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Protective effects of grape seed proanthocyanidins on cardiovascular remodeling in DOCA-salt hypertension rats
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ScienceDirect Journal of Nutritional Biochemistry 26 (2015) 841 – 849
Protective effects of grape seed proanthocyanidins on cardiovascular remodeling in DOCA-salt hypertension rats Ling-ling Huang a, b, 1 , Chen Pan c, 1, Li Wang a, 1 , Ling Ding a , Kun Guo a , Hong-zhi Wang b , A-man Xu d,⁎, Shan Gao a,⁎ a Department of Pharmacology, Basic Medical College, Anhui Medical University, Hefei 230032, China Cancer Hospital, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, China c Department of Clinical Pharmacy, Lishui People's Hospital, Zhejiang 323000, China d Department of General Surgery, the Fourth Affiliated Hospital, Anhui Medical University, Hefei 230032, China b
Received 11 October 2014; received in revised form 25 February 2015; accepted 6 March 2015
Abstract Cardiovascular remodeling, as a hallmark of hypertension-induced pathophysiology, causes substantial cardiovascular morbidity and mortality. There is increasing evidence that has demonstrated a broad spectrum of pharmacological and therapeutic benefits of grape seed proanthocyanidins (GSP) against oxidative stress and cardiovascular diseases. In this study, 180- to 200-g SD rats treated with DOCA (120 mg/week sc with 1% NaCl and 0.2% KCl in drinking water) and GSP (150, 240, 384 mg/kg) or amlodipine (ALM) (5 mg/kg) for 4 weeks were recruited. The protective effects of GSP on blood pressure and cardiovascular remodeling in rats with DOCAsalt-induced hypertension were investigated. Our results indicated that DOCA-salt could induce hypertension, cardiovascular remodeling and dysfunction, oxidative stress and the release of endothelin-1 (ET-1) and could increase JNK1/2 and p38MAPK phosphorylation. GSP or ALM treatments significantly improved hypertension, cardiovascular remodeling and dysfunction and oxidative stress, restrained the release of ET-1 and down-regulated the JNK1/2 and p38MAPK phosphorylation. These findings demonstrate that GSP has protective effects against increase of blood pressure induced by DOCA-salt hypertension and cardiovascular remodeling by inhibiting the reactive oxygen species/mitogen-activated protein kinase pathway via restraining the release of ET-1. © 2015 Elsevier Inc. All rights reserved. Keywords: Grape seed proanthocyanidins; DOCA-salt hypertension; Cardiovascular remodeling; Antioxidant; p38; JNK
1. Introduction Hypertension is one of the most important risk factors for cardiovascular diseases. American Heart Association has redefined it as a progressive, multiple cardiovascular risk factors syndrome in 2009 [1]. The arterial wall in a state of long-term hemodynamic changes under hypertension could lead to vascular remodeling and functional imbalance. Vascular remodeling is an active process of vascular structure change, which is involved in cell proliferation, migration, apoptosis and extracellular matrix formation and degradation [2]. Moreover, vascular remodeling is the original pathological mechanism of the hypertension organ damage. Research showed that 30–40% of the patients with essential hypertension were associated with left ventricular hypertension (LVH), and 2.5–5% of the general population was associated with LVH [3,4]. Studies also have shown that the mechanism of hypertensive ⁎ Corresponding authors. A. M. Xu is to be contacted at: Department of General Surgery, the Fourth Affiliated Hospital, Anhui Medical University, Hefei 230022, China; S. Gao, Department of Pharmacology, Basic Medical College, Anhui Medical University, Hefei 230032, China. Tel.: +86-55165161133. E-mail addresses: [email protected] (A. Xu), [email protected] (S. Gao). 1 Contributed equally to this work. http://dx.doi.org/10.1016/j.jnutbio.2015.03.007 0955-2863/© 2015 Elsevier Inc. All rights reserved.
LVH was complex, and the possible mechanism of LVH was related to mechanical factors, neurohumoral regulation, the activation of intracellular signal transduction pathways, endocrine factors and genetic factors [5–7]. A previous study proved that reactive oxygen species (ROS) mediated myocardial hypertrophy response induced by ET-1 [8]. In hypertension, ROS performance on vascular remodeling and function, the main focus, included endothelial cells, smooth muscle cells and noncellular components. Hence, the antioxidant has become a hot spot in the antihypertensive treatment in recent years. Grape seed polyphenols [grape seed proanthocyanidin (GSP)], a polyphenols compound extracted from grape seed, belong to the bioflavonoids. Recent studies have shown that GSP down-regulates blood sugar and exerts the antioxidation, antitumor, antiaging, antiatherosclerosis and other pharmacological effects as well, which is associated with multiple system disorders like cardiovascular, endocrine, respiratory diseases and so on [9]. In our previous studies, it was found that GSP had a protective effect on ventricular remodeling induced by isoproterenol, which might be related to the improvement of the body's antioxidant capacity [10]. We also found that GSP inhibited heart, kidney and thoracic aorta damage in DOCA-salt mice, further suggesting that the protective effects of GSP on cardiovascular injury may be related to the role of its antioxidative stress and endothelial dysfunction (ED) [11].
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formal test, all experimental animals adapted to breeding 1 week. Each test value is the average of the five measured at least. 2.4. Hemodynamics, blood and tissue sampling
Fig. 1. Effects of GSP on DOCA-salt hypertensive rats' SBP (mean±S.D., n=6–8).⁎⁎Pb.01 vs. Sham group; #Pb.05, ##Pb.01 vs. DOCA-salt group.
Herein, we have attempted to identify whether chronic treatment with GSP reverses DOCA-salt-induced cardiovascular remodeling and, if so, to explore the potential mechanisms, focusing on the involvement of oxidative stress and the ET-1. 2. Materials and Methods 2.1. Animals Fifty-four male SD rats, weighing 180–200 g, provided by the animal center of Nanjing Medical University were recruited in the study. All procedures were performed in accordance with the protocol outlined in the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No. 85-23, revised 1996) and approved by the Committee on the Ethics of Animal Experiments of Anhui Medical University. Rats were housed under a 12-h light–dark cycle at 18–25°C and received commercial rat chew ad libitum. High salt (1% NaCl and 0.2% KCl) or tap water was administered as drink ad libitum for 4 weeks.
At the end of 4 weeks, all animals were anesthetized with 10% chloral hydrate (3 ml/kg, ip), the right carotid artery was cannulated with a polyethylene catheter connected to a Statham transducer and the mean carotid artery pressure was measured. Then the catheter was inserted along the right coronary artery into the left ventricle, and the signals were recorded on a fourchannel direct-writing oscillograph (BL420S; Chengdu Taimeng Software Co. Ltd.) and digitally sampled (1 kHz) on a personal computer equipped with an analogue to digital interface (BL420S biological function experiment system). The left ventricular systolic pressures (LVSP), left ventricular end-diastolic pressures (LVEDP), the maximal rate of left ventricular systolic and diastolic pressure (±dp/dtmax) were recorded. Thereafter, blood samples were collected from the heart into tubes containing anticoagulant. Blood samples were immediately centrifuged, and the plasma was stored at −80°C until being analyzed. Then animals were killed by exsanguination. The thoracic cavity was opened to expose the still-beating heart. Hearts and aortas were collected. Hearts were rinsed in ice-cold 0.9% NaCl solution, blotted and weighed. The heart weight index (HW/BW) was calculated by dividing heart weight by body weight. Then the heart samples were divided into several parts for various analyses, and aorta samples were employed for further study. 2.5. Isolated aorta preparations Isolated aorta preparations were performed as previously described [12] with minor modification. The thorax was opened, and the descending aorta was immediately excised. After the removal of loose connective tissue, transverse ring (about 4 mm in length) was cut and mounted in individual organ chambers filled with Krebs buffer of the following composition (in mM/L): NaCl, 118; KCl, 4.75; NaHCO3, 25; MgSO4, 1.2; CaCl2, 2; KH2PO4, 1.2; glucose, 11. The solution was continuously aerated with a mixture of 95% O2 and 5% CO2 and maintained at 37°C. Rings were stretched to 1.0 g of resting tension by means of two L-shaped stainless steel wires, which were inserted into the lumen and attached to the chamber and to an isometric force-displacement transducer. Tissues were stretched and washed every 15 min with warm Krebs solution. After an equilibration period of 1 h, the contractile function of the vessel was tested by replacing the Krebs solution with 80 mM K+ solution that was prepared by replacing NaCl with an equimolar amount of KCl and the result was used as the reference contraction. After the washout and restoration of vessel tension to the baseline levels, the rings were exposed to phenylephrine (10−5 M) to reach maximal tension, and acetylcholine (ACh) (10−8 to 10−5 M) was added cumulatively to measure the relaxation response. 2.6. Histological analysis
2.2. DOCA-salt hypertension and experiment protocol DOCA-salt hypertension was performed as described in our previous study [11]. Briefly, rats were anesthetized with 10% chloral hydrate (3 ml/kg, ip), then left kidney was uninephrectomized. They were treated with 120 mg/kg deoxycorticosterone acetate per week [DOCA, soluble in oil/ethanol injection (5/1, v/v); Sigma-Aldrich] and given high salt (1% NaCl and 0.2% KCl) in drinking water for 4 weeks. Animals were randomly divided into 7 groups (n=6–8): Sham rats; UnX-Sham rats; DOCA-salt hypertension rats; DOCA-salt hypertension rats treated with GSP at 150, 240, 384 mg/kg/day; DOCA-salt hypertension rats treated with amlodipine (ALM) at 5 mg/kg/day. The UnX-Sham rats only received left kidney removed. GSP and ALM were given by intragastric gavage dissolved in distilled water.
After weighed, the hearts were immersion-fixed in neutral 10% buffered formalin for histological analysis. Paraffin sections (5 μm) were cut and stained with hematoxylin and eosin (HE) and Van Gieson (VG). Thereafter, the myocyte cross-sectional area (CSA), perivascular collagen area (PVCA) and collagen volume fraction (CVF) were quantitatively analyzed with NIH Image 1.61 software (National Institutes of Health Service Branch) in digitalized microscopic images as has been previously described [13]. Thoracic aorta was removed from rats and fixed in neutral 10% buffered formalin. Paraffin-embedded thoracic aorta (5 μm) was cut, dewaxed and stained with HE. The structural changes of aorta were investigated using a light microscope. Total aorta area (TAA), CSA, aorta radius (AR) and media thickness (MT) of aorta were recorded under a light microscope as has been previously reported [12].
2.3. Tail artery systolic blood pressure (SBP) measurement
2.7. Nitric oxide (NO), endothelin-1 (ET-1), brain natriuretic peptide (BNP) and hydroxyproline (Hyp) measurement
The tail artery systolic pressure of rats was measured by the tail-cuff apparatus (ALC-NIBP, Shanghai Alcott Biotech Co. Ltd.) weekly in the state of awareness. Before
Due to the short half-life and low concentration of NO in vivo, we evaluated plasma − NO levels by measuring its stable metabolites, nitrite (NO− 2 ) and nitrate (NO3 ), by the
Table 1 Effects of GSP on DOCA-salt hypertensive rats' cardiac function (mean±S.D., n=6–8). Group
n
LVSP (mmHg)
LVEDP (mmHg)
+dp/dtmax (mmHg/s)
−dp/dtmax (mmHg/s)
Sham UnX-Sham DOCA-salt GSP 150 GSP 240 GSP 384 ALM
7 8 8 7 6 7 6
162.63±20.25 159.72±30.25 102.36±9.94 ⁎⁎ 114.01±23.82 143.01±35.69 # 145.07±37.15 # 150.29±20.28 ##
−40.50±6.76 −39.4±20.14 −4.27±19.96 ⁎⁎ −10.51±16.01 −16.38±6.87 −23.35±25.13 −25.54±17.15
5910.19±767.23 5487.37±2487.13 3654.47±1338.87 ⁎ 4236.82±1697.73 4145.63±1743.03 4352.83±2129.62 4530.0±1030.17
−5397.01±1041.48 −5258.56±2658.35 −3407.85±1372.55 ⁎ −3930.14±952.29 −3949.94±1774.85 −4020.78±1851.20 −4400.98±909.65
+dp/dtmax is the maximal rate of LVSP; −dp/dtmax is the maximal rate of left ventricular diastolic pressure. ⁎ Pb.05. ⁎⁎ Pb.01 vs. Sham group. # ##
Pb.05. Pb.01 vs. DOCA-salt group.
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Fig. 2. Effects of GSP on DOCA-salt hypertensive rats' HW/BW. (A) Representative figure of heart macroscopic imagines; (B) statistic results (mean±S.D., n=6–8). (1) Sham group; (2) UnX-Sham group; (3) DOCA-salt hypertension group; (4) GSP 150 mg/kg group; (5) GSP 240 mg/kg group; (6) GSP 384 mg/kg group; (7) ALM 5 mg/kg group. ⁎⁎Pb.01 vs. Sham group; #Pb.05, ##Pb.01 vs. DOCA-salt group.
NO Detection Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instruction. Briefly, nitrate was converted to nitrite with aspergillus nitrite reductase, and the total nitrite was measured with the Griess reagent. The absorbance was determined at 540 nm with a spectrophotometer. Both ET-1 levels in plasma and heart tissue were assayed by using the commercial radioimmunoassay kit (Endothelin RIA Kit; Eastern Asia Radioimmunity Research Institute, Beijing, China) according to the instructions provided by manufacturer. Briefly, cardiac tissue were homogenized with 1% bovine serum albumin solution and centrifuged at 3000 rpm, 4°C for 10 min. The supernatant was used to measure ET-1 content. The ET-1 levels were expressed as picograms per milligram of protein and protein concentration was determined by the method of BCA Protein Assay (Beyotime Institute of Biotechnology). The content of Hyp in cardiac tissue and BNP in plasma was measured using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and enzyme-linked immunosorbent assay kit (Biocalvin Co. Ltd., Suzhou, China) according to the instructions provided by manufacturer, respectively.
2.8. Superoxide dismutase (SOD) activities and malonaldehyde (MDA) levels SOD activities in plasma and heart tissue were measured using xanthine oxidase method that measured the absorbance value at 550 nm with the commercial kit (Nanjing Jiancheng Bioengineering, Nanjing Institute, China), and the activity of SOD was expressed as units per milliliter (plasma) and units per milligram protein (tissue). MDA content in plasma and cardiac tissue was measured by thiobarbituric-acidreactive substance method following the manufacturer's instruction (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) that measured the absorbance value at the wavelength of 532 nm, and MDA levels were expressed as nanomoles of MDA per milliliter (plasma) and nanomoles of MDA per milligram protein (tissue). Cardiac tissue were homogenized with 0.9% saline solution and centrifuged at 3000 rpm, 4°C for 10 min, and then the supernatant was removed to detect the SOD activity and MDA content. Cardiac tissue protein concentration was determined by the same method mentioned in Section 2.7.
2.9. Western blot analysis Western blot was performed as previously described [14]. Protein was isolated from freshly pulverized tissue and quantified with BCA Protein Assay. Gel electrophoresis was performed using Bis-Tris gels and transferred to PVDF membranes. Membranes were probed overnight at 4°C with antibodies recognizing the following antigens: GAPDH (1:600, Zhong Shan Gold Bridge Biotechnology, TA-08), JNK1/2 (1:500, Santa Cruz Biotechnology, sc 7345), p-JNK1/2 (1:1000, Cell Signaling Technology, #4668), p38MAPK (1:1000, Cell Signaling Technology, #9212) and p-p38MAPK (1:1000, Cell Signaling Technology, #4511). Goat antirabbit/mouse horseradish peroxidase-conjugated secondary antibody was used (1:10000, Zhong Shan Gold Bridge Biotechnology, ZDR-5118/5117) and antibody–antigen complexes were visualized by enhanced chemiluminescence. Results of Western blots were quantified by the NIH Image J program.
2.10. Statistical analyses The experimental results are expressed as means±S.D.. Statistical analysis was performed by two-tailed and two-sample Student's t test; statistical significance was accepted at Pb.05.
3. Results During the experiment, three rats died of weak physique, and the death of another two rats was due to the errors in the process of blood pressure measurement that caused choke to death. Thus, at the end of the experiment, the data of Sham (n=7), UnX-Sham (n=8), DOCAsalt (n=8), GSP 150 (n=7), GSP 240 (n=6), GSP 384 (n=7) and ALM (n=6) groups were evaluated, respectively. There was no significant difference between Sham group and UnX-Sham group including hemodynamics and cardiovascular remodeling index, biochemical and molecular biology indicator and so on. 3.1. Effects of GSP on tail arterial SBP in DOCA-salt hypertensive rats As shown in Fig. 1, rats in different groups presented similar SBP before operation treatment (PN.05). After treatment, the DOCA-salt group rats presented a significantly increased SBP on week 1 (168.53± 10.81 vs. 143.26±4.36 mmHg, in Sham group; Pb.01). SBP was considerably lower in hypertensive rats treated with GSP and ALM as compared to DOCA-salt hypertensive rats. A progressive reduction in SBP was observed in GSP 150 mg/kg treatment group from week 3 (Pb.05), GSP 240, 384 mg/kg treatment group from week 1 (Pb.01) and ALM treatment group from week 2 (Pb.01). 3.2. Effects of GSP on hemodynamics in DOCA-salt hypertensive rats As shown in Table 1, LVSP and ±dp/dtmax of DOCA-salt group hypertensive rats were significantly lower (Pb.01), and LVEDP increased obviously (Pb.01), indicating that the DOCA-salt hypertensive rats' cardiac function was significantly impaired; through the GSP and ALM treatment for 4 weeks, LVSP increased significantly (Pb.05, Pb.01), and LVEDP and ±dp/dtmax were also considerably higher. 3.3. Effects of GSP on cardiac hypertrophy in DOCA-salt hypertensive rats Results for all groups 4 weeks after DOCA-salt hypertensive induction are shown in Fig. 2. Morphological hypertrophy of heart was characterized by significant increase of HW/BW ratio in DOCA-salt group (Pb.01), whereas the body weight showed no significant difference between groups (data not shown). Similarly, HE staining of cardiac tissue from DOCA-salt group rats showed that myocyte CSA and longitudinal diameter increased significantly compared with the Sham group rats (Fig. 3) (Pb.01). GSP at all doses treatment in DOCAsalt hypertensive rats for 4 weeks, however, reversed those pathological changes, as well as ALM. In keeping with these data, plasma
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BNP levels and the molecular marker of cardiac hypertrophy in DOCAsalt group increased significantly (Fig. 4B). A reduction in plasma BNP levels was found in GSP and ALM treatment groups. This reduced effect of GSP, which attained statistical significance at 384 mg/kg (Pb.05).
— increased by 35.06% ±18.79 (Pb.01) (Fig. 4A). GSP at all doses for 4 weeks' treatment could markedly reverse cardiac collagen deposition in DOCA-salt hypertensive rats, as well as ALM (Pb.05, Pb.01).
3.5. Effects of GSP on ED in DOCA-salt hypertensive rats 3.4. Effects of GSP on cardiac collagen deposition in DOCA-salt hypertensive rats Four weeks after the induction of hypertension, the significantly increased levels of myocardial and perivascular fibrosis were observed in DOCA-salt group rats (Fig. 5). Moreover, the Hyp content — reflecting the collagen level in cardiac tissue and the extent of myocardial fibrosis
Aortic rings from DOCA-salt-treated animals showed strongly reduced endothelium- dependent vasodilator responses to ACh in arteries stimulated by phenylephrine compared to the Sham aortic rings (Fig. 6). The aortic rings obtained from DOCA-salt rats treated with both GSP and ALM showed a significant increase in vasodilatation induced by ACh compared to the animals from DOCA-salt group.
Fig. 3. Effects of GSP on DOCA-salt hypertensive rats' cardiomyocyte CSA and cardiomyocyte long axis (HE stain, magnification ×400). (A) Representative images of histological section of cardiomyocyte cross-section (HE stain, magnification ×400); (B) representative images of histological section of cardiomyocyte long axis (HE stain, magnification ×400); (C) quantitative analyses results (mean±S.D., n=6–8). (1) Sham group; (2) UnX-Sham group; (3) DOCA-salt hypertension group; (4) GSP 150 mg/kg group; (5) GSP 240 mg/kg group; (6) GSP 384 mg/kg group; (7) ALM 5 mg/kg group. ⁎⁎Pb.01 vs. Sham group; ##Pb.01 vs. DOCA-salt group.
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Fig. 4. Effects of GSP on DOCA-salt hypertensive rats' Hyp content in cardiac tissue and BNP content in serum (mean±S.D., n=6–8). ⁎⁎Pb.01 vs. Sham group; #Pb.05, ##Pb.01 vs. DOCA-salt group.
Fig. 5. Effects of GSP on DOCA-salt hypertensive rats' PVCA and CVF in cardiac (VG stain, magnification ×400). (A) Representative images of histological section of CVF (VG stain, magnification ×400); (B) representative images of histological section of PVCA (VG stain, magnification ×400); (C) quantitative analyses of CVF (mean±S.D., n=6–8); (D) quantitative analyses of PVCA (mean±S.D., n=6–8); (1) Sham group; (2) UnX-Sham group; (3) DOCA-salt hypertension group; (4) GSP 150 mg/kg group; (5) GSP 240 mg/kg group; (6) GSP 384 mg/kg group; (7) ALM 5 mg/kg group. **Pb.01 vs. Sham group; ##Pb.01 vs. DOCA-salt group.
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3.8. Effects of GSP on the phosphorylation of JNK1/2 and p38MAPK in DOCA-salt hypertensive rats Fig. 10 showed that JNK1/2 and p38MAPK phosphorylated obviously in DOCA-salt hypertension-induced animal's heart (Pb.05). GSP (150, 240, 384 mg/kg) significantly inhibited the expression of p-JNK1/2 in a dose-dependent manner. In addition, GSP exhibited stronger inhibitory effects on p-p38MAPK expression at 384 mg/kg. 4. Discussion Fig. 6. Effects of GSP on DOCA-salt hypertensive rats' aorta vasodilation (mean±S.D., n=6–8). ⁎⁎Pb.01 vs. Sham group; ##Pb.01 vs. DOCA-salt group.
As expected, DOCA-salt group animals presented a significantly lower NO (8.11±1.92 μmol/L) and higher ET-1 (60.83±8.14 pg/ml) release in plasma and cardiac tissue as compared to Sham animals (14.34±3.05 μmol/L, 43.26±9.20 pg/ml, 5.01±1.30 pg/mg protein; Pb.01). Surprisingly, the reduced NO and increased ET-1 levels were controlled after GSP and ALM treatment. GSP performed significantly higher NO at 240, 384 mg/kg (12.30±1.92, 14.92±3.91 μmol/L; Pb.05, Pb.01), as well as ALM (13.53±5.11 μmol/L; Pb.05). GSP almost completely suppressed ET-1 plasma and cardiac tissue levels (43.46± 9.06, 2.65±1.38; Pb.01, Pb.05) at 384 mg/kg, reaching similar levels of Sham group (Fig. 7). 3.6. Effects of GSP on aortic remodeling in DOCA-salt hypertensive rats The vascular remodeling of the upper thoracic aorta exposed to DOCA-salt was observed at the end of 4 weeks. Compared with Sham group rats, the values of the area of the TAA, CSA, AR and MT of the aorta in DOCA-salt rats were significantly increased. These changes could be prevented by the treatment with GSP at all doses for 4 weeks, as well as ALM (Table 2 and Fig. 8). 3.7. Effects of GSP on SOD activities and MDA levels in DOCA-salt hypertensive rats SOD activities were significantly decreased in DOCA-salt group in comparison with the Sham group (Fig. 9A and C) (Pb.01), and GSP 150, 240, 384 mg/kg and ALM 5 mg/kg administration for 4 weeks obviously increased SOD activities (Pb.05, Pb.01). MDA levels was significantly increased in DOCA-salt group on week 4 compared with the Sham group. Treatment with GSP 150, 240, 384 mg/kg and ALM 5 mg/kg for 4 weeks inhibited the increase of serum and cardiac tissue MDA markedly (Fig. 9B and D; Pb.01).
DOCA can be subcutaneously administered, NaCl can be given in drinking fluids and the arterial blood pressure has then been shown to increase significantly in rats [15]. Hypertension is a complex disease, which is not caused by a single factor, but is rather an interactional regulation of the environment, the genetic and epigenetic [16,17]. DOCA-salt hypertension model characterizes a variety of mechanisms, involving sodium retention, the RAS system activate [18] and vascular active peptides increase [19,20], including ET-1, vasopressin, serotonin and so on; thus, it has been regarded as a classic experimental hypertension animal model. Pathologic cardiac hypertrophy is a maladaptive response of the heart; however, persistence of hypertrophy for long periods can be detrimental, resulting in heart failure, cardiac dysfunction and even sudden death [21,22]. Pathologic hypertrophy is a result of volume or pressure overload or other hormonal or cytokine stimuli, characterized by inadequate myocyte hypertrophy and amount of collagen deposition in the cardiac interstitium, which may contribute to the compromise of both diastolic and systolic function [23]. In line with these previous studies, the present study reveals that DOCA-salt treatment results in marked cardiac hypertrophy, manifested as an elevation of the HW/BW ratio and CSA, an increase of collagen deposition, an increase of LVEDP and impairments of LVSP and ±dp/dtmax. The results also demonstrate that oral therapy with GSP prevents progressive left ventricular dysfunction and attenuates progressive cardiac remodeling in this rat model. The beneficial effects of GSP on cardiac remodeling are shown by attenuation of (1) progressive heart dysfunction as evidenced by normalized LVEDP and ±dp/dtmax, (2) cardiac hypertrophy as evidenced by reduced HW/BW ratio and myocyte CSA and (3) reduced reactive interstitial fibrosis as evidenced by less accumulation of collagen in the cardiac interstitium, as well as ALM. Interestingly, we also find that GSP exert protective effects on blood pressure with a dose-dependent manner although lowest dose was enough for its protective effects on cardiac hypertrophy, myocyte CSA and aortic remodeling. Recently, the role of oxidative stress in hypertension has been identified with characteristics. DOCA-salt hypertension exhibits low
Fig. 7. Effects of GSP on DOCA-salt hypertensive rats' ET-1 and NO release (mean±S.D., n=6–8). (A and B) The ET-1 levels in serum and heart tissue; (C) the NO levels in serum ⁎⁎Pb.01 vs. Sham group; #Pb.05, ##Pb.01 vs. DOCA-salt group.
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Table 2 Effects of GSP on DOCA-salt hypertensive rats' thoracic aorta remodeling (mean±S.D.) Group
n
TAA (×103 μm2)
CSA (×103 μm2)
AR (μm)
MT (μm)
Sham UnX-Sham DOCA-salt GSP 150 GSP 240 GSP 384 ALM
7 8 8 7 6 7 6
1061.58±67.86 1142.04±125.72 3394.40±243.14 ⁎⁎ 1127.98±126.46 ## 939.23±155.03 ## 944.45±8.43 ## 1019.71±230.46 ##
249.58±21.38 279.39±42.60 1447.71±113.00 ⁎⁎ 337.44±62.34 ## 215.209±57.20 ## 229.098±9.19 ## 259.90±61.19 ##
581.23±18.36 602.299±24.798 1147.15±51.23 ⁎⁎ 598.54±34.33 ## 545.34±44.86 ## 548.43±2.45 ## 566.88±65.18 ##
72.94±5.45 78.70±8.79 186.48±9.513 ⁎⁎ 97.54±15.36 ## 66.16±12.70 ## 71.13±3.00 ## 77.96±12.72 ##
⁎⁎ Pb.01 vs. Sham group. ##
Pb.01 vs. DOCA-salt group.
rennin, salt sensitivity but elevated arterial ET-1 and oxidative stress [24]. Elevated arterial ET-1 levels in DOCA-salt rats led to NADPH oxidase activation and superoxide formation via the ETA receptors, resulting in excessive ROS production and ED [25]. This NADPH oxidase-derived ROS function as secondary messengers activated myriad redox-sensitive downstream targets, such as RAS, c-src, mitogen-activated protein kinases (MAPKs), the PI3 kinase/Akt pathway, NF-κB, AP-1, HIF-1 and others, the significant role of which was confirmed in vascular remodeling [26]. ED is identified as the imbalances in the production of vasodilator and vasoconstrictor agents; however, the most commonly accepted ED alteration pertains to abnormalities in the regulation of the lumen of vessels, decreased production in the bioavailability of NO and augmented ET-1 synthesis, release or activity [27]. In our present study, ED has been defined by blunting of endothelium-dependent relaxation response to ACh in noradrenalin precontracted aortic rings and decreasing NO content and increasing ET-1 content in plasma, accompanied with OS in this DOCA-salt hypertensive rats. In addition, hypertrophic remodeling of aortic with increased CSA was found in this model as well. This study also demonstrates that chronic treatment with GSP prevents OS, ED and aortic remodeling in a dose-dependent manner, as well as ALM. Abundant researches indicated that there is a variety of signal mechanisms involved in the evolution of the cardiac hypertrophy, including MAPK signaling pathways. Numerous experimental studies have reported that exogenous ROS can modulate many signaling pathways known to be involved in cardiomyocyte hypertrophy, such as the activation of ERK1/2, JNK, p38MAPK, Akt, PKCs and NF-κB [28– 30]. It is now evident that the ET-1, a potent vasoconstrictive peptide
produced by endothelial cells, is also produced by cardiomyocytes and contributes to the development of cardiac hypertrophy [31,32]. ET-1 production is increased in salt-dependent model, as has already been mentioned, which by activation of renal ETB receptors inhibits sodium reabsorption [33]. Cheng et al. reported that ET-1 activated JNK and p38 phosphorylation, which contributed to the myocardial hypertrophy development [34]. Furthermore, ET-1 has been demonstrated to increase intracellular ROS in cardiomyocytes and antioxidant pretreatment on ET-1-induced cardiac hypertrophy and MAPKs phosphorylation were reduced [35]. Moreover, the ET-1-induced cardiomyocyte hypertrophy was suppressed by the specific ETA receptor antagonist, which was in accordance with the alteration of the molecular markers for hypertrophy, expression of ANP [31]. As shown in our results, OS has been proved by decreasing SOD activity and increasing MDA levels in plasma and local cardiac tissue, and in line with previous reports, excessive release of ET-1 (both plasma and cardiac tissue) was found in these DOCA-salt-induced hypertensive rats. Moreover, we also find that treatment with GSP can prevent cardiac hypertrophy by ameliorating DOCA-salt-induced oxidative stress, which in turn inhibits the downstream events, such as the activation of MAPKs (e.g., JNK and p38-MAPK), and can reduce the levels of BNP. Taken together, this in vivo study provides evidence that marked OS exists in the DOCA-salt rat model, which participates in CR and ED, at least in part. Long-term treatment with GSP prevents these pathologic changes as well as positive drugs — ALM. The protective effect is most likely due to the ability of GSP to inhibit the ROS/MAPK pathway via restraining the release of ET-1.
Fig. 8. Representative figure of rats' thoracic aorta remodeling in different groups (HE stain, magnification ×40). (1) Sham group; (2) UnX-Sham group; (3) DOCA-salt hypertension group; (4) GSP 150 mg/kg group; (5) GSP 240 mg/kg group; (6) GSP 384 mg/kg group; (7) ALM 5 mg/kg group.
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Fig. 9. Effects of GSP on DOCA-salt hypertensive rats' SOD activity and MDA content in serum and cardiac tissue (mean±S.D., n=6–8). (A and C) The SOD activity in serum and heart tissue; (B and D) the MDA content in serum and heart tissue. **Pb.01 vs. Sham group; #Pb.05, ##Pb.01 vs. DOCA-salt group.
Acknowledgments This work was supported by PhD Programs Foundation of Ministry of Education of China (No. 20103420120002), Anhui Medical University Foundation for Middle-aged and Young Scientist Leaders of Disciplines in Science (No.201324) and
National Natural Science Foundation of China (No. 81073088; No. 81373774). The authors' responsibilities were as follows: G.S., H.L.L., P.C. and X.A.M. participated in the design of data analyses and manuscript preparation. H.L.L., W.L., D.L. and W.H.Z. conducted the assays and analyses. All authors read and approved the final manuscript.
Fig. 10. Effects of GSP on phosphorylation of JNK1/2 and p38MAPK in DOCA-salt hypertensive rats (mean±S.D., n=3). (1) Sham group; (2) UnX-Sham group; (3) DOCA-salt hypertension group; (4) GSP 150 mg/kg group; (5) GSP 240 mg/kg group; (6) GSP 384 mg/kg group; (7) ALM 5 mg/kg group. ⁎Pb.05 vs. Sham group; #Pb.05 vs. DOCA-salt group.
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ScienceDirect Journal of Nutritional Biochemistry 26 (2015) 841 – 849
Protective effects of grape seed proanthocyanidins on cardiovascular remodeling in DOCA-salt hypertension rats Ling-ling Huang a, b, 1 , Chen Pan c, 1, Li Wang a, 1 , Ling Ding a , Kun Guo a , Hong-zhi Wang b , A-man Xu d,⁎, Shan Gao a,⁎ a Department of Pharmacology, Basic Medical College, Anhui Medical University, Hefei 230032, China Cancer Hospital, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, China c Department of Clinical Pharmacy, Lishui People's Hospital, Zhejiang 323000, China d Department of General Surgery, the Fourth Affiliated Hospital, Anhui Medical University, Hefei 230032, China b
Received 11 October 2014; received in revised form 25 February 2015; accepted 6 March 2015
Abstract Cardiovascular remodeling, as a hallmark of hypertension-induced pathophysiology, causes substantial cardiovascular morbidity and mortality. There is increasing evidence that has demonstrated a broad spectrum of pharmacological and therapeutic benefits of grape seed proanthocyanidins (GSP) against oxidative stress and cardiovascular diseases. In this study, 180- to 200-g SD rats treated with DOCA (120 mg/week sc with 1% NaCl and 0.2% KCl in drinking water) and GSP (150, 240, 384 mg/kg) or amlodipine (ALM) (5 mg/kg) for 4 weeks were recruited. The protective effects of GSP on blood pressure and cardiovascular remodeling in rats with DOCAsalt-induced hypertension were investigated. Our results indicated that DOCA-salt could induce hypertension, cardiovascular remodeling and dysfunction, oxidative stress and the release of endothelin-1 (ET-1) and could increase JNK1/2 and p38MAPK phosphorylation. GSP or ALM treatments significantly improved hypertension, cardiovascular remodeling and dysfunction and oxidative stress, restrained the release of ET-1 and down-regulated the JNK1/2 and p38MAPK phosphorylation. These findings demonstrate that GSP has protective effects against increase of blood pressure induced by DOCA-salt hypertension and cardiovascular remodeling by inhibiting the reactive oxygen species/mitogen-activated protein kinase pathway via restraining the release of ET-1. © 2015 Elsevier Inc. All rights reserved. Keywords: Grape seed proanthocyanidins; DOCA-salt hypertension; Cardiovascular remodeling; Antioxidant; p38; JNK
1. Introduction Hypertension is one of the most important risk factors for cardiovascular diseases. American Heart Association has redefined it as a progressive, multiple cardiovascular risk factors syndrome in 2009 [1]. The arterial wall in a state of long-term hemodynamic changes under hypertension could lead to vascular remodeling and functional imbalance. Vascular remodeling is an active process of vascular structure change, which is involved in cell proliferation, migration, apoptosis and extracellular matrix formation and degradation [2]. Moreover, vascular remodeling is the original pathological mechanism of the hypertension organ damage. Research showed that 30–40% of the patients with essential hypertension were associated with left ventricular hypertension (LVH), and 2.5–5% of the general population was associated with LVH [3,4]. Studies also have shown that the mechanism of hypertensive ⁎ Corresponding authors. A. M. Xu is to be contacted at: Department of General Surgery, the Fourth Affiliated Hospital, Anhui Medical University, Hefei 230022, China; S. Gao, Department of Pharmacology, Basic Medical College, Anhui Medical University, Hefei 230032, China. Tel.: +86-55165161133. E-mail addresses: [email protected] (A. Xu), [email protected] (S. Gao). 1 Contributed equally to this work. http://dx.doi.org/10.1016/j.jnutbio.2015.03.007 0955-2863/© 2015 Elsevier Inc. All rights reserved.
LVH was complex, and the possible mechanism of LVH was related to mechanical factors, neurohumoral regulation, the activation of intracellular signal transduction pathways, endocrine factors and genetic factors [5–7]. A previous study proved that reactive oxygen species (ROS) mediated myocardial hypertrophy response induced by ET-1 [8]. In hypertension, ROS performance on vascular remodeling and function, the main focus, included endothelial cells, smooth muscle cells and noncellular components. Hence, the antioxidant has become a hot spot in the antihypertensive treatment in recent years. Grape seed polyphenols [grape seed proanthocyanidin (GSP)], a polyphenols compound extracted from grape seed, belong to the bioflavonoids. Recent studies have shown that GSP down-regulates blood sugar and exerts the antioxidation, antitumor, antiaging, antiatherosclerosis and other pharmacological effects as well, which is associated with multiple system disorders like cardiovascular, endocrine, respiratory diseases and so on [9]. In our previous studies, it was found that GSP had a protective effect on ventricular remodeling induced by isoproterenol, which might be related to the improvement of the body's antioxidant capacity [10]. We also found that GSP inhibited heart, kidney and thoracic aorta damage in DOCA-salt mice, further suggesting that the protective effects of GSP on cardiovascular injury may be related to the role of its antioxidative stress and endothelial dysfunction (ED) [11].
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formal test, all experimental animals adapted to breeding 1 week. Each test value is the average of the five measured at least. 2.4. Hemodynamics, blood and tissue sampling
Fig. 1. Effects of GSP on DOCA-salt hypertensive rats' SBP (mean±S.D., n=6–8).⁎⁎Pb.01 vs. Sham group; #Pb.05, ##Pb.01 vs. DOCA-salt group.
Herein, we have attempted to identify whether chronic treatment with GSP reverses DOCA-salt-induced cardiovascular remodeling and, if so, to explore the potential mechanisms, focusing on the involvement of oxidative stress and the ET-1. 2. Materials and Methods 2.1. Animals Fifty-four male SD rats, weighing 180–200 g, provided by the animal center of Nanjing Medical University were recruited in the study. All procedures were performed in accordance with the protocol outlined in the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No. 85-23, revised 1996) and approved by the Committee on the Ethics of Animal Experiments of Anhui Medical University. Rats were housed under a 12-h light–dark cycle at 18–25°C and received commercial rat chew ad libitum. High salt (1% NaCl and 0.2% KCl) or tap water was administered as drink ad libitum for 4 weeks.
At the end of 4 weeks, all animals were anesthetized with 10% chloral hydrate (3 ml/kg, ip), the right carotid artery was cannulated with a polyethylene catheter connected to a Statham transducer and the mean carotid artery pressure was measured. Then the catheter was inserted along the right coronary artery into the left ventricle, and the signals were recorded on a fourchannel direct-writing oscillograph (BL420S; Chengdu Taimeng Software Co. Ltd.) and digitally sampled (1 kHz) on a personal computer equipped with an analogue to digital interface (BL420S biological function experiment system). The left ventricular systolic pressures (LVSP), left ventricular end-diastolic pressures (LVEDP), the maximal rate of left ventricular systolic and diastolic pressure (±dp/dtmax) were recorded. Thereafter, blood samples were collected from the heart into tubes containing anticoagulant. Blood samples were immediately centrifuged, and the plasma was stored at −80°C until being analyzed. Then animals were killed by exsanguination. The thoracic cavity was opened to expose the still-beating heart. Hearts and aortas were collected. Hearts were rinsed in ice-cold 0.9% NaCl solution, blotted and weighed. The heart weight index (HW/BW) was calculated by dividing heart weight by body weight. Then the heart samples were divided into several parts for various analyses, and aorta samples were employed for further study. 2.5. Isolated aorta preparations Isolated aorta preparations were performed as previously described [12] with minor modification. The thorax was opened, and the descending aorta was immediately excised. After the removal of loose connective tissue, transverse ring (about 4 mm in length) was cut and mounted in individual organ chambers filled with Krebs buffer of the following composition (in mM/L): NaCl, 118; KCl, 4.75; NaHCO3, 25; MgSO4, 1.2; CaCl2, 2; KH2PO4, 1.2; glucose, 11. The solution was continuously aerated with a mixture of 95% O2 and 5% CO2 and maintained at 37°C. Rings were stretched to 1.0 g of resting tension by means of two L-shaped stainless steel wires, which were inserted into the lumen and attached to the chamber and to an isometric force-displacement transducer. Tissues were stretched and washed every 15 min with warm Krebs solution. After an equilibration period of 1 h, the contractile function of the vessel was tested by replacing the Krebs solution with 80 mM K+ solution that was prepared by replacing NaCl with an equimolar amount of KCl and the result was used as the reference contraction. After the washout and restoration of vessel tension to the baseline levels, the rings were exposed to phenylephrine (10−5 M) to reach maximal tension, and acetylcholine (ACh) (10−8 to 10−5 M) was added cumulatively to measure the relaxation response. 2.6. Histological analysis
2.2. DOCA-salt hypertension and experiment protocol DOCA-salt hypertension was performed as described in our previous study [11]. Briefly, rats were anesthetized with 10% chloral hydrate (3 ml/kg, ip), then left kidney was uninephrectomized. They were treated with 120 mg/kg deoxycorticosterone acetate per week [DOCA, soluble in oil/ethanol injection (5/1, v/v); Sigma-Aldrich] and given high salt (1% NaCl and 0.2% KCl) in drinking water for 4 weeks. Animals were randomly divided into 7 groups (n=6–8): Sham rats; UnX-Sham rats; DOCA-salt hypertension rats; DOCA-salt hypertension rats treated with GSP at 150, 240, 384 mg/kg/day; DOCA-salt hypertension rats treated with amlodipine (ALM) at 5 mg/kg/day. The UnX-Sham rats only received left kidney removed. GSP and ALM were given by intragastric gavage dissolved in distilled water.
After weighed, the hearts were immersion-fixed in neutral 10% buffered formalin for histological analysis. Paraffin sections (5 μm) were cut and stained with hematoxylin and eosin (HE) and Van Gieson (VG). Thereafter, the myocyte cross-sectional area (CSA), perivascular collagen area (PVCA) and collagen volume fraction (CVF) were quantitatively analyzed with NIH Image 1.61 software (National Institutes of Health Service Branch) in digitalized microscopic images as has been previously described [13]. Thoracic aorta was removed from rats and fixed in neutral 10% buffered formalin. Paraffin-embedded thoracic aorta (5 μm) was cut, dewaxed and stained with HE. The structural changes of aorta were investigated using a light microscope. Total aorta area (TAA), CSA, aorta radius (AR) and media thickness (MT) of aorta were recorded under a light microscope as has been previously reported [12].
2.3. Tail artery systolic blood pressure (SBP) measurement
2.7. Nitric oxide (NO), endothelin-1 (ET-1), brain natriuretic peptide (BNP) and hydroxyproline (Hyp) measurement
The tail artery systolic pressure of rats was measured by the tail-cuff apparatus (ALC-NIBP, Shanghai Alcott Biotech Co. Ltd.) weekly in the state of awareness. Before
Due to the short half-life and low concentration of NO in vivo, we evaluated plasma − NO levels by measuring its stable metabolites, nitrite (NO− 2 ) and nitrate (NO3 ), by the
Table 1 Effects of GSP on DOCA-salt hypertensive rats' cardiac function (mean±S.D., n=6–8). Group
n
LVSP (mmHg)
LVEDP (mmHg)
+dp/dtmax (mmHg/s)
−dp/dtmax (mmHg/s)
Sham UnX-Sham DOCA-salt GSP 150 GSP 240 GSP 384 ALM
7 8 8 7 6 7 6
162.63±20.25 159.72±30.25 102.36±9.94 ⁎⁎ 114.01±23.82 143.01±35.69 # 145.07±37.15 # 150.29±20.28 ##
−40.50±6.76 −39.4±20.14 −4.27±19.96 ⁎⁎ −10.51±16.01 −16.38±6.87 −23.35±25.13 −25.54±17.15
5910.19±767.23 5487.37±2487.13 3654.47±1338.87 ⁎ 4236.82±1697.73 4145.63±1743.03 4352.83±2129.62 4530.0±1030.17
−5397.01±1041.48 −5258.56±2658.35 −3407.85±1372.55 ⁎ −3930.14±952.29 −3949.94±1774.85 −4020.78±1851.20 −4400.98±909.65
+dp/dtmax is the maximal rate of LVSP; −dp/dtmax is the maximal rate of left ventricular diastolic pressure. ⁎ Pb.05. ⁎⁎ Pb.01 vs. Sham group. # ##
Pb.05. Pb.01 vs. DOCA-salt group.
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Fig. 2. Effects of GSP on DOCA-salt hypertensive rats' HW/BW. (A) Representative figure of heart macroscopic imagines; (B) statistic results (mean±S.D., n=6–8). (1) Sham group; (2) UnX-Sham group; (3) DOCA-salt hypertension group; (4) GSP 150 mg/kg group; (5) GSP 240 mg/kg group; (6) GSP 384 mg/kg group; (7) ALM 5 mg/kg group. ⁎⁎Pb.01 vs. Sham group; #Pb.05, ##Pb.01 vs. DOCA-salt group.
NO Detection Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instruction. Briefly, nitrate was converted to nitrite with aspergillus nitrite reductase, and the total nitrite was measured with the Griess reagent. The absorbance was determined at 540 nm with a spectrophotometer. Both ET-1 levels in plasma and heart tissue were assayed by using the commercial radioimmunoassay kit (Endothelin RIA Kit; Eastern Asia Radioimmunity Research Institute, Beijing, China) according to the instructions provided by manufacturer. Briefly, cardiac tissue were homogenized with 1% bovine serum albumin solution and centrifuged at 3000 rpm, 4°C for 10 min. The supernatant was used to measure ET-1 content. The ET-1 levels were expressed as picograms per milligram of protein and protein concentration was determined by the method of BCA Protein Assay (Beyotime Institute of Biotechnology). The content of Hyp in cardiac tissue and BNP in plasma was measured using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and enzyme-linked immunosorbent assay kit (Biocalvin Co. Ltd., Suzhou, China) according to the instructions provided by manufacturer, respectively.
2.8. Superoxide dismutase (SOD) activities and malonaldehyde (MDA) levels SOD activities in plasma and heart tissue were measured using xanthine oxidase method that measured the absorbance value at 550 nm with the commercial kit (Nanjing Jiancheng Bioengineering, Nanjing Institute, China), and the activity of SOD was expressed as units per milliliter (plasma) and units per milligram protein (tissue). MDA content in plasma and cardiac tissue was measured by thiobarbituric-acidreactive substance method following the manufacturer's instruction (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) that measured the absorbance value at the wavelength of 532 nm, and MDA levels were expressed as nanomoles of MDA per milliliter (plasma) and nanomoles of MDA per milligram protein (tissue). Cardiac tissue were homogenized with 0.9% saline solution and centrifuged at 3000 rpm, 4°C for 10 min, and then the supernatant was removed to detect the SOD activity and MDA content. Cardiac tissue protein concentration was determined by the same method mentioned in Section 2.7.
2.9. Western blot analysis Western blot was performed as previously described [14]. Protein was isolated from freshly pulverized tissue and quantified with BCA Protein Assay. Gel electrophoresis was performed using Bis-Tris gels and transferred to PVDF membranes. Membranes were probed overnight at 4°C with antibodies recognizing the following antigens: GAPDH (1:600, Zhong Shan Gold Bridge Biotechnology, TA-08), JNK1/2 (1:500, Santa Cruz Biotechnology, sc 7345), p-JNK1/2 (1:1000, Cell Signaling Technology, #4668), p38MAPK (1:1000, Cell Signaling Technology, #9212) and p-p38MAPK (1:1000, Cell Signaling Technology, #4511). Goat antirabbit/mouse horseradish peroxidase-conjugated secondary antibody was used (1:10000, Zhong Shan Gold Bridge Biotechnology, ZDR-5118/5117) and antibody–antigen complexes were visualized by enhanced chemiluminescence. Results of Western blots were quantified by the NIH Image J program.
2.10. Statistical analyses The experimental results are expressed as means±S.D.. Statistical analysis was performed by two-tailed and two-sample Student's t test; statistical significance was accepted at Pb.05.
3. Results During the experiment, three rats died of weak physique, and the death of another two rats was due to the errors in the process of blood pressure measurement that caused choke to death. Thus, at the end of the experiment, the data of Sham (n=7), UnX-Sham (n=8), DOCAsalt (n=8), GSP 150 (n=7), GSP 240 (n=6), GSP 384 (n=7) and ALM (n=6) groups were evaluated, respectively. There was no significant difference between Sham group and UnX-Sham group including hemodynamics and cardiovascular remodeling index, biochemical and molecular biology indicator and so on. 3.1. Effects of GSP on tail arterial SBP in DOCA-salt hypertensive rats As shown in Fig. 1, rats in different groups presented similar SBP before operation treatment (PN.05). After treatment, the DOCA-salt group rats presented a significantly increased SBP on week 1 (168.53± 10.81 vs. 143.26±4.36 mmHg, in Sham group; Pb.01). SBP was considerably lower in hypertensive rats treated with GSP and ALM as compared to DOCA-salt hypertensive rats. A progressive reduction in SBP was observed in GSP 150 mg/kg treatment group from week 3 (Pb.05), GSP 240, 384 mg/kg treatment group from week 1 (Pb.01) and ALM treatment group from week 2 (Pb.01). 3.2. Effects of GSP on hemodynamics in DOCA-salt hypertensive rats As shown in Table 1, LVSP and ±dp/dtmax of DOCA-salt group hypertensive rats were significantly lower (Pb.01), and LVEDP increased obviously (Pb.01), indicating that the DOCA-salt hypertensive rats' cardiac function was significantly impaired; through the GSP and ALM treatment for 4 weeks, LVSP increased significantly (Pb.05, Pb.01), and LVEDP and ±dp/dtmax were also considerably higher. 3.3. Effects of GSP on cardiac hypertrophy in DOCA-salt hypertensive rats Results for all groups 4 weeks after DOCA-salt hypertensive induction are shown in Fig. 2. Morphological hypertrophy of heart was characterized by significant increase of HW/BW ratio in DOCA-salt group (Pb.01), whereas the body weight showed no significant difference between groups (data not shown). Similarly, HE staining of cardiac tissue from DOCA-salt group rats showed that myocyte CSA and longitudinal diameter increased significantly compared with the Sham group rats (Fig. 3) (Pb.01). GSP at all doses treatment in DOCAsalt hypertensive rats for 4 weeks, however, reversed those pathological changes, as well as ALM. In keeping with these data, plasma
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BNP levels and the molecular marker of cardiac hypertrophy in DOCAsalt group increased significantly (Fig. 4B). A reduction in plasma BNP levels was found in GSP and ALM treatment groups. This reduced effect of GSP, which attained statistical significance at 384 mg/kg (Pb.05).
— increased by 35.06% ±18.79 (Pb.01) (Fig. 4A). GSP at all doses for 4 weeks' treatment could markedly reverse cardiac collagen deposition in DOCA-salt hypertensive rats, as well as ALM (Pb.05, Pb.01).
3.5. Effects of GSP on ED in DOCA-salt hypertensive rats 3.4. Effects of GSP on cardiac collagen deposition in DOCA-salt hypertensive rats Four weeks after the induction of hypertension, the significantly increased levels of myocardial and perivascular fibrosis were observed in DOCA-salt group rats (Fig. 5). Moreover, the Hyp content — reflecting the collagen level in cardiac tissue and the extent of myocardial fibrosis
Aortic rings from DOCA-salt-treated animals showed strongly reduced endothelium- dependent vasodilator responses to ACh in arteries stimulated by phenylephrine compared to the Sham aortic rings (Fig. 6). The aortic rings obtained from DOCA-salt rats treated with both GSP and ALM showed a significant increase in vasodilatation induced by ACh compared to the animals from DOCA-salt group.
Fig. 3. Effects of GSP on DOCA-salt hypertensive rats' cardiomyocyte CSA and cardiomyocyte long axis (HE stain, magnification ×400). (A) Representative images of histological section of cardiomyocyte cross-section (HE stain, magnification ×400); (B) representative images of histological section of cardiomyocyte long axis (HE stain, magnification ×400); (C) quantitative analyses results (mean±S.D., n=6–8). (1) Sham group; (2) UnX-Sham group; (3) DOCA-salt hypertension group; (4) GSP 150 mg/kg group; (5) GSP 240 mg/kg group; (6) GSP 384 mg/kg group; (7) ALM 5 mg/kg group. ⁎⁎Pb.01 vs. Sham group; ##Pb.01 vs. DOCA-salt group.
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Fig. 4. Effects of GSP on DOCA-salt hypertensive rats' Hyp content in cardiac tissue and BNP content in serum (mean±S.D., n=6–8). ⁎⁎Pb.01 vs. Sham group; #Pb.05, ##Pb.01 vs. DOCA-salt group.
Fig. 5. Effects of GSP on DOCA-salt hypertensive rats' PVCA and CVF in cardiac (VG stain, magnification ×400). (A) Representative images of histological section of CVF (VG stain, magnification ×400); (B) representative images of histological section of PVCA (VG stain, magnification ×400); (C) quantitative analyses of CVF (mean±S.D., n=6–8); (D) quantitative analyses of PVCA (mean±S.D., n=6–8); (1) Sham group; (2) UnX-Sham group; (3) DOCA-salt hypertension group; (4) GSP 150 mg/kg group; (5) GSP 240 mg/kg group; (6) GSP 384 mg/kg group; (7) ALM 5 mg/kg group. **Pb.01 vs. Sham group; ##Pb.01 vs. DOCA-salt group.
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3.8. Effects of GSP on the phosphorylation of JNK1/2 and p38MAPK in DOCA-salt hypertensive rats Fig. 10 showed that JNK1/2 and p38MAPK phosphorylated obviously in DOCA-salt hypertension-induced animal's heart (Pb.05). GSP (150, 240, 384 mg/kg) significantly inhibited the expression of p-JNK1/2 in a dose-dependent manner. In addition, GSP exhibited stronger inhibitory effects on p-p38MAPK expression at 384 mg/kg. 4. Discussion Fig. 6. Effects of GSP on DOCA-salt hypertensive rats' aorta vasodilation (mean±S.D., n=6–8). ⁎⁎Pb.01 vs. Sham group; ##Pb.01 vs. DOCA-salt group.
As expected, DOCA-salt group animals presented a significantly lower NO (8.11±1.92 μmol/L) and higher ET-1 (60.83±8.14 pg/ml) release in plasma and cardiac tissue as compared to Sham animals (14.34±3.05 μmol/L, 43.26±9.20 pg/ml, 5.01±1.30 pg/mg protein; Pb.01). Surprisingly, the reduced NO and increased ET-1 levels were controlled after GSP and ALM treatment. GSP performed significantly higher NO at 240, 384 mg/kg (12.30±1.92, 14.92±3.91 μmol/L; Pb.05, Pb.01), as well as ALM (13.53±5.11 μmol/L; Pb.05). GSP almost completely suppressed ET-1 plasma and cardiac tissue levels (43.46± 9.06, 2.65±1.38; Pb.01, Pb.05) at 384 mg/kg, reaching similar levels of Sham group (Fig. 7). 3.6. Effects of GSP on aortic remodeling in DOCA-salt hypertensive rats The vascular remodeling of the upper thoracic aorta exposed to DOCA-salt was observed at the end of 4 weeks. Compared with Sham group rats, the values of the area of the TAA, CSA, AR and MT of the aorta in DOCA-salt rats were significantly increased. These changes could be prevented by the treatment with GSP at all doses for 4 weeks, as well as ALM (Table 2 and Fig. 8). 3.7. Effects of GSP on SOD activities and MDA levels in DOCA-salt hypertensive rats SOD activities were significantly decreased in DOCA-salt group in comparison with the Sham group (Fig. 9A and C) (Pb.01), and GSP 150, 240, 384 mg/kg and ALM 5 mg/kg administration for 4 weeks obviously increased SOD activities (Pb.05, Pb.01). MDA levels was significantly increased in DOCA-salt group on week 4 compared with the Sham group. Treatment with GSP 150, 240, 384 mg/kg and ALM 5 mg/kg for 4 weeks inhibited the increase of serum and cardiac tissue MDA markedly (Fig. 9B and D; Pb.01).
DOCA can be subcutaneously administered, NaCl can be given in drinking fluids and the arterial blood pressure has then been shown to increase significantly in rats [15]. Hypertension is a complex disease, which is not caused by a single factor, but is rather an interactional regulation of the environment, the genetic and epigenetic [16,17]. DOCA-salt hypertension model characterizes a variety of mechanisms, involving sodium retention, the RAS system activate [18] and vascular active peptides increase [19,20], including ET-1, vasopressin, serotonin and so on; thus, it has been regarded as a classic experimental hypertension animal model. Pathologic cardiac hypertrophy is a maladaptive response of the heart; however, persistence of hypertrophy for long periods can be detrimental, resulting in heart failure, cardiac dysfunction and even sudden death [21,22]. Pathologic hypertrophy is a result of volume or pressure overload or other hormonal or cytokine stimuli, characterized by inadequate myocyte hypertrophy and amount of collagen deposition in the cardiac interstitium, which may contribute to the compromise of both diastolic and systolic function [23]. In line with these previous studies, the present study reveals that DOCA-salt treatment results in marked cardiac hypertrophy, manifested as an elevation of the HW/BW ratio and CSA, an increase of collagen deposition, an increase of LVEDP and impairments of LVSP and ±dp/dtmax. The results also demonstrate that oral therapy with GSP prevents progressive left ventricular dysfunction and attenuates progressive cardiac remodeling in this rat model. The beneficial effects of GSP on cardiac remodeling are shown by attenuation of (1) progressive heart dysfunction as evidenced by normalized LVEDP and ±dp/dtmax, (2) cardiac hypertrophy as evidenced by reduced HW/BW ratio and myocyte CSA and (3) reduced reactive interstitial fibrosis as evidenced by less accumulation of collagen in the cardiac interstitium, as well as ALM. Interestingly, we also find that GSP exert protective effects on blood pressure with a dose-dependent manner although lowest dose was enough for its protective effects on cardiac hypertrophy, myocyte CSA and aortic remodeling. Recently, the role of oxidative stress in hypertension has been identified with characteristics. DOCA-salt hypertension exhibits low
Fig. 7. Effects of GSP on DOCA-salt hypertensive rats' ET-1 and NO release (mean±S.D., n=6–8). (A and B) The ET-1 levels in serum and heart tissue; (C) the NO levels in serum ⁎⁎Pb.01 vs. Sham group; #Pb.05, ##Pb.01 vs. DOCA-salt group.
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Table 2 Effects of GSP on DOCA-salt hypertensive rats' thoracic aorta remodeling (mean±S.D.) Group
n
TAA (×103 μm2)
CSA (×103 μm2)
AR (μm)
MT (μm)
Sham UnX-Sham DOCA-salt GSP 150 GSP 240 GSP 384 ALM
7 8 8 7 6 7 6
1061.58±67.86 1142.04±125.72 3394.40±243.14 ⁎⁎ 1127.98±126.46 ## 939.23±155.03 ## 944.45±8.43 ## 1019.71±230.46 ##
249.58±21.38 279.39±42.60 1447.71±113.00 ⁎⁎ 337.44±62.34 ## 215.209±57.20 ## 229.098±9.19 ## 259.90±61.19 ##
581.23±18.36 602.299±24.798 1147.15±51.23 ⁎⁎ 598.54±34.33 ## 545.34±44.86 ## 548.43±2.45 ## 566.88±65.18 ##
72.94±5.45 78.70±8.79 186.48±9.513 ⁎⁎ 97.54±15.36 ## 66.16±12.70 ## 71.13±3.00 ## 77.96±12.72 ##
⁎⁎ Pb.01 vs. Sham group. ##
Pb.01 vs. DOCA-salt group.
rennin, salt sensitivity but elevated arterial ET-1 and oxidative stress [24]. Elevated arterial ET-1 levels in DOCA-salt rats led to NADPH oxidase activation and superoxide formation via the ETA receptors, resulting in excessive ROS production and ED [25]. This NADPH oxidase-derived ROS function as secondary messengers activated myriad redox-sensitive downstream targets, such as RAS, c-src, mitogen-activated protein kinases (MAPKs), the PI3 kinase/Akt pathway, NF-κB, AP-1, HIF-1 and others, the significant role of which was confirmed in vascular remodeling [26]. ED is identified as the imbalances in the production of vasodilator and vasoconstrictor agents; however, the most commonly accepted ED alteration pertains to abnormalities in the regulation of the lumen of vessels, decreased production in the bioavailability of NO and augmented ET-1 synthesis, release or activity [27]. In our present study, ED has been defined by blunting of endothelium-dependent relaxation response to ACh in noradrenalin precontracted aortic rings and decreasing NO content and increasing ET-1 content in plasma, accompanied with OS in this DOCA-salt hypertensive rats. In addition, hypertrophic remodeling of aortic with increased CSA was found in this model as well. This study also demonstrates that chronic treatment with GSP prevents OS, ED and aortic remodeling in a dose-dependent manner, as well as ALM. Abundant researches indicated that there is a variety of signal mechanisms involved in the evolution of the cardiac hypertrophy, including MAPK signaling pathways. Numerous experimental studies have reported that exogenous ROS can modulate many signaling pathways known to be involved in cardiomyocyte hypertrophy, such as the activation of ERK1/2, JNK, p38MAPK, Akt, PKCs and NF-κB [28– 30]. It is now evident that the ET-1, a potent vasoconstrictive peptide
produced by endothelial cells, is also produced by cardiomyocytes and contributes to the development of cardiac hypertrophy [31,32]. ET-1 production is increased in salt-dependent model, as has already been mentioned, which by activation of renal ETB receptors inhibits sodium reabsorption [33]. Cheng et al. reported that ET-1 activated JNK and p38 phosphorylation, which contributed to the myocardial hypertrophy development [34]. Furthermore, ET-1 has been demonstrated to increase intracellular ROS in cardiomyocytes and antioxidant pretreatment on ET-1-induced cardiac hypertrophy and MAPKs phosphorylation were reduced [35]. Moreover, the ET-1-induced cardiomyocyte hypertrophy was suppressed by the specific ETA receptor antagonist, which was in accordance with the alteration of the molecular markers for hypertrophy, expression of ANP [31]. As shown in our results, OS has been proved by decreasing SOD activity and increasing MDA levels in plasma and local cardiac tissue, and in line with previous reports, excessive release of ET-1 (both plasma and cardiac tissue) was found in these DOCA-salt-induced hypertensive rats. Moreover, we also find that treatment with GSP can prevent cardiac hypertrophy by ameliorating DOCA-salt-induced oxidative stress, which in turn inhibits the downstream events, such as the activation of MAPKs (e.g., JNK and p38-MAPK), and can reduce the levels of BNP. Taken together, this in vivo study provides evidence that marked OS exists in the DOCA-salt rat model, which participates in CR and ED, at least in part. Long-term treatment with GSP prevents these pathologic changes as well as positive drugs — ALM. The protective effect is most likely due to the ability of GSP to inhibit the ROS/MAPK pathway via restraining the release of ET-1.
Fig. 8. Representative figure of rats' thoracic aorta remodeling in different groups (HE stain, magnification ×40). (1) Sham group; (2) UnX-Sham group; (3) DOCA-salt hypertension group; (4) GSP 150 mg/kg group; (5) GSP 240 mg/kg group; (6) GSP 384 mg/kg group; (7) ALM 5 mg/kg group.
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Fig. 9. Effects of GSP on DOCA-salt hypertensive rats' SOD activity and MDA content in serum and cardiac tissue (mean±S.D., n=6–8). (A and C) The SOD activity in serum and heart tissue; (B and D) the MDA content in serum and heart tissue. **Pb.01 vs. Sham group; #Pb.05, ##Pb.01 vs. DOCA-salt group.
Acknowledgments This work was supported by PhD Programs Foundation of Ministry of Education of China (No. 20103420120002), Anhui Medical University Foundation for Middle-aged and Young Scientist Leaders of Disciplines in Science (No.201324) and
National Natural Science Foundation of China (No. 81073088; No. 81373774). The authors' responsibilities were as follows: G.S., H.L.L., P.C. and X.A.M. participated in the design of data analyses and manuscript preparation. H.L.L., W.L., D.L. and W.H.Z. conducted the assays and analyses. All authors read and approved the final manuscript.
Fig. 10. Effects of GSP on phosphorylation of JNK1/2 and p38MAPK in DOCA-salt hypertensive rats (mean±S.D., n=3). (1) Sham group; (2) UnX-Sham group; (3) DOCA-salt hypertension group; (4) GSP 150 mg/kg group; (5) GSP 240 mg/kg group; (6) GSP 384 mg/kg group; (7) ALM 5 mg/kg group. ⁎Pb.05 vs. Sham group; #Pb.05 vs. DOCA-salt group.
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