ŀ
1
Li-Fu formula prevents the IL-6 mediated cardiac hypertrophy in hyper
cholesterol-fed hamsters
Yi-Chang Cheng1, Chieh-HisWu2, Wei-Wen Kuo2, James A Lin3, Hsueh-Fang Wang5, Fuu-Jen Tsai4,6, Chang-Hai Tsai7, Chih-Yang Huang3,4,8,*, Tsai-Ching Hsu9,*, Bor-Show Tzang10,11,12,*
1
Emergency Department of Taichung Veterans General Hospital, Taichung, Taiwan;
2
Department of Biological Science and Technology, China Medical University, Taichung, Taiwan;
3
Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan;
4
Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan;
5
Department of Food and Nutrition, Huang-Kuang University, Taichung, Taiwan;
6
Department of Pediatrics, Medical Research and Medical Genetics, China Medical College Hospital, Taichung, Taiwan;
7
Department of Healthcare Administration, Asia University, Taichung, Taiwan;
8
Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan;
9
Institute of Immunology, Chung Shan Medical University, Taichung, Taiwan;
10
Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan
11
Department of Biochemistry, School of Medicine, Chung Shan Medical University, Taichung, Taiwan
12
Clinical Laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan
* These authors share equal contributions
Address reprint requests to: Bor-Show Tzang, Ph.D., Institute of Biochemistry and
Biotechnology, Chung Shan Medical University, Taichung, Taiwan. No.110, Sec.1,
Jianguo N. Rd., Taichung 402,Taiwan R.O.C. Tel: 886-4-24730022 ext. 11680; Fax:
886-4-23248195; Email: [email protected]
3 ABSTRACT
Hypercholesterolemia diets are considered as major sources to cause cardiac
hypertrophy. This study intends to evaluate the effects of Li-Fu formula on cardiac
hypertrophy induced by hypercholesterolemia diet. Twenty-four male Golden Syrian
hamsters at 3 months of age were randomly divided into Control, Cholesterol and
Li-Fu formula groups and fed with different experimental diets for 2 months.
Histopathological analysis and western blotting were performed to measure the
myocardial architecture, and expressions of different cardiac hypertrophy-associated
molecules in the excised left ventricle from hamsters. The ratios of Whole heart
weight (WHW)/Body weight (BW) and Left ventricle weight (LVW)/BW were
significantly higher in the Cholesterol group but significantly lower in the Li-Fu
formula group. The protein levels of both ANP and BNP were significantly increased
in the Cholesterol group but significantly reduced in the Li-Fu formula group.
Additionally, significantly increased interleukin (IL)-6, STAT3, MEK5, p-ERK5 and
non-cardiomyocyte proliferate signal molecules such as p-MEK and p-ERK, were
detected in the Cholesterol group but significantly reduced in the Li-Fu formula group.
Notably, no significant variations of inflammatory signaling molecules, including
p-P38 and p-JNK, were detected in all groups. Our experimental results demonstrate
signaling, non-cardiomyocyte proliferate signaling in the excised left ventricle of
hamsters from the Li-Fu formula. We suggested the protective effects of Li-Fu
formula on cardiac hypertrophy that may be useful in prevention or treatment of
hypertrophy-associated cardiovascular diseases.
5 Introduction
Hypercholesterol diets are the major sources to cause cardiac hypertrophy (1). Cardiac
hypertrophy is recognized as a cardiac adaptive response to any stress that can exist in
a state of compensation or progress to a decompensated state over time (2). Prolonged
hypertrophy of the cardiomyocytes is demonstrated as the main cause of sudden
cardiac death (3). A number of studies indicated that various diseases have been
associated with cardiac hypertrophy including occlusive atherosclerotic coronary
heart disease (CHD), associated myocardial infarction (MI), heart failure hypertension,
endocrine disorders, toxicants, and bacterial endocarditis (4-7).
Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are known as the
cardiac hormones in normal adults that were secreted by the atria and ventricles.
Higher levels of ANP and BNP expression are expressed in the fetal ventricles than
adult ventricles (8). Cardiac ANP and BNP levels are increased in myocardial
infarction of animal models (9), heart failure (10), hypertrophy (11), and also in
human cardiac diseases (12). Increased expressions of ANP and BNP are observed in
ventricular during the molecular process of cardiac hypertrophy, which are recognized
as markers of the induction of the embryonic gene program in ventricular hypertrophy
Interleukin (IL)-6 is known as a potent hypertrophic factor of cardiomyocytes (14-15).
The IL-6 receptor system consists of various signaling pathways including
inflammatory related p38 MAPK, and hypertrophy involved STAT1-STAT3
heterodimer pathway, STAT3 homodimer pathway, and non-cardiomyocyte
proliferative related MAPK extracellular signal regulated kinase (ERK)s pathway that
are activated by the dimerization of gp130 (16-19). The activation of
STAT3-dependent signaling pathway by gp130 was reported to promote cardiac
myocyte hypertrophy (20), herein the STAT1 and the STAT 3 were shown to be
chronically phosphorylated in the failing heart (21). Moreover, the ERK5 molecule
plays a critical role in post-natal eccentric hypertrophy of the heart (9, 22). ERK5 and
its upstream MAPK-kinase 5 (MEK5) reveals a specific role in transduction of
cytokine signals that regulate serial sarcomere assembly and in the induction of
eccentric cardiac hypertrophy resulting in dilated cardiomyopathy and sudden death
(22). Therefore, it is crucial to investigate the pathologic role of IL-6-MEK5-ERK5
signaling pathway under cardiac hypertrophy. Additionally, various molecules have
been elucidated responsible for the development of cardiac hypertrophy, including
mitogen activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and
7
N-terminal kinases (JNK), and the p38 MAPK cascades (p38) enrolled in the MAPK
pathway also play crucial roles in the development of cardiac hypertrophy (24).
To avoid the side effects by administration of western drugs, growing studies were
performed to investigate the natural products for the cardiac protection that have been
used as drugs or diet supplements for a long history in many medical-experiences.
Recent studies reported the cardioprotective effect of various oriental herb extracts or
dietary supplements including Fructus Crataegi, Salvia miltiorrhiza and Astragali
radix. The quercetin is the main ingredient in Fructus crataegi that has been
demonstrated as an anti-inflammatory substance by inhibiting TNF-α release from
macrophages and recognized to have cardiac protective effect (25,26). Salvia
miltiorrhiza is mainly composed of sodium tanshinone IIA sulfonate (STS), a
derivative of tanshinone IIA that can reduce myocardial infarct size and prolong the
survival cardiac cell in rabbit and human (15,27,28). Astragali radix contains many
isoflavones, isoflavonoids, and many saponins, which have been demonstrated to
have protective effects on heart by reducing inflammation, oxidant and cardiac
ischemia-reperfusion injury (29-34) In our recent publication, we also demonstrated
the protective effect of Li-Fu formula composing of Celery, Black fungus, Mushroom,
To further understand the effects and possible mechanisms of Li-Fu formula on
cardiac hypertrophy, we performed the histopathological analysis and Western
blotting assay to examine the expression of hyper-trophic associated molecules in the
cardiac tissues from hamsters that were fed with hypercholesterol diets. We suggest
ŀ
9 Material and Methods
Animals and diet
A total of 24 male Golden Syrian hamsters weighting 135 to 170 gram at the age of 8
weeks were purchased from National Laboratory Animal Center, Taipei, Taiwan, and
housed in an animal room at 22 ± 2 °C with a 12/12 h light−dark cycle under
supervision of Institutional Animal Care and Use Committee of China Medical
University, Taichung, Taiwan. Hamsters were acclimatized for 2 weeks while
receiving free access to water and were fed chow diet (Lab Diet 5001; PMI Nutrition
International Inc., Brentwood, MO, USA) ad libitum. The hamsters were then
randomized into 3 groups as control, cholesterol and Li-Fu formula groups and
switched to experimental diets. The control, cholesterol and Li-Fu formula groups
received chow diet, chow diet with 0.2% cholesterol (Sigma, Saint Louis Mo, USA),
and chow diet with 0.2 % cholesterol and 2% Li-Fu formula for 8 weeks, respectively.
Celery and Black fungus are obtained from common supermarket and Mushroom, Saliva miltior rhiza, Crataegi cuneata and Stragali radix are purchased from traditional Chinese pharmacy. The Li-Fu formula was firstly created and provided by Dr Li-Fu Chen, China Medical University, Taichung, Taiwan. To make Li-Fu formula, every component of desired weight was crushed and mixed with a blender, then placed in 1000ml distilled water and boiled for 1 h under reflux. The resultant solution was
divided into several parts and stored in a –80 °C freezer for further use (35). The Li-Fu formula is composed of Celery, Black fungus, Mushroom, Saliva miltior rhiza, Crataegi cuneata, and Astragali radix as shown in Table 1 and the experimental dietary composition is shown in Table 2. The Ambient temperature was maintained at 25°C. Diets were prepared weekly and stored at -80°C. All experimental procedures were performed according to the NIH Guide for the Care and Use of Laboratory Animals.
All protocols were approved by the Institutional Animal Care and Use Committee of
China Medical University, Taichung, Taiwan. Food intake and food spillage were
measured daily, and body weight was recorded every 3 days.
Cardiac characteristics
Three groups of hamsters at age of 8-9 month old were weighed and decapitated after
receiving 8 weeks of experimental diets. The hearts of animals were excised and
cleaned with distilled H2O. The left and right atrium and ventricle were separated and
weighed. The body weight (BW), left ventricle weight (LVW), the ratios of the whole
heart weight (WHW) to body weight (BW) and the ratios of the left ventricular weight
(LVW) to body weight (BW), were measured and calculated.
11
The hearts of animals were excised and were soaked in formalin and covered with
wax. Slides were prepared by deparaffinization and dehydration. They were passed
through a series of graded alcohols (100%, 95% and 75%), 15 minutes of each. The
slides were then dyed with hematoxylin. After gently rinsing with water, each slide
was then soaked with 85% alcohol, 100% alcohol I and II for 15 minutes each. At the
end, they were soaked with Xylene I and Xylene II. Photomicrographs were obtained
using Zeiss Axiophot microscopes.
Tissue Extraction
Cardiac tissue extracts were obtained by homogenizing the left ventricle samples in a
PBS buffer (0.14 M NaCl, 3 mM KCl, 1.4 mM KH2PO4, 14 mM K2HPO4) at a ratio
of 100 mg tissue/0.5ml PBS for 5 min. The homogenates were placed on ice for 10
min and then centrifuged at 12,000 g for 30 min. The supernatant was collected and
stored at -70°C for further experiments. Protein concentration was determined using a BioRad Protein Assay (BioRad Laboratories, Hercules, CA, USA) and were quantified by absorbance at 595 nm using a spectrophotometer (Beckman Coulter, Palo Alto, CA, USA).
Electrophoresis and Western Blot
sulfate-polyacrylamide gel electrophoresis was performed with 10% polyacrylamide
gels. The samples were electrophoresed at 140 V for 3.5 hours and equilibrated for 15
min in 25 mM Tris-HCl, pH 8.3, containing 192 mM glycine and 20% (V/V)
methanol. Electrophoresed proteins were transferred to nitrocellulose membranes
(Amersham, Hybond-C Extra Supported, 0.45µm pore size) with a Bio-Rad Scientific
Instruments Transphor Unit at 100 mA for 14 h. Nitrocellulose membranes were
incubated at room temperature for 2 hours in blocking buffer containing 100 mM
Tris-HCl, pH 7.5, 0.9% (w/v) NaCl, 0.1% (v/v) fetal bovine serum. Antibodies
including ANP, BNP, IL-6, STAT3, MEK5, p-EKR5, MEK, p-MEK, p-ERK, p-P38,
JNK, p-JNK and α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were
diluted to 1:200 in antibody binding buffer containing 100 mM Tris-HCL, pH 7.5,
0.9% (w/v) NaCl, 0.1% (v/v) Tween-20 and 1% (v/v) fetal bovine serum. Incubations
were performed at room temperature for 3.5 hours. The immunoblots were washed
three times in 50 ml blotting buffer for 10 min and then immersed in the second
antibody solution containing horseradish peroxidase (HRP) conjugated goat
anti-hamster IgG (Promega Corp., Madison, WI, USA) for 1 hour that was diluted
1000-fold in binding buffer. The immunoblots were then washed in blotting buffer for
10 min three times. Pierce's Supersignal West Dura HRP Detection Kit (Pierce
13
The blots were scanned and quantified by densitometry (Appraise, Beckman-Coulter,
Brea, California, USA).
Statistical Analysis
All of the statistical analyses were performed using SPSS 10.0 software (SPSS Inc.,
Chicago, IL). Three independent experiments were repeated. Statistical analyses were
performed using the analysis of variance plus posterior multiple comparison test to
test the difference. The data between two experimental animal groups was compared
by Student’s t-test for two independent samples. In all cases, a difference at P<0.05
RESULTS
Experimental diets and cardiac characteristics
To investigate the effect of Li-Fu formula on hypertrophy in cardiac cells, we
examined the body weight and cardiac characteristics. Firstly, Li-Fu formula was
prepared as described in materials and methods and the compositions of the Li-Fu
formula was shown in Table 1. Table 2 presents the ingredients of experimental diets
for different groups of hamsters. Body weight (BW), left ventricle weight (LVW), the
ratios of whole heart weight (WHW) to body weight (BW) and the ratios of left
ventricular weight (LVW) to body weight (BW) of hamsters from Control,
Cholesterol and Li-Fu formula groups were detected (Table 3). The ratios of
WHW/BW and LVW/BW were significantly higher in hamsters of the Cholesterol
group compared to the Control group. Notably, the ratios of WHW/BW and LVW/BW
were significantly reduced in the hamsters from the Li-Fu formula group compared to
the Cholesterol group (Table 3).
Cardiac architecture changes
To further confirm the effect of Li-Fu formula on the cardiac hypertrophy, we did a
cross section of whole heart and histopathological analysis of ventricular tissue
15
significantly increased in the Cholesterol group but significantly decreased in the
Li-Fu formula group (Fig. 1A). The ventricular myocardium in the Control group
showed normal architecture with normal interstitial space. In contrast, the abnormal
myocardial architecture and the increased interstitial space were observed in the
Cholesterol group that shows structural disorganization and cardiomyocyte disarray
but significantly decreased in the Li-Fu formula group in 400X magnification images
(Fig. 1B). Moreover, the protein levels of both ANP and BNP were significantly
increased in hearts of the Cholesterol group compared to the Control group. In
contrast, significantly reduced ANP and BNP protein expressions were detected in
hearts of hamsters from the Li-Fu formula group (Fig. 2).
Effect of Li-Fu formula on cardiac hypertrophy associated signaling pathways
In order to identify the hyper-trophic factor IL-6, signal transducer and activator of
transcription STAT-3 and mitogen-activated protein kinase/ERK (MEK) signaling
pathways associated with the cardiac hypertrophy induced by hypercholesterol diet,
the protein products of IL-6, STAT3, MEK5, and p-ERK5 were measured by western
blotting. In hearts of the Cholesterol group, the protein products of IL-6, STAT3,
MEK5, and p-ERK5 showed significant increase potency compared to the hearts of
and p-ERK5 protein expression was observed in hearts of the Li-Fu formula group
(Fig 3). We further detected the protein levels of MEK and p-MEK. As shown in
figure four, significantly increased MEK and p-MEK protein levels were detected in
hearts of the Cholesterol group compared to the Control group (Fig. 4). Notably,
significantly decreased p-MEK was observed in hearts of the Li-Fu formula group
compared to the Cholesterol group (Fig 4). Additionally, significantly increased
p-ERK protein was detected in hearts of the Cholesterol group compared to the
Control group. In contrast, significantly reduced p-ERK protein was observed in
hearts of hamsters from the Li-Fu formula group compared to those from the
Cholesterol group (Fig. 5). However, no significant variations in p-P38 and p-JNK
protein levels were detected between hearts of the Control and the Cholesterol groups
17 DISCUSSION
Hypercholesterolmia diets have been recognized as the major sources to cause cardiac
hypertrophy and associated with numbers of heart diseases (4,5,7,32). Because of the
side effects of western drugs in treatment of cardiac diseases, the investigations of
natural product such as dietary supplements or oriental herbs on cardiac protection are
performed. In the current study, we intend to elucidate the effect of a formula
composed of dietary supplements and oriental herbs on cardiac hypertrophy. Our
experimental results indicated the significant reduction of the WHW/BW and
LVW/BW ratios in hamsters from Li-Fu formula group compared to those from the
Cholesterol group. Moreover, the hypertrophic marker protein such as ANP, BNP,
eccentric hypertrophic related factors such as IL-6, STAT3, MEK5, p-ERK5, p-MEK
and p-ERK were significantly increased in the Cholesterol group whereas significant
reduction of all these proteins were observed in the Li-Fu formula group.
The interleukin (IL)-6 is known as a pleiotypic factor that has been associated with
various cardiac diseases (14,36,37). Elevated IL-6 mRNA is observed in patients of
cardiac hypertrophy with hypertrophic cardiomyopathy (37). As figure 7 shows,
various signaling molecules, including p38 MAPK, STAT1-STAT3 heterodimer
kinase (ERK)s pathway, were induced by IL-6 receptor signaling systems and
contributed to the cardiac hypertrophy.1,9,17,18-20,22 In our experimental results,
significant elevation of IL-6 expression was detected in the excised ventricle of
hamsters from the Cholesterol group as well as those hypertrophic related signaling
molecules including STAT3, MEK5, p-ERK5. Notably, the significant reduction of
these hypertrophic factors and signaling molecules were detected in the excised
ventricle of hamsters from the Li-Fu formula group. To further clarify the involved
signaling pathway, we further examined the MAPK pathway that is important in
cardiac hypertrophy and consists three major cascades including the
non-cardiomyocyte proliferative extracellular-regulated kinase (ERK), and the
inflammatory related c-Jun N-terminal kinases (JNK), and the p38 MAPK cascades
(p38) (24). As revealed in current study, significant increase of phosphorylated ERK
(p-ERK) was observed in the excised ventricle of hamster from the Cholesterol group
and the p-ERK level was significantly reduced in the excised ventricle of hamster
from the Li-Fu formula group. Moreover, higher increase of p-MEK, the upstream
kinase activator of EKR, was also detected in hamsters from the Li-Fu group
compared to the Cholesterol group. However, no significant differences in protein
levels of p-P38 and p-JNK were detected between hamsters from the Cholesterol and
19
against cardiac hypertrophy via attenuation of non-cardiomyocyte proliferation related
p-ERK cascade but not P38 or JNK cascade.
Because of the moderated side effects than western drugs, more than half of the
population in the world relies on traditional medicine for therapeutic needs. Indeed,
herbal remedies and alternative medicines are used throughout the world and in the
past herbs often represented the original sources of most drugs (38,39,40). The Li-Fu
formula was firstly created by Dr. Li-Fu Chen, China Medical University, Taichung,
Taiwan, and composed of various dietary supplements and oriental herbs, including
Celery, Black fungus, Mushroom, Saliva miltior rhiza, Crataegi cuneata, and Astragali
radix that were routinely used as traditional medicine in oriental worlds. For instance,
a major ingredient of Li-Fu formula, Salvia miltiorrhiza, is known as "Danshen" and
mainly composed sodium tanshinone IIA sulfonate (STS), a derivative of tanshinone
IIA that is also known to protect cardio-vascular ischemia-reperfusion and oxidant
injuries (15,27,28,30,32,33,34,39,40). To elucidate the effect and possible mechanism
of Li-Fu formula on hypercholesterolmia induced cardiac hypertrophy, we performed
the histopathological analysis and western blotting to measure the myocardial
architecture, and expression of different cardiac hypertrophy associated molecules in
WHW/BW and LVW/BW were observed in hamsters from the Li-Fu formula group
compared to those from the Cholesterol group. These findings did suggest the
protective effects of Li-Fu formula on cardiac hypertrophy.
In the world, more than half of the population relies on traditional medicine for
therapeutic needs either by stewing or solution extracting (39-41). Although the
precise mechanism of most herbal medicine or dietary supplement has not been fully
understood, the experience of the traditional use over the years cannot be neglected”.
Altogether, our experimental results revealed that Li-Fu formula, the traditional
oriental herbs and diet supplements formula, have significant protective effects
against cardiac hypertrophy. Besides the attenuated expression of ANP and BNP, the
effect against cardiac hypertrophy of Li-Fu formula is probably via the reduction of
eccentric hypertrophy related IL-6 receptor pathway and non-cardiomyocyte
proliferation involved ERK signaling cascade but not JNK and P38 cascades.
Therefore, the Li-Fu formula could provide an alternative regimen for the prevention
21 ACKNOWLEDGEMENTS
The paper is supported by grant CMU96-100 from the China Medical University,
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29 Figure legends
Figure 1. Cardiac cross sections and cardiomyopathic changes in hamsters of control,
cholesterol and Li-Fu formula groups. (A) The cross section of whole heart in the
three groups. Arrows indicate that the left ventricular lumen diameters increased in
the cholesterol group but decreased in the Li-Fu formula group. (B) Representative
histopathological analysis of cardiac tissue sections with Hematoxylin and eosin
staining in hamsters of control, cholesterol and Li-Fu formula groups. The images of
myocardial architecture were magnified by 100 times.
Figure 2. (A) The representative protein products of ANP and BNP extracted from the
left ventricles of excised hearts in hamsters of Control, Cholesterol and Li-Fu formula
groups were measured by Western Blotting analysis. (B)(C) Bars represent the
relative protein quantification of ANP and BNP on the basis of α-tubulin. All bars
indicate mean values±SD (n=6 in each group). **P<0.01, significant differences
between Control and Cholesterol group. #P<0.05 and ##P<0.01, significant
differences between Cholesterol and Li-Fu formula groups.
Figure 3. (A) The representative protein products of IL-6, STAT3, MEK5 and p-ERK5
and Li-Fu formula groups were measured by Western Blotting analysis. (B)(C)(D)(E)
Bars represent the relative protein quantification of IL-6, STAT3, MEK5 and p-ERK5
on the basis of α-tubulin. All bars indicate mean values±SD (n=6 in each group).
**P<0.01, significant differences between Control and Cholesterol group. ##P<0.01,
significant differences between Cholesterol and Li-Fu formula groups.
Figure 4. (A) The representative protein products of p-MEK and MEK extracted from
the left ventricles of excised hearts in hamsters of Control, Cholesterol and Li-Fu
formula groups were measured by Western Blotting analysis. (B)(C) Bars represent
the relative protein quantification of p-MEK and MEK on the basis of α-tubulin. All
bars indicate mean values±SD (n=6 in each group). **P<0.01, significant differences
between Control and Cholesterol group. ##P<0.01, significant differences between
Cholesterol and Li-Fu formula groups.
Figure 5. (A) The representative protein product of p-EKR extracted from the left
ventricles of excised hearts in hamsters of Control, Cholesterol and Li-Fu formula
groups were measured by Western Blotting analysis. (B) Bars represent the relative
protein quantification of p-ERK on the basis of α-tubulin. All bars indicate mean
31
and Cholesterol group. ##P<0.01, significant differences between Cholesterol and
Li-Fu formula groups.
Figure 6. Our proposed hypothesis that cardiac IL-6, MEK-5-ERK-5 and STAT3
hypertrophic pathways and MEK1/2-ERK1/2 non-cardiacmyocyte proliferative
pathway are more activated in hyper cholesterol-fed hamaster hearts. The eccentric
hypertrophy related pathway, IL-6 related MEK5-ERK5 pathways and
MEK1/2-ERK1/2 non-cardiacmyocyte proliferative pathway may play a part of role
for developing eccentric cardiac hypertrophy and pathological changes in hyper
cholesterol-fed hamaster hearts. Dash lines represent possible theoretical pathways
but is still unconfirmed. Up arrows and down arrows on the right side represent
