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Cardioprotective effect of total paeony glycosides against isoprenaline-induced myocardial ischemia in rats
Phytomedicine 19 (2012) 672–676
Contents lists available at SciVerse ScienceDirect
Phytomedicine journal homepage: www.elsevier.de/phymed
Cardioprotective effect of total paeony glycosides against isoprenaline-induced myocardial ischemia in rats 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
a r t i c l e
i n f o
Keywords: Paeonia rubra Total paeony glycosides Myocardial ischemia Myocardial enzymes Free radicals
a b s t r a c t 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 cardiomyopathy. 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/8 h 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. © 2012 Elsevier GmbH. All rights reserved.
Introduction 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 lactiflora pall, contain 96.2% of paeoniflorin (PF) and other
∗ 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: mo [email protected] (X. Mo). 0944-7113/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2012.03.004
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 (ISO)-induced animal model (Cheng et al. 2006), which displayed typical myocardinal ischemia. We observed that the activities of myocardial enzymes and oxidative stress were
J. Long et al. / Phytomedicine 19 (2012) 672–676
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Table 1 Comparison of the 50% free radical scavenging concentration of TPGs with previously known specific scavengers (mg/ml). Radicals
TPG
SOD
Hydroxyl Superoxide
0.236 0.003893
0.000657
Mannitol 21.84
Hydroxyl and superoxide were detected using spin trapping agents with an ESR spectrometer. The half-maximal inhibitory concentrations (IC50 ) of TPGs and other scavengers were calculated by linear regression of log-dose vs radical yields.
Fig. 1. RP-HPLC spectrum of total paeony glycosides (TPG) (Mo et al. 2011). The major peaks at 1.801, 2.403, 4.437 and 7.116 were assigned as oxypaeoniflorin, albiflorin, paeoniflorin and benzoyloxypaeoniflorin, respectively. The HPLC analysis was performed using a ZORBAX80 Extend C18 reverse phase column (4.6 mm × 150 mm, 5 m) with acetonitrile–water (1:4) as mobile phase at a rate of 1 ml/min at room temperature.
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 (Piao 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-HCl) was obtained from Sigma. Glutamic oxaloacetic transaminase (GOT), creatine kinase (CK), lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) were obtained from Nanjing Jiancheng Biotechnology Institute (Nanjing, China).
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 H2 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. Serum preparation 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 ◦ C at 3000 r/min for 10 min. Serum was thawed and centrifuged at 4 ◦ 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. Statistical analyses Data are presented as means ± SE. Statistical significance was determined by t-test. P values <0.05 were considered as being statistically significant. Results
In vitro free radical scavenging by TPGs TPG effects on free radical scavenging Hydroxyl, superoxide, and lipid radicals were detected using spin trapping agents with an electron spin resonance (ESR) spectrometer with minor modifications (Long et al. 2009). Briefly, hydroxyl radicals were detected in a 50 l reaction mixture containing 50 mM Cr2+ , 100 mM H2 O2 , 10 mM NADPH and 100 mM DMPO. The reaction was initiated by Cr2+ 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 l sample. Spectra were measured 3 min after addition of XOD to initiate the reaction.
TPGs dose-dependently scavenged both hydroxyl radicals (Fig. 2A) and superoxide radicals (Fig. 2B). The 50% free radical scavenging concentrations (IC50 ) 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 IC50 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).
Animals and ISO-induced myocardial ischemia in rats
Beneficial effects of TPGs in preventing ischemic myocardial dysfunction induced by ISO
Male Sprague Dawley rats (60 days old, 30 male and 30 female, weighing 200–250 g) 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
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 post-treatment only (TPG therapy),
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Fig. 2. Effect of TPGs on free radicals scavenging. (A) Left: ESR spectra of DMPO-trapped hydroxyl radicals recorded 5 min after reaction initiation in PBS solution (0.2 M, pH 7.4) containing 100 mM DMPO, 50 mM Cr2+ , 10 mM NADPH and 100 mM H2 O2 . Right: Relationship between the signal intensity of DMPO-OH and various concentrations of TPGs. (B) Left: ESR spectra of 5,5-dimethyl-pyrroline N-oxide (DMPO)-trapped superoxide radicals recorded 3 min after reaction initiation in PBS solution (0.1 M, pH 7.4) containing 0.33 mM hypoxanthine (HPX), 0.83 mM DTPA, 0.15 M DMPO, and 0.083 units/ml xanthine oxidase (XOD). Right: Relationship between the signal intensity of DMPO-OOH and the various concentrations of TPGs.
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
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). Discussion
20
##
15 10
##
## ##
5 0
Control
ISO
TPG
TPG therapy
U/ml U
**
25
Myocardial ischemic injury occurs following inhibition of aerobic oxidation, augmentation of anaerobic glycolysis and accumulation of lactic acid dehydrogenase, accompanied by reduction
CK K activity
GOT activity (Carmen's unit)
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
Propranolol
treatment
Fig. 3. Effect of TPGs on myocardial GOT in ISO-induced myocardial ischemia in rats. **p < 0.01 vs. normal control; ## p < 0.01 vs. ISO injury, n = 10).
20 18 16 14 12 10 8 6 4 2 0
** ##
Control
##
##
##
ISO
TPG treatment
TPG therapy
Propranolol
Fig. 4. Effect of TPGs on myocardial CK activity in ISO-induced myocardial ischemia in rats. **p < 0.01 vs. normal control; ## p < 0.01 vs. ISO injury, n = 10).
J. Long et al. / Phytomedicine 19 (2012) 672–676
**
7000 6000
##
5000 4000
##
##
##
3000 2000 1000 0 Control
ISO
TPG treatment
TPG therapy
Propranolol
Fig. 5. Effect of TPGs on myocardial LDH activity in ISO-induced myocardial ischemia in rats. **p < 0.01 vs. normal control; ## p < 0.01 vs. ISO injury, n = 10). 70 60
## ##
50 40
#
**
30
#
20 10 0
Control
ISO
TPG treatment
TPG therapy
Propranolol
Fig. 6. Effect of TPGs on myocardial SOD activity in ISO-induced myocardial ischemia in rats. **p < 0.01 vs. normal control; # p < 0.05, ## p < 0.01 vs. ISO injury, n = 10).
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
Fig. 7. Effect of TPGs on myocardial MDA level in ISO-induced myocardial ischemia in rats. **p < 0.01 vs. normal control; # p < 0.05, ## p < 0.01 vs. ISO injury, n = 10).
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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 3NT 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. Acknowledgements Authors thank Dr. Edward Sharman, University of California for his critical reading and language editing. This work was partially supported by Foundation of Xi’an Jiaotong University, Program for New Century Excellent Talents in University, and the National Natural Science Foundation of China (Grant No. 31070740). References Chang, Y., Wei, W., Zhang, L., Xu, H.M., 2009. Effects and mechanisms of total glucosides of paeony on synoviocytes activities in rat collagen-induced arthritis. J. Ethnopharmacol. 121 (1), 43–48. Cheng, Y., Ping, J., Xu, L.M., 2006. Currucmin inhibits the activation marker of hepatic stellate cells by up-regulating the peroxisome proliferator-activated receptor gama. Chin. J. Pract. Inter. Med. 26 (24), 1937–1940. Hagiwara, S., Teshima, Y., Takahashi, N., Koga, H., Saikawa, T., Noguchi, T., 2011. New lipoic acid derivative drug sodium zinc dihydrolipoylhistidinate prevents cardiac dysfunction in an isolated perfused rat heart model. Crit. Care Med. 39 (3), 506–511. Jiang, B., Qiao, J., Yang, Y., Lu, Y., 2009. Inhibitory effect of paeoniflorin on the inflammatory vicious cycle between adipocytes and macrophages. J. Cell. Biochem. (April) (Epub ahead of print). Li, J., Chen, C.X., Shen, Y.H., 2011. Effects of total glucosides from paeony (Paeonia lactiflora Pall) roots on experimental atherosclerosis in rats. J. Ethnopharmacol. 135 (2), 469–475. Liu, D.Z., Xie, K.Q., Ji, X.Q., Ye, Y., Jiang, C.L., Zhu, X.Z., 2005. Neuroprotective effect of paeoniflorin on cerebral ischemic rat by activating adenosine A1 receptor in a manner different from its classical agonists. Br. J. Pharmacol. 146 (4), 604–611. Long, J., Gao, H., Sun, L., Liu, J., Zhao-Wilson, X., 2009. Grape extract protects mitochondria from oxidative damage and improves locomotor dysfunction and extends lifespan in a Drosophila Parkinson’s disease model. Rejuvenation Res. 12 (5), 321–331. Mo, X., Zhao, N., Du, X., Bai, L., Liu, J., 2011. The protective effect of peony extract on acute myocardial infarction in rats. Phytomedicine 18 (6), 451–457. Piao, T., Mo, X.Y., Lin, R.Z., 2007. Study on the free radical-scavenging and anticoagulant activities of total paeony glycoside in vitro. Chin. Pharm. 18 (9), 643–646. Su, J., Zhang, P., Zhang, J.J., Qi, X.M., Wu, Y.G., Shen, J.J., 2010. Effects of total glucosides of paeony on oxidative stress in the kidney from diabetic rats. Phytomedicine 17 (3–4), 254–260. Tang, Y., Wang, M., Le, X., Meng, J., Huang, L., Yu, P., Chen, J., Wu, P., 2011. Antioxidant and cardioprotective effects of Danshensu (3-(3, 4-dihydroxyphenyl)
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-2-hydroxy-propanoic acid from Salvia miltiorrhiza) on isoproterenol-induced myocardial hypertrophy in rats. Phytomedicine. Wang, Q.T., Zhang, L.L., Wu, H.X., Wei, W., 2011. The expression change of beta-arrestins in fibroblast-like synoviocytes from rats with collagen-induced arthritis and the effect of total glucosides of paeony. J. Ethnopharmacol. 133 (2), 511–516. Wu, C.F., 1985. A review on the pharmacology of Paeonia lactiflora and its chemical components. Zhong Yao Tong Bao 10 (6), 43–45. Wu, H., Zhu, Z., Zhang, G., Zhao, L., Zhang, H., Zhu, D., Chai, Y., 2009a. Comparative pharmacokinetic study of paeoniflorin after oral administration of pure paeoniflorin, extract of Cortex Moutan and Shuang-Dan prescription to rats. J. Ethnopharmacol. 125 (3), 444–449. Wu, Y., Ren, K., Liang, C., Yuan, L., Qi, X., Dong, J., Shen, J., Lin, S., 2009b. Renoprotective effect of total glucosides of paeony (TGP) and its mechanism in experimental diabetes. J. Pharmacol. Sci. 109 (1), 78–87.
Xu, H.M., Wei, W., Jia, X.Y., Chang, Y., Zhang, L., 2007. Effects and mechanisms of total glucosides of paeony on adjuvant arthritis in rats. J. Ethnopharmacol. 109 (3), 442–448. Zhang, P., Zhang, J.J., Su, J., Qi, X.M., Wu, Y.G., Shen, J.J., 2009. Effect of total glucosides of paeony on the expression of nephrin in the kidneys from diabetic rats. Am. J. Chin. Med. 37 (2), 295–307. Zheng, L.Y., Pan, J.Q., Lv, J.H., 2008. Effects of total glucosides of paeony on enhancing insulin sensitivity and antagonizing nonalcoholic fatty liver in rats. Zhongguo Zhong Yao Za Zhi 33 (20), 2385–2390. Zheng, Y.Q., Wei, W., 2005. Total glucosides of paeony suppresses adjuvant arthritis in rats and intervenes cytokine-signaling between different types of synoviocytes. Int. Immunopharmacol. 5 (10), 1560–1573. Zhu, L., Wei, W., Zheng, Y.Q., Jia, X.Y., 2005. Effects and mechanisms of total glucosides of paeony on joint damage in rat collagen-induced arthritis. Inflamm. Res. 54 (5), 211–220.
Contents lists available at SciVerse ScienceDirect
Phytomedicine journal homepage: www.elsevier.de/phymed
Cardioprotective effect of total paeony glycosides against isoprenaline-induced myocardial ischemia in rats 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
a r t i c l e
i n f o
Keywords: Paeonia rubra Total paeony glycosides Myocardial ischemia Myocardial enzymes Free radicals
a b s t r a c t 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 cardiomyopathy. 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/8 h 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. © 2012 Elsevier GmbH. All rights reserved.
Introduction 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 lactiflora pall, contain 96.2% of paeoniflorin (PF) and other
∗ 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: mo [email protected] (X. Mo). 0944-7113/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2012.03.004
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 (ISO)-induced animal model (Cheng et al. 2006), which displayed typical myocardinal ischemia. We observed that the activities of myocardial enzymes and oxidative stress were
J. Long et al. / Phytomedicine 19 (2012) 672–676
673
Table 1 Comparison of the 50% free radical scavenging concentration of TPGs with previously known specific scavengers (mg/ml). Radicals
TPG
SOD
Hydroxyl Superoxide
0.236 0.003893
0.000657
Mannitol 21.84
Hydroxyl and superoxide were detected using spin trapping agents with an ESR spectrometer. The half-maximal inhibitory concentrations (IC50 ) of TPGs and other scavengers were calculated by linear regression of log-dose vs radical yields.
Fig. 1. RP-HPLC spectrum of total paeony glycosides (TPG) (Mo et al. 2011). The major peaks at 1.801, 2.403, 4.437 and 7.116 were assigned as oxypaeoniflorin, albiflorin, paeoniflorin and benzoyloxypaeoniflorin, respectively. The HPLC analysis was performed using a ZORBAX80 Extend C18 reverse phase column (4.6 mm × 150 mm, 5 m) with acetonitrile–water (1:4) as mobile phase at a rate of 1 ml/min at room temperature.
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 (Piao 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-HCl) was obtained from Sigma. Glutamic oxaloacetic transaminase (GOT), creatine kinase (CK), lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) were obtained from Nanjing Jiancheng Biotechnology Institute (Nanjing, China).
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 H2 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. Serum preparation 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 ◦ C at 3000 r/min for 10 min. Serum was thawed and centrifuged at 4 ◦ 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. Statistical analyses Data are presented as means ± SE. Statistical significance was determined by t-test. P values <0.05 were considered as being statistically significant. Results
In vitro free radical scavenging by TPGs TPG effects on free radical scavenging Hydroxyl, superoxide, and lipid radicals were detected using spin trapping agents with an electron spin resonance (ESR) spectrometer with minor modifications (Long et al. 2009). Briefly, hydroxyl radicals were detected in a 50 l reaction mixture containing 50 mM Cr2+ , 100 mM H2 O2 , 10 mM NADPH and 100 mM DMPO. The reaction was initiated by Cr2+ 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 l sample. Spectra were measured 3 min after addition of XOD to initiate the reaction.
TPGs dose-dependently scavenged both hydroxyl radicals (Fig. 2A) and superoxide radicals (Fig. 2B). The 50% free radical scavenging concentrations (IC50 ) 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 IC50 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).
Animals and ISO-induced myocardial ischemia in rats
Beneficial effects of TPGs in preventing ischemic myocardial dysfunction induced by ISO
Male Sprague Dawley rats (60 days old, 30 male and 30 female, weighing 200–250 g) 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
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 post-treatment only (TPG therapy),
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Fig. 2. Effect of TPGs on free radicals scavenging. (A) Left: ESR spectra of DMPO-trapped hydroxyl radicals recorded 5 min after reaction initiation in PBS solution (0.2 M, pH 7.4) containing 100 mM DMPO, 50 mM Cr2+ , 10 mM NADPH and 100 mM H2 O2 . Right: Relationship between the signal intensity of DMPO-OH and various concentrations of TPGs. (B) Left: ESR spectra of 5,5-dimethyl-pyrroline N-oxide (DMPO)-trapped superoxide radicals recorded 3 min after reaction initiation in PBS solution (0.1 M, pH 7.4) containing 0.33 mM hypoxanthine (HPX), 0.83 mM DTPA, 0.15 M DMPO, and 0.083 units/ml xanthine oxidase (XOD). Right: Relationship between the signal intensity of DMPO-OOH and the various concentrations of TPGs.
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
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). Discussion
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Myocardial ischemic injury occurs following inhibition of aerobic oxidation, augmentation of anaerobic glycolysis and accumulation of lactic acid dehydrogenase, accompanied by reduction
CK K activity
GOT activity (Carmen's unit)
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
Propranolol
treatment
Fig. 3. Effect of TPGs on myocardial GOT in ISO-induced myocardial ischemia in rats. **p < 0.01 vs. normal control; ## p < 0.01 vs. ISO injury, n = 10).
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Fig. 4. Effect of TPGs on myocardial CK activity in ISO-induced myocardial ischemia in rats. **p < 0.01 vs. normal control; ## p < 0.01 vs. ISO injury, n = 10).
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Fig. 5. Effect of TPGs on myocardial LDH activity in ISO-induced myocardial ischemia in rats. **p < 0.01 vs. normal control; ## p < 0.01 vs. ISO injury, n = 10). 70 60
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Fig. 6. Effect of TPGs on myocardial SOD activity in ISO-induced myocardial ischemia in rats. **p < 0.01 vs. normal control; # p < 0.05, ## p < 0.01 vs. ISO injury, n = 10).
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
Fig. 7. Effect of TPGs on myocardial MDA level in ISO-induced myocardial ischemia in rats. **p < 0.01 vs. normal control; # p < 0.05, ## p < 0.01 vs. ISO injury, n = 10).
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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 3NT 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. Acknowledgements Authors thank Dr. Edward Sharman, University of California for his critical reading and language editing. This work was partially supported by Foundation of Xi’an Jiaotong University, Program for New Century Excellent Talents in University, and the National Natural Science Foundation of China (Grant No. 31070740). References Chang, Y., Wei, W., Zhang, L., Xu, H.M., 2009. Effects and mechanisms of total glucosides of paeony on synoviocytes activities in rat collagen-induced arthritis. J. Ethnopharmacol. 121 (1), 43–48. Cheng, Y., Ping, J., Xu, L.M., 2006. Currucmin inhibits the activation marker of hepatic stellate cells by up-regulating the peroxisome proliferator-activated receptor gama. Chin. J. Pract. Inter. Med. 26 (24), 1937–1940. Hagiwara, S., Teshima, Y., Takahashi, N., Koga, H., Saikawa, T., Noguchi, T., 2011. New lipoic acid derivative drug sodium zinc dihydrolipoylhistidinate prevents cardiac dysfunction in an isolated perfused rat heart model. Crit. Care Med. 39 (3), 506–511. Jiang, B., Qiao, J., Yang, Y., Lu, Y., 2009. Inhibitory effect of paeoniflorin on the inflammatory vicious cycle between adipocytes and macrophages. J. Cell. Biochem. (April) (Epub ahead of print). Li, J., Chen, C.X., Shen, Y.H., 2011. Effects of total glucosides from paeony (Paeonia lactiflora Pall) roots on experimental atherosclerosis in rats. J. Ethnopharmacol. 135 (2), 469–475. Liu, D.Z., Xie, K.Q., Ji, X.Q., Ye, Y., Jiang, C.L., Zhu, X.Z., 2005. Neuroprotective effect of paeoniflorin on cerebral ischemic rat by activating adenosine A1 receptor in a manner different from its classical agonists. Br. J. Pharmacol. 146 (4), 604–611. Long, J., Gao, H., Sun, L., Liu, J., Zhao-Wilson, X., 2009. Grape extract protects mitochondria from oxidative damage and improves locomotor dysfunction and extends lifespan in a Drosophila Parkinson’s disease model. Rejuvenation Res. 12 (5), 321–331. Mo, X., Zhao, N., Du, X., Bai, L., Liu, J., 2011. The protective effect of peony extract on acute myocardial infarction in rats. Phytomedicine 18 (6), 451–457. Piao, T., Mo, X.Y., Lin, R.Z., 2007. Study on the free radical-scavenging and anticoagulant activities of total paeony glycoside in vitro. Chin. Pharm. 18 (9), 643–646. Su, J., Zhang, P., Zhang, J.J., Qi, X.M., Wu, Y.G., Shen, J.J., 2010. Effects of total glucosides of paeony on oxidative stress in the kidney from diabetic rats. Phytomedicine 17 (3–4), 254–260. Tang, Y., Wang, M., Le, X., Meng, J., Huang, L., Yu, P., Chen, J., Wu, P., 2011. Antioxidant and cardioprotective effects of Danshensu (3-(3, 4-dihydroxyphenyl)
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