rich Saline on Isoproterenol-induced Myocardial Infarction in Rats. Heart, Lung and Circulation (2014), http://dx.doi.org/10.1016/j.hlc.2014.11.018
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Figure 3 Effects of hydrogen-rich saline on SOD activity, MDA, and 8-OHdG concentrations in serum and cardiac tissue. Levels of MDA (A and B), 8-OHdG (C and D), and SOD activity (E and F) in serum and cardiac tissue. Control = normal control group; MI = ISO-treated group; 5, 7.5, and 10 = hydrogen-rich saline (5, 7.5, and 10 mL/kg body weight i.p.) pretreated group; Vc = Vitamin C pretreated group. MDA = malondialdehyde; 8-OHdG = 8-hydroxy-desoxyguanosine; SOD = superoxide dismutase. ~P < 0.05, ~~P < 0.01 versus control; *P < 0.05, **P < 0.01 versus MI; # P < 0.05 versus Vc.
with hydrogen-rich saline, these pathological changes were ameliorated (Fig. 6C-Fig. 6E).
Discussion This study is the first to demonstrate the protective effects of hydrogen-rich saline on ISO-induced MI in rats. The results suggest that hydrogen-rich saline exerts strong cardioprotective effects against ISO-induced MI in rats. This is supported by the results of haemodynamic studies, histological findings and measurements of biochemical parameters, enzymes, and cytokines. In addition, we used Vitamin C as a positive
control drug in this study due to its multiple antioxidant properties[15,19,20], and found that the effect of hydrogenrich saline (7.5 or 10 mL/kg body weight) was similar to or better than that of Vitamin C. In our study, the diagnosis of a successful model was dependent upon obvious infarct size of the myocardium, histopathological changes of the myocardium, impaired left ventricular function, and increased serum levels of myocardial marker enzymes [1]. Many enzymes contained in the myocardium are considered diagnostic markers for MI because they leak from intracellular into the extracellular fluid when the myocardium is metabolically damaged [21]. Therefore, changes in membrane integrity or permeability may be reflected by diversities
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Figure 4 Effects of hydrogen-rich saline on TNF-a and IL-6 levels in serum. (A) Level of TNF-a in serum. (B) IL-6 level in serum. Control = normal control group; MI = ISO-treated group; 5, 7.5, and 10 = hydrogen-rich saline (5, 7.5, and 10 mL/kg body weight i.p.) pretreated group; Vc = Vitamin C pretreated group. TNF-a = tumour necrosis factor-a; IL-6 = interleukin-6. ~~ P < 0.01 versus control; *P < 0.05, **P < 0.01 versus MI; ## P < 0.01 versus Vc.
in the levels of these enzymes in serum. In our study, the serum levels of AST and CK-MB were elevated significantly in ISOtreated rats, which was similar to previous reports [1]. Pretreatment with hydrogen-rich saline significantly lowered levels of these diagnostic marker enzymes in serum, and decreased infarct size, which suggests that hydrogen-rich saline can maintain membrane integrity, thereby reducing enzyme leakage. It has been confirmed that supramaximal dosages of ISO lead to ventricular dysfunction, and that it may be prevented by antioxidants [22]. We also used other haemodynamic parameters to fully appraise left ventricular function [6]. The present study demonstrates that hydrogen-rich saline improves ISO-induced left ventricular dysfunction, supported by increased LVSP, +dP/dt max, -dP/dt max, and lower LVEDP.
There also is proof that administration of large doses of ISO causes severe oxidative stress [23]. Overproduction of ROS induces lipid peroxidation and DNA damage, and leads to tissue injury [24]. MDA is a stable ultimate product of lipid peroxidation, and its increased content indicates activation of lipid peroxidation, which results in cardiac damage in ISOtreated rats [25]. 8-Hydroxy- deoxyguanosine is produced when ROS causes DNA oxidative damage, and its level cannot be affected by diet or cell renewal. Therefore, 8-OHdG is considered a biomarker to evaluate the degree of DNA oxidative damage and the level of oxidative stress [26]. SOD catalyses the dismutation of free radicals into harmless substances in the body, and has been regarded as an important cell defense enzyme. In this study, ISO induced increased levels of 8-OHdG and MDA, and decreased SOD activity in cardiac tissue and serum. However, hydrogen-rich saline
Figure 5 Effects of hydrogen-rich saline on Ca2+-ATPase and Na+-K+-ATPase in cardiac tissue. (A) Ca2+-ATPase. (B) Na+K+-ATPase. Control = normal control group; MI = ISO-treated group; 5, 7.5, and 10 = hydrogen-rich saline (5, 7.5, and 10 mL/kg body weight i.p.) pretreated group; Vc = Vitamin C pretreated group. ~~P < 0.01 versus control; *P < 0.05, ** P < 0.01 versus MI; # P < 0.05, ## P < 0.01 versus Vc.
Please cite this article in press as: Jing L, et al. Cardioprotective Effect of Hydrogen-rich Saline on Isoproterenol-induced Myocardial Infarction in Rats. Heart, Lung and Circulation (2014), http://dx.doi.org/10.1016/j.hlc.2014.11.018
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Figure 6 Representative photomicrographs of left ventricular sections (stained with haematoxylin and eosin, 200 ). Bar indicates 100 mm. (A) Control group shows normal cardiac fibres and no inflammatory cell infiltration. (B) MI group showed marked infarction with splitting of cardiac muscle fibers and inflammatory cells indicated by the arrow. (C-E) Hydrogen-rich saline (5, 7.5, and 10 mL/kg body weight i.p.) pretreated groups and (F) Vc pretreated group showed ameliorated pathological changes indicated by the arrow.
pretreatment lowered 8-OHdG and MDA content and enhanced SOD activity, which is in accordance with its antioxidant effect. In the infarcted myocardium and surrounding region, inflammatory cytokines, including IL-6 and TNF-a, are produced as a result of host reaction and display a magnifying effect on the inflammatory response [27]. ROS promotes formation of inflammatory cytokines, and the latter further stimulate the generation of ROS [28]. Levels of IL-6 and TNF-a in serum were increased significantly in the ISO-treated group,
while these cytokines were down-regulated by hydrogen-rich saline. These results demonstrate that hydrogen-rich saline may exert its protective effects on ISO-induced MI, at least in part, by regulating some cytokines. Moreover, this further demonstrates the anti-inflammatory activity of hydrogen-rich saline, consistent with previous reports [29]. As members of membrane-bound enzymes, Ca2+-ATPase and Na+-K+-ATPase play an important role in sustaining normal transmembrane ionic distribution. Any change in the character of these ion pumps may influence the heart
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function and be involved in the pathological process of ischaemic lesions [30]. In our ISO-treated hearts, Na+-K+ATPase activity decreased and Ca2+-ATPase activity increased. Na+-K+-ATPase is a lipid-dependent enzyme, which contains a ‘‘SH’’ group. Protein oxidation induced by excess lipid peroxidation may make this enzyme inactive [31]. In the myocardium, suppression of Na+-K+-ATPase promotes Na+ and Ca2+ cation exchange, which influences cellular calcium levels and Ca2+-ATPase activity [32]. Our results indicate that the regulatory effect of hydrogen-rich saline on Na+-K+-ATPase and Ca2+-ATPase activity may be achieved by protecting the ‘‘SH’’ groups from oxidation and reducing lipid peroxidation. This indicates that hydrogenrich saline may display membrane-stabilising effects via its antioxidant activity. Histopathological examination found that ISO-induced disorders in myocardial structure included infarct areas with oedema and inflammatory cell infiltration. However, hydrogen-rich saline pretreatment ameliorated these pathological changes, which confirmed the cardioprotective role of hydrogen-rich saline. In conclusion, our results indicate the protective effects of hydrogen-rich saline on ISO-induced MI in rats, which may be related to its antioxidant and anti-inflammatory activities.
Conflict of interests The author(s) declare(s) that there is no conflict of interests to disclose regarding the publication of this article.
Acknowledgements This work was supported partly by the National Natural Science Foundation of China (81173061), the Excellent Young Research Award Fund of Shandong Province, China (BS2011YY059) and Science and Technology Project of Taian City (20113021).
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ORIGINAL ARTICLE
Heart, Lung and Circulation (2014) xx, 1–9 1443-9506/04/$36.00 http://dx.doi.org/10.1016/j.hlc.2014.11.018
Cardioprotective Effect of Hydrogen-rich Saline on Isoproterenol-induced Myocardial Infarction in Rats Lei Jing a,b,d,1, Yun Wang a,1, Xiao-Min Zhao a,b*, Bing Zhao a,b,e, Ji-Ju Han a, Shu-Cun Qin a, Xue-Jun Sun c a
Key Laboratory of Atherosclerosis in Universities of Shandong (Taishan Medical University), Taian, 271000, China Department of Pharmocology, Taishan Medical University, Taian, 271000, China Department of Diving Medicine, the Second Military Medical University, Shanghai, 200433, China d Department of Pharmacy, Dongping County People’s Hospital, Dongping, 271500, China e Department of Pharmacy, Boshan District Hospital of Zibo, Boshan, 255200, China b c
Received 30 July 2014; received in revised form 18 November 2014; accepted 24 November 2014; online published-ahead-of-print xxx
Background
Infusion with hydrogen gas-saturated saline has recently been reported to exert antioxidant and antiinflammatory activity that may protect against organ damage induced by oxidative stress. Therefore because oxidative stress plays a significant role in the pathophysiology of myocardial infarction (MI), the aim of our study was to investigate whether hydrogen-rich saline has cardioprotective effects against isoproterenol-induced MI in rats.
Methods
An acute MI model was induced in male Wistar rats by subcutaneous injection of isoproterenol. Different doses of hydrogen-rich saline (5, 7.5, and 10 mL/kg body weight i.p.) or Vitamin C (250 mg/kg body weight i.g.) were administered to the rats. Oxidative stress indices including levels of myocardial marker enzymes, inflammatory cytokines, membrane-bound myocardial enzymes and histopathological changes were measured.
Results
Compared with those in isoproterenol-MI group, hydrogen-rich saline decreased malondialdehyde and 8-hydroxy-desoxyguanosine concentrations, enhanced superoxide dismutase and Na+-K+-ATPase activity, lowered Ca2+-ATPase activity and decreased interleukin-6 and tumour necrosis factor-a levels in the serum and/or cardiac tissue of rats. Hydrogen-rich saline pretreatment also diminished infarct size, improved left heart function, and ameliorated pathological changes of the left heart.
Conclusion
From these results, hydrogen-rich saline exerts cardiovascular protective effects against isoproterenolinduced MI at least in part via interactions which evoke antioxidant and anti-inflammatory activities.
Keywords
Hydrogen Inflammation Isoproterenol Myocardial infarction Oxidative stress
Introduction As an acute condition, myocardial infarction (MI) occurs as the result of unbalanced coronary artery supply and myocardial demand [1]. It is still the main cause of death worldwide, although therapeutic modalities and clinical care have
improved [2]. Extensive previous studies have shown that the production of toxic, reactive oxygen species (ROS), such as hydrogen peroxide, hydroxyl radicals and superoxide radicals in ischaemic tissue, induce damage to myocardial cells, leading to oxidative damage to membrane lipids, proteins and DNA [3]. To date the major challenge remains in
*Corresponding author at: Key Laboratory of Atherosclerosis in Universities of Shandong, Taishan Medical University, Taian, 271000, China. Tel.: +86 538 6225275; fax: +86 538 6225275, Email: [email protected] 1
These two authors contributed equally to the study.
© 2014 Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ). Published by Elsevier Inc. All rights reserved.
Please cite this article in press as: Jing L, et al. Cardioprotective Effect of Hydrogen-rich Saline on Isoproterenol-induced Myocardial Infarction in Rats. Heart, Lung and Circulation (2014), http://dx.doi.org/10.1016/j.hlc.2014.11.018
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finding a clinically practical and efficacious agent to limit this post-MI related injury. Isoproterenol (ISO) is a potent mixed beta-adrenergic agonist that at high acute dose has been reported to cause severe myocardial stress and induce infarct-like necrosis [4]. Isoproterenol-induced MI in a rat model replicates the acute myocardial necrosis, which is followed by increased release of cardiac enzymes, accumulation of lipid peroxidases, and impaired cardiac function [5]. The pathophysiological and morphologic alterations in this model mimic those of human MI [6]. Thus, to evaluate MI originating from oxidative stress, we considered the ISO-induced MI to be the most suitable. Hydrogen gas (H2) has been used to prevent decompression sickness [7]. In 2007, Ohsawa et al. found that hydrogen could be used as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals [8]. Several other studies have shown that H2 had therapeutic antioxidant activity and fought against organ damage, such as neonatal cerebral hypoxia-ischaemia, pulmonary hypertension, lung, hepatic or myocardial injury induced by ischaemia/reperfusion [9–13]. Taking convenience and safety into account, we used hydrogen-rich saline instead of hydrogen gas. This study was therefore designed to explore possible pharmacological effects, as well as the mechanism of hydrogen-rich saline, on ISO-induced MI in rats.
Materials and Methods Animals Male Wistar rats, weighing 220 20 g, were supplied by the Experimental Animal Center of Shandong University of Traditional Chinese Medicine (Shandong, China). Rats were acclimated for five days before any operation. All rats received humane care according to the Chinese Academy of Science’s Guide for the Care and Use of Laboratory Animals.
Drugs and Materials The method for the preparation of hydrogen-rich saline was described previously [14]. Briefly, hydrogen was dissolved in physiological saline for six hours under high pressure of 0.4 megapascal (MPa) to the supersaturated level. The saturated hydrogen saline was stored in an aluminum bag with no dead volume at 4 8C under atmospheric pressure. To ensure the hydrogen concentration of more than 0.6 mM, hydrogen-rich saline was made every week. Isoproterenol (ISO) was obtained from Sigma-Aldrich (Sigma, St. Louis, MO, USA). Malondialdehyde (MDA), superoxide dismutase (SOD), aspartate aminotransferase (AST), Na+-K+ATPase and Ca2+-ATPase assay reagents were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Interleukin-6 (IL-6), 8-hydroxy-desoxyguanosine (8-OHdG) and tumour necrosis factor-a (TNF-a) enzymelinked immunosorbent assay (ELISA) kits were obtained from Shanghai Bluegene Biotech Co., Ltd. (Shanghai, China). Creatine kinase isoenzyme (CK-MB) ELISA kits
were from R&D systems (Beijing, China). All the other chemicals used were of analytical grade.
Experimental Design Rats were divided randomly into six groups: Group 1 (n = 14): normal control rats pretreated with physiological saline intraperitoneally [i.p.] before physiological saline was given subcutaneously; Group 2 (n = 22): MI group in which rats pretreated with physiological saline i.p. before ISO was administered; Groups 3 to 5 (n = 22 in each group): rats pretreated with hydrogen-rich saline (5, 7.5, and 10 mL/kg body weight i.p.) before ISO was administered. Group 6 (n = 22): rats pretreated with Vitamin C (250 mg/kg body weight intragastrically [i.g.]) [15] before ISO was administered. ISO was given twice subcutaneously at an interval of 24 hours at a dose of 200 mg/kg body weight in 1 mL 0.9% saline [16,17], and physiological saline was given in the same way in group 1. Haemodynamic studies were performed before animals were sacrificed and cardiac tissues were collected from eight rats in each group for measurements of infarct area. Hearts from an additional six animals in each group were used for histopathological examination, and eight animals were used for preparation of homogenate. Infarct area was not measured in the normal control group, thus all 14 control animals were assessed for other measures. All animal experiments were approved by the Animal Ethics Committee of the Taishan Medical University.
Haemodynamic Studies At the end of the experiment (12 h after the second injection of ISO) [17], rats were anaesthetised i.p. with 1% pentobarbital sodium (40 mg/kg), and placed in the supine position. A PE-50 polyethylene tube was advanced into the left ventricle through the right carotid artery. Haemodynamic parameters, including left ventricular systolic pressure (LVSP), the maximal rate of pressure rise (+dP/dt max) and pressure fall (-dP/dt max) of the left ventricular, and left ventricle end-diastolic pressure (LVEDP) were recorded on an MP150 system (BIOPAC, USA).
Measurement of Infarct Area A triphenyl tetrazolium chloride (TTC) test was applied to evaluate infarct size [18]. After haemodynamic studies, the heart was removed, washed in cold saline to eliminate excess blood, trimmed of excess epicardial fat, sliced transversely into 2 mm thick sections, stained with 1% TTC solution, and scanned; the percentage of infarct size was calculated by dividing the infarct (white) area by the total (white and red) area using the Image-Pro1 Plus Version 6.0.
Determination of Biochemical Parameters, Enzymes, and Cytokines Blood was taken from the abdominal aorta for preparation of serum. Heart homogenate was prepared using cardiac apex
Please cite this article in press as: Jing L, et al. Cardioprotective Effect of Hydrogen-rich Saline on Isoproterenol-induced Myocardial Infarction in Rats. Heart, Lung and Circulation (2014), http://dx.doi.org/10.1016/j.hlc.2014.11.018
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tissues and was centrifuged. Protein concentration in the supernatant was determined using the Bradford method. Levels of CK-MB, AST, IL-6 and TNF-a in serum, MDA, SOD and 8-OHdG in serum and cardiac tissues, and Na+-K+-ATPase and Ca2+-ATPase activities in heart tissues, were measured according to the manufacturers’ instructions.
Histopathological Examination The cardiac apex tissues obtained from different groups were washed with cold saline, fixed in 10% buffered formalin solution, embedded in paraffin, sectioned and stained with haematoxylin and eosin (H & E). The sections were viewed under a high power microscope (200 ) and photomicrographs were taken.
Statistical Analysis Statistical analysis was carried out by a one-way analysis of variance (ANOVA) using SPSS software package version 11.5. Results were expressed as mean S.D. A P-value of less than 0.05 was considered statistically significant.
Results Hydrogen-rich Saline Diminished Infarct Area and Lowered Serum CK-MB and AST Levels Photographs (Fig. 1A) showed a large infarct area in the ISOtreated (MI) group. Groups pretreated with hydrogen-rich saline (5, 7.5, or 10 mL/kg) had reduced infarct size compared to the MI group. The percentage of infarct size is shown in Figure 1B. Figures 1C and 1D show the activity of CK-MB and AST in the serum of normal and experimental rats. The activity of these enzymes was enhanced significantly in ISO-treated rats. Pretreatment with hydrogen-rich saline at doses of 5, 7.5, or 10 mL/kg showed decreased CK-MB and AST activity.
Hydrogen-rich Saline Improved Cardiac Function Compared to the control group, ISO treatment led to left ventricular dysfunction, which presented an increase in
Figure 1 Hydrogen-rich saline reduced the infarct area and CK-MB and AST levels in serum. (A) Representative photographs of heart tissue in different groups stained with triphenyl tetrazolium chloride (TTC). The heart was sliced transversely into sequential sections and stained with TTC. White and red areas represent infarct and noninfarct areas respectively. (B) Infarct size (%). (C) and (D) Changes in serum CK-MB and AST levels. Control = normal control group; MI = ISO-treated group; 5, 7.5, and 10 = hydrogen-rich saline (5, 7.5, and 10 mL/kg body weight i.p.) pretreated group; Vc = Vitamin C pretreated group. AST = aspartate aminotransferase; CK-MB = creatine kinase isoenzyme. ~~P < 0.01 versus control; *P < 0.05, **P < 0.01 versus MI; ## P < 0.01 versus Vc.
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LVEDP, a decrease in LVSP, and a steep decline in +dP/dt max and -dP/dt max (Fig. 2). The elevation in LVEDP and decreases in LVSP, +dP/dt max, and -dP/dt max were partially or fully reversed by pretreatment with hydrogen-rich saline (Fig. 2).
Effect of Hydrogen-rich Saline on SOD Activity, 8-OHdG and MDA Concentrations in Serum and Cardiac Tissue ISO-treated rats showed higher concentrations of 8-OHdG and MDA and lower SOD activity in the serum and cardiac tissue when compared to the control group. After hydrogen-rich saline pretreatment, 8-OHdG and MDA levels decreased and SOD activity increased compared to the MI group (Fig. 3).
Hydrogen-rich Saline Reduced IL-6 and TNF-a Levels in Serum Results showed that the serum levels of IL-6 and TNF-a in the MI group significantly increased compared to the control
group (Fig. 4). Hydrogen-rich saline pretreatment reduced the increases in IL-6 and TNF-a.
Effects of Hydrogen-rich Saline on Ca2+-ATPase and Na+-K+-ATPase Activity in Cardiac Tissue The activity of two kinds of membrane-bound enzymes, including Ca2+-ATPase and Na+-K+-ATPase in the heart, are shown in Fig. 5. The activity of Na+-K+-ATPase decreased, and Ca2+-ATPase increased markedly in the heart of ISO-administered rats. Hydrogen-rich saline pretreatment at different doses significantly increased Na+-K+-ATPase activity, and decreased Ca2+-ATPase activity.
Histopathological Examination Histopathological findings of control myocardium showed normal cardiac fibres and no inflammatory cell infiltration (Fig. 6A). ISO caused the formation of infarction areas, characterised by disordered cardiac muscle fibres and infiltration of inflammatory cells (Fig. 6B). However, after pretreatment
Figure 2 Effect of hydrogen-rich saline on cardiac function. (A) LVSP. (B) LVEDP. (C) +dP/dt max. (D) -dP/dt max. Control = normal control group; MI = ISO-treated group; 5, 7.5, and 10 = hydrogen-rich saline (5, 7.5, and 10 mL/kg body weight i.p.) pretreated group; Vc = Vitamin C pretreated group. LVSP = left ventricular systolic pressure; LVEDP = left ventricular end diastolic pressure; +dP/dt max = maximal rate of pressure rise; -dP/dt max = maximal rate of pressure fall. ~~ P < 0.01 versus control; *P < 0.05, **P < 0.01 versus MI; ## P < 0.01 versus Vc.
Please cite this article in press as: Jing L, et al. Cardioprotective Effect of Hydrogen-rich Saline on Isoproterenol-induced Myocardial Infarction in Rats. Heart, Lung and Circulation (2014), http://dx.doi.org/10.1016/j.hlc.2014.11.018
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Figure 3 Effects of hydrogen-rich saline on SOD activity, MDA, and 8-OHdG concentrations in serum and cardiac tissue. Levels of MDA (A and B), 8-OHdG (C and D), and SOD activity (E and F) in serum and cardiac tissue. Control = normal control group; MI = ISO-treated group; 5, 7.5, and 10 = hydrogen-rich saline (5, 7.5, and 10 mL/kg body weight i.p.) pretreated group; Vc = Vitamin C pretreated group. MDA = malondialdehyde; 8-OHdG = 8-hydroxy-desoxyguanosine; SOD = superoxide dismutase. ~P < 0.05, ~~P < 0.01 versus control; *P < 0.05, **P < 0.01 versus MI; # P < 0.05 versus Vc.
with hydrogen-rich saline, these pathological changes were ameliorated (Fig. 6C-Fig. 6E).
Discussion This study is the first to demonstrate the protective effects of hydrogen-rich saline on ISO-induced MI in rats. The results suggest that hydrogen-rich saline exerts strong cardioprotective effects against ISO-induced MI in rats. This is supported by the results of haemodynamic studies, histological findings and measurements of biochemical parameters, enzymes, and cytokines. In addition, we used Vitamin C as a positive
control drug in this study due to its multiple antioxidant properties[15,19,20], and found that the effect of hydrogenrich saline (7.5 or 10 mL/kg body weight) was similar to or better than that of Vitamin C. In our study, the diagnosis of a successful model was dependent upon obvious infarct size of the myocardium, histopathological changes of the myocardium, impaired left ventricular function, and increased serum levels of myocardial marker enzymes [1]. Many enzymes contained in the myocardium are considered diagnostic markers for MI because they leak from intracellular into the extracellular fluid when the myocardium is metabolically damaged [21]. Therefore, changes in membrane integrity or permeability may be reflected by diversities
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Figure 4 Effects of hydrogen-rich saline on TNF-a and IL-6 levels in serum. (A) Level of TNF-a in serum. (B) IL-6 level in serum. Control = normal control group; MI = ISO-treated group; 5, 7.5, and 10 = hydrogen-rich saline (5, 7.5, and 10 mL/kg body weight i.p.) pretreated group; Vc = Vitamin C pretreated group. TNF-a = tumour necrosis factor-a; IL-6 = interleukin-6. ~~ P < 0.01 versus control; *P < 0.05, **P < 0.01 versus MI; ## P < 0.01 versus Vc.
in the levels of these enzymes in serum. In our study, the serum levels of AST and CK-MB were elevated significantly in ISOtreated rats, which was similar to previous reports [1]. Pretreatment with hydrogen-rich saline significantly lowered levels of these diagnostic marker enzymes in serum, and decreased infarct size, which suggests that hydrogen-rich saline can maintain membrane integrity, thereby reducing enzyme leakage. It has been confirmed that supramaximal dosages of ISO lead to ventricular dysfunction, and that it may be prevented by antioxidants [22]. We also used other haemodynamic parameters to fully appraise left ventricular function [6]. The present study demonstrates that hydrogen-rich saline improves ISO-induced left ventricular dysfunction, supported by increased LVSP, +dP/dt max, -dP/dt max, and lower LVEDP.
There also is proof that administration of large doses of ISO causes severe oxidative stress [23]. Overproduction of ROS induces lipid peroxidation and DNA damage, and leads to tissue injury [24]. MDA is a stable ultimate product of lipid peroxidation, and its increased content indicates activation of lipid peroxidation, which results in cardiac damage in ISOtreated rats [25]. 8-Hydroxy- deoxyguanosine is produced when ROS causes DNA oxidative damage, and its level cannot be affected by diet or cell renewal. Therefore, 8-OHdG is considered a biomarker to evaluate the degree of DNA oxidative damage and the level of oxidative stress [26]. SOD catalyses the dismutation of free radicals into harmless substances in the body, and has been regarded as an important cell defense enzyme. In this study, ISO induced increased levels of 8-OHdG and MDA, and decreased SOD activity in cardiac tissue and serum. However, hydrogen-rich saline
Figure 5 Effects of hydrogen-rich saline on Ca2+-ATPase and Na+-K+-ATPase in cardiac tissue. (A) Ca2+-ATPase. (B) Na+K+-ATPase. Control = normal control group; MI = ISO-treated group; 5, 7.5, and 10 = hydrogen-rich saline (5, 7.5, and 10 mL/kg body weight i.p.) pretreated group; Vc = Vitamin C pretreated group. ~~P < 0.01 versus control; *P < 0.05, ** P < 0.01 versus MI; # P < 0.05, ## P < 0.01 versus Vc.
Please cite this article in press as: Jing L, et al. Cardioprotective Effect of Hydrogen-rich Saline on Isoproterenol-induced Myocardial Infarction in Rats. Heart, Lung and Circulation (2014), http://dx.doi.org/10.1016/j.hlc.2014.11.018
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Figure 6 Representative photomicrographs of left ventricular sections (stained with haematoxylin and eosin, 200 ). Bar indicates 100 mm. (A) Control group shows normal cardiac fibres and no inflammatory cell infiltration. (B) MI group showed marked infarction with splitting of cardiac muscle fibers and inflammatory cells indicated by the arrow. (C-E) Hydrogen-rich saline (5, 7.5, and 10 mL/kg body weight i.p.) pretreated groups and (F) Vc pretreated group showed ameliorated pathological changes indicated by the arrow.
pretreatment lowered 8-OHdG and MDA content and enhanced SOD activity, which is in accordance with its antioxidant effect. In the infarcted myocardium and surrounding region, inflammatory cytokines, including IL-6 and TNF-a, are produced as a result of host reaction and display a magnifying effect on the inflammatory response [27]. ROS promotes formation of inflammatory cytokines, and the latter further stimulate the generation of ROS [28]. Levels of IL-6 and TNF-a in serum were increased significantly in the ISO-treated group,
while these cytokines were down-regulated by hydrogen-rich saline. These results demonstrate that hydrogen-rich saline may exert its protective effects on ISO-induced MI, at least in part, by regulating some cytokines. Moreover, this further demonstrates the anti-inflammatory activity of hydrogen-rich saline, consistent with previous reports [29]. As members of membrane-bound enzymes, Ca2+-ATPase and Na+-K+-ATPase play an important role in sustaining normal transmembrane ionic distribution. Any change in the character of these ion pumps may influence the heart
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function and be involved in the pathological process of ischaemic lesions [30]. In our ISO-treated hearts, Na+-K+ATPase activity decreased and Ca2+-ATPase activity increased. Na+-K+-ATPase is a lipid-dependent enzyme, which contains a ‘‘SH’’ group. Protein oxidation induced by excess lipid peroxidation may make this enzyme inactive [31]. In the myocardium, suppression of Na+-K+-ATPase promotes Na+ and Ca2+ cation exchange, which influences cellular calcium levels and Ca2+-ATPase activity [32]. Our results indicate that the regulatory effect of hydrogen-rich saline on Na+-K+-ATPase and Ca2+-ATPase activity may be achieved by protecting the ‘‘SH’’ groups from oxidation and reducing lipid peroxidation. This indicates that hydrogenrich saline may display membrane-stabilising effects via its antioxidant activity. Histopathological examination found that ISO-induced disorders in myocardial structure included infarct areas with oedema and inflammatory cell infiltration. However, hydrogen-rich saline pretreatment ameliorated these pathological changes, which confirmed the cardioprotective role of hydrogen-rich saline. In conclusion, our results indicate the protective effects of hydrogen-rich saline on ISO-induced MI in rats, which may be related to its antioxidant and anti-inflammatory activities.
Conflict of interests The author(s) declare(s) that there is no conflict of interests to disclose regarding the publication of this article.
Acknowledgements This work was supported partly by the National Natural Science Foundation of China (81173061), the Excellent Young Research Award Fund of Shandong Province, China (BS2011YY059) and Science and Technology Project of Taian City (20113021).
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