Original Papers
Cardioprotective Effect of Polydatin on Ventricular Remodeling after Myocardial Infarction in Coronary Artery Ligation Rats
Authors
Yan Gao 1, 2, Jianping Gao 1, Changxun Chen 1, Huilin Wang 1, Juan Guo 1, Rong Wu 1
Affiliations
1 2
Department of Pharmacology, Shanghai University of Traditional Chinese Medicine, Shanghai, China Present address: Jiading Hospital of Traditional Chinese Medicine, Shanghai, China
Key words " polydatin l " myocardial infarction l " ventricular remodeling l " coronary artery ligation l " renin‑angiotensin‑aldosterl one system " peroxidation l
Abstract
received revised accepted
Introduction
February 26, 2014 February 12, 2015 March 2, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1545907 Published online April 23, 2015 Planta Med 2015; 81: 568–577 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Jianping Gao Department of Pharmacology Shanghai University of Traditional Chinese Medicine No. 1200 Cailun Road Shanghai, 201203 China Phone: + 86 21 51 32 22 13 [email protected]
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The purpose of this study was to explore the effect of polydatin on ventricular remodeling after myocardial infarction in coronary artery ligation rats and to elucidate the underlying mechanisms. A rat model of ventricular remodeling after myocardial infarction was established by left coronary artery ligation. Rats with coronary artery ligation were randomly divided into five groups: control, plus 40 mg/kg captopril, plus 25 mg/kg polydatin, plus 50 mg/kg polydatin, and plus 100 mg/kg polydatin. The sham-operated group was used as a negative control. Rats were administered intragastrically with the corresponding drugs or drinking water for seven weeks. At the end of the treatment, the left ventricular weight index and heart weight index were assessed. The cross-sectional size of cardiomyocytes was measured by staining myocardium tissue with hematoxylin and eosin. Collagen content was counted by Sirius red in aqueous saturated picric acid. The concentrations
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Chronic heart failure (CHF) is known as the cardiac function insufficiency in which the heart cannot put out commensurate blood to supply venous return and tissue metabolism and then causes some syndromes [1]. CHF is a serious threat to human health and the incidence and mortality rate of this disease are rising [2]. CHF is considered to be an irreversible and progressive process characterized by ventricular remodeling and a number of neurohormonal perturbations. It is well known that ventricular remodeling is considered one of the main pathophysiological mechanisms in the process of the development of chronic cardiac insufficiency [3, 4]. Therefore, inhibiting ventricular remodeling early may be an effective way to postpone heart failure induced
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of angiotensin I, angiotensin II, aldosterone, and endothelin 1 in myocardium or serum were determined by radioimmunoassay. Hydroxyproline and nitric oxide concentrations and glutathione peroxidase and catalase activities in serum were measured by ultraviolet spectrophotometry. Our results showed that seven weeks of polydatin treatment resulted in a significantly reduced left ventricular weight index, heart weight index, serum concentrations of hydroxyproline and aldosterone, an increased concentration of nitric oxide as well as enhanced activities of glutathione peroxidase and catalase. Myocardial angiotensin I, angiotensin II, and endothelin 1 levels were also reduced. The cardiomyocyte cross-sectional area and collagen deposition diminished. This study suggests that polydatin may attenuate ventricular remodeling after myocardial infarction in coronary artery ligation rats through restricting the excessive activation of the renin-angiotensin-aldosterone system and inhibiting peroxidation.
by myocardial infarction (MI) and other cardiovascular diseases. Many researchers try to seek effective components from traditional Chinese medicine to control or relieve CHF. Polydatin (C20H22O8, 3,4′,5-trihydroxystibene-3" Fig. 1) is one of the natuβ-mono-D-glucoside, l ral hydroxy-diphenyl ethylene compounds isolated from traditional Chinese medicine, Polygonum cuspidatum Sieb. et Zucc. Modern pharmacological studies have demonstrated that polydatin has multiple biological activities and important clinical value, such as anti-cerebral hemorrhage [5], antithrombosis, antioxidation, affecting ion channels [6], and regulating protein kinase and nitric oxide [7]. Polydatin can also markedly lower the serum levels of total cholesterol, triglyceride, and low-density lipoprotein cholesterol in hyperlipidemic rabbits [8]. It inhibits neutrophil
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Fig. 1 Structure of polydatin.
chemotaxis in inflammatory reactions [9]. Regarding the cardiac protective effect, previous studies have shown that polydatin can attenuate myocardial hypertrophy induced by isopropyl adrenaline in mice and ventricular remodeling caused by abdominal aorta ligation in rats [10]. Zhang et al. also proved that polydatin attenuates ischemia/reperfusion-induced apoptosis in the myocardium of rats [11]. In this study, we adopted the method of coronary artery ligation (CAL) to establish an MI model, and then we investigated the effect and underlying mechanisms of polydatin on ventricular remodeling after MI.
Results !
Similar to a previous report [12], the immediate postoperative mortality rate in our study was about 25 %. Unrecovered breathing, acute left ventricular failure, and fatal ventricular arrhythmia were the main causes of death in the rats. One week after the operation, surviving rats were randomly assigned to different groups. The number of rats was 9 in the sham-operated control group, 15 in the CAL control group, and 11 in each of the rest of the groups. During the seven-week treatment process, five rats died (mortality 33.3 %) in the CAL control group, two rats died (mortality 18.2 %) in the CAL plus 100 mg/kg polydatin group, and one rat died in each of the following groups: sham-operated group (mortality 11.1 %), CAL plus 40 mg/kg captopril, CAL plus 25 mg/kg polydatin, and CAL plus 50 mg/kg polydatin (mortality 9.1 %). After treatment for seven weeks, the left ventricular wall below the ligation part became thinner and showed fibrosis with a pale color in the CAL control rats. The whole heart photographs of all " Fig. 2 A–F. The hearts of the sham-opergroups are shown in l ated rats appeared normal without an infarction area, myocardial fibrosis, or any other abnormalities. On the contrary, a small area of fibrosis appeared in the hearts of the captopril- or polydatin" Fig. 2 G, the left ventricular weight treated groups. As shown in l index (LVWI) and heart weight index (HWI) were significantly greater in the CAL control group than in the sham-operated control group (p < 0.01). After polydatin and captopril treatment for seven weeks, LVWI and HWI were significantly reduced (p < 0.05, " Fig. 3, the cardiomyocyte p < 0.01, respectively). As shown in l cross-sectional area in the CAL control group was significantly larger than that in the sham-operated control group (p < 0.01). Polydatin- and captopril-treated groups showed a decreased average cross-sectional area of cardiomyocytes compared to the CAL control group (p < 0.05, p < 0.01, respectively). Under a microscope, cardiomyocytes appeared orange, and myocardial interstitial and perivascular collagen fibers appeared red or deep red stained with Sirius red. In the rat myocardium of the sham-operated group, a little amount of collagen was found in the interstitial and perivascular space. In the CAL control rats, there was a large accumulation of collagen in the interstitial and perivascular space of the ventricle. Conversely, less collagen depositions were found in both the CAL plus polydatin and captopril
" Fig. 4a1 and 4a3). Under groups than in the CAL control group (l the polarized light microscope, type I collagen fibers appeared red or yellow and type III collagen fibers appeared green. In the rat myocardium of the sham-operated control, fewer amounts of " Fig. 4a2 and 4a4). Nevertheless, collagen fibers were found (l collagen distributions were significantly increased in the CAL control rats compared to the sham-operated rats (p < 0.01). On the contrary, collagen fibers were significantly decreased in the " Fig. 4 b) polydatin- and captopril-treated groups (p < 0.01, l compared to the CAL control group. In addition, as shown in " Fig. 5, the hydroxyproline (Hyp) concentration in the serum l was increased in the CAL control group compared with that in the sham-operated group (p < 0.01). Interestingly, the polydatintreated groups, both at 25 and 50 mg/kg, as well as the captopriltreated groups exhibited a significantly reduced Hyp concentra" Fig. 5). tion compared to the CAL control group (p < 0.01, l Left ventricular systolic pressure (LVSP) and the maximal rate of rise of the left ventricular pressure (+ dp/dtmax) were significantly lower in the CAL control group than in the sham-operated control group (p < 0.01, p < 0.05). The left ventricular end-diastolic pressure (LVEDP) was slightly increased, and the maximal rate of fall of the left ventricular pressure (-dp/dtmax) was slightly reduced in the CAL control group. However, there was no significant difference compared with the sham-operated group. Nevertheless, polydatin- and captopril-treated groups showed a significantly increased LVSP and +dp/dtmax (p < 0.05, p < 0.01, respectively). In addition, both captopril- and polydatin-treated groups showed a tendency of reducing LVEDP and increasing-dp/dtmax, however, no significant difference compared with the CAL con" Table 1). trol group was found (l " As shown in l Fig. 6, in the CAL control group, angiotensin I (Ang I) and angiotensin II (Ang II) concentrations of ventricular tissue and aldosterone (ALD) concentration in the serum were significantly higher than those in the sham-operated control group (p < 0.05, p < 0.01, respectively). Treatment with polydatin or captopril for seven weeks significantly reduced Ang I, Ang II, and ALD concentrations (p < 0.05, p < 0.01, respectively). The low activity of glutathione peroxidase (GSH‑Px) in the serum of the CAL rats was enhanced by polydatin and captopril, and low catalase (CAT) activity in the CAL rats was attenuated by treatment with both polydatin 25 mg/kg and captopril (p < 0.05, " Fig. 7). p < 0.01, l " Fig. 8, the endothelin 1 (ET-1) concentration of the As shown in l ventricular tissue was significantly higher, and the serum nitric oxide (NO) concentration was lower in the CAL control group than in the sham-operated group (p < 0.01). Treatment with polydatin or captopril for seven weeks significantly reduced the ET-1 concentration and increased the NO concentration (p < 0.01).
Discussion !
Left ventricular remodeling is a complex process involving cardiac myocyte growth and death, fibrosis, inflammation, and electrophysiological remodeling [13]. The main causes of ventricular remodeling are hypertension and MI. After MI, abnormal myocardial blood flow can impair myocardial O2 delivery, resulting in maladaptive remodeling. It in turn induces the migration of macrophages, monocytes, and neutrophils into the infarct zone in left ventricle, which initiates intracellular signaling and neurohormonal activation, and localizes the inflammatory response.
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Fig. 2 Some of the whole heart photographs of different groups. A Aham-operated control; B coronary artery ligation (CAL) control; C CAL plus 40 mg/kg captopril; D CAL plus 25 mg/kg polydatin; E CAL plus 50 mg/kg polydatin; F CAL plus 100 mg/kg polydatin. G Effects of polydatin on cardiac weight indexes. Compared with the shamoperated control group, ##p < 0.01. Compared with the CAL control group, *p < 0.05, **p < 0.01. LVWI, left ventricular weight/body weight index; HWI, heart weight/body weight index. Data are presented as the mean ± SD. (Color figure available online only.)
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There will be a lot of compensatory responses of the body after MI, but it is not sufficient to satisfy the needs of heart [14]. The MI model induced by CAL in rats is similar to clinical MI caused by coronary artery occlusion. Following acute MI, the ventricular remodeling is the result of the infarction expansion process, followed by the hypertrophy process. Our experimental results indicate that after ligation for eight weeks, a stable and successful animal model of myocardial hypertrophy is established. In the ventricular remodeling animal model, the heart weight index or mass and the transverse area of the cardiac myocyte are the main indicators of the degree of cardiac hypertrophy [15, 16]. Our study showed that LVWI, HWI, and the average crosssectional area of cardiomyocytes were significantly larger in the CAL control group. Polydatin treatment for seven weeks significantly decreased the cardiac mass index of the CAL rats.
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Myocyte apoptosis and necrosis activate multiple biochemical intracellular signaling that initiates left ventricular dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. As a consequence, inhibition of myocardial fibrosis can improve cardiac function. Treatment with polydatin for seven weeks significantly reduced intercellular and perivascular collagen accumulation. Hyp is one of the major components of collagen in the body, accounting for about 13.4 % of normal collagen and very little of elastin. Therefore, the serum Hyp concentration can be used as an important index for measuring collagen metabolism and judging the degree of fibrosis. It can also promote the myocardial collagen protein synthesis, myocardial interstitial proliferation, and fibrosis [17]. Our results showed that CAL resulted in a significantly increased serum Hyp concentration, while treatment with polydatin significantly reduced the serum Hyp concentration. Based on these results, we speculate that
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polydatin may delay and block fibrosis by decreasing the synthesis and secretion of collagen, and then slow down or inhibit the process of ventricular remodeling. In addition, the effect of polydatin on restraining ventricular remodeling may be partly due to smaller infarctions induced by polydatin, as previous studies suggest that polydatin can lower myocardial infarct size significantly in rats [7, 18]. Hemodynamic disorder is one of the factors that lead to myocardial hypertrophy and reconstruction [19]. Epidemiological studies have reported that approximately 40 % of myocardial infarctions are accompanied by left ventricular systolic dysfunction [20]. In our study, the untreated animals developed cardiac function insufficiency in seven weeks, evidenced by a decreased LVSP and +dp/dtmax compared to the sham-operated rats. Polydatin significantly increased LVSP and +dp/dtmax. Although the cardiac function was compromised by CAL, it did not lead to heart failure, which was evidenced by the mild elevation of LVEDP and the mild reduction of-dp/dtmax. Pharmacologic inhibition of the renin-angiotensin-aldosterone and sympathetic nervous systems has been evaluated in a large number of post-MI clinical trials [21]. In patients with heart failure, the neuroendocrine activation is expressed more excessively
[22]. The renin-angiotensin-aldosterone system (RAAS) plays an important role in the pathophysiology of ventricular remodeling [23]. Therefore, restraining RAAS activation is an important means to treat ventricular remodeling. With regard to the initiation and development of CHF, the main active materials in the RAAS are Ang II and ALD. Ang II plays a vital role in promoting cardiac hypertrophy. It is also a potent vasoconstrictor, which can affect cardiac structure and fibrosis [24]. Inhibition of Ang II synthesis has been demonstrated to be beneficial in preventing left ventricular remodeling and reducing post-MI mortality. Not only does it promote ALD and the retention of water and sodium, it also causes the activation of the sympathetic nerve, collagen synthesis, and interstitial abnormality [25, 26]. Consequently, all these effects mentioned above promote myocardial fibrosis, damage the endothelial function, and induce ventricular remodeling. Our results indicate that the function of polydatin in antagonizing ventricular remodeling after MI may be correlated with its inhibition of the activated RAAS in rats, including significantly reduced myocardial tissue, Ang I, Ang II, and serum ALD concentrations. Endothelial activity factors, such as ET-1 and NO, lead to the alteration of the balance between vasodilatation and vasoconstric-
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Fig. 3 Effect of polydatin on the cardiomyocyte cross-sectional area in rats with ventricular remodeling after myocardial infarction induced by left coronary artery ligation (CAL). The histopathological observations of the heart (hematoxylin and eosin stain, 400×). A Sham-operated control; B coronary artery ligation (CAL) control; C CAL plus 40 mg/kg captopril; D CAL plus 25 mg/kg polydatin; E CAL plus 50 mg/kg polydatin; F CAL plus 100 mg/kg polydatin. G Effect of polydatin on the cardiomyocyte cross-sectional area. Compared with the sham-operated control group, ##p < 0.01. Compared with the CAL control group, *p < 0.05, **p < 0.01. Data are presented as the mean ± SD. (Color figure available online only.)
Original Papers
Fig. 4 a Effects of polydatin on cardiac collagen accumulation in the interstitial and perivascular space of the left ventricle in rats with ventricular remodeling after myocardial infarction induced by left coronary artery ligation (CAL). Numbers 1 and 3 show the images of the Sirius red stained section under normal light illumination; cardiomyocytes appear orange and myocardial interstitial and perivascular collagen fibers appear red or deep red stained
with Sirius red. Numbers 2 and 4 show the polarized light Sirius red images; type I collagen fibers appear red or yellow and type III collagen fibers appear green. Magnification: 400×. A Sham-operated control; B coronary artery ligation (CAL) control; C CAL plus 40 mg/kg captopril; D CAL plus 25 mg/kg polydatin; E CAL plus 50 mg/kg polydatin; F CAL plus 100 mg/kg polydatin. (Color figure available online only.)
Fig. 4 b The effects of polydatin on the interstitial collagen volume fraction (ICVF) and perivascular collagen volume fraction (PVCF) of the left ventricle. Compared with the sham-operated control, ## p < 0.01. Compared with the CAL control, **p < 0.01. Data are presented as the mean ± SD.
tion in systemic circulation. ET-1 is a potent vasoconstrictor that is produced by smooth muscle cells, macrophages, airway epitheliums, and alveolar epithelial cells. ET-1 can increase the intracellular Ca2+ concentration, which leads to cardiac mast cell degranulation; it also promotes the synthesis of collagen and induces cardiomyocyte hypertrophy and inflammation, which leads to
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heart failure [27]. The ET-1 level is increased in symptomatic patients with ischemic heart disease and MI [28, 29]. NO, which plays an important protective role in the cardiovascular system, inhibits smooth muscle cell proliferation and migration. It also enhances proliferation and migration of endothelial cells as well as inhibits apoptosis, suppresses platelet aggregation, and pre-
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Fig. 6 Effect of polydatin on RAAS in rats with ventricular remodeling after myocardial infarction induced by left coronary artery ligation (CAL). a shows the concentrations of angiotensin I and aldosterone, respectively. b shows the concentration of angiotensin II. Compared with the sham-operated control group, #p < 0.05, ##p < 0.01. Compared with the CAL control group, *p < 0.05, **p < 0.01. Data are presented as the mean ± SD.
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Fig. 5 Effect of polydatin on hydroxyproline concentration in rats with ventricular remodeling after myocardial infarction induced by left coronary artery ligation (CAL). Compared with the sham-operated control group, ##p < 0.01. Compared with the CAL control group, **p < 0.01. Data are presented as the mean ± SD.
Original Papers
Table 1 Effects of polydatin on hemodynamic parameters in rats with ventricular remodeling after myocardial infarction caused by left coronary artery ligation (CAL) (mean ± SD). Group
n
Dose (mg/kg)
LVSP (mmHg)
LVEDP (mmHg)
+dP/dtmax (mmHg/s)
−dP/dtmax (mmHg/s)
Sham-operated control CAL control CAL plus captopril CAL plus polydatin CAL plus polydatin CAL plus polydatin
8 10 10 10 10 9
– –
119.65 ± 11.88 95.68 ± 8.56## 109.44 ± 9.30** 116.02 ± 12.25** 114.14 ± 19.27* 118.32 ± 16.48**
0.97 ± 1.71 2.42 ± 2.35 1.06 ± 2.70 0.56 ± 3.80 0.91 ± 7.53 0.33 ± 4.38
9433.45 ± 1915.83 7038.29 ± 601.50# 8769.02 ± 1253.99* 8521.04 ± 1000.75* 8047.74 ± 790.82* 8466.21 ± 924.94*
− 6262.70 ± 964.33 − 5678.89 ± 674.58 − 6339.34 ± 524.21 − 6190.66 ± 1009.03 − 6039.54 ± 594.66 − 5703.71 ± 785.82
40 25 50 100
Note: Compared with the sham-operated control group, #p < 0.05; ##p < 0.01. Compared with the CAL control group, *p < 0.05; **p < 0.01. LVSP, left ventricular systolic pressure; LVEDP, left ventricular end diastolic pressure; +dp/dtmax, the maximal rate of rise of left ventricular pressure; −dp/dtmax, the maximal rate of fall of left ventricular pressure
Fig. 7 Effect of polydatin on oxidative stress in rats with ventricular remodeling after myocardial infarction induced by left coronary artery ligation (CAL). a shows glutathione peroxidase activity. b shows catalase activity. Compared with the sham-operated control group, #p < 0.05. Compared with the CAL control group, *p < 0.05, **p < 0.01. Data are presented as the mean ± SD.
vents platelet, leukocyte, and monocyte adhesion to the endothelium [30]. In our study, CAL increased the ET-1 concentration of the left ventricular tissue and reduced the serum NO concentration. Polydatin treatment resulted in a decreased ET-1 level and
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an increased NO concentration. These results indicate that polydatin may protect myocardial tissue and antagonize ventricular remodeling through adjusting the release of vasoactive factors from endothelial cells.
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Fig. 8 Effect of polydatin on endothelial activity factors in rats with ventricular remodeling after myocardial infarction induced by left coronary artery ligation (CAL). a shows the concentration of endothelin-1. b shows the concentration of nitric oxide. Compared with the sham-operated control group, ##p < 0.01. Compared with the CAL control group, **p < 0.01. Data are presented as the mean ± SD.
GSH‑Px can directly eliminate oxygen free radicals and also react with myeloperoxidase to prevent it from further involvement in the oxygen free radicals reaction [31]. GSH‑Px was also shown to inhibit the resulting peroxide-induced cell apoptosis and necrosis, and natural steady state change [32]. CAT plays an important role in cellular antioxidant responses and has one of the highest turnover numbers of all enzymes. One catalase molecule can convert millions of molecules of hydrogen peroxide into water and oxygen each second [33]. Our experimental results showed that GSH‑Px and CAT activities were significantly lower in the CAL group; however, treatment with polydatin for seven weeks significantly increased GSH‑Px activity. Polydatin at 25 mg/kg enhanced the activity of CAT. These results indicate that polydatin may contribute to protect myocardial tissue by increasing GSH‑Px and CAT activities. In conclusion, treatment with polydatin can improve cardiac function and attenuate ventricular remodeling in MI rats caused by CAL. The underlying mechanism may involve restraining the
excessive activation of RAAS and inhibiting peroxidation. Therefore, polydatin may be a potential candidate for the prevention and treatment of cardiac dysfunction and ventricular remodeling after MI.
Materials and Methods !
Animals Male Sprague-Dawley rats (weight approximately 200–220 g) were supplied by Shanghai Slac laboratory animal Co., Ltd. All animals were maintained in a 12-h light/dark cycle constant condition with the temperature at 23 ± 1 °C and the humidity at 40 ± 5 %. These rats received humane care and had free access to a standard diet and distilled water. The animal experiments were approved by the Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine (approval No. 12 009, February 10, 2012) and conformed to the Guide for the Care and
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Use of Laboratory Animals, published by the US National Institutes of Health (NIH publication No. 85–23, revised in 1996).
Drugs and reagents Polydatin (with a purity of 98.7 %, Lot NO. ZL20120907YY) was supplied by Nanjing Zelang Pharmaceutical Co., Ltd. Captopril tablets (Lot NO. 120 505) were obtained from Shanghai Hengshan Pharmaceutical Co., Ltd. They were suspended in distilled water before use.
Experimental protocols The rats were anesthetized with the intraperitoneal administration of sodium pentobarbital (35 mg/kg), and then endotracheal intubated and ventilated with an animal respirator type HX300s (Chengdu Technology & Market Co., Ltd). A left thoracotomy was performed, and the heart was exposed. The left coronary artery was ligated approximately 2 mm from its origin with a 4–0 silk suture. Coronary artery occlusion MI was demonstrated by grossly visible scarring of the change in color of the left ventricle, and ischemia was confirmed by the raising of ST (ECG-6511 Electrocardiograph, Shanghai Nihon Kohden). Then the thorax was closed immediately and the skin sutured. Sham-operated rats underwent a similar process without CAL as described above. After surgical operation, each rat was intraperitoneally administered tramadol (20 mg/kg) for three days to ease pain and intramuscularly injected benzyl penicillin for four days to prevent infection. A week after the operation, the surviving CAL rats were randomly divided into five groups: CAL control, CAL plus 40 mg/kg captopril, CAL plus 25 mg/kg polydatin, CAL plus 50 mg/kg polydatin, and CAL plus 100 mg/kg polydatin. The rats in the treatment groups orally received polydatin or captopril at doses described above once a day, while the rats in the sham-operated control and CAL control groups were treated in the same manner with distilled water. The treatment continued for seven weeks.
Hemodynamic measurement Seven weeks after treatment, rat body weight (BW) was recorded after fasting for 12 h and anesthetized with an intraperitoneal injection of urethane (1.0 g/kg). Then a polypropylene catheter was inserted into the left ventricle. The polypropylene catheter was filled with heparin saline solution and connected to a pressure transducer. After an equilibrium period for about 3 min, LVSP, LVEDP, and ±dp/dtmax were recorded with a multichannel biological signal analysis system (RM6240C type, Chengdu Technology & Market Co., Ltd.).
Cardiac weight indexes and morphological examinations After the hemodynamic parameters were recorded, blood samples were collected from the abdominal aorta into 10-mL centrifugal tubes and centrifuged (4 °C, 1780 × g, 10 min) to get the serum, which was stored immediately in a − 80 °C freezer until being assayed. The hearts were taken out, rinsed with cold saline solution, and the left ventricles were separated from the atria, aorta, and adipose tissue. The left ventricle weight (LVW, mg) and heart weight (HW, mg) were measured, and then LVWI (mg/g) and HWI (mg/g) were estimated by calculating the ratios of the LVW to the BW and the HW to the BW. The left ventricular tissue was divided into two parts. The upper part was immersed in 10% formalin. The lower part was separated into three sections and immediately frozen in liquid nitrogen and then stored in a − 80 °C freezer until being assayed. Gao Y et al. Cardioprotective Effect of …
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The fixed part of the ventricle in formalin was dehydrated and embedded in paraffin, and then cut into 5-µm thick slices and heated overnight in a 60 °C incubator. The sections were stained with hematoxylin and eosin (H&E) for measurement of the cardiomyocyte cross-sectional area. Sirius red in aqueous saturated picric acid was used for examination of perivascular and interstitial fibrosis in the myocardium. Each sample slice was photographed (400× magnification) under a microscope (Olympus BX51). All photos were analyzed with Image-Pro Plus 6.3 analyzing software (Media Cybernetics) by computer. Three fields of every sample slice stained with H&E were examined, and 30 myocardial cells of each field were randomly chosen to calculate the average cross-sectional area of myocytes. The percentage of collagen area per field was calculated as the myocardial interstitial collagen volume fraction (ICVF). The ratio of the perivascular collagen area to the vessel lumen area was calculated as the perivascular collagen volume fraction (PCVF) in the myocardium. For each sample, three fields were randomly selected to photograph and calculate the above parameters. Collagen accumulation as assessed by polarized light microscopy in the interstitial and perivascular space of the left ventricle was analyzed, respectively, with Image-Pro Plus 6.3 analyzing software.
Radioimmunoassay determination The ventricular tissue (100 mg) was homogenized with 1 mL cold 0.9 % NaCl. The homogenized tissue was centrifuged (4 °C, 1780 × g, 15 min), and the supernatant was collected for measurement. A radioimmunoassay was used to detect Ang I, Ang II, and ET-1 concentrations of the ventricular tissue and ALD concentration in the serum. Their concentrations were respectively analyzed with the corresponding iodine [125I] kits (Beijing North Institute of Biological Technology).
Measurement of hydroxyproline and nitric oxide concentrations as well as glutathione peroxidase and catalase activities in serum Hyp and NO concentrations, and GSH‑Px and CAT activities were detected by ultraviolet spectrophotometry with the corresponding kits (Nanjing Jiancheng Institute of Bioengineering). They were expressed as the concentration or activity per milliliter of serum sample of the corresponding rat.
Statistical analysis SPSS 19.0 software was used to analyze and present the data. Numeric variable values are expressed as mean ± SD. One-way analysis of variance and the Student-Neuman-Keuls or Dunnettʼs T3 and Tamhaneʼs T2 tests were used to compare the statistical significances between groups. Differences were considered to be statistically significant when p < 0.05.
Acknowledgements !
This project was supported by the Shanghai Committee of Education Foundation (NO: 12YZ059).
Conflict of Interest !
The authors declare no conflict of interest.
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Original Papers
Cardioprotective Effect of Polydatin on Ventricular Remodeling after Myocardial Infarction in Coronary Artery Ligation Rats
Authors
Yan Gao 1, 2, Jianping Gao 1, Changxun Chen 1, Huilin Wang 1, Juan Guo 1, Rong Wu 1
Affiliations
1 2
Department of Pharmacology, Shanghai University of Traditional Chinese Medicine, Shanghai, China Present address: Jiading Hospital of Traditional Chinese Medicine, Shanghai, China
Key words " polydatin l " myocardial infarction l " ventricular remodeling l " coronary artery ligation l " renin‑angiotensin‑aldosterl one system " peroxidation l
Abstract
received revised accepted
Introduction
February 26, 2014 February 12, 2015 March 2, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1545907 Published online April 23, 2015 Planta Med 2015; 81: 568–577 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Jianping Gao Department of Pharmacology Shanghai University of Traditional Chinese Medicine No. 1200 Cailun Road Shanghai, 201203 China Phone: + 86 21 51 32 22 13 [email protected]
!
The purpose of this study was to explore the effect of polydatin on ventricular remodeling after myocardial infarction in coronary artery ligation rats and to elucidate the underlying mechanisms. A rat model of ventricular remodeling after myocardial infarction was established by left coronary artery ligation. Rats with coronary artery ligation were randomly divided into five groups: control, plus 40 mg/kg captopril, plus 25 mg/kg polydatin, plus 50 mg/kg polydatin, and plus 100 mg/kg polydatin. The sham-operated group was used as a negative control. Rats were administered intragastrically with the corresponding drugs or drinking water for seven weeks. At the end of the treatment, the left ventricular weight index and heart weight index were assessed. The cross-sectional size of cardiomyocytes was measured by staining myocardium tissue with hematoxylin and eosin. Collagen content was counted by Sirius red in aqueous saturated picric acid. The concentrations
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Chronic heart failure (CHF) is known as the cardiac function insufficiency in which the heart cannot put out commensurate blood to supply venous return and tissue metabolism and then causes some syndromes [1]. CHF is a serious threat to human health and the incidence and mortality rate of this disease are rising [2]. CHF is considered to be an irreversible and progressive process characterized by ventricular remodeling and a number of neurohormonal perturbations. It is well known that ventricular remodeling is considered one of the main pathophysiological mechanisms in the process of the development of chronic cardiac insufficiency [3, 4]. Therefore, inhibiting ventricular remodeling early may be an effective way to postpone heart failure induced
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of angiotensin I, angiotensin II, aldosterone, and endothelin 1 in myocardium or serum were determined by radioimmunoassay. Hydroxyproline and nitric oxide concentrations and glutathione peroxidase and catalase activities in serum were measured by ultraviolet spectrophotometry. Our results showed that seven weeks of polydatin treatment resulted in a significantly reduced left ventricular weight index, heart weight index, serum concentrations of hydroxyproline and aldosterone, an increased concentration of nitric oxide as well as enhanced activities of glutathione peroxidase and catalase. Myocardial angiotensin I, angiotensin II, and endothelin 1 levels were also reduced. The cardiomyocyte cross-sectional area and collagen deposition diminished. This study suggests that polydatin may attenuate ventricular remodeling after myocardial infarction in coronary artery ligation rats through restricting the excessive activation of the renin-angiotensin-aldosterone system and inhibiting peroxidation.
by myocardial infarction (MI) and other cardiovascular diseases. Many researchers try to seek effective components from traditional Chinese medicine to control or relieve CHF. Polydatin (C20H22O8, 3,4′,5-trihydroxystibene-3" Fig. 1) is one of the natuβ-mono-D-glucoside, l ral hydroxy-diphenyl ethylene compounds isolated from traditional Chinese medicine, Polygonum cuspidatum Sieb. et Zucc. Modern pharmacological studies have demonstrated that polydatin has multiple biological activities and important clinical value, such as anti-cerebral hemorrhage [5], antithrombosis, antioxidation, affecting ion channels [6], and regulating protein kinase and nitric oxide [7]. Polydatin can also markedly lower the serum levels of total cholesterol, triglyceride, and low-density lipoprotein cholesterol in hyperlipidemic rabbits [8]. It inhibits neutrophil
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Fig. 1 Structure of polydatin.
chemotaxis in inflammatory reactions [9]. Regarding the cardiac protective effect, previous studies have shown that polydatin can attenuate myocardial hypertrophy induced by isopropyl adrenaline in mice and ventricular remodeling caused by abdominal aorta ligation in rats [10]. Zhang et al. also proved that polydatin attenuates ischemia/reperfusion-induced apoptosis in the myocardium of rats [11]. In this study, we adopted the method of coronary artery ligation (CAL) to establish an MI model, and then we investigated the effect and underlying mechanisms of polydatin on ventricular remodeling after MI.
Results !
Similar to a previous report [12], the immediate postoperative mortality rate in our study was about 25 %. Unrecovered breathing, acute left ventricular failure, and fatal ventricular arrhythmia were the main causes of death in the rats. One week after the operation, surviving rats were randomly assigned to different groups. The number of rats was 9 in the sham-operated control group, 15 in the CAL control group, and 11 in each of the rest of the groups. During the seven-week treatment process, five rats died (mortality 33.3 %) in the CAL control group, two rats died (mortality 18.2 %) in the CAL plus 100 mg/kg polydatin group, and one rat died in each of the following groups: sham-operated group (mortality 11.1 %), CAL plus 40 mg/kg captopril, CAL plus 25 mg/kg polydatin, and CAL plus 50 mg/kg polydatin (mortality 9.1 %). After treatment for seven weeks, the left ventricular wall below the ligation part became thinner and showed fibrosis with a pale color in the CAL control rats. The whole heart photographs of all " Fig. 2 A–F. The hearts of the sham-opergroups are shown in l ated rats appeared normal without an infarction area, myocardial fibrosis, or any other abnormalities. On the contrary, a small area of fibrosis appeared in the hearts of the captopril- or polydatin" Fig. 2 G, the left ventricular weight treated groups. As shown in l index (LVWI) and heart weight index (HWI) were significantly greater in the CAL control group than in the sham-operated control group (p < 0.01). After polydatin and captopril treatment for seven weeks, LVWI and HWI were significantly reduced (p < 0.05, " Fig. 3, the cardiomyocyte p < 0.01, respectively). As shown in l cross-sectional area in the CAL control group was significantly larger than that in the sham-operated control group (p < 0.01). Polydatin- and captopril-treated groups showed a decreased average cross-sectional area of cardiomyocytes compared to the CAL control group (p < 0.05, p < 0.01, respectively). Under a microscope, cardiomyocytes appeared orange, and myocardial interstitial and perivascular collagen fibers appeared red or deep red stained with Sirius red. In the rat myocardium of the sham-operated group, a little amount of collagen was found in the interstitial and perivascular space. In the CAL control rats, there was a large accumulation of collagen in the interstitial and perivascular space of the ventricle. Conversely, less collagen depositions were found in both the CAL plus polydatin and captopril
" Fig. 4a1 and 4a3). Under groups than in the CAL control group (l the polarized light microscope, type I collagen fibers appeared red or yellow and type III collagen fibers appeared green. In the rat myocardium of the sham-operated control, fewer amounts of " Fig. 4a2 and 4a4). Nevertheless, collagen fibers were found (l collagen distributions were significantly increased in the CAL control rats compared to the sham-operated rats (p < 0.01). On the contrary, collagen fibers were significantly decreased in the " Fig. 4 b) polydatin- and captopril-treated groups (p < 0.01, l compared to the CAL control group. In addition, as shown in " Fig. 5, the hydroxyproline (Hyp) concentration in the serum l was increased in the CAL control group compared with that in the sham-operated group (p < 0.01). Interestingly, the polydatintreated groups, both at 25 and 50 mg/kg, as well as the captopriltreated groups exhibited a significantly reduced Hyp concentra" Fig. 5). tion compared to the CAL control group (p < 0.01, l Left ventricular systolic pressure (LVSP) and the maximal rate of rise of the left ventricular pressure (+ dp/dtmax) were significantly lower in the CAL control group than in the sham-operated control group (p < 0.01, p < 0.05). The left ventricular end-diastolic pressure (LVEDP) was slightly increased, and the maximal rate of fall of the left ventricular pressure (-dp/dtmax) was slightly reduced in the CAL control group. However, there was no significant difference compared with the sham-operated group. Nevertheless, polydatin- and captopril-treated groups showed a significantly increased LVSP and +dp/dtmax (p < 0.05, p < 0.01, respectively). In addition, both captopril- and polydatin-treated groups showed a tendency of reducing LVEDP and increasing-dp/dtmax, however, no significant difference compared with the CAL con" Table 1). trol group was found (l " As shown in l Fig. 6, in the CAL control group, angiotensin I (Ang I) and angiotensin II (Ang II) concentrations of ventricular tissue and aldosterone (ALD) concentration in the serum were significantly higher than those in the sham-operated control group (p < 0.05, p < 0.01, respectively). Treatment with polydatin or captopril for seven weeks significantly reduced Ang I, Ang II, and ALD concentrations (p < 0.05, p < 0.01, respectively). The low activity of glutathione peroxidase (GSH‑Px) in the serum of the CAL rats was enhanced by polydatin and captopril, and low catalase (CAT) activity in the CAL rats was attenuated by treatment with both polydatin 25 mg/kg and captopril (p < 0.05, " Fig. 7). p < 0.01, l " Fig. 8, the endothelin 1 (ET-1) concentration of the As shown in l ventricular tissue was significantly higher, and the serum nitric oxide (NO) concentration was lower in the CAL control group than in the sham-operated group (p < 0.01). Treatment with polydatin or captopril for seven weeks significantly reduced the ET-1 concentration and increased the NO concentration (p < 0.01).
Discussion !
Left ventricular remodeling is a complex process involving cardiac myocyte growth and death, fibrosis, inflammation, and electrophysiological remodeling [13]. The main causes of ventricular remodeling are hypertension and MI. After MI, abnormal myocardial blood flow can impair myocardial O2 delivery, resulting in maladaptive remodeling. It in turn induces the migration of macrophages, monocytes, and neutrophils into the infarct zone in left ventricle, which initiates intracellular signaling and neurohormonal activation, and localizes the inflammatory response.
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Fig. 2 Some of the whole heart photographs of different groups. A Aham-operated control; B coronary artery ligation (CAL) control; C CAL plus 40 mg/kg captopril; D CAL plus 25 mg/kg polydatin; E CAL plus 50 mg/kg polydatin; F CAL plus 100 mg/kg polydatin. G Effects of polydatin on cardiac weight indexes. Compared with the shamoperated control group, ##p < 0.01. Compared with the CAL control group, *p < 0.05, **p < 0.01. LVWI, left ventricular weight/body weight index; HWI, heart weight/body weight index. Data are presented as the mean ± SD. (Color figure available online only.)
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There will be a lot of compensatory responses of the body after MI, but it is not sufficient to satisfy the needs of heart [14]. The MI model induced by CAL in rats is similar to clinical MI caused by coronary artery occlusion. Following acute MI, the ventricular remodeling is the result of the infarction expansion process, followed by the hypertrophy process. Our experimental results indicate that after ligation for eight weeks, a stable and successful animal model of myocardial hypertrophy is established. In the ventricular remodeling animal model, the heart weight index or mass and the transverse area of the cardiac myocyte are the main indicators of the degree of cardiac hypertrophy [15, 16]. Our study showed that LVWI, HWI, and the average crosssectional area of cardiomyocytes were significantly larger in the CAL control group. Polydatin treatment for seven weeks significantly decreased the cardiac mass index of the CAL rats.
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Myocyte apoptosis and necrosis activate multiple biochemical intracellular signaling that initiates left ventricular dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. As a consequence, inhibition of myocardial fibrosis can improve cardiac function. Treatment with polydatin for seven weeks significantly reduced intercellular and perivascular collagen accumulation. Hyp is one of the major components of collagen in the body, accounting for about 13.4 % of normal collagen and very little of elastin. Therefore, the serum Hyp concentration can be used as an important index for measuring collagen metabolism and judging the degree of fibrosis. It can also promote the myocardial collagen protein synthesis, myocardial interstitial proliferation, and fibrosis [17]. Our results showed that CAL resulted in a significantly increased serum Hyp concentration, while treatment with polydatin significantly reduced the serum Hyp concentration. Based on these results, we speculate that
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polydatin may delay and block fibrosis by decreasing the synthesis and secretion of collagen, and then slow down or inhibit the process of ventricular remodeling. In addition, the effect of polydatin on restraining ventricular remodeling may be partly due to smaller infarctions induced by polydatin, as previous studies suggest that polydatin can lower myocardial infarct size significantly in rats [7, 18]. Hemodynamic disorder is one of the factors that lead to myocardial hypertrophy and reconstruction [19]. Epidemiological studies have reported that approximately 40 % of myocardial infarctions are accompanied by left ventricular systolic dysfunction [20]. In our study, the untreated animals developed cardiac function insufficiency in seven weeks, evidenced by a decreased LVSP and +dp/dtmax compared to the sham-operated rats. Polydatin significantly increased LVSP and +dp/dtmax. Although the cardiac function was compromised by CAL, it did not lead to heart failure, which was evidenced by the mild elevation of LVEDP and the mild reduction of-dp/dtmax. Pharmacologic inhibition of the renin-angiotensin-aldosterone and sympathetic nervous systems has been evaluated in a large number of post-MI clinical trials [21]. In patients with heart failure, the neuroendocrine activation is expressed more excessively
[22]. The renin-angiotensin-aldosterone system (RAAS) plays an important role in the pathophysiology of ventricular remodeling [23]. Therefore, restraining RAAS activation is an important means to treat ventricular remodeling. With regard to the initiation and development of CHF, the main active materials in the RAAS are Ang II and ALD. Ang II plays a vital role in promoting cardiac hypertrophy. It is also a potent vasoconstrictor, which can affect cardiac structure and fibrosis [24]. Inhibition of Ang II synthesis has been demonstrated to be beneficial in preventing left ventricular remodeling and reducing post-MI mortality. Not only does it promote ALD and the retention of water and sodium, it also causes the activation of the sympathetic nerve, collagen synthesis, and interstitial abnormality [25, 26]. Consequently, all these effects mentioned above promote myocardial fibrosis, damage the endothelial function, and induce ventricular remodeling. Our results indicate that the function of polydatin in antagonizing ventricular remodeling after MI may be correlated with its inhibition of the activated RAAS in rats, including significantly reduced myocardial tissue, Ang I, Ang II, and serum ALD concentrations. Endothelial activity factors, such as ET-1 and NO, lead to the alteration of the balance between vasodilatation and vasoconstric-
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Fig. 3 Effect of polydatin on the cardiomyocyte cross-sectional area in rats with ventricular remodeling after myocardial infarction induced by left coronary artery ligation (CAL). The histopathological observations of the heart (hematoxylin and eosin stain, 400×). A Sham-operated control; B coronary artery ligation (CAL) control; C CAL plus 40 mg/kg captopril; D CAL plus 25 mg/kg polydatin; E CAL plus 50 mg/kg polydatin; F CAL plus 100 mg/kg polydatin. G Effect of polydatin on the cardiomyocyte cross-sectional area. Compared with the sham-operated control group, ##p < 0.01. Compared with the CAL control group, *p < 0.05, **p < 0.01. Data are presented as the mean ± SD. (Color figure available online only.)
Original Papers
Fig. 4 a Effects of polydatin on cardiac collagen accumulation in the interstitial and perivascular space of the left ventricle in rats with ventricular remodeling after myocardial infarction induced by left coronary artery ligation (CAL). Numbers 1 and 3 show the images of the Sirius red stained section under normal light illumination; cardiomyocytes appear orange and myocardial interstitial and perivascular collagen fibers appear red or deep red stained
with Sirius red. Numbers 2 and 4 show the polarized light Sirius red images; type I collagen fibers appear red or yellow and type III collagen fibers appear green. Magnification: 400×. A Sham-operated control; B coronary artery ligation (CAL) control; C CAL plus 40 mg/kg captopril; D CAL plus 25 mg/kg polydatin; E CAL plus 50 mg/kg polydatin; F CAL plus 100 mg/kg polydatin. (Color figure available online only.)
Fig. 4 b The effects of polydatin on the interstitial collagen volume fraction (ICVF) and perivascular collagen volume fraction (PVCF) of the left ventricle. Compared with the sham-operated control, ## p < 0.01. Compared with the CAL control, **p < 0.01. Data are presented as the mean ± SD.
tion in systemic circulation. ET-1 is a potent vasoconstrictor that is produced by smooth muscle cells, macrophages, airway epitheliums, and alveolar epithelial cells. ET-1 can increase the intracellular Ca2+ concentration, which leads to cardiac mast cell degranulation; it also promotes the synthesis of collagen and induces cardiomyocyte hypertrophy and inflammation, which leads to
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heart failure [27]. The ET-1 level is increased in symptomatic patients with ischemic heart disease and MI [28, 29]. NO, which plays an important protective role in the cardiovascular system, inhibits smooth muscle cell proliferation and migration. It also enhances proliferation and migration of endothelial cells as well as inhibits apoptosis, suppresses platelet aggregation, and pre-
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Fig. 6 Effect of polydatin on RAAS in rats with ventricular remodeling after myocardial infarction induced by left coronary artery ligation (CAL). a shows the concentrations of angiotensin I and aldosterone, respectively. b shows the concentration of angiotensin II. Compared with the sham-operated control group, #p < 0.05, ##p < 0.01. Compared with the CAL control group, *p < 0.05, **p < 0.01. Data are presented as the mean ± SD.
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Fig. 5 Effect of polydatin on hydroxyproline concentration in rats with ventricular remodeling after myocardial infarction induced by left coronary artery ligation (CAL). Compared with the sham-operated control group, ##p < 0.01. Compared with the CAL control group, **p < 0.01. Data are presented as the mean ± SD.
Original Papers
Table 1 Effects of polydatin on hemodynamic parameters in rats with ventricular remodeling after myocardial infarction caused by left coronary artery ligation (CAL) (mean ± SD). Group
n
Dose (mg/kg)
LVSP (mmHg)
LVEDP (mmHg)
+dP/dtmax (mmHg/s)
−dP/dtmax (mmHg/s)
Sham-operated control CAL control CAL plus captopril CAL plus polydatin CAL plus polydatin CAL plus polydatin
8 10 10 10 10 9
– –
119.65 ± 11.88 95.68 ± 8.56## 109.44 ± 9.30** 116.02 ± 12.25** 114.14 ± 19.27* 118.32 ± 16.48**
0.97 ± 1.71 2.42 ± 2.35 1.06 ± 2.70 0.56 ± 3.80 0.91 ± 7.53 0.33 ± 4.38
9433.45 ± 1915.83 7038.29 ± 601.50# 8769.02 ± 1253.99* 8521.04 ± 1000.75* 8047.74 ± 790.82* 8466.21 ± 924.94*
− 6262.70 ± 964.33 − 5678.89 ± 674.58 − 6339.34 ± 524.21 − 6190.66 ± 1009.03 − 6039.54 ± 594.66 − 5703.71 ± 785.82
40 25 50 100
Note: Compared with the sham-operated control group, #p < 0.05; ##p < 0.01. Compared with the CAL control group, *p < 0.05; **p < 0.01. LVSP, left ventricular systolic pressure; LVEDP, left ventricular end diastolic pressure; +dp/dtmax, the maximal rate of rise of left ventricular pressure; −dp/dtmax, the maximal rate of fall of left ventricular pressure
Fig. 7 Effect of polydatin on oxidative stress in rats with ventricular remodeling after myocardial infarction induced by left coronary artery ligation (CAL). a shows glutathione peroxidase activity. b shows catalase activity. Compared with the sham-operated control group, #p < 0.05. Compared with the CAL control group, *p < 0.05, **p < 0.01. Data are presented as the mean ± SD.
vents platelet, leukocyte, and monocyte adhesion to the endothelium [30]. In our study, CAL increased the ET-1 concentration of the left ventricular tissue and reduced the serum NO concentration. Polydatin treatment resulted in a decreased ET-1 level and
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an increased NO concentration. These results indicate that polydatin may protect myocardial tissue and antagonize ventricular remodeling through adjusting the release of vasoactive factors from endothelial cells.
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Fig. 8 Effect of polydatin on endothelial activity factors in rats with ventricular remodeling after myocardial infarction induced by left coronary artery ligation (CAL). a shows the concentration of endothelin-1. b shows the concentration of nitric oxide. Compared with the sham-operated control group, ##p < 0.01. Compared with the CAL control group, **p < 0.01. Data are presented as the mean ± SD.
GSH‑Px can directly eliminate oxygen free radicals and also react with myeloperoxidase to prevent it from further involvement in the oxygen free radicals reaction [31]. GSH‑Px was also shown to inhibit the resulting peroxide-induced cell apoptosis and necrosis, and natural steady state change [32]. CAT plays an important role in cellular antioxidant responses and has one of the highest turnover numbers of all enzymes. One catalase molecule can convert millions of molecules of hydrogen peroxide into water and oxygen each second [33]. Our experimental results showed that GSH‑Px and CAT activities were significantly lower in the CAL group; however, treatment with polydatin for seven weeks significantly increased GSH‑Px activity. Polydatin at 25 mg/kg enhanced the activity of CAT. These results indicate that polydatin may contribute to protect myocardial tissue by increasing GSH‑Px and CAT activities. In conclusion, treatment with polydatin can improve cardiac function and attenuate ventricular remodeling in MI rats caused by CAL. The underlying mechanism may involve restraining the
excessive activation of RAAS and inhibiting peroxidation. Therefore, polydatin may be a potential candidate for the prevention and treatment of cardiac dysfunction and ventricular remodeling after MI.
Materials and Methods !
Animals Male Sprague-Dawley rats (weight approximately 200–220 g) were supplied by Shanghai Slac laboratory animal Co., Ltd. All animals were maintained in a 12-h light/dark cycle constant condition with the temperature at 23 ± 1 °C and the humidity at 40 ± 5 %. These rats received humane care and had free access to a standard diet and distilled water. The animal experiments were approved by the Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine (approval No. 12 009, February 10, 2012) and conformed to the Guide for the Care and
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Use of Laboratory Animals, published by the US National Institutes of Health (NIH publication No. 85–23, revised in 1996).
Drugs and reagents Polydatin (with a purity of 98.7 %, Lot NO. ZL20120907YY) was supplied by Nanjing Zelang Pharmaceutical Co., Ltd. Captopril tablets (Lot NO. 120 505) were obtained from Shanghai Hengshan Pharmaceutical Co., Ltd. They were suspended in distilled water before use.
Experimental protocols The rats were anesthetized with the intraperitoneal administration of sodium pentobarbital (35 mg/kg), and then endotracheal intubated and ventilated with an animal respirator type HX300s (Chengdu Technology & Market Co., Ltd). A left thoracotomy was performed, and the heart was exposed. The left coronary artery was ligated approximately 2 mm from its origin with a 4–0 silk suture. Coronary artery occlusion MI was demonstrated by grossly visible scarring of the change in color of the left ventricle, and ischemia was confirmed by the raising of ST (ECG-6511 Electrocardiograph, Shanghai Nihon Kohden). Then the thorax was closed immediately and the skin sutured. Sham-operated rats underwent a similar process without CAL as described above. After surgical operation, each rat was intraperitoneally administered tramadol (20 mg/kg) for three days to ease pain and intramuscularly injected benzyl penicillin for four days to prevent infection. A week after the operation, the surviving CAL rats were randomly divided into five groups: CAL control, CAL plus 40 mg/kg captopril, CAL plus 25 mg/kg polydatin, CAL plus 50 mg/kg polydatin, and CAL plus 100 mg/kg polydatin. The rats in the treatment groups orally received polydatin or captopril at doses described above once a day, while the rats in the sham-operated control and CAL control groups were treated in the same manner with distilled water. The treatment continued for seven weeks.
Hemodynamic measurement Seven weeks after treatment, rat body weight (BW) was recorded after fasting for 12 h and anesthetized with an intraperitoneal injection of urethane (1.0 g/kg). Then a polypropylene catheter was inserted into the left ventricle. The polypropylene catheter was filled with heparin saline solution and connected to a pressure transducer. After an equilibrium period for about 3 min, LVSP, LVEDP, and ±dp/dtmax were recorded with a multichannel biological signal analysis system (RM6240C type, Chengdu Technology & Market Co., Ltd.).
Cardiac weight indexes and morphological examinations After the hemodynamic parameters were recorded, blood samples were collected from the abdominal aorta into 10-mL centrifugal tubes and centrifuged (4 °C, 1780 × g, 10 min) to get the serum, which was stored immediately in a − 80 °C freezer until being assayed. The hearts were taken out, rinsed with cold saline solution, and the left ventricles were separated from the atria, aorta, and adipose tissue. The left ventricle weight (LVW, mg) and heart weight (HW, mg) were measured, and then LVWI (mg/g) and HWI (mg/g) were estimated by calculating the ratios of the LVW to the BW and the HW to the BW. The left ventricular tissue was divided into two parts. The upper part was immersed in 10% formalin. The lower part was separated into three sections and immediately frozen in liquid nitrogen and then stored in a − 80 °C freezer until being assayed. Gao Y et al. Cardioprotective Effect of …
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The fixed part of the ventricle in formalin was dehydrated and embedded in paraffin, and then cut into 5-µm thick slices and heated overnight in a 60 °C incubator. The sections were stained with hematoxylin and eosin (H&E) for measurement of the cardiomyocyte cross-sectional area. Sirius red in aqueous saturated picric acid was used for examination of perivascular and interstitial fibrosis in the myocardium. Each sample slice was photographed (400× magnification) under a microscope (Olympus BX51). All photos were analyzed with Image-Pro Plus 6.3 analyzing software (Media Cybernetics) by computer. Three fields of every sample slice stained with H&E were examined, and 30 myocardial cells of each field were randomly chosen to calculate the average cross-sectional area of myocytes. The percentage of collagen area per field was calculated as the myocardial interstitial collagen volume fraction (ICVF). The ratio of the perivascular collagen area to the vessel lumen area was calculated as the perivascular collagen volume fraction (PCVF) in the myocardium. For each sample, three fields were randomly selected to photograph and calculate the above parameters. Collagen accumulation as assessed by polarized light microscopy in the interstitial and perivascular space of the left ventricle was analyzed, respectively, with Image-Pro Plus 6.3 analyzing software.
Radioimmunoassay determination The ventricular tissue (100 mg) was homogenized with 1 mL cold 0.9 % NaCl. The homogenized tissue was centrifuged (4 °C, 1780 × g, 15 min), and the supernatant was collected for measurement. A radioimmunoassay was used to detect Ang I, Ang II, and ET-1 concentrations of the ventricular tissue and ALD concentration in the serum. Their concentrations were respectively analyzed with the corresponding iodine [125I] kits (Beijing North Institute of Biological Technology).
Measurement of hydroxyproline and nitric oxide concentrations as well as glutathione peroxidase and catalase activities in serum Hyp and NO concentrations, and GSH‑Px and CAT activities were detected by ultraviolet spectrophotometry with the corresponding kits (Nanjing Jiancheng Institute of Bioengineering). They were expressed as the concentration or activity per milliliter of serum sample of the corresponding rat.
Statistical analysis SPSS 19.0 software was used to analyze and present the data. Numeric variable values are expressed as mean ± SD. One-way analysis of variance and the Student-Neuman-Keuls or Dunnettʼs T3 and Tamhaneʼs T2 tests were used to compare the statistical significances between groups. Differences were considered to be statistically significant when p < 0.05.
Acknowledgements !
This project was supported by the Shanghai Committee of Education Foundation (NO: 12YZ059).
Conflict of Interest !
The authors declare no conflict of interest.
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