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Effects of ethanolic extract from radix scrophulariae on ventricular remodeling rats.

ARTICLE INFO

Keywords: Radix Scrophulariae Ethanolic extract Rat Coronary artery ligation Ventricular remodeling

ABSTRACT

Purpose: To explore the effects of ethanolic extract of Radix Scrophulariae (EERS) on ventricular remodeling in rats.

Methods: Rats with coronary artery ligation (CAL) were randomly assigned to 5 groups: CAL model; CAL plus 40 mg/kg captopril; CAL plus 60 mg/kg, 120 mg/kg, 240 mg/kg EERS. Sham operation rats were randomly assigned to 2 groups, sham-operated control and sham-operated plus 120 mg/kg EERS. The rats were orally administered with the corresponding drugs or drinking water for 14 weeks. 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 hydroxyproline (Hyp), matrix metalloproteinase 2 (MMP-2), angiotensin II (Ang II), aldosterone (ALD), endothelin 1 (ET-1), atrial natriuretic peptide (ANP), tumor necrosis factor [alpha] (TNF-[alpha]) and renin activity (RA) in myocardium or serum were determined. Real-time RT-PCR was used to detect the mRNA expressions of angiotensin converting enzyme (ACE), ET-1 and ANP.

Results: EERS could significantly reduce the LVWI and HWI, decrease heart tissue concentrations of Hyp and collagen deposition, diminish cardiomyocyte cross-section area, reduce the tissue level of Ang II, ET-1, ANP and TNF-[alpha]. EERS could also down regulate the mRNA expression of ACE, ET-1 and ANP in myocardium.

Conclusion: EERS attenuates ventricular remodeling. The mechanisms may be related to restraining the excessive activation of RAAS, INF-[alpha] and modulating some gene expressions associated with cardiac hypertrophy.

Introduction

Chronic heart failure (CHF) is the ultimate consequence of a vast number of cardiovascular diseases and constitutes one of the worldwide leading causes of morbidity and mortality. It is considered to be an irreversible and progressive process characterized by ventricular remodeling (VR), diminished pump performance and a number of neurohormonal perturbations (Rong et al. 2009). Most of all, ventricular remodeling is considered as the basic mechanism in the process of CHF (Nagase and Woessner 1999). Therefore, inhibiting ventricular remodeling early may be an effective way to postpone heart failure induced by myocardial infarction, hypertension and other cardiovascular diseases.

Radix Scrophulariae, a traditional Chinese herb medicine derives from the Scrophularia ningpoensis (Xuans hen), has long been used in clinic to treat febrile diseases with impairment of Yin manifested by deep red tongue and dire thirst or with eruptions, constipation due to impairment of body fluid; phtisis with cough, conjunctivitis, sore throat, scrofula, diphteria, boils and sores, internal bleeding (Wagner et al. 2011). The previous study reveals that total rough extracts of Radix Scrophulariae has beneficial effect against ventricular remodeling induced by ligating the left coronary artery of the rats (Gu et al. 2010). The present study was aimed at investigating the midterm effects of ethanolic extract of Radix Scrophulariae on ventricular remodeling in rats, and the underlying mechanisms.

Materials and methods

Drugs and reagents

Preparation of ethanolic extract of Radix Scrophulariae (EERS): 60 kg of dried rough powdered roots of Scrophularia ningpoensis was refluxed with 600170% ethanol for 2 h, after taking the filtered solution, the residue was refluxed with the same solvent in the same condition for two more times. The combined extract solution was concentrated under reduced pressure to remove the ethanol. Then, the concentrated solution was subjected to D-1400 macro resin chromatograph column eluted with water, 10% ethanol water solution and 50% ethanol water solution respectively. The eluent parts (the 50% EtOH eluent part was removal of solvent under vacuum at 50 [degrees] C first) were subjected to spray dryer with inlet air temperature of 100 [degrees] C and outlet air temperature of 60 [degrees] C. The extract yield after spray drying was 360g.

HPLC analyses of the principal constituents of EERS: A HPLC method was developed to check the principal constituents of the extract. 10 mg EERS sample powder was put into a 10 ml of volumetric flask, 8 ml of distilled water was added, sonicated for 3 min, cooled to room temperature and diluted to 10 ml. The solution was filtered for HPLC injection.

Chromatographic analyses were performed on an Agilent l200 series HPLC instrument, quaternary HPLC pump, column heater, diode array detector, and Agilent Chem-Station for data collection and manipulation. Reverse phase separations of the procyanidin oligomers were performed on a 5 [micro]m silica column (250 x 4.6 mm) (Phenomenex, Torrance, CA). Samples were analyzed with a linear gradient from 97% solvent A (water with 0.03% phosphoric acid) and 3% solvent B (100% acetonitrile) to 50% solvent A and 50% solvent B in 42 min at a flow rate of 1.0 ml/min. UV data were collected by using a diode array detector set at 210 nm, 280 nm and 330 nm.

The HPLC chromatograms of EERS were shown in Fig. 1. Four main peaks were determined by comparing with the standard compounds. They were Harpagide with the remain time at 10.1 min under 210 nm, Harpagoside with the remain time at 33.3 min under 280 nm, Cinnamic acid with the remain time at 35.4 min under 280 nm, Angoroside C with the remain time at 27.9 min under 330 nm. After setting up the regression equations of these four standards respectively, the contents of them in the EERS were assayed as Harpagide 18.7%, Harpagoside 13.4%, Cinnamic acid 5.7%, Angoroside C 14.6%. Their chemical formulas were shown in Fig. 2 (Wagner et al. 2011). The other main constituents in EERS were saccharides which had no peaks under UV detector.

The standard compounds of Harpagide, Harpagoside, Angoroside C and Cinnamic acid were purchased from Shanghai Hotmed Sciences Co., Ltd. (Shanghai, China).

Captopril tablets (Lot number: 090727) were from Jiangsu Huanghe River Pharmaceutical Co., Ltd. (Jiangsu, China). They were dissolved in distilled water before use.

Animals and experimental protocols

Male SD rats (180-200 g, grade of specific pathogen free) were supplied by Shanghai Slac laboratory animal Co., Ltd. All animals were maintained in a 12 h light/dark cycle room with the temperature at 22-24 [degrees] C and the humidity at 40 [+ or -] 5%. The rats received humane care and had free access to a standard diet and drinking water. The animal experiments were approved by 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).

Left ventricular remodeling was created by left coronary artery ligation (CAL) in rats. In brief, rats were anesthetized by intraperitoneal administration of sodium pentobarbital (40 mg/kg), and the chest wall was shaved. Animals were then intubated, ventilated with a type HX-300s animal respirator (Chengdu Technology & Market Co., Ltd.). A left thoracotomy was performed, the heart was exposed and pericardiotomy was then performed, the left coronary artery was ligated approximately 2 mm from its origin with a 4-0 silk suture. Coronary artery occlusion with myocardial infarction (MI) was demonstrated by grossly visible scarring of the change in colour of the left ventricle and ischaemia was confirmed by the raising of ST (ECG-6511 Electrocardiograph, Shanghai Nihon Kohden). Then the thorax was closed immediately and the skin sutured.

Sham-operated rats underwent a similar procedure, but no coronary ligation was performed. After cardiac surgery, each rat was given benzylpenicillin by intramuscular injection for three days to prevent infection. The operated rats were burdened by elevated mortality during the initial 24h after CAL (Agnoletti et al. 2006). In our study, the survival rate from the surgery was about 55%.

On the second day after operation, the rats were randomly divided into seven groups: sham-operated control, sham-operated plus 120 mg/kg EERS, CAL plus drinking water (model), CAL plus 40 mg/kg captopril, CAL plus 60 mg/kg EERS, CAL plus 120 mg/kg EERS and CAL plus 240 mg/kg EERS.

The rats were orally administrated with EERS or captopril at above described doses once a day. And drinking water was administered in the same manner to the sham-operated control and model groups. Treatment started from 1 day after operation and continued for 14 weeks.

Hemodynamic parameters measurements

Cages were inspected daily in all groups, 14 weeks after treatment, rat body weight (BW) was recorded after fasting for 12 h and then anesthetized with intraperitoneal injection of urethane (1.0 g/kg). A polypropylene catheter was inserted into the right carotid artery. The arterial catheter was filled with heparinized saline solution and connected to a pressure transducer. After an equilibrium period for about 5 min, systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and heart rate (FIR) were recorded with a multi-channel biological signal analysis system. The catheter was advanced into the left ventricle to measure the left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP) and the maximal rate of rise and fall of the ventricular pressure ([+ or -] dp/dtmax).

Cardiac weight indexes calculation and histological examination

After hemodynamic parameters being recorded, the blood sample was collected from carotid artery and centrifuged (4 [degrees] C, 2325 x g, 10 min) to recover serum which was stored immediately in a - 70 [degrees]C freezer until being assayed. The heart was taken out, rinsed with cold saline solution, and the left ventricle was separated from the atria, aorta and adipose tissue. The left ventricle weight (LVW) and heart weight (HW) 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 two parts. The upper part was immersed in formalin (10% formaldehyde). The lower part was separated into several sections and rapidly frozen in liquid nitrogen and then stored in a 70 - [degrees] C freezer until being assayed.

The fixed part of ventricle in formalin was dehydrated and embedded in paraffin, then cut into 5 pirn thick slices and heated overnight in a 60 [degrees] C incubator. The sections were stained with hematoxylin and eosin (HE) for measurement of cardiomyocyte cross section area, and with Sirius red in aqueous saturated picric acid for examination of interstitial and perivascular fibrosis in myocardium. Each sample slice was photographed (400x magnification) under the microscope (Olympus BX51, Japan). All photos were analyzed with the image-Pro Plus 6.3 analyzing software (Media Cybernetics, Bethesda, MD, USA) by computer.

At least 3 fields per sample slice were randomly selected and 20 myocardial cells per field were randomly chosen to calculate the mean cross section area of myocardial cells. The percentage of collagen area in each field was calculated as the myocardial interstitial collagen volume fraction (ICVF). The area ratio of perivascular collagen to vessel lumen was calculated as the perivascular collagen volume fraction (PCVF) in myocardium (Shinzato et al. 2007).

Type I and type III collagen accumulation in the interstitial space of the left ventricle was assessed by polarized light microscopy. Type I, type III collagen volume fraction (CVF) was also determined with Image-Pro 6.3 analyzing software.

Hydroxyproline (Hyp) and matrix metalloproteinase 2 (MMP-2) determination

Hyp concentrations were detected by ultraviolet spectrophotometry with the Hydroxyproline kit (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China). MMP-2 concentration of ventricular tissue was measured with a rat matrix metalloproteinase 2 (MMP-2) enzyme-linked immunosorbent assay (ELISA) kit (R&D). Protein concentrations of myocardial homogenates were assayed with the Coomassie Brilliant Blue Kit (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China). Tissue Hyp and MMP-2 were expressed as concentration per milligram protein of ventricular tissue.

Radioimmunoassay determination

Radioimmunoassay was used to detect angiotensin II (Ang II), aldosterone (ALD), endothelia 1 (ET-1), atrial natriuretic peptide (ANP), tumor necrosis factor a (TNF-[alpha]) and resin activity (RA) concentrations of ventricular tissue or serum. The homogenized tissue was centrifuged (4 [degrees] C, 1780 x g, 15 min) and the supernatant was collected for measurement. Ang II, ALD, ET-1, ANP, TNF-[alpha] concentrations and RA were respectively analyzed with Iodine [125I] Ang II kit, Iodine [125I] ALD kit, Iodine [125I] ET-1 kit, Iodine [125I] ANP kit, Iodine [125I] TNF-[alpha] kit and Iodine [125I] RA kit (all purchased from Beijing North Institute of Biological Technology, Beijing, China). Tissue Ang II, ET-1 and ANP concentration was expressed as per milligram protein of ventricular tissue. Serum ALD and TNF-[alpha] were expressed as concentration per milliliter serum sample. Tissue RA was expressed as the amount of angiotensin I per milligram protein of ventricular tissue in an hour.

Real-time RT-PCR determination

The mRNA expressions of angiotensin converting enzyme (ACE), ET-1 and ANP were determined by real time RT-PCR. Total RNA was extracted from the tissues by using the Trizol reagent (Invitrogen) according to the manufacturer's instructions. RNA yields and purity were assessed by spectrophotometric analysis (Nano Drop 1000, Thermo). Total RNA (1 [micro]g) from each well was subjected to reverse transcription with random hexamer primers, deoxynucle-oside triphosphates (dNTPs), and Maloney murine leukemia virus (M-MLV) reverse transcriptase in a total reaction volume of 20 [micro]l. The real-time RT-PCRs (20 [micro]l) consisted of 3.7 [micro]l SYBR Green Mix,

1 [micro]l mixed primers, 1 [micro]l cDNA and 14.3 [micro]l double-distilled water. A typical protocol included incubation at 50 [degrees] C for 2 min, and tact activation at 95 [degrees] C for 2 min, followed by 40 cycles with 95 [degrees] C denaturation for 15 s, 59 [degrees] C annealing for 20 s, and 72 [degrees] C extension for 20 s. The sequences of primer were as follows:

  ACE mRNA sense: 5'-ATGAGGCTATTGGAGATGTTTTG-3',
  ACE mRNA anti-sense: 5'-TCCTIGGTGATGCTTCCGT-3';
  ANP mRNA sense: 5'-GGGAAGTCAACCCGTCTCAG-3',
  ANP mRNA anti-sense: 5'-CGGAAGCTGTTGCAGCCTAG-3';
  ET-1 mRNA sense: 5'-CTGGACATCATCTGGGTCAACA-3',
  ET-1 mRNA anti-sense: 5'-GCTCGAGTTCTTTGTCTGCTT-3';
  GAPDH sense: 5'-TGGCATGGACTGTGGTCATG-3',
  GAPDH anti-sense: 5'-TGGGTGTGAACCACGAGAAA-3'.


Real-time RT-PCR was carried out with Mastercycler ep realplex (4) (Eppendorf AG-22331 Hamburg, Germany), and data analyzed by Mastercycler ep realplex assay 2.2 Software.

All values obtained with ACE, ANP or ET-1 primers were normalized to the values obtained with the GAPDH primers. The results were expressed as the relative integrated intensity.

Statistical analysis

All values were expressed as mean [+ or -] standard deviation (SD). Statistical analysis was performed by one-way analysis of variance for multiple comparisons, followed by the post hoc test to evaluate the difference between two groups. Values of p < 0.05 were considered as a statistically significant difference for all analyses.

Results

Effects on hernodynamic data

As shown in Figs. 3 and 4 respectively, there was no significant difference of SBP, DBP, MAP and HR between sham-operated control group and sham-operated plus EERS group. In the model group, SBP and MAP were reduced and HR increased when compared with that in the sham-operated control group (p < 0.05). EERS did not influence the blood pressure very much, except that at 120 mg/kg, it increased SBP (p < 0.05). EERS at 60 mg/kg and 120 mg/kg significantly reduced HR (p < 0.01) and captopril reduced HR as well (p <0.05).

LVSP and +dp/dtmax were significantly lower in the model group than that in the sham-operated control group (p < 0.01 or p < 0.05). As a tendency, LVEDP was increased, and - dp/dtmax reduced in the model group, however there were no significant differences between the sham-operated control and the model groups (Table 1). EERS at 120 mg/kg or 240 mg/1<g increased LVSP and +dp/dtmax obviously (p < 0.05 or p < 0.01). Captopril increased LVSP, +dp/dtmax as well (p < 0.05 or p < 0.01). However, both captopril and EERS did not show obvious influence on LVEDP and - dp/dt max (Table 1).

Table 1
Effects of ethanolic extract of Radix Scrophulariae (EERS) on
hemodynamic parameters of rats with ventricular remodeling by
left coronary artery ligation (CAL) ([x.bar] [+ or -] SD,
n = 8-9).

Group                      LVSP      LVEDP    +dp/dtmax  -dp/dtmax
                          (mmHg)    (mmHg)    (mmHg/s)   (mmHg/s)

Sham-operated control       111.2       0.3     13522.8   -12985.5
                         [+ or -]  [+ or -]    [+ or -]   [+ or -]
                             11.8       7.3      1016.5     1085.5

Sham-operated plus 120       96.3       0.6     13545.9   -12919.8
mg/kg EERS               [+ or -]  [+ or -]    [+ or -]   [+ or -]
                             44.1       4.1      1255.6     1994.9

Model                        59.9       6.7  11734.8 [+   -12723.0
                         [+ or -]  [+ or -]    [+ or -]   [+ or -]
                           31.2##       9.2      700.2#     1289.3

CAL plus 40 mg/kg            99.1       5.2     13408.3   -13095.5
captopril                [+ or -]  [+ or -]    [+ or -]   [+ or -]
                           15.7**       7.7      516.1*      414.5

CAL plus 60 mg/kg EERS       91.3       6.3     13138.1    12831.4
                         [+ or -]  [+ or -]    [+ or -]   [+ or -]
                             23.6       3.9       449.7      620.5

CAL plus 120 mg/kg EERS     111.1       5.9     13320.7   -12972.8
                         [+ or -]  [+ or -]    [+ or -]   [+ or -]
                           22.1**       6.0     1523.8*     1498.2

CAL plus 240 mg/kg EERS     110.9       7.7     13700.3   -13289.9
                         [+ or -]  [+ or -]    [+ or -]   [+ or -]
                           16.5**       4.0     833.3**      989.2

Note: Compared with sham-operated control, * p < 0.05, ## p < 0.01.
Compared with model, *p < 0.05, **p < 0.01. LVSP, left ventricular
systolic pressure; LVEDP, left ventricular end diastolic pressure;
+dp/dtmax, the maximal rate of rise; -dp/dtmax, the maximal rate
of fall.


Effects on cardiac weight indexes

As shown in Fig. 5, there was no significant difference of LVWI and HWI between sham-operated control group and sham-operated plus EERS group. The LVWI and HWI were significantly greater in the model group than those in the sham-operated control group (p < 0.01). EERS and captopril treatment significantly decreased LVWI and HWI (p < 0.05).

Effects on cardiomyocyte cross-section area and collagen accumulation

As shown in Figs. 6 and 7, there was no significant difference of the average cross-section area of cardiomyocytes between sham-operated control group and sham-operated plus EERS group. However, in the model group it was significantly larger than that in the sham-operated control group (p <0.01). EERS (60, 120 and 240 mg/kg) or captopril attenuated the increase of average cross-section area of cardiomyocytes (p < 0.01).

Under microscope, myocardial interstitial and perivascular collagen fibers appeared red or deep red, cardiomyocytes appeared pink or orange in these sections stained with Sirius red. In rat myocardium of sham-operated and sham-operated plus EERS group, little amount of collagen was found in the interstitial and perivascular space. After CAL, there was a large accumulation of collagen in the interstitial and perivascular space of ventricle in model rats. Less collagen deposition was found in CAL plus EERS or captopril group than that in model group (Figs. 8-10).

As shown in Figs. 11 and 12, 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 and sham-operated plus EERS groups, less amount of type I and III collagen fibers was found. Collagen distributions of type I and III were significantly increased in the model rats compared with that in the sham-operated rats (p < 0.01). However, these were significantly decreased in EERS or captopril treated groups (p < 0.01). The ratio of Ifill collagen in the model group was higher than that in the sham-operated group (p< 0.05). EERS and captopril significantly decreased the ratio of Ifill collagen (Table 2, and Figs. 11 and 12).

Effects on hydroxyproline and MMP-2

As shown in Fig. 13, Hyp concentration was lower in the sham-operated control and sham-operated plus EERS groups. It was significantly increased in the model group (p < 0.05). EERS and captopril decreased Hyp concentration obviously (p < 0.01 or p < 0.05).

Table 2
Effects of ethanolic extract of Radix Scrophulariae (EERS) on
type I, type III collagen, interstitial collagen volume fraction
and 1/111 collagen ratio of the left ventricle in rats with
ventricular remodeling by left coronary artery ligation (CAL)
(x [+ or -] SD, n = 8).

Group                         Collagen I  Collagen    I/III
                                  (%)       III (%)   collagen
                                                      ratio

Sham-operated control               0.60      0.10      8.45
                                [+ or -]  [+ or -]  [+ or -]
                                    0.41      0.08      5.86

Sham-operated plus 120 mg/kg        0.31      0.17      2.73
EERS                            [+ or -]  [+ or -]  [+ or -]
                                    0.13      0.16     1.68#

Model                              16.64      0.83     25.56
                                [+ or -]  [+ or -]  [+ or -]
                                  5.27##    0.42##    17.72#

CAL plus 40 mg/kg captopril         0.48      0.08      8.08
                                [+ or -]  [+ or -]  [+ or -]
                                  0.15**    0.04**     4.77*

CAL plus 60 mg/kg EERS              1.12      0.12      9.33
                                [+ or -]  [+ or -]  [+ or -]
                                  0.87**    0.08**     4.51*

CAL plus 120 mg/kg EERS             0.74      0.15      6.50
                                [+ or -]  [+ or -]  [+ or -]
                                  0.26**    0.07**     5.61*

CAL plus 240 mg/kg EERS             0.86      0.15      6.67
                                [+ or -]  [+ or -]  [+ or -]
                                  0.48**    0.11**     3.52*


Note: Compared with sham-operated control, *p<0.05, fr#p<0.01.
Compared with model, *p < 0.05, **p< 0.01.


As shown in Fig. 14, MMP-2 concentration was lower in the sham-operated control and sham-operated plus EERS groups. It was significantly increased in the model group (p < 0.01), EERS and captopril reduced MMP-2 concentration significantly (p < 0.01 or p < 0.05).

Effects on Ang II, ALD, ET-1, ANP, TNF-[alpha] and RA

As shown in Table 3, there was no significant difference of Ang II concentration between sham-operated control and sham-operated plus EERS groups. Ang 11 concentration was significantly higher in the model group than that in the sham-operated control group (p < 0.05). Compared with model control, EERS at 120, 240 mg/1<g and captopril significantly reduced Ang II concentration (p < 0.05 or p < 0.01). However, there was no significant difference of ALD concentration among all the groups. RA was significantly higher in the model group than that in the sham-operated control group (p < 0.05). EERS and captopril did not show obvious effects on RA.

Table 3
Effects ofethanolic extract of Radix Scrophulariae(EERS) on srum
aldosterone (ALD), cardiac renin activity (RA) and angiotensin
II (Ang II) concentration in rats with ventricular remodeling by
left coronary artery ligation (CAL) (x [+ or -] SD.    n = 13).

Group                              RA         ALD        Ang II
                              pg/mg proth)  (ng/ml)   (pg/mg prot)

Sham-operated control               0.0382     0.291        67.80
                                  [+ or -]  [+ or -]     [+ or -]
                                    0.0160     0.048        12.23

Sham-operated plus 120mg/kg         0.0490     0.273        67.00
EERS                              [+ or -]  [+ or -]     [+ or -]
                                    0.0187     0.025        11.54

Model                               0.0546     0.313        78.42
                                  [+ or -]  [+ or -]     [+ or -]
                                    0.0136     0.055       13.96 #

CAL plus 40 rng/kg captopril        0.0545      0321        63.17
                                  [+ or -]  [+ or -]     [+ or -]
                                    0.0123     0.036       20.39*

CAL plus 60 mg/kg EERS              0.0518     0.315        69.50
                                  [+ or -]  [+ or -]     [+ or -]
                                    0.0156     0.063        14.21

CAL plus 120 mg/kg EERS             0.0514     0.284        68.59
                                  [+ or -]  [+ or -]     [+ or -]
                                    0.0084     0.058       10.87 #

CAL plus 240 mg/kg HERS             0.0527     0.274        60.78
                                  [+ or -]  [+ or -]     [+ or -]
                                    0.0077     0.063       9.70 **

Note: Compared with sham-operated control, #p< 0.05. Compared with
model, *p<0.05, **p<0.01.


ET-1 and ANP concentrations were significantly higher in the model control group than that in the sham-operated control group (p < 0.01). When compared to model group, EERS at 60, 120, 240 mg/kg significantly reduced ET-1 and ANP concentrations (p < 0.05 or p < 0.01). Captopril decreased ANP concentration as well (p < 0.05). There was no significant difference of ET-1 and ANP concentrations between sham-operated control and sham-operated plus EERS groups (Table 4).

As shown in Fig. 15, there was no significant difference of TNF-[alpha] concentration between sham-operated control and sham-operated plus EERS groups. TNF-[alpha] concentration was significantly increased in the model group. EERS significantly lowered the concentration when compared to model control (p < 0.05 or p < 0.01).

Table 4
Effects of ethanolic extract of Radix Scrophulariae (EERS)
on endothelin 1 (ET-1) and atrial natriuretic peptide (ANP)
concentrations of the left ventricle in rats with ventricular
remodeling by left coronary artery ligation (CAL) (x [+ or -]
SD, n = 13).

Group                    ET-1 (pg/mg prot)  ANP (pg/mgprot)

Sham-operated control               12.33           2517.7
                                 [+ or -]         [+ or -]
                                     3.33           1202.1

Sham-operated plus 120              13.81           3279.7
mg/kg EERS                       [+ or -]         [+ or -]
                                     1.99            909.2

Model                               16.05           4185.1
                                 [+ or -]         [+ or -]
                                  2.02 ##         1223.5 ##

CAL plus 40 mg/kg                   15.75           3274.5
captopril                        [+ or -]         [+ or -]
                                    2.16           828.5 *

CAL plus 60 mg/kg EERS              13.46           3437.5
                                 [+ or -]         [+ or -]
                                   3.07 *            870.4

CAL plus 120 mg/kg EERS             14.14           2730.5
                                 [+ or -]         [+ or -]
                                   2.16 *         672.3 **

CAL plus 240 mg/kg EERS             13.96           2909.2
                                 [+ or -]         [+ or -]
                                   2.36 *         934.7 **

Note: Compared with sham-operated control, ##p<0.01. Compared
with model, *p < 0.05, *xp < 0.01.


Effects on ACE, ET-1 and ANP mRNA expression

The mRNA expressions of ACE, ET-1 and ANP in model rats were significantly higher than those in the sham-operated control rats (p < 0.05 or p < 0.01). Treatment with EERS at 60-240 mg/kg inhibited mRNA over expression of ACE and ET-1 (p < 0.05 or p < 0.01). Captopril decreased mRNA expression of ACE and ET-1 as well. However, EERS only at 240 mg/kg decreased ANP mRNA expression (Table 5).

Discussion

Based on population attributable risks to HF, hypertension has the greatest impact, myocardial infarction also has a high attributable risk (Kannel 2000). CAL offers the possibility to study the midterm effects of myocardial infarction. It usually causes a significant reduction in SBP, DBP and high in HR (Agnoletti et al. 2006; Sharma et al. 2004). LVEDP is often used to assess the degree of cardiac dysfunction in rats (Grossman 1990; Pfeffer et al. 1979). Some data indicated that 6 weeks after CAL, the untreated animals have developed severe CHF, evidenced by decreased LVSP and dp/dtmax and increased LVEDP compared to the sham-operated rats (Xia et al. 2006; Tao et al. 2005). Our results were consistent with those in the literature indicating that the cardiac hypertrophy model induced by MI was successful. EERS significantly increased SBP, LVSP, +dp/dtmax and slowed down HR, demonstrating that it could improve cardiac function to some extent.

Following acute myocardial infarction the ventricular remodeling is the result of the infarction expansion process, whereas late is the result of the hypertrophy process (Zornoff et al. 2009). The heart weight index or mass and the transverse area of cardiomy-ocytes are the main indicators of the degree of cardiac hypertrophy (Anversa et al. 1985; Ji et al. 2008). In our study, the degree of cardiac hypertrophy was displayed by the increase of LVWI, HWI and demonstrable microscopically with an up to 100% increase in cell transverse area. Treatment with EERS for 14 weeks attenuated cardiac hypertrophy.

With the stimulation of ventricular remodeling, a large amount of collagen secretes and accumulates in the myocardial intercellular and perivascular space. It is known that, after the acute phase of MI, fibroblast proliferation and collagen deposition occur in a long-term period. Depending on the species, the whole process is completed within weeks to months (Fishbein et al. 1978). The increase of collagen secretion and aggregation will lead to the increase of myocardial stiffness, the change of cardiac structure, the worsen of ventricular remodeling, and even ventricular dysfunction (Brower et al. 2006; Swan 1994). Thus inhibition of myocardial fibrosis may improve the cardiac function. Our observation showed that EERS significantly reduced intercellular and perivascular collagen accumulation. According to the results, we deduce that EERS blocks myocardial fibrosis by reducing synthesis and secretion of collagen, and then slows down or inhibits the process of ventricurlar remodeling and myocardial failure.

Table 5
Effects of ethanolic extract of Radix Scrophulariae (EERS)
on mRNA expression of angiotensin converting enzyme (ACE),
endothelin 1 (ET-1) and atrial natriuretic peptide (ANP)
in rats with ventricular remodeling by left coronary artery
ligation (CAL) (x [+ or -] SD, n = 5).

Croup                            ACE       ET-1      ANP

Sham-operated control            0.94      1.03      1.44
                             [+ or -]  [+ or -]  [+ or -]
                                 0.17      0.26      1.07

Sham-operated plus 120mg/kg      0.32      1.26      2.14
EERS                         [+ or -]  [+ or -]  [+ or -]
                              0.12 **      0.77      1.90

Model                            1.46      3.34     17.20
                             [+ or -]  [+ or -]  [+ or -]
                               0.45 #   0.99 ##   13.10 #

CAL plus 40 mg/kg captopril      0.51      1.80     10.47
                             [+ or -]  [+ or -]  [+ or -]
                              0.33 **    -1.33 *     12.28

CAL plus 60 mg/kg EERS           0.40      1.60     12.87
                             [+ or -]  [+ or -]  [+ or -]
                              0.09 **    0.49 **     10.40

CAL plus 120 mg/kg EERS          0.62      1.61      6.80
                             [+ or -]  [+ or -]  [+ or -]
                              0.27 **     1.01 *      5.31

CAL plus 240 mg/kg EERS          0.56      1.66      2.82
                             [+ or -]  [+ or -]  [+ or -]
                              0.31 **     1.12"     2.77 *

Note: Compared with sham-operated control, #p < 0.05, ##p < 0.01.
Compared with model, *p < 0.05, **p < 0.01.


Collagen type I and III are major fibrillar collagens involved in tissue repair (Sun and Weber 1996). In particular, type 1 collagen characterized by tensile strength (Weber 1989), comprises approximately 85% of the total (Heeneman et al. 2003). Its accumulation may contribute to myocardial stiffness by limiting the motion of cardiomyocytes, and may promote arrhythmias by electrical isolation of adjacent cardiomyocytes. Type III collagen, with little tensile strength, is deposited during healing. A shift in the balance between synthesis and degradation of collagen type I and HI plays a key role in the alters of tissue stiffness (Burlew and Weber 2002). Our results demonstrated that EERS reduced type I and III collagen accumulation and the ratio of This may be beneficial to improving myocardial structure and function.

Matrix metalloproteinase (MMPs) plays a key role in the process of CHF, its' increased activity could aggravate myocardial fibrosis (Peterson 2004). Hydroxyproline is a characteristic amino acids in collagen of fibrous tissue, total collagen content determined in cardiac myofibroblasts could use hydroxyproline assay (Wang et al. 2007). In our study, following CAL MMP-2 and hydroxyproline content increased and EERS or captopril significantly decreased the contents.

Many reports underscored the importance of the interaction between inflammatory cytokines and the renin-angiotensin-aldosterone system (RAAS) in ventricular remodeling in the progression of CHF (Fronds et al. 2001; Gurlek et al. 2001). Angiotensin II can provoke inflammatory responses, whereas TNF can provoke activation of the RAAS, the overexpression of them is sufficient to contribute to the disease progression by worsening left ventricular remodeling and progression in heart failure (Sekiguchi et al. 2004). Recent clinical and experimental studies have noted that increased release of TNF-[alpha], can contribute to LV myocardial remodeling (Bradham et al. 2002). In our study, EERS significantly attenuated the increase of local synthesis of Ang II and TNF-[alpha] in the ventricular myocardium, confirming a favorable effect of EERS on postpone the process to CHF.

There was study reported that cardiac ACE activity in CHF patients was higher than those with healthy hearts (Riegger 1994). ACE inhibitors could attenuate left VR and have become an integral component of the treatment of heart failure (Brower et al. 2007; Pfeffer et al. 1992). EERS significantly down-regulated mRNA expression of ACE, it may be one of the mechanisms of its action against cardiac remodeling.

ANP has received particular attention because of its effects on blood pressure regulation and cardiac function (Rubattu et al. 2008). Sakai et al. found that ET-1 was involved in myocardial hypertrophy and ventricular remodeling after myocardial infarction (Sakai et al. 1996). After combining with its receptors, ET-'1 could stimulate ANP mRNA expression and secretion, which is one of the characteristics associated with poor long-term prognosis in myocardial hypertrophy (Hall et al. 1994; Remes 1994). In our study, the results showed that EERS significantly attenuated the increase of local ET-1 and ANP synthesis in the ventricular myocardium and down-regulated the expression of corresponding mRNA.

In conclusion, EERS prevented cardiac fibrosis and attenuated left VR. These indicate that EERS may have therapeutical potential in the early treatment of ventricular remodeling and heart failure. The mechanism may be related to its restraining the excessive activation of RAAS, decreasing the excretion of ET-1 and TNF-[alpha], attenuating the mRNA expressions of ACE, ANP and ET-1.

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* Corresponding author. Tel.: +86 21 51322200; fax: +86 21 51322200. E-mail address: cxchen6@126.com (C.X. Chen).

Xiao Yan Huang (a), Chang Xun Chen (a), (*), Xue Mei Zhang (b), Ying Liu (a), Xi Mill Wu (b), Yi Ming Li (b)

(a.) Department of Pharmacology, Shanghai University of Traditional Chinese Medicine, 1200 Calm Road, Shanghai 201203, China

(b.) Department of Natural Product Chemistry, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

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doi:10.1016/j.phyrned.2011.09.079
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Author:Huang, Xiao Yan; Chen, Chang Xun; Zhang, Xue Mei; Liu, Ying; Wu, Xi Min; Li, Yi Ming
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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