European Journal of Pharmacology 740 (2014) 200–208
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Beneficial effects of houttuynin on ventricular remodeling induced by coronary artery ligation in rats Yan Gao, Jian Ping Gao n, Chang Xun Chen, Hui Lin Wang, Juan Guo, Rong Wu Department of Pharmacology, Shanghai University of Traditional Chinese Medicine, NO. 1200 Cailun Road, Shanghai 201203, China
art ic l e i nf o
a b s t r a c t
Article history: Received 4 March 2014 Received in revised form 4 July 2014 Accepted 9 July 2014 Available online 18 July 2014
To examine the effects of houttuynin on ventricular remodeling induced by coronary artery ligation in rats and the underlying mechanisms. A rat model of ventricular remodeling was established by left coronary artery ligation (CAL). Rats were randomly divided into four groups: CAL control, CAL plus 40 mg/kg captopril, CAL plus 100 mg/kg houttuynin and sham-operated control. The rats were administered intragastrically with the corresponding drugs or distilled water for 7 weeks. At the end of the experiment, the left ventricular weight index (LVWI) and heart weight index (HWI) were determined. Myocardium tissue was stained with hematoxylin and eosin or picric acid/Sirius red for cardiomyocyte cross-section area or collagen content measurements respectively. The concentrations of angiotensin I (Ang I), angiotensin II (Ang II), aldosterone (ALD) and endothelin-1 (ET-1) in myocardium or serum were detected by radioimmunoassay. The hydroxyproline (Hyp) concentration was measured by alkali hydrolysis. Ultraviolet spectrophotometry was used to determine glutathione peroxidase (GSH-Px) and catalase (CAT) activities in serum. Houttuynin significantly diminished LVWI and HWI, decreased Ang I, Ang II, ALD, ET-1 and Hyp concentrations in myocardium or serum, increased NO concentration and GSH-Px, CAT activities after 7 weeks of treatment. Houttuynin could also reduce cardiomyocyte cross-section area and collagen deposition. Houttuynin attenuates ventricular remodeling in coronary artery ligation rats by restricting the excessive activation of rennin-angiotensin-aldosterone system (RAAS) and the peroxidation. & 2014 Elsevier B.V. All rights reserved.
Keywords: Houttuynin Ventricular remodeling Coronary artery ligation Myocardial ischemia RAAS Peroxidase
1. Introduction Chronic heart failure (CHF) is the final common pathway of a process of myocardial cell death triggered by varied etiologies and characterized by myocardial dysfunction and inadequate remodeling, which is well known that the heart cannot put out commensurate blood to supply venous return and tissue metabolism. It is a complex disease process connected with cardiovascular system as well as other organs and skeletal muscles, from cardiomyopathy of ischemic origin to other infectious, inflammatory or infiltrative processes (Sánchez et al., 2013). Heart failure is common, increasing in prevalence, and causes substantial morbidity and mortality (Shah, 2013). It is well known that ventricular remodeling is considered as one of the main pathophysiological mechanisms in the process of the development of chronic cardiac insufficiency (Messerli and Ketelhut, 1991; Katz and Zile, 2006). As a result, the need for new therapeutic advances is urgent. Many researchers try to seek for effective components from traditional Chinese medicine to control or relieve ventricular remodeling.
Houttuynin [CH3(CH2)8COCH2CHO] is one of the main ingredients in the volatile oil of the Chinese herb Houttuynia cordata Thunb. Sodium houttuyfonate has been used in China for the clinical treatment of bronchitis and upper respiratory infection for many years. It is reported that houttuynin has widely anti-pathogenic effect on pathogenic biofilm formation of either bacteria or fungus (Shao et al., 2013). And houttuynin analogs may be potentially useful ingredients for the formulation of antihypertensive products (Yuan et al., 2006). Gao et al. (2009) have demonstrated that sodium houttuyfonate can inhibit myocardial hypertrophy induced by isoproterenol in mice and by L-thyroxine in rats , attenuate ventricular remodeling induced by abdominal aortic banding in rats (Gao et al., 2010). Nevertheless, the effects of houttuynin on ventricular remodeling after myocardial infarction have not been reported. In this study, we investigated the effects of houttuynin on ventricular remodeling induced by left coronary artery ligation and the related mechanisms in rats.
2. Materials and methods 2.1. Animals
n
Corresponding author. Tel.: þ 86 21 51322213; fax: þ 86 21 51322213. E-mail address: [email protected] (J.P. Gao).
http://dx.doi.org/10.1016/j.ejphar.2014.07.015 0014-2999/& 2014 Elsevier B.V. All rights reserved.
The study was conducted on mature male Sprague-Dawley rats weighing approximately 200–220 g. All animals were obtained
Y. Gao et al. / European Journal of Pharmacology 740 (2014) 200–208
from Shanghai Slac laboratory animal Co., Ltd. The rats were housed in colony cages (four to five rats per cage) at an ambient temperature of 237 1 1C and the humidity at 4075% with alternating 12 h cycles of light and dark. These rats received humane care and had free access to standard food and water ad libitum for 5 days to adapt to the environment. The Principles of Laboratory Animal Care were followed according to the Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine and conformed to the Guide for the Care and Use of Laboratory Animals, published by the US National Institute of Health (NIH publication no. 85-23, revised in 1996). 2.2. Drugs and reagents Sodium houttuyfonate (houttuynin, Lot number: 110703 and 111003) was supplied by Shanghai Qingping Pharmaceutical Co., Ltd. (Shanghai, China). It was suspended in distilled water before administration of rats. Captopril (Lot number: 110705) was obtained from Shanghai Hengshan Pharmaceutical Co., Ltd. (Shanghai, China). It was suspended in distilled water before using. 2.3. Experimental protocols A rat model of ventricular remodeling was induced by subjecting the animals to the left coronary artery ligating (CAL) using a 4-0 silk suture under anesthesia induced by intraperitoneal administration of sodium pentobarbital 35 mg /kg, in the volume of 1.5 ml/kg. At the same time of operating, the rats were tracheally intubated and ventilated with a type HX-300s animal respirator (Chengdu Technology & Market Co., Ltd.). Coronary artery occlusion with myocardial infarction (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). After surgical operation, each rat was given benzyl-penicillin by intramuscular injection for four days to prevent infection. A week after operation, the survived rats were randomly divided into four groups. CAL control group: rats underwent CAL and received distilled water. Captopril group: rats underwent CAL and received captopril at 40 mg/kg/day. Houttuynin group: rats underwent CAL and received houttuynin at 100 mg/kg/day. The treatment was orally administrated and continued for 7 weeks. Sham-operated rats underwent the same surgical procedure, except for CAL as described above and received distilled water for 7 weeks. Each rat of different groups was given drug or distilled water in the volume of 10 ml/kg. 2.4. Hemodynamic measurement At the end of the above experiments, rat body weight (BW) was recorded after fasting for 12 h, and then the rats were anesthetized with urethane 1 g/kg intraperitoneally, in the volume of 4 ml/kg. To evaluate left ventricular function, a polypropylene catheter filled with heparin saline solution was inserted into the carotid artery. The polypropylene catheter was connected to a pressure transducer. After an equilibrium period for about 3 min, systolic blood pressure (SBP), diastolic blood pressure (DBP), pulse pressure and heart rate (HR) were recorded with a multi-channel biological signal analysis system (RM6240C type, Chengdu Technology & Market Co., Ltd.). The catheter was advanced into the left ventricle to measure the left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) as well as maximal and minimal rates of developed left ventricular pressure ( þdP/ dtmax and dP/dtmax) respectively.
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2.5. Cardiac weight indexes and morphological examination After recording the hemodynamic parameters, the blood sample was collected from abdominal aorta into 10 ml centrifugal tube and centrifuged (4 1C, 1780 g, 10 min) to get serum which was stored immediately in a 80 1C refrigerator until being used for biochemical analysis. Hearts were excised, rinsed with cold saline solution, and the left ventricle was separated from the atria, aorta and adipose tissue. The left ventricle weight (LVW, mg) and heart weight (HW, mg) were measured, and then left ventricular weight index (LVWI, mg/g) and heart weight index (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 2 parts. The upper part was immersed in formalin (10% formaldehyde). The lower part was separated into three sections and immediately frozen in liquid nitrogen and then stored in a 80 1C refrigerator until ready for further use and analysis. The fixed part of ventricle immersed in formalin was dehydrated and embedded in paraffin wax, and then cut into 5-μm thick slices and heated overnight in a 60 1C incubator. Myocardium tissue was stained with hematoxylin and eosin (H&E) for cardiomyocyte crosssection area measurement or with Sirius red in aqueous saturated picric acid for examination of perivascular and interstitial fibrosis in myocardium. Each sample slice was photographed (400 magnification) under the microscope (Olympus BX51, Olympus, Tokyo, Japan). All photos were analyzed with the image-pro plus 6.3 analyzing software (Media Cybernetics, Bethesda, MD, USA) by computer. Three fields of every sample slice stained with H&E were examined and 30 myocardial cells of each field were chosen randomly to calculate the average cross section area of cardiomyocytes. The percentage of collagen area per field was calculated as the myocardial interstitial collagen volume fraction (ICVF). The perivascular collagen volume fraction (PCVF) in myocardium was calculated by estimating the area ratio of perivascular collagen to vessel lumen area. For each sample three fields were randomly selected to calculate the above parameters. Collagen accumulation that assessed by polarized light microscopy in the interstitial and perivascular space of the left ventricle was analyzed with imagepro plus 6.3 analyzing software. 2.6. Radioimmunoassay determination Ventricular tissue (100 mg) was homogenized with 1 ml cold 0.9% NaCl. The homogenized tissue was centrifuged (4 1C, 1780 g, 15 min) and the supernatant was collected for measurement. Angiotensin I (Ang I), angiotensin II (Ang II), endothelin 1 (ET-1) concentrations of ventricular tissue and aldosterone (ALD) concentration in serum were measured by radioimmunoassay. Ang I, Ang II, ALD and ET-1 concentrations were respectively detected with the Iodine [125I] Ang I kit, Iodine [125I] Ang II kit, Iodine [125I] ALD kit and Iodine [125I] ET-1 kit. Diagnostic kits were provided by Northern Biotechnology Research Institute (Beijing, China). All measurements were performed according to the manufacturers' protocols. 2.7. Measurement of hydroxyproline (Hyp) and NO concentrations, glutathione peroxidase (GSH-Px) and catalase (CAT) activities in serum Because of instability of NO in physiological solutions, most of the NO being rapidly converted to nitrite (NO2 ) and further to nitrate (NO3 ), serum levels of NO2 /NO3 were measured using NO Detection Kit. Briefly, nitrate was converted to nitrite with aspergillus nitrite reductase, and the total nitrite was measured with the Griess reagent. The absorbance was determined with a spectrophotometer. Hyp concentration, GSH-Px and CAT activities were detected by ultraviolet spectrophotometry with the Hydroxyproline kit, Glutathione
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peroxidase kit and Catalase kit respectively (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China). 2.8. Statistical analysis All values were expressed as mean7standard deviation (x 7S.D.). Statistical analysis was performed by one-way analysis of variance (ANOVA) for multiple comparisons, followed by the Student–Newman–Keuls test or dunnettt's T3 test (according to whether the variance is homogeneous or not) to evaluate the difference between two comparison groups through the software of SPSS 19.0 version. Probabilities of 0.05 or less were considered to be statistically significant.
weight among the groups observed, although CAL animals tended to show a slight decrease in body weight. The LVWI and HWI were significantly greater in the CAL control group than those in the shamoperated control group. Treatment for 7 weeks, houttuynin and captopril decreased LVWI and HWI significantly.
3.4. Effects on cardiomyocyte cross-section area and collagen accumulation On H&E light micrographs for histopathological study, normal control group rats showed normal architecture. As shown in
3. Results 3.1. Gross examination The whole heart photograph of each group is shown in Fig. 1, through macroscopic anatomical observation, the wall of left ventricular infarction area under CAL became thinner, most part of the heart in the CAL control group developed fibrosis. Heart in sham-operated rat had no abnormal phenomenon. A slight myocardial fibrosis was observed in the hearts of houttuynin and captopril groups after treatment for seven weeks. 3.2. Effects on hemodynamic parameters
Fig. 2. Effects of houttuynin on blood pressure in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x 7S.D.). Compared with shamoperated control group, #Po0.05, ##Po0.01. Compared with CAL control, nnPo0.01. SBP, systolic blood pressure; DBP, diastolic blood pressure.
As shown in Figs. 2 and 3 respectively, in the CAL control group, SBP and pulse pressure were reduced; HR was increased when compared with those in the sham-operated control group. Houttuynin increased SBP and DBP obviously, whereas, it did not influence the heart rate very much. As shown in Table1, LVSP and þ dp/dtmax were significantly lower in the CAL control group than those in the sham-operated control group. As a tendency, LVEDP was increased, and dp/dtmax was reduced in the CAL control group. However, there were no significant differences between the sham-operated and the CAL control groups. Both houttuynin and captopril increased LVSP and þdp/dtmax obviously. Houttuynin increased dp/dtmax as well. Meanwhile, both captopril and houttuynin reduced LVEDP as a tendency. In brief, these results demonstrate that houttuynin and captopril improve cardiac function to some extent. 3.3. Effects on cardiac weight indexes The effects of houttuynin on HWI (HW/BW) and LVWI (LVW/BW) are shown in Fig. 4. There was no significant difference in body
Fig. 3. Effect of houttuynin on heart rate in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x 7S.D.). Compared with sham-operated control group, #Po0.05.
Fig. 1. The whole heart photograph of different groups. A, Sham-operated control; B, Coronary artery ligation (CAL) control; C, CAL plus 40 mg/kg captopril; D, CAL plus 100 mg/kg houttuynin.
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Table 1 Effects of houttuynin on hemodynamic parameters in rats with ventricular remodeling caused by left coronary artery ligation (CAL) (x7 S.D.). Group
n
Dose (mg/kg)
LVSP (mmHg)
LVEDP (mmHg)
þ dP/dtmax (mmHg/s)
-dP/dtmax (mmHg/s)
Sham-operated control CAL control CAL plus captopril CAL plus houttuynin
8 10 10 8
– – 40 100
119.65 7 11.88 95.687 8.56a 109.447 9.30c 133.057 9.22c
0.977 1.71 2.42 7 2.35 1.067 2.70 0.22 7 5.36
9433.457 1915.83 7038.297 601.50b 8769.02 7 1253.99d 10,260.45 7 1627.98c
6262.707 964.33 5678.897 674.58 6339.347 524.21 7202.377 857.39c
Note: Compared with sham-operated control group, aP o 0.01; bP o 0.05. Compared with CAL control group, cP o 0.01; dP o 0.05. LVSP, left ventricular systolic pressure; LVEDP, left ventricular end diastolic pressure; þ dp/dtmax, the maximal rate of rise; dp/dtmax, the maximal rate of fall.
group. Treatment with houttuynin or captopril for 7 weeks significantly reduced Ang I, Ang II, ET-1 and ALD concentrations, as shown in Tables 2 and 3. These data show that houttuynin and captopril may antagonize myocardial remodeling by inhibiting the RAAS. 3.6. Effects on Hyp and NO concentrations, GSH-Px and CAT activities
Fig. 4. Effects of houttuynin on cardiac weight indexes in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x7 S.D.). Compared with sham-operated control group, ##P o0.01. Compared with CAL control group, nP o 0.05, nnPo 0.01. LVWI, left ventricular weight/body weight index; HWI, heart weight/body weight index.
As shown in Figs. 12 and 13 and Table 4, Hyp concentration is increased, the activities of CAT and GSH-Px in the serum are lower in CAL control group than those in sham-operated group. Houttuynin and captopril significantly enhanced the activities of GSHPx and CAT, decreased Hyp concentration obviously. In a word, houttuynin and captopril could eliminate oxygen free radical by increasing the activities of GSH-Px and CAT. As shown in Table 5, NO concentration was lower in serum of CAL control group than that in sham-operation group. Houttuynin and captopril significantly increased NO concentration. This result indicates that to a certain extent, houttuynin and captopril may protect myocardial tissue by improving endothelial function.
4. Discussion Figs. 5 and 6, in CAL group rats, many myocardial structure disorders of muscle fibers as well as infiltration of acute inflammatory cells, along with extravasations of red blood cells were observed. Cardiomyocyte cross-section area in the CAL control group was significantly larger than that in the sham-operated control group. Houttuynin and captopril decreased the average cross-section area of cardiomyocytes. Under microscope, cardiomyocytes, stained with Sirius red, appeared orange, myocardial interstitial and perivascular collagen fibers appeared red or deep red. In rat myocardium of shamoperated group, little amount of collagen was found in the interstitial and perivascular space. There was a large accumulation of collagen in the ventricle of CAL control group. Less collagen deposition was found in CAL plus houttuynin or captopril groups than that in CAL control group (Figs. 7 and 8). As shown in Figs. 9–11, under the polarized light microscope, type I collagen fibers appeared red or yellow, type III collagen fibers appeared green. In rat myocardium of sham-operated control, fewer amounts of collagen fibers were found. Collagen distributions were significantly increased in the CAL control rats compared with those in the sham-operated rats. However, these were significantly decreased in houttuynin and captopril treated groups. The results indicate that houttuynin and captopril block myocardial fibrosis by decreasing collagen synthesis and accumulation, and then attenuate ventricular remodeling. 3.5. Effects on Ang I, Ang II, ALD and ET-1 concentrations In the CAL control group, Ang I, Ang II and ET-1 concentrations in left ventricular tissue, ALD concentration in serum were significantly higher than those in the sham-operated control
Cardiovascular disease is a major public health problem that imposes a huge economic burden on health systems around the world (Teuteberg et al., 2006; Levy et al., 2006). Heart failure is the final common pathway of an irreversible process associated with loss of myocardial cells. In this process, the capacity for renewal and repair of myocardial tissue is inadequate, which leads to ventricular remodeling ultimately. Therefore, inhibiting ventricular remodeling early may be an effective way to postpone heart failure induced by myocardial infarction and other cardiovascular diseases. Left coronary artery ligation-induced cardiac ischemia is known to exhibit many metabolic and morphologic abnormalities in experimental animal models, similar to those in humans. Acute myocardial infarction (MI) is associated with a rapid loss of cardiomyocytes. Cell death after ischemic injury can occur by apoptosis, necrosis and to some degree by autophagy (Whelan et al., 2010). Although with improved clinical care and greater public awareness, MI remains the leading cause of death worldwide (Aronow, 2006). Following acute MI, the hypertrophy process is the result of the infarction expansion. In our experiment, some results indicate that 8 weeks after left coronary artery ligation, the untreated animals develop severe myocardial hypertrophy. In ventricular remodeling animal model, the heart weight index and the transverse area of cardiac myocytes are the main indicators to measure the degree of cardiac hypertrophy (Shao et al., 2012; Bai et al., 2013). In our study, the degree of cardiac hypertrophy was displayed by the increase of LVWI, HWI and demonstrable microscopically with an up to approximately 90% increase in average cross-section area of cardiomyocyte in the CAL
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Fig. 5. Effect of houttuynin on cardiomyocyte cross-section area in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (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 100 mg/kg houttuynin.
Fig. 6. Effects of houttuynin on cardiomyocyte cross-section area in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x 7S.D.). Compared with sham-operated control group, ##Po 0.01. Compared with CAL control group, **Po 0.01.
control group than those in the sham-operated group. After 7 weeks treatment, houttuynin at 100 mg/kg and captopril significantly decreased cardiac weight index and average crosssection area of cardiomyocyte. Hyp, accounted for 13% of the total amino acid, is the main composition of collagen in tissues, which can promote the myocardial collagen protein synthesis, myocardial interstitial proliferation and fibrosis (Brown et al., 2005; Koshman et al., 2013). The experimental results showed that the left anterior descending coronary artery ligation could increase serum Hyp concentration significantly in the CAL control group. Houttuynin at 100 mg/kg and captopril obviously reduced serum Hyp concentration. We deduce that houttuynin may delay and block fibrosis by decreasing synthesis and secretion of collagen, and then slow down or inhibit the process of ventricular remodeling according to the results.
RAAS is composed of Ang I, Ang II, ALD and Angiotensinconverting enzyme (ACE) (Adams, 2004). The activation of the RAAS is a well-known pathway following myocardial infarction, leading to adverse left ventricular (LV) remodeling, heart failure and cardiac death (Pitt et al., 2003). Currently, the levels of Ang II and ALD have become the key indicators for diagnosis, treatment and research about heart failure. Aldosterone, the last component of the RAAS, plays a pivotal role in the pathophysiology of cardiovascular diseases primarily through mineralocorticoid-receptor dependent actions (Connell and Davies, 2005). Aldosterone that was previously thought to be synthesized solely in the adrenal cortex is also produced in extra-adrenal tissues, such as the heart, particularly in pathological states (Fujisaki et al., 2013). Our result indicates that the function of houttuynin in antagonizing myocardial remodeling correlated with its inhibition of the RAAS in rats including reducing myocardial tissue Ang I, Ang II and serum ALD concentrations significantly. In systemic circulation, endothelial cells play a crucial role in the regulation of vascular tone. The endothelial dysfunction, such as Endothelin-1 or NO, leads to the alteration of vasodilatation and vasoconstriction balance (Budhiraja et al., 2004; Rebic et al., 2014). ET-1 is a marker of endothelial dysfunction in a spectrum of cardiovascular diseases ranging from coronary artery disease to heart failure (Paulus et al., 2011). It is a potent vasoconstrictor and released following various injurious stimuli, including shear stress, thrombin, angiotensin II, cytokines, and free radicals. It has shown that ET-1 can induce fibroblast proliferation and differentiation, collagen synthesis and fibroblast apoptosis (Kulasekaran et al., 2009). ET-1 level in plasma is increased in patients with the severity of heart failure (Yin et al., 2011). Nitric oxide (NO) is a key signaling molecule to modulate downstream proteins and is produced endogenously by nitric oxide synthase. In heart failure disease conditions, dysfunction of NO contributes to the contractile dysfunction, adverse remodeling, and finally myocardial hypertrophy (Tang et al., 2014; Pall, 2013). In our study, coronary artery ligation induced a significant higher ET-1 concentration and
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Fig. 7. Effects of houttuynin on cardiac collagen accumulation in the interstitial space of the left ventricle in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (Sirius red stain, 400 ). A, Sham-operated control; B, Coronary artery ligation (CAL) control; C, CAL plus 40 mg/kg captopril; D, CAL plus 100 mg/kg houttuynin.
Fig. 8. Effects of houttuynin on cardiac collagen accumulation in the perivascular space of the left ventricle in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (Sirius red stain, 400 ). A, Sham-operated control; B, Coronary artery ligation (CAL) control; C, CAL plus 40 mg/kg captopril; D, CAL plus 100 mg/kg houttuynin.
lower NO concentration. Houttuynin decreased ET-1 level and increased NO concentration indicating that the function of houttuynin in protecting myocardial tissues and antagonizing ventricular remodeling correlates with its improvement of endothelial function.
GSH-Px inhibits the peroxide induced cell apoptosis and necrosis, which is used as a cofactor in the removal of hydrogen peroxide and lipoperoxides (Crawford et al., 2011). CAT destroys intracellular Peroxide (H2O2) to form water and molecular oxygen. The excess oxidative stress either via the increased production of
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Fig. 9. Effects of houttuynin on collagen accumulation in the interstitial space of the left ventricle in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (Sirius red stain and polarized light, 400 ). A, Sham-operated control; B, Coronary artery ligation (CAL) control; C, CAL plus 40 mg/kg captopril; D, CAL plus 100 mg/kg houttuynin.
Fig. 10. Effects of houttuynin on collagen in the perivascular space of the left ventricle in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (Sirius red stain and polarized light, 400 ). A, Sham-operated control; B, Coronary artery ligation (CAL) control; C, CAL plus 40 mg/kg captopril; D, CAL plus 100 mg/kg houttuynin.
peroxides or via the decreased inactivation process of scavenger enzymes attacks the lipid component of cellular membrane and gives rise to the increased lipid peroxidation (Lim et al., 2013). In our experiment, GSH-Px and CAT activities were lower in serum of CAL control group than those in sham-operated group, indicating that the function of houttuynin on improving myocardial remodeling correlates with increasing the GSH-Px and CAT activities, and eliminating oxygen free radical as well.
5. Conclusions In summary, our in vivo results indicate that houttuynin plays an antifibrotic role in inhibiting coronary artery ligation-induced ventricular remodeling. The mechanism may be related to restraining the excessive activation of RAAS and inhibiting peroxidation. Thus, houttuynin may be potentially as a therapeutic tool for ventricular remodeling and even heart failure.
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Fig. 11. Effects of houttuynin on interstitial collagen volume fraction (ICVF) and perivascular collagen volume fraction (PVCF) of the left ventricle in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x7S.D.). Compared with sham-operated control, ##Po0.01. Compared with CAL control, nnPo0.01.
Fig. 13. Effect of houttuynin on GSH-Px activity in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x7 S.D.). Compared with shamoperated control group, ##Po 0.01. Compared with CAL control group, nnPo 0.01. GSH-Px, glutathione peroxidase.
Table 2 Effects of houttuynin on angiotensin I (Ang I) and angiotensin II (Ang II) concentrations in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x7 S.D.).
Table 4 Effect of houttuynin on CAT activity in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x7 S.D.).
Group
n
Dose(mg/kg)
Ang I (ng/ml)
Ang II (pg/ml)
Group
n
Dose(mg/kg)
CAT(U/ml)
Sham-operated control CAL control CAL plus captopril CAL plus houttuynin
8 10 10 8
– – 40 100
1.38 70.29 1.7070.17b 1.38 70.13c 1.43 70.21d
116.93 7 22.29 150.87 7 19.12a 99.98 7 16.72c 104.797 22.07c
Sham-operated control CAL control CAL plus captopril CAL plus houttuynin
8 10 10 8
– – 40 100
6.02 73.42 1.97 71.01b 3.62 71.29d 4.52 71.96d
Note: Compared with sham-operated control group, aP o 0.01; Compared with CAL control group, cP o 0.01; dP o 0.05.
b
P o 0.05.
Note: Compared with sham-operated control group, bP o 0.05. Compared with CAL control group, dP o 0.05. CAT, catalase
Table 3 Effects of houttuynin on aldosterone (ALD) and endothelin-1 (ET-1) concentrations in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x7 S.D.).
Table 5 Effect of houttuynin on NO concentration in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x 7 S.D.).
Group
n
Dose(mg/kg)
ALD(ng/ml)
ET-1(pg/mg prot)
Sham-operated control CAL control CAL plus captopril CAL plus houttuynin
8 10 10 8
– – 40 100
1.02 7 0.32 1.53 7 0.35b 1.147 0.35c 1.047 0.37c
62.85 7 9.62 108.92 7 29.56a 58.79 7 28.15d 60.62 7 12.15d
Note: Compared with sham-operated contro groupl, aP o 0.01; Compared with CAL control group, cP o 0.01; dP o 0.05.
b
P o 0.05.
Group
n
Dose(mg/kg)
NO(μmol/l)
Sham-operated control CAL control CAL plus captopril CAL plus houttuynin
8 10 10 8
– – 40 100
14.727 7.53 6.687 2.94a 13.047 6.25c 19.067 7.71c
Note: Compared with sham-operated control group, aP o 0.01. Compared with CAL control group, cP o 0.01.
Acknowledgment Project supported by the Shanghai Committee of Education Foundation (No. 12YZ059). References
Fig. 12. Effect of houttuynin on Hyp concentration in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x 7 S.D.). Compared with sham-operated control group, ##Po 0.01. Compared with CAL control group, n P o0.05, nnPo 0.01. Hyp, hydroxyproline.
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Beneficial effects of houttuynin on ventricular remodeling induced by coronary artery ligation in rats Yan Gao, Jian Ping Gao n, Chang Xun Chen, Hui Lin Wang, Juan Guo, Rong Wu Department of Pharmacology, Shanghai University of Traditional Chinese Medicine, NO. 1200 Cailun Road, Shanghai 201203, China
art ic l e i nf o
a b s t r a c t
Article history: Received 4 March 2014 Received in revised form 4 July 2014 Accepted 9 July 2014 Available online 18 July 2014
To examine the effects of houttuynin on ventricular remodeling induced by coronary artery ligation in rats and the underlying mechanisms. A rat model of ventricular remodeling was established by left coronary artery ligation (CAL). Rats were randomly divided into four groups: CAL control, CAL plus 40 mg/kg captopril, CAL plus 100 mg/kg houttuynin and sham-operated control. The rats were administered intragastrically with the corresponding drugs or distilled water for 7 weeks. At the end of the experiment, the left ventricular weight index (LVWI) and heart weight index (HWI) were determined. Myocardium tissue was stained with hematoxylin and eosin or picric acid/Sirius red for cardiomyocyte cross-section area or collagen content measurements respectively. The concentrations of angiotensin I (Ang I), angiotensin II (Ang II), aldosterone (ALD) and endothelin-1 (ET-1) in myocardium or serum were detected by radioimmunoassay. The hydroxyproline (Hyp) concentration was measured by alkali hydrolysis. Ultraviolet spectrophotometry was used to determine glutathione peroxidase (GSH-Px) and catalase (CAT) activities in serum. Houttuynin significantly diminished LVWI and HWI, decreased Ang I, Ang II, ALD, ET-1 and Hyp concentrations in myocardium or serum, increased NO concentration and GSH-Px, CAT activities after 7 weeks of treatment. Houttuynin could also reduce cardiomyocyte cross-section area and collagen deposition. Houttuynin attenuates ventricular remodeling in coronary artery ligation rats by restricting the excessive activation of rennin-angiotensin-aldosterone system (RAAS) and the peroxidation. & 2014 Elsevier B.V. All rights reserved.
Keywords: Houttuynin Ventricular remodeling Coronary artery ligation Myocardial ischemia RAAS Peroxidase
1. Introduction Chronic heart failure (CHF) is the final common pathway of a process of myocardial cell death triggered by varied etiologies and characterized by myocardial dysfunction and inadequate remodeling, which is well known that the heart cannot put out commensurate blood to supply venous return and tissue metabolism. It is a complex disease process connected with cardiovascular system as well as other organs and skeletal muscles, from cardiomyopathy of ischemic origin to other infectious, inflammatory or infiltrative processes (Sánchez et al., 2013). Heart failure is common, increasing in prevalence, and causes substantial morbidity and mortality (Shah, 2013). It is well known that ventricular remodeling is considered as one of the main pathophysiological mechanisms in the process of the development of chronic cardiac insufficiency (Messerli and Ketelhut, 1991; Katz and Zile, 2006). As a result, the need for new therapeutic advances is urgent. Many researchers try to seek for effective components from traditional Chinese medicine to control or relieve ventricular remodeling.
Houttuynin [CH3(CH2)8COCH2CHO] is one of the main ingredients in the volatile oil of the Chinese herb Houttuynia cordata Thunb. Sodium houttuyfonate has been used in China for the clinical treatment of bronchitis and upper respiratory infection for many years. It is reported that houttuynin has widely anti-pathogenic effect on pathogenic biofilm formation of either bacteria or fungus (Shao et al., 2013). And houttuynin analogs may be potentially useful ingredients for the formulation of antihypertensive products (Yuan et al., 2006). Gao et al. (2009) have demonstrated that sodium houttuyfonate can inhibit myocardial hypertrophy induced by isoproterenol in mice and by L-thyroxine in rats , attenuate ventricular remodeling induced by abdominal aortic banding in rats (Gao et al., 2010). Nevertheless, the effects of houttuynin on ventricular remodeling after myocardial infarction have not been reported. In this study, we investigated the effects of houttuynin on ventricular remodeling induced by left coronary artery ligation and the related mechanisms in rats.
2. Materials and methods 2.1. Animals
n
Corresponding author. Tel.: þ 86 21 51322213; fax: þ 86 21 51322213. E-mail address: [email protected] (J.P. Gao).
http://dx.doi.org/10.1016/j.ejphar.2014.07.015 0014-2999/& 2014 Elsevier B.V. All rights reserved.
The study was conducted on mature male Sprague-Dawley rats weighing approximately 200–220 g. All animals were obtained
Y. Gao et al. / European Journal of Pharmacology 740 (2014) 200–208
from Shanghai Slac laboratory animal Co., Ltd. The rats were housed in colony cages (four to five rats per cage) at an ambient temperature of 237 1 1C and the humidity at 4075% with alternating 12 h cycles of light and dark. These rats received humane care and had free access to standard food and water ad libitum for 5 days to adapt to the environment. The Principles of Laboratory Animal Care were followed according to the Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine and conformed to the Guide for the Care and Use of Laboratory Animals, published by the US National Institute of Health (NIH publication no. 85-23, revised in 1996). 2.2. Drugs and reagents Sodium houttuyfonate (houttuynin, Lot number: 110703 and 111003) was supplied by Shanghai Qingping Pharmaceutical Co., Ltd. (Shanghai, China). It was suspended in distilled water before administration of rats. Captopril (Lot number: 110705) was obtained from Shanghai Hengshan Pharmaceutical Co., Ltd. (Shanghai, China). It was suspended in distilled water before using. 2.3. Experimental protocols A rat model of ventricular remodeling was induced by subjecting the animals to the left coronary artery ligating (CAL) using a 4-0 silk suture under anesthesia induced by intraperitoneal administration of sodium pentobarbital 35 mg /kg, in the volume of 1.5 ml/kg. At the same time of operating, the rats were tracheally intubated and ventilated with a type HX-300s animal respirator (Chengdu Technology & Market Co., Ltd.). Coronary artery occlusion with myocardial infarction (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). After surgical operation, each rat was given benzyl-penicillin by intramuscular injection for four days to prevent infection. A week after operation, the survived rats were randomly divided into four groups. CAL control group: rats underwent CAL and received distilled water. Captopril group: rats underwent CAL and received captopril at 40 mg/kg/day. Houttuynin group: rats underwent CAL and received houttuynin at 100 mg/kg/day. The treatment was orally administrated and continued for 7 weeks. Sham-operated rats underwent the same surgical procedure, except for CAL as described above and received distilled water for 7 weeks. Each rat of different groups was given drug or distilled water in the volume of 10 ml/kg. 2.4. Hemodynamic measurement At the end of the above experiments, rat body weight (BW) was recorded after fasting for 12 h, and then the rats were anesthetized with urethane 1 g/kg intraperitoneally, in the volume of 4 ml/kg. To evaluate left ventricular function, a polypropylene catheter filled with heparin saline solution was inserted into the carotid artery. The polypropylene catheter was connected to a pressure transducer. After an equilibrium period for about 3 min, systolic blood pressure (SBP), diastolic blood pressure (DBP), pulse pressure and heart rate (HR) were recorded with a multi-channel biological signal analysis system (RM6240C type, Chengdu Technology & Market Co., Ltd.). The catheter was advanced into the left ventricle to measure the left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) as well as maximal and minimal rates of developed left ventricular pressure ( þdP/ dtmax and dP/dtmax) respectively.
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2.5. Cardiac weight indexes and morphological examination After recording the hemodynamic parameters, the blood sample was collected from abdominal aorta into 10 ml centrifugal tube and centrifuged (4 1C, 1780 g, 10 min) to get serum which was stored immediately in a 80 1C refrigerator until being used for biochemical analysis. Hearts were excised, rinsed with cold saline solution, and the left ventricle was separated from the atria, aorta and adipose tissue. The left ventricle weight (LVW, mg) and heart weight (HW, mg) were measured, and then left ventricular weight index (LVWI, mg/g) and heart weight index (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 2 parts. The upper part was immersed in formalin (10% formaldehyde). The lower part was separated into three sections and immediately frozen in liquid nitrogen and then stored in a 80 1C refrigerator until ready for further use and analysis. The fixed part of ventricle immersed in formalin was dehydrated and embedded in paraffin wax, and then cut into 5-μm thick slices and heated overnight in a 60 1C incubator. Myocardium tissue was stained with hematoxylin and eosin (H&E) for cardiomyocyte crosssection area measurement or with Sirius red in aqueous saturated picric acid for examination of perivascular and interstitial fibrosis in myocardium. Each sample slice was photographed (400 magnification) under the microscope (Olympus BX51, Olympus, Tokyo, Japan). All photos were analyzed with the image-pro plus 6.3 analyzing software (Media Cybernetics, Bethesda, MD, USA) by computer. Three fields of every sample slice stained with H&E were examined and 30 myocardial cells of each field were chosen randomly to calculate the average cross section area of cardiomyocytes. The percentage of collagen area per field was calculated as the myocardial interstitial collagen volume fraction (ICVF). The perivascular collagen volume fraction (PCVF) in myocardium was calculated by estimating the area ratio of perivascular collagen to vessel lumen area. For each sample three fields were randomly selected to calculate the above parameters. Collagen accumulation that assessed by polarized light microscopy in the interstitial and perivascular space of the left ventricle was analyzed with imagepro plus 6.3 analyzing software. 2.6. Radioimmunoassay determination Ventricular tissue (100 mg) was homogenized with 1 ml cold 0.9% NaCl. The homogenized tissue was centrifuged (4 1C, 1780 g, 15 min) and the supernatant was collected for measurement. Angiotensin I (Ang I), angiotensin II (Ang II), endothelin 1 (ET-1) concentrations of ventricular tissue and aldosterone (ALD) concentration in serum were measured by radioimmunoassay. Ang I, Ang II, ALD and ET-1 concentrations were respectively detected with the Iodine [125I] Ang I kit, Iodine [125I] Ang II kit, Iodine [125I] ALD kit and Iodine [125I] ET-1 kit. Diagnostic kits were provided by Northern Biotechnology Research Institute (Beijing, China). All measurements were performed according to the manufacturers' protocols. 2.7. Measurement of hydroxyproline (Hyp) and NO concentrations, glutathione peroxidase (GSH-Px) and catalase (CAT) activities in serum Because of instability of NO in physiological solutions, most of the NO being rapidly converted to nitrite (NO2 ) and further to nitrate (NO3 ), serum levels of NO2 /NO3 were measured using NO Detection Kit. Briefly, nitrate was converted to nitrite with aspergillus nitrite reductase, and the total nitrite was measured with the Griess reagent. The absorbance was determined with a spectrophotometer. Hyp concentration, GSH-Px and CAT activities were detected by ultraviolet spectrophotometry with the Hydroxyproline kit, Glutathione
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peroxidase kit and Catalase kit respectively (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China). 2.8. Statistical analysis All values were expressed as mean7standard deviation (x 7S.D.). Statistical analysis was performed by one-way analysis of variance (ANOVA) for multiple comparisons, followed by the Student–Newman–Keuls test or dunnettt's T3 test (according to whether the variance is homogeneous or not) to evaluate the difference between two comparison groups through the software of SPSS 19.0 version. Probabilities of 0.05 or less were considered to be statistically significant.
weight among the groups observed, although CAL animals tended to show a slight decrease in body weight. The LVWI and HWI were significantly greater in the CAL control group than those in the shamoperated control group. Treatment for 7 weeks, houttuynin and captopril decreased LVWI and HWI significantly.
3.4. Effects on cardiomyocyte cross-section area and collagen accumulation On H&E light micrographs for histopathological study, normal control group rats showed normal architecture. As shown in
3. Results 3.1. Gross examination The whole heart photograph of each group is shown in Fig. 1, through macroscopic anatomical observation, the wall of left ventricular infarction area under CAL became thinner, most part of the heart in the CAL control group developed fibrosis. Heart in sham-operated rat had no abnormal phenomenon. A slight myocardial fibrosis was observed in the hearts of houttuynin and captopril groups after treatment for seven weeks. 3.2. Effects on hemodynamic parameters
Fig. 2. Effects of houttuynin on blood pressure in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x 7S.D.). Compared with shamoperated control group, #Po0.05, ##Po0.01. Compared with CAL control, nnPo0.01. SBP, systolic blood pressure; DBP, diastolic blood pressure.
As shown in Figs. 2 and 3 respectively, in the CAL control group, SBP and pulse pressure were reduced; HR was increased when compared with those in the sham-operated control group. Houttuynin increased SBP and DBP obviously, whereas, it did not influence the heart rate very much. As shown in Table1, LVSP and þ dp/dtmax were significantly lower in the CAL control group than those in the sham-operated control group. As a tendency, LVEDP was increased, and dp/dtmax was reduced in the CAL control group. However, there were no significant differences between the sham-operated and the CAL control groups. Both houttuynin and captopril increased LVSP and þdp/dtmax obviously. Houttuynin increased dp/dtmax as well. Meanwhile, both captopril and houttuynin reduced LVEDP as a tendency. In brief, these results demonstrate that houttuynin and captopril improve cardiac function to some extent. 3.3. Effects on cardiac weight indexes The effects of houttuynin on HWI (HW/BW) and LVWI (LVW/BW) are shown in Fig. 4. There was no significant difference in body
Fig. 3. Effect of houttuynin on heart rate in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x 7S.D.). Compared with sham-operated control group, #Po0.05.
Fig. 1. The whole heart photograph of different groups. A, Sham-operated control; B, Coronary artery ligation (CAL) control; C, CAL plus 40 mg/kg captopril; D, CAL plus 100 mg/kg houttuynin.
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Table 1 Effects of houttuynin on hemodynamic parameters in rats with ventricular remodeling caused by left coronary artery ligation (CAL) (x7 S.D.). Group
n
Dose (mg/kg)
LVSP (mmHg)
LVEDP (mmHg)
þ dP/dtmax (mmHg/s)
-dP/dtmax (mmHg/s)
Sham-operated control CAL control CAL plus captopril CAL plus houttuynin
8 10 10 8
– – 40 100
119.65 7 11.88 95.687 8.56a 109.447 9.30c 133.057 9.22c
0.977 1.71 2.42 7 2.35 1.067 2.70 0.22 7 5.36
9433.457 1915.83 7038.297 601.50b 8769.02 7 1253.99d 10,260.45 7 1627.98c
6262.707 964.33 5678.897 674.58 6339.347 524.21 7202.377 857.39c
Note: Compared with sham-operated control group, aP o 0.01; bP o 0.05. Compared with CAL control group, cP o 0.01; dP o 0.05. LVSP, left ventricular systolic pressure; LVEDP, left ventricular end diastolic pressure; þ dp/dtmax, the maximal rate of rise; dp/dtmax, the maximal rate of fall.
group. Treatment with houttuynin or captopril for 7 weeks significantly reduced Ang I, Ang II, ET-1 and ALD concentrations, as shown in Tables 2 and 3. These data show that houttuynin and captopril may antagonize myocardial remodeling by inhibiting the RAAS. 3.6. Effects on Hyp and NO concentrations, GSH-Px and CAT activities
Fig. 4. Effects of houttuynin on cardiac weight indexes in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x7 S.D.). Compared with sham-operated control group, ##P o0.01. Compared with CAL control group, nP o 0.05, nnPo 0.01. LVWI, left ventricular weight/body weight index; HWI, heart weight/body weight index.
As shown in Figs. 12 and 13 and Table 4, Hyp concentration is increased, the activities of CAT and GSH-Px in the serum are lower in CAL control group than those in sham-operated group. Houttuynin and captopril significantly enhanced the activities of GSHPx and CAT, decreased Hyp concentration obviously. In a word, houttuynin and captopril could eliminate oxygen free radical by increasing the activities of GSH-Px and CAT. As shown in Table 5, NO concentration was lower in serum of CAL control group than that in sham-operation group. Houttuynin and captopril significantly increased NO concentration. This result indicates that to a certain extent, houttuynin and captopril may protect myocardial tissue by improving endothelial function.
4. Discussion Figs. 5 and 6, in CAL group rats, many myocardial structure disorders of muscle fibers as well as infiltration of acute inflammatory cells, along with extravasations of red blood cells were observed. Cardiomyocyte cross-section area in the CAL control group was significantly larger than that in the sham-operated control group. Houttuynin and captopril decreased the average cross-section area of cardiomyocytes. Under microscope, cardiomyocytes, stained with Sirius red, appeared orange, myocardial interstitial and perivascular collagen fibers appeared red or deep red. In rat myocardium of shamoperated group, little amount of collagen was found in the interstitial and perivascular space. There was a large accumulation of collagen in the ventricle of CAL control group. Less collagen deposition was found in CAL plus houttuynin or captopril groups than that in CAL control group (Figs. 7 and 8). As shown in Figs. 9–11, under the polarized light microscope, type I collagen fibers appeared red or yellow, type III collagen fibers appeared green. In rat myocardium of sham-operated control, fewer amounts of collagen fibers were found. Collagen distributions were significantly increased in the CAL control rats compared with those in the sham-operated rats. However, these were significantly decreased in houttuynin and captopril treated groups. The results indicate that houttuynin and captopril block myocardial fibrosis by decreasing collagen synthesis and accumulation, and then attenuate ventricular remodeling. 3.5. Effects on Ang I, Ang II, ALD and ET-1 concentrations In the CAL control group, Ang I, Ang II and ET-1 concentrations in left ventricular tissue, ALD concentration in serum were significantly higher than those in the sham-operated control
Cardiovascular disease is a major public health problem that imposes a huge economic burden on health systems around the world (Teuteberg et al., 2006; Levy et al., 2006). Heart failure is the final common pathway of an irreversible process associated with loss of myocardial cells. In this process, the capacity for renewal and repair of myocardial tissue is inadequate, which leads to ventricular remodeling ultimately. Therefore, inhibiting ventricular remodeling early may be an effective way to postpone heart failure induced by myocardial infarction and other cardiovascular diseases. Left coronary artery ligation-induced cardiac ischemia is known to exhibit many metabolic and morphologic abnormalities in experimental animal models, similar to those in humans. Acute myocardial infarction (MI) is associated with a rapid loss of cardiomyocytes. Cell death after ischemic injury can occur by apoptosis, necrosis and to some degree by autophagy (Whelan et al., 2010). Although with improved clinical care and greater public awareness, MI remains the leading cause of death worldwide (Aronow, 2006). Following acute MI, the hypertrophy process is the result of the infarction expansion. In our experiment, some results indicate that 8 weeks after left coronary artery ligation, the untreated animals develop severe myocardial hypertrophy. In ventricular remodeling animal model, the heart weight index and the transverse area of cardiac myocytes are the main indicators to measure the degree of cardiac hypertrophy (Shao et al., 2012; Bai et al., 2013). In our study, the degree of cardiac hypertrophy was displayed by the increase of LVWI, HWI and demonstrable microscopically with an up to approximately 90% increase in average cross-section area of cardiomyocyte in the CAL
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Fig. 5. Effect of houttuynin on cardiomyocyte cross-section area in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (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 100 mg/kg houttuynin.
Fig. 6. Effects of houttuynin on cardiomyocyte cross-section area in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x 7S.D.). Compared with sham-operated control group, ##Po 0.01. Compared with CAL control group, **Po 0.01.
control group than those in the sham-operated group. After 7 weeks treatment, houttuynin at 100 mg/kg and captopril significantly decreased cardiac weight index and average crosssection area of cardiomyocyte. Hyp, accounted for 13% of the total amino acid, is the main composition of collagen in tissues, which can promote the myocardial collagen protein synthesis, myocardial interstitial proliferation and fibrosis (Brown et al., 2005; Koshman et al., 2013). The experimental results showed that the left anterior descending coronary artery ligation could increase serum Hyp concentration significantly in the CAL control group. Houttuynin at 100 mg/kg and captopril obviously reduced serum Hyp concentration. We deduce that houttuynin may delay and block fibrosis by decreasing synthesis and secretion of collagen, and then slow down or inhibit the process of ventricular remodeling according to the results.
RAAS is composed of Ang I, Ang II, ALD and Angiotensinconverting enzyme (ACE) (Adams, 2004). The activation of the RAAS is a well-known pathway following myocardial infarction, leading to adverse left ventricular (LV) remodeling, heart failure and cardiac death (Pitt et al., 2003). Currently, the levels of Ang II and ALD have become the key indicators for diagnosis, treatment and research about heart failure. Aldosterone, the last component of the RAAS, plays a pivotal role in the pathophysiology of cardiovascular diseases primarily through mineralocorticoid-receptor dependent actions (Connell and Davies, 2005). Aldosterone that was previously thought to be synthesized solely in the adrenal cortex is also produced in extra-adrenal tissues, such as the heart, particularly in pathological states (Fujisaki et al., 2013). Our result indicates that the function of houttuynin in antagonizing myocardial remodeling correlated with its inhibition of the RAAS in rats including reducing myocardial tissue Ang I, Ang II and serum ALD concentrations significantly. In systemic circulation, endothelial cells play a crucial role in the regulation of vascular tone. The endothelial dysfunction, such as Endothelin-1 or NO, leads to the alteration of vasodilatation and vasoconstriction balance (Budhiraja et al., 2004; Rebic et al., 2014). ET-1 is a marker of endothelial dysfunction in a spectrum of cardiovascular diseases ranging from coronary artery disease to heart failure (Paulus et al., 2011). It is a potent vasoconstrictor and released following various injurious stimuli, including shear stress, thrombin, angiotensin II, cytokines, and free radicals. It has shown that ET-1 can induce fibroblast proliferation and differentiation, collagen synthesis and fibroblast apoptosis (Kulasekaran et al., 2009). ET-1 level in plasma is increased in patients with the severity of heart failure (Yin et al., 2011). Nitric oxide (NO) is a key signaling molecule to modulate downstream proteins and is produced endogenously by nitric oxide synthase. In heart failure disease conditions, dysfunction of NO contributes to the contractile dysfunction, adverse remodeling, and finally myocardial hypertrophy (Tang et al., 2014; Pall, 2013). In our study, coronary artery ligation induced a significant higher ET-1 concentration and
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Fig. 7. Effects of houttuynin on cardiac collagen accumulation in the interstitial space of the left ventricle in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (Sirius red stain, 400 ). A, Sham-operated control; B, Coronary artery ligation (CAL) control; C, CAL plus 40 mg/kg captopril; D, CAL plus 100 mg/kg houttuynin.
Fig. 8. Effects of houttuynin on cardiac collagen accumulation in the perivascular space of the left ventricle in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (Sirius red stain, 400 ). A, Sham-operated control; B, Coronary artery ligation (CAL) control; C, CAL plus 40 mg/kg captopril; D, CAL plus 100 mg/kg houttuynin.
lower NO concentration. Houttuynin decreased ET-1 level and increased NO concentration indicating that the function of houttuynin in protecting myocardial tissues and antagonizing ventricular remodeling correlates with its improvement of endothelial function.
GSH-Px inhibits the peroxide induced cell apoptosis and necrosis, which is used as a cofactor in the removal of hydrogen peroxide and lipoperoxides (Crawford et al., 2011). CAT destroys intracellular Peroxide (H2O2) to form water and molecular oxygen. The excess oxidative stress either via the increased production of
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Fig. 9. Effects of houttuynin on collagen accumulation in the interstitial space of the left ventricle in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (Sirius red stain and polarized light, 400 ). A, Sham-operated control; B, Coronary artery ligation (CAL) control; C, CAL plus 40 mg/kg captopril; D, CAL plus 100 mg/kg houttuynin.
Fig. 10. Effects of houttuynin on collagen in the perivascular space of the left ventricle in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (Sirius red stain and polarized light, 400 ). A, Sham-operated control; B, Coronary artery ligation (CAL) control; C, CAL plus 40 mg/kg captopril; D, CAL plus 100 mg/kg houttuynin.
peroxides or via the decreased inactivation process of scavenger enzymes attacks the lipid component of cellular membrane and gives rise to the increased lipid peroxidation (Lim et al., 2013). In our experiment, GSH-Px and CAT activities were lower in serum of CAL control group than those in sham-operated group, indicating that the function of houttuynin on improving myocardial remodeling correlates with increasing the GSH-Px and CAT activities, and eliminating oxygen free radical as well.
5. Conclusions In summary, our in vivo results indicate that houttuynin plays an antifibrotic role in inhibiting coronary artery ligation-induced ventricular remodeling. The mechanism may be related to restraining the excessive activation of RAAS and inhibiting peroxidation. Thus, houttuynin may be potentially as a therapeutic tool for ventricular remodeling and even heart failure.
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Fig. 11. Effects of houttuynin on interstitial collagen volume fraction (ICVF) and perivascular collagen volume fraction (PVCF) of the left ventricle in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x7S.D.). Compared with sham-operated control, ##Po0.01. Compared with CAL control, nnPo0.01.
Fig. 13. Effect of houttuynin on GSH-Px activity in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x7 S.D.). Compared with shamoperated control group, ##Po 0.01. Compared with CAL control group, nnPo 0.01. GSH-Px, glutathione peroxidase.
Table 2 Effects of houttuynin on angiotensin I (Ang I) and angiotensin II (Ang II) concentrations in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x7 S.D.).
Table 4 Effect of houttuynin on CAT activity in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x7 S.D.).
Group
n
Dose(mg/kg)
Ang I (ng/ml)
Ang II (pg/ml)
Group
n
Dose(mg/kg)
CAT(U/ml)
Sham-operated control CAL control CAL plus captopril CAL plus houttuynin
8 10 10 8
– – 40 100
1.38 70.29 1.7070.17b 1.38 70.13c 1.43 70.21d
116.93 7 22.29 150.87 7 19.12a 99.98 7 16.72c 104.797 22.07c
Sham-operated control CAL control CAL plus captopril CAL plus houttuynin
8 10 10 8
– – 40 100
6.02 73.42 1.97 71.01b 3.62 71.29d 4.52 71.96d
Note: Compared with sham-operated control group, aP o 0.01; Compared with CAL control group, cP o 0.01; dP o 0.05.
b
P o 0.05.
Note: Compared with sham-operated control group, bP o 0.05. Compared with CAL control group, dP o 0.05. CAT, catalase
Table 3 Effects of houttuynin on aldosterone (ALD) and endothelin-1 (ET-1) concentrations in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x7 S.D.).
Table 5 Effect of houttuynin on NO concentration in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x 7 S.D.).
Group
n
Dose(mg/kg)
ALD(ng/ml)
ET-1(pg/mg prot)
Sham-operated control CAL control CAL plus captopril CAL plus houttuynin
8 10 10 8
– – 40 100
1.02 7 0.32 1.53 7 0.35b 1.147 0.35c 1.047 0.37c
62.85 7 9.62 108.92 7 29.56a 58.79 7 28.15d 60.62 7 12.15d
Note: Compared with sham-operated contro groupl, aP o 0.01; Compared with CAL control group, cP o 0.01; dP o 0.05.
b
P o 0.05.
Group
n
Dose(mg/kg)
NO(μmol/l)
Sham-operated control CAL control CAL plus captopril CAL plus houttuynin
8 10 10 8
– – 40 100
14.727 7.53 6.687 2.94a 13.047 6.25c 19.067 7.71c
Note: Compared with sham-operated control group, aP o 0.01. Compared with CAL control group, cP o 0.01.
Acknowledgment Project supported by the Shanghai Committee of Education Foundation (No. 12YZ059). References
Fig. 12. Effect of houttuynin on Hyp concentration in rats with ventricular remodeling induced by left coronary artery ligation (CAL) (x 7 S.D.). Compared with sham-operated control group, ##Po 0.01. Compared with CAL control group, n P o0.05, nnPo 0.01. Hyp, hydroxyproline.
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