Cardioprotective effect of total paeony glycosides against isoprenaline-induced myocardial ischemia in rats.
Total paeony glycosides
Paeoniae radix is a traditional Chinese medicinal herb for treating some diseases; important components are total paeony glycosides (TPGs), an approved drug by the State Food and Drug Administration (SFDA) for the therapy of rheumatoid arthritis (RA). We firstly reported myocardial benefits of TPGs previously, and the present study is to further investigate the underlying mechanisms for preventing oxidative damage in card iomyopathy. We measured the capacity of TPGs to scavenge free radicals in vitro. Then 60 SD rats were randomly divided into five groups: (1) a normal control group, (2) an isoprenaline (ISO)-induced myocardial ischemic model group, (3) a TPG treatment group (TPGs 269.4 mg/kg delivered by intragastric administration for 3 days before ISO administration and TPGs 449 mg/kg delivered for 3 days after ISO administration), (4) a TPG therapy group (TPGs 449 mg/kg delivered for 3 days after ISO administration), and (5) a positive control group (propranolol 15 mg/kg for 3 days after ISO administration). The ISO-induced myocardial ischemic model was established by subcutaneous injection of 1 mg/kg/8h ISO (2 times). The activities of myocardial enzymes, including glutamic oxaloacetic transaminase (GOT), creatine kinase (CK), lactate dehydrogenase (LDH), antioxidant enzyme superoxide dismutase (SOD) as well as the content of lipid peroxidation product malondialdehyde (MDA) were detected. We found that TPGs potently eliminated hydroxyl radicals and superoxide in vitro using ESR assays. Compared with model rats, TPG treatment, TPG therapy and the positive control treatment exhibited significantly reduced activities of GOT. LDH, and CK (p <0.01), increased activity of SOD (p <0.01) and lower levels of MDA (p < 0.05). More interestingly, the protective effect of TPG treatment was even better than that of propranolol. These results suggest that TPGs significantly ameliorate ISO-induced myocardial ischemia and their action might be through reducing oxidative stress in ischemic myocardium.
[c] 2012 Elsevier GmbH. All rights reserved.
Radix Paeoniae rubrae is a medicinal herb that has been used in China and other Asian countries for thousands of years for treating various diseases, including obesity and diabetes (Jiang et al. 2009; Su et al. 2010; Wu et al. 2009a, b; Zhang et al. 2009; Zheng et al. 2008), hepatitis, arthritis (Zheng and Wei 2005), artherosclerosis (Li et al. 2011; Chang et al. 2009), dementia, and traumatic injuries (Liu et al. 2005). Total paeony glycosides (TPGs), extracted from the root of Paeonia lacriflora pall, contain 96.2% of paeoniflorin (PF) and other components such as hydroxyl-paeoniflorin, paeonin, albiflorin, and benzoylpaeoniflorin (Wu 1985). TPGs were reported being having anti-allergic, anti-inflammatory (Chang et al. 2009), anti-oxidant (Su et al. 2010), and immunoregulatory effects (Jiang et al. 2009; Su et al. 2010; Zheng et al. 2008). In 1998, TPGs were approved by the State Food and Drug Administration (SFDA) to enter the market as a disease-modifying drug for rheumatoid arthritis (RA).
In a previous study, for the first time, we found that TPGs effectively protect against acute myocardial ischemia (AMI) by the regulation of cardiac enzymes and apoptosis (Mo et al. 2011). As oxidative stress is highly implicated in cardiomyoathy (Hagiwara et al. 2011), to further understand the antioxidative effect of TPGs in myocardial ischemia, in present study, we employed the isoprenaline (IS0)-induced animal model (Cheng et al. 2006), which displayed typical myocardinal ischemia. We observed that the activities of myocardial enzymes and oxidative stress were effectively ameliorated by TPG intervention in ISO-induced myocardial ischemia rats.
Materials and methods
TPG extract, chemicals and reagents
TPG extract was prepared from the dried peony plant as previously described (Pico et al. 2007). Qualitative and quantitative analyses of paeoniflorin, the major component of TPGs, were performed by RP-HPLC with UV detection (Wu et al. 2009a, b) and a UV spectrometer. As shown in Fig. 1, paeoniflorin is the most abundant glycoside in the extract (the retention time at 4.437 min) and the other minor components are oxypaeoniflorin (the retention time at 1.801 min), albiflorin (the retention at 2.403) and benzoyloxypaeoniflorin (the retention time at 7.116) as previously described (Mo et al. 2011 ). The stock solution contained 44.9 mg/ml of paeoniflorin in water. Isoprenaline hydrochloride (ISO-HCI) was obtained from Sigma. Glutamic oxaloacetic transaminase (GOT), creative kinase (a), lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) were obtained from Nanjing Jiancheng Biotechnology Institute (Nanjing, China).
In vitro free radical scavenging by TPGs
Hydroxyl, superoxide, and lipid radicals were detected using spin trapping agents with an electron spin resonance (ESR) specrometer with minor modifications (Long et al. 2009). Briefly, hydroxyl radicals were detected in a 50[micro]l reaction mixture containing 50 [micro]M [Cr.sup.2+], 100 mM [H.sub.2][0.sub.2], 10 mM NADPH and 100 mM DMPO. The reaction was initiated by [Cr.sup.2+] and then ESR spectra were recorded 5 min after reaction start.
For superoxide, ESR spectra were obtained in a reaction mixture containing 0.33 mM hypoxanthine, 0.83 mM DTPA, 0.15 M DMPO, 0.083 units/ml xanthine oxidase, and 20 [micro]l sample. Spectra were measured 3 min after addition of XOD to initiate the reaction.
Animals and ISO-induced myocardial ischemia in rats
Male Sprague Dawley rats (60 days old, 30 male and 30 female, weighing 200-250g) were bought from the experimental animal center of the Medical School, Xi'an jiaotong University (Xi'an, China). Protocols adhered to the Animal Care guidelines of Xi'an Jiaotong University. 60 SD rats were randomly divided into five groups, including normal controls, an ISO injury (ISO-induced myocardial ischemia) group, a TPG treatment group, a TPG therapy group and positive controls. The myocardial ischemic rat model was established by subcutaneous injection of 1 mg/kg/8 h ISO (2 times). The normal control group received water ad libitum, the ISO group received 10 mg/kg [H.sub.2]O by intragastric administration for 3 days after ISO injection, the TPG treatment group received 269.4 mg/kg TPGs by intragastric administration for 3 days before ISO injection and again received 449 mg/kg TPGs for 3 days following ISO injection. The TPG therapy group received 449 mg/kg TPGs for 3 days only after ISO administration. The positive control group received 15 mg/kg propranolol for 3 days after ISO injection.
Blood was obtained from the abdominal aorta of rats that had been anesthetized with diethyl ether at the end of 7 days of treatment. Serum was pipetted and saved at -20 C following centrifugation at 4 [degrees]C at 3000 r/min for 10 min. Serum was thawed and centrifuged at 4 [degrees]C at 3000 r/min for 5 min before assays.
Assays for enzyme activities
Activities of GOT, GK, LDH, MDA and SOD were measured using kits (Nanjing Jiancheng biotechnology institute, Nanjing, China) according to the manufacturer's instructions.
Data are presented as means [+ or -] SE. Statistical significance was determined by t-test. P values <0.05 were considered as being statistically significant.
TPG effects on free radical scavenging
TPGs close-dependently scavenged both hydroxyl radicals (Fig. 2A) and superoxide radicals (Fig. 2B). The 50% free radical scavenging concentrations ([IC.sub.50]) are 0.236, and 0.003892 for hydroxyl and superoxide, respectively. The free radical scavenging effects were compared with those of some well-known specific scavengers as shown in Table 1. The [IC.sub.50] of TPGs for hydroxyl is 92-fold lower than that of mannital, a well-known hydroxyl radical scavenger, but they are less potent than SOD for superoxide radicals (with 16.9% the efficiency of SOD).
Table 1 Comparison of the 50% free radical scavenging concentration of TPGs with previously known specific scavengers (mg/ml) Radicals TPG SOD Mannitol Hydroxyl 0.236 21.84 Superoxide 0.003893 0.000657 Hydroxyl and superoxide were detected using spin trapping agents with an ESR spectrometer. The half-maximal inhibitory concentrations ([IC.sub.50]) of TPGs and other scavengers were calculated by linear regression of log-dose vs radical yields.
Beneficial effects of TPGs in preventing ischernic myocardial dysfunction induced by ISO
Key myocardial enzymes for evaluating myocardial function, such as GOT, CK, LDH, were measured in serum from experimental group animals (Figs. 3-5). Compared with levels in the normal control group, the activities of GOT, CK and LDH were significantly increased in ISO rats, clearly suggesting that ISO produced myocardial damage. TPG intervention, either pre-treatment + posttreatment (TPG treatment), or posttreatment only (TPG therapy), significantly returned the enzyme activities elevated by ISO back to normal levels (Figs. 3-5), just as propranolol did.
TPGs enhancement of the antioxidant defense system in ISO-induced ischemic myocardium
The SOD activity and MDA levels in experimental group animals were determined (Figs. 6 and 7). SOD activity was significantly decreased in ISO-exposed rats compared with that in normal control rats (p < 0.01). The content of MDA in serum of ISO-exposed rats was significantly increased (p <0.01). More interestingly, TPGs, as effectively as propranolol, improved SOD activity and MDA levels (p <0.01 or p <0.05) in serum of ISO-induced ischemic myocardium (Figs. 6 and 7).
Myocardial ischemic injury occurs following inhibition of aerobic oxidation, augmentation of anaerobic glycolysis and accumulation of lactic acid dehydrogenase, accompanied by reduction of ATP production, ion gradient disruption and degradation of membrane integrity leading to outleakage of cardiac muscle enzymes. Consequently, the content of myocardial enzymes in blood serum increase, so that variation of myocardial zymogram in blood serum constitutes an index to define damage due to myocardial ischemia. Compared with normal control group, the activities of GOT, CK and LDH were significantly increased in ISO-treated rats, consistent with a previous report (Tang et al. 2011), which demonstrated that myocardial damage was induced by isoprenaline, while TPGs successfully restored serum myocardial enzyme activities in ISO-injured rats.
TPGs are an approved drug in China that are associated with few side-effects. Their pharmacological roles have been investigated extensively in animal models of arthritis and diabetes (Chang et al. 2009; Su et al. 2010; Wang et al. 2011: Wu et al. 2009a, b; Xu et al. 2007; Zhang et al. 2009; Zhu et al. 2005). However, there has been little experimental evidence demonstrating cardiologic protection by TPGs; herein we explored its role in experimental myocardial ischemia in rats and interestingly, found that TPGs showed dose-dependent protection against acute myocardial infarction (AMI)-induced alterations in cardiac enzymes, cytokines, oxidative stress, and coagulation (Mo et al. 2011). Multiple lines of evidence indicate that damage due to myocardial ischemia leads to production of oxygen-containing free radicals and lipid peroxidation. The products of lipid peroxidation react with free amino and nucleic acids to form Schiff bases that crosslink the biological macromolecules that contain them. Consequently, the membrane integrity of the myocardial cell is damaged, and thereby the physiological functions of the heart are inhibited, leading to serious arrhythmia and cellular necrosis. In a previous study, TPGs were found dose-dependently to decrease the levels of renal 3-NT proteins. These levels increased under diabetic conditions, and pathological levels of T-AOC, SOD and CAT in experimental diabetic rats were normalized by TPG treatment (Su et al. 2010). In the present study, ESR gave direct evidence that TPGs function as promising scavengers of superoxide and hydroxyl radials, especially being much more potent hydroxyl radial scavengers than mannitol. This was reflected in the lower MDA levels measured in TPG-treated animals, suggesting that less lipid peroxidation formed in ISO-treated animals due to amelioration of oxidative stress by TPGs. Besides the direct destruction of free radicals by TPGs, oxidative defense enzymes such as SOD that are damaged in ISO-treated animals are also activated by TPGs. Compared with the clinical drug propranolol, TPG treatment acted more potently both to lower MDA levels and to restore SOD activity. In view of in the lower incidence of side effects associated with TPG administration, TPGs thus present an advantage in clinical application.
In conclusion, consistent with our previous finding, TPGs have protective effects on experimental myocardial ischemia induced by isoprenaline in rats. The underlying mechanism is likely that TPGs prevent oxidative damage in ischemia by scavenging free radicals.
Authors thank Dr. Edward Sharman, University of California for his critical reading and language editing. This work was partially supported by Foundation of Van Jiaotong University, Program for New Century Excellent Talents in University, and the National Natural Science Foundation of China (Grant No. 31070740).
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Jiangang Long (a), Meili Gao (a), Yu Kong (a), Xian Shen (a), Xiaoyang Du (b), Young-Ok Son (c), Xianglin Shi (c), Jiankang Liu (a), Xiaoyan Mo (a), *
(a.) Department of Biological Science and Engineering, Institute of Mitochondrial Biology and Medicine, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, Xi'an Jiaotong University School of Life Science and Technology. Xi'an 710049. China
(b.) Medical School, Xi'an Jiaotong University, Xi'an 710061, China
(c.) Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY 40536-0305, USA
* Corresponding author at: Department of Biological Science and Engineering, Xi'an Jiaotong University School of Life Science and Technology, Xi'an 710049, China. Tel.: +86 29 82668463.
E-mail address: email@example.com (X. Mo).
0944-7113/$--see front matter [c] 2012 Elsevier GmbH. All rights reserved.
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|Author:||Long, Jiangang; Gao, Meili; Kong, Yu; Shen, Xian; Du, Xiaoyang; Son, Young-Ok; Shi, Xianglin; Liu, J|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Jun 15, 2012|
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