RetractionRetracted: Herbal Supplement Ameliorates Cardiac Hypertrophyin Rats with CCl4-Induced Liver Cirrhosis

Evidence-Based Complementary and Alternative Medicine

Received 8 August 2017; Accepted 8 August 2017; Published 22 November 2017

Copyright © 2017 Evidence-Based Complementary and Alternative Medicine. This is an open access article distributed under theCreative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, providedthe original work is properly cited.

Evidence-Based Complementary and Alternative Medicinehas retracted the article titled “Herbal Supplement Amelio-rates Cardiac Hypertrophy in Rats with CCl

4-Induced Liver

Cirrhosis” [1]. The article was found to contain duplicatedimages in Figure 4(a), in which the IL6 blots are identicalto the p-ERK5 blots. The authors provided a correctedfigure, with replacements for p-ERK5, which is available asSupplementary Materials. However, they could not providethe underlying blots.

References

[1] P.-C. Li, Y.-W. Chiu, Y.-M. Lin et al., “Herbal supplementameliorates cardiac hypertrophy in rats with CCl

4-induced

liver cirrhosis,” Evidence-Based Complementary and AlternativeMedicine, vol. 2012, Article ID 139045, 9 pages, 2012.

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017, Article ID 5276749, 1 pagehttps://doi.org/10.1155/2017/5276749

RETRACTED

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2012, Article ID 139045, 9 pagesdoi:10.1155/2012/139045

Research Article

Herbal Supplement Ameliorates Cardiac Hypertrophy inRats with CCl4-Induced Liver Cirrhosis

Ping-Chun Li,1, 2 Yung-Wei Chiu,3, 4 Yueh-Min Lin,5 Cecilia Hsuan Day,6

Guang-YuhHwang,2 Peiying Pai,7 Fuu-Jen Tsai,8 Chang-Hai Tsai,9 Yu-Chun Kuo,10

Hsiao-Chuan Chang,11 Jer-Yuh Liu,12, 13 and Chih-Yang Huang8, 10, 14

1Division of Cardiovascular Surgery, China Medical University Hospital, Taichung 40402, Taiwan2Department of Life Science, Tunghai University, Taichung 40704, Taiwan3Emergency Department and Center of Hyperbaric Oxygen Therapy, Tungs’ Taichung MetroHarbor Hospital,Taichung 43503, Taiwan

4Institute of Medicine, Chung Shan Medical University, Taichung 40201, Taiwan5Department of Pathology, Changhua Christian Hospital, Changhua 50006, Taiwan6Department of Nursing, MeiHo University, Pingtung 91202, Taiwan7Division of Cardiology, China Medical University Hospital, Taichung 40402, Taiwan8Graduate Institute of Chinese Medical Science, China Medical University, Taichung 40402, Taiwan9Department of Healthcare Administration, Asia University, Taichung 41354, Taiwan10Graduate Institute of Basic Medical Science, China Medical University, Taichung 40402, Taiwan11Department of Biotechnology, Asia University, Taichung 41354, Taiwan12Center for Molecular Medicine, China Medical University Hospital, Taichung 40402, Taiwan13Graduate Institute of Cancer Biology, China Medical University, Taichung 40402, Taiwan14Department of Health and Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan

Correspondence should be addressed to Jer-Yuh Liu, jyl@mail.cmu.edu.tw and Chih-Yang Huang, cyhuang@mail.cmu.edu.tw

Received 31 May 2012; Revised 31 July 2012; Accepted 7 August 2012

Academic Editor: Y. Ohta

Copyright © 2012 Ping-Chun Li et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

We used the carbon tetrachloride (CCl4) induced liver cirrhosis model to test the molecular mechanism of action involved incirrhosis-associated cardiac hypertrophy and the effectiveness ofOcimum gratissimum extract (OGE) and silymarin against cardiachypertrophy. We treated male wistar rats with CCl4 and either OGE (0.02 g/kg B.W. or 0.04 g/kg B.W.) or silymarin (0.2 g/kg B.W.).Cardiac eccentric hypertrophy was induced by CCl4 along with cirrhosis and increased expression of cardiac hypertrophy relatedgenes NFAT, TAGA4, and NBP, and the interleukin-6 (IL-6) signaling pathway related genes MEK5, ERK5, JAK, and STAT3. OGEor silymarin co-treatment attenuated CCl4-induced cardiac abnormalities, and lowered expression of genes which were elevatedby this hepatotoxin. Our results suggest that the IL-6 signaling pathway may be related to CCl4-induced cardiac hypertrophy.OGE and silymarin were able to lower liver fibrosis, which reduces the chance of cardiac hypertrophy perhaps by lowering theexpressions of IL-6 signaling pathway related genes. We conclude that treatment of cirrhosis using herbal supplements is a viableoption for protecting cardiac tissues against cirrhosis-related cardiac hypertrophy.

1. Introduction

Patients with advanced cirrhosis have consistently beendiagnosed with cardiac dysfunction under the conditionof hyperdynamic circulation [1]. Increased cardiac outputand reduced systemic vascular resistance are both signsof this condition [2–4]. Although cardiac dysfunction in

patients with cirrhosis and potential clinical implicationshave long been known [5], little is understood regardingthe molecular mechanism of action involved in cirrhosis-associated alteration in cardiac structure and function,especially cardiac hypertrophy.

Cirrhosis is known as a possible cause of portal veinconstriction which may induce the activation of vasopressin,

RETRACTED

2 Evidence-Based Complementary and Alternative Medicine

angiotensin II (Ang II), and the sympathetic nervous system[6]. Cardiac hypertrophy is induced by such direct mechan-ical wall stress as well as paracrine/autocrine factors such asAng II, which in turn activates specific signaling pathways,for instance, mitogen-activated protein kinases (MAPKs)and calcineurin. These can cause cardiac hypertrophy andincrease of related gene expressions, such as proto-oncogenesc-Fos and c-JUN, genes which encode atrial natriureticpeptide (ANP) and B-type natriuretic peptide (BNP), andstructural genes β-myosin heavy chain (β-MHC) and skeletalα-actin [7]. Ang II is associated with increased plasma levelsof proinflammatory cytokines such as interleukin-6 (IL-6)[8], which is an effective stimulator of the Janus kinase/signaltransducer and activator of transcription (JAK/STAT) path-way in cardiac hypertrophy [7]. However, the role ofthese protein markers and transcriptional factors in cardiachypertrophy and remodeling in vivo has not been examinedin cirrhosis-associated hypertrophy.

Carbon tetrachloride (CCl4) is frequently used to induceexperimental cirrhosis in rats [9]. This model has recentlybeen used to investigate the role of lipophilic bile acidsand examine cardiac gene expression profiles in cirrhoticcardiomyopathy [10, 11]. Silymarin, a standardized extractof the milk thistle (Silybum marianum L. Gaertner), containsthree biochemicals: silybin, silydianin, and silychristin andhas a long tradition as a herbal remedy [12]. Ocimumgratissimum extract (OGE), a commonly used herb in folkmedicine, is rich in antioxidants and possesses many ther-apeutic functions [13–21]. Both herbal extracts have beenshown using the CCl4 model to inhibit liver cirrhosis [22].Therefore the motive for this experiment is to use the CCl4-induced liver cirrhosis model to understand the molecularmechanism of action involved in cirrhosis-associated cardiachypertrophy, as well as to test effectiveness of silymarin andOGE against cardiac damage and hypertrophy.

2. Materials andMethods

2.1. Preparation of OGE. Leaves of Ocimum gratissimumwere harvested and washed with distilled water followedby homogenization with distilled water using polytron.The homogenate was incubated at 95◦C for 1 hour (h)and then filtered through two layers of gauze. The filtratewas centrifuged at 20,000 g, 4◦C for 15 minutes (min)to remove insoluble pellets and the supernatant (OGE)was thereafter collected, lyophilized, and stored at −20◦Cuntil use. The final extract (OGE) was composed of 11.1%polyphenol (including 0.03% catechins, 0.27% caffeic acid,0.37% epicatechin, and 3.27% rutin).

2.2. Animals and Treatment. Forty male wistar rats weighing200–240 g were purchased from the National Animal Centerand housed in conventional cages with free access to waterand rodent chow at 20–22◦C with a 12-hour light-dark cycle.All procedures involving laboratory animal use were in accor-dance with the guidelines of the Instituted Animal Care andUse Committee of Chung Shan Medical University (IACUC,CSMU) for the care and use of laboratory animals. The ratswere divided evenly into five groups of 8 rats and treated

intraperitoneally with CCl4 (8%CCl4/corn oil, 1mL/kg bodyweight (BW) twice a week, Monday and Thursday) for 8weeks, as described by Hernandez-Muoz et al. [23], withsome modifications. At the same time, the rats were treatedwith various dosages of OGE (0–0.04 g/kg BW), or silymarinorally (0.2 g/kg BW, four times a week, Tuesday, Wednesday,Friday, and Saturday) [24, 25]. The control rats were treatedwith corn oil (1mL/kg BW) and fed a normal diet. At theend of the experiment, blood and heart were immediatelyobtained after the animals were sacrificed.

2.3. Histological Examinations. The heart was fixed in 10%formalin, processed using routine histology procedures,embedded in paraffin, cut in 5 μm sections, and mounted ona slide. The samples were stained with hematoxylin and eosinfor histopathological examination.

2.4. Preparation of Tissue Extract. All procedures were per-formed at 4◦C. The heart samples were lysed by 30 strokesusing a Kontes homogenizer at a ratio of 100mg tissue/1mL lysis buffer. The lysis buffer consisted of 50mM Tris-HCl (pH 7.4), 2mM EDTA, 2mM EGTA, 150mM NaCl,1mM PMSF, 10 μg/mL leupeptin, 1mM sodium orthovana-date, 1% (v/v) 2-mercaptoethanol, 1% (v/v) Nonidet P40,and 0.3% sodium deoxycholate. These homogenates werecentrifuged at 100,000 g for 1 h at 4◦C. The supernatant wasstored at −70◦C for Western blot assay.

2.5. Electrophoresis and Western Blot. Tissue extract sam-ples were prepared as described above. Sodium do decosulfate-polyacrylamide gel electrophoresis is carried out asdescribed by Laemmli [26] using 10% polyacrylamide gels.After samples are electrophoresed at 140 V for 3.5 h, thegels are equilibrated for 15min in 25mM Tris-HCl, pH8.3, containing 192mM glycine and 20% (v/v) methanol.Electrophoresed proteins are transferred to nitrocellulosepaper (Amersham, Hybond-C Extra Supported, 0.45 Micro)using Hoefer Scientific Instruments Transpher Units at100mA for 14 h. The nitrocellulose paper was incubatedat room temperature for 2 h in blocking buffer containing100mM Tris-HCl, pH 7.5, 0.9% (w/v) NaCl, 0.1% (v/v)Tween 20, and 3% (v/v) fetal bovine serum. AntibodiesBNP, phospho-GATA binding protein 4 (p-GATA4), nuclearfactor of activated T cells (NFAT), mitogen-activated proteinkinase kinase 5 (MEK5), extracellular signal-regulated pro-tein kinase 5 (ERK5), phospho-extracellular signal-regulatedprotein kinase 5 (p-ERK5), phospho-Janus kinase (p-JAK),signal transducer and activator of transcription 3 (STAT3),α-tubulin purchased from Santa Cruz Biotechnology, Inc.(CA, USA), and IL-6 purchased fromAbcam Inc. (MA, USA)are diluted to 1 : 2000 in antibody binding buffer containing100mM Tris-HCl, pH 7.5, 0.9% (w/v) NaCl, 0.1% (v/v)Tween 20, and 1% (v/v) fetal bovine serum. Incubations wereperformed at room temperature for 3.5 h. The immunoblotswere washed three times in 50 mL blotting buffer for10min and then immersed for 1 h in the second antibodysolution containing horseradish peroxidase goat anti-rabbitor anti-mouse IgG (Promega, WI, USA), which were dilutedin binding buffer to 1000-fold, for various antibodies.

RETRACTED

Evidence-Based Complementary and Alternative Medicine 3

Table 1: Changes in body weight and organ weight of CCl4-induced cirrhosis-related cardiac hypertrophy.

Aa B C D E

(n = 8) (n = 8) (n = 8) (n = 8) (n = 8)

BW (g) 425 ± 16.475 402 ± 8.920 385 ± 6.547 388 ± 10.823 420 ± 19.272

WHW (g) 1.041 ± 0.015 1.173 ± 0.031∗ 0.975 ± 0.023# 1.089 ± 0.026 1.023 ± 0.015#

LVW (g) 0.813 ± 0.010 0.898 ± 0.018∗ 0.745 ± 0.028# 0.777 ± 0.021 0.767 ± 0.023#

WHW/BW (%) 2.467 ± 0.095 2.918 ± 0.093∗ 2.535 ± 0.065# 2.813 ± 0.067 2.461 ± 0.101#

LVW/BW (%) 1.922 ± 0.050 2.233 ± 0.045∗ 1.933 ± 0.054# 2.190 ± 0.062 1.844 ± 0.085#

LVW/WHW (%) 0.781 ± 0.011 0.767 ± 0.012 0.764 ± 0.020 0.779 ± 0.015 0.751 ± 0.030aGroup A is given olive oil and water, Group B is given CCl4 and water, Group C is given CCl4 and 0.02 g/kg of OGE, Group D is given CCl4 and 0.04 g/kg ofOGE, and Group E is given CCl4 and 0.2 g/kg of silymarin. The individual severity rates in rats were expressed as mean ± SE. BW: body weight; WHW: wholeheart weight; LVW: left ventricle weight. ∗Significant differences from Group A, P < 0.05. #Significant differences from Group B, P < 0.05.

Table 2: Changes in diameter and thickness of left heart ventricle of CCl4-induced cirrhosis-related cardiac hypertrophy.

Aa B C D E

(n = 8) (n = 8) (n = 8) (n = 8) (n = 8)

Diameter of LV (mm) 8.17 ± 0.00 10.67 ± 0.22∗ 8.50 ± 0.19# 9.33 ± 0.22# 8.83 ± 0.11#

Thickness of LV (mm) 3.83 ± 0.11 4.43 ± 0.15∗ 3.87 ± 0.12# 4.17 ± 0.11 3.83 ± 0.11#

Thickness/diameter (mm) 0.42 ± 0.01 0.42 ± 0.01 0.46 ± 0.02 0.45 ± 0.02 0.43 ± 0.01aGroup A is given olive oil and water, Group B is given CCl4 and water, Group C is given CCl4 and 0.02 g/kg of OGE, Group D is given CCl4 and 0.04 g/kg ofOGE, and Group E is given CCl4 and 0.2/kg g of silymarin. The individual severity rates in rats were expressed as mean ± SE. LV: left ventricle. ∗Significantdifferences from Group A, P < 0.05. #Significant differences from Group B, P < 0.05.

After washing with blocking buffer, the membrane wasvisualized using chemiluminescence (Amersham PharmaciaBiotech, Piscataway, NJ, USA).

2.6. Statistical Analysis. The experimental results are expre-ssed as the mean ± SE. Data were assessed using analysisof variance (ANOVA) followed by a Student-Newman-Keulscorrection to adjust the significance level to avoid a type Ierror. Student’s t-test was used in the comparison betweengroups. A P value less than 0.05 was considered statisticallydifferent.

3. Results

3.1. Changes in Heart Weight of CCl4-Induced Cirrhosis-Associated Cardiac Hypertrophy. Throughout the experi-mental period of 8 weeks, there was no difference in bodyweight of rats within the 5 groups. At the end of theexperiments when rat livers were measured, liver fibrosiswas observed in the CCl4-treated group, as compared tothe control group which was given olive oil. And for thegroups treated with OGE or silymarin, a protective effect wasobserved: liver fibrosis was significantly ameliorated com-pared to the CCl4-treated group (data pending publication).In comparison, Table 1 shows that the whole heart weight(WHW), left ventricle weight (LVW), and their ratio to thebody weight of the CCl4-treated group were significantlyhigher than the control group. For groups treated with0.02 g/kg BW OGE and treated with 0.2 g/kg BW silymarin,weights of the heart remained equal to the control group.However, for the group treated with 0.04 g/kg BW OGE, theweight values had a less significant decrease compared to theCCl4-treated only group.

3.2. Changes in Diameter and Thickness and HistologicalStructure of Left Heart Ventricle of CCl4-Induced Cirrhosis-Associated Cardiac Hypertrophy. The left ventricle diameterof the CCl4-treated group was significantly larger andthe walls were moderately thicker than the control group(Figure 1 upper panel and Table 2), but a change of thatscale in ventricle diameter was not present in the OGE andsilymarin cotreated groups.

The left most picture in Figure 1 (lower panel) showsthe appearance of a normal heart: one with a unified tissuepattern. However, hearts treated with CCl4 had clearly lost itstissue integrity, but such a change was clearly not observed ingroups cotreated with 0.02 g/kg BW OGE and silymarin.

3.3. The Expression of Cardiac Hypertrophy Related Genes inthe Heart of CCl4-Treated Rats. The expression of cardiachypertrophy related genes, such as BNP, p-GATA4, andNFAT4, were also tested [7]. Their figures were increased inthe CCl4-treated group as compared to the control group(Figures 2 and 3). In the groups cotreated with 0.02 g/kgBW OGE or silymarin, the expression of BNP, p-GATA4,and NFAT returned to control level. The results of the0.04 g/kg BW OGE-treated group were consistent with theabove figures, in that their expressions were decreased, butnot back to control levels.

3.4. The Expression of IL-6 Signaling Pathway Related Genesin the Heart of CCl4-Treated Rats. We wanted to test for IL-6signaling pathways because studies have shown that cardiachypertrophy can be attributed to IL-6 related cytokines[7]. Western blotting analysis shows that the expressionsof IL-6, MEK5, ERK5, and p-ERK5 were increased inthe CCl4-treated group as compared to the control group

RETRACTED

4 Evidence-Based Complementary and Alternative Medicine

RVRVRVRVRV LVLVLV

LVLV

CCl4 + 0.2 g/kgsilymarin

CCl4CCl4 + 0.02 g/kgOGE

CCl4 + 0.04 g/kgOGE

Control

00 0 0 010 10 10 10 10(cm)

Figure 1: Cardiac pathologic analysis in the heart of CCl4-treated rats. Herbs and CCl4 were given as described in Materials and Methods.The top panels show the heart of the macroscopic cross-section. The bottom panels show high magnification (×400) of tissue structure. LV:left heart ventricle; RV: right heart ventricle.

BNP

α-tubulin

36 kDa

57 kDa

CCl4

CCl4+

0.02 g/kg

OGE

CCl4+ CCl4+0.02 g/kg

OGE

0.04 g/kgsilymarinControl

(a)

0.1

0.12

0.08

0.06

0.04

0.02

0

∗

#

#

BN

P/α

-tu

bulin

CCl4+

0.02 g/kg

OGE

CCl4+ CCl4+0.02 g/kg

OGE

0.04 g/kgsilymarin

CCl4Control

(b)

Figure 2: The expressions of BNP by Western blotting analysis (a) and quantitative analysis (b) in the heart of CCl4-treated rats. Theindividual severity rates in rats were expressed as mean ± SE, n = 8. ∗P < 0.05 as compared with control group. #P < 0.05 as compared withthe CCl4-treated group.

(Figure 4). In the groups cotreated with 0.02 g/kg BW OGEor silymarin, the expression of IL-6, MEK5, ERK5, and p-ERK5 returned to control level. The expressions were alsolowered in the 0.04 g/kg BW OGE-treated group, but notback to the levels of the control group.

The expressions of other IL-6 signaling pathway genes, p-JAK and STAT3, were tested, the data shows that both their

expressions were increased in the CCl4-treated group as com-pared to the control group (Figure 5). In the groups cotreatedwith 0.02 g/kg BW OGE or silymarin, the expressions of p-JAK and STAT3 returned to control levels, except for the0.04 g/kg BW OGE group, which were lowered but not backto the control levels. This result is consistent with the dataabove.

RETRACTED

Evidence-Based Complementary and Alternative Medicine 5

120 kDa

57 kDa

92 kDap-GATA4

NFAT3

α-tubulin

CCl4+0.02 g/kg

OGE

CCl4+ CCl4+0.02 g/kg

OGE0.04 g/kg

silymarinCCl4Control

(a)

CCl4+0.02 g/kg

OGE

CCl4+ CCl4+0.02 g/kg

OGE0.04 g/kg

silymarin

CCl4ControlCCl4+0.02 g/kg

OGE

CCl4+ CCl4+0.02 g/kg

OGE0.04 g/kg

silymarin

CCl4Control

0.3

0.25

0.2

0.15

0.1

0.05

0

0.3

0.25

0.2

0.15

0.1

0.05

0

∗

∗

# #

##

p-G

ATA

4/α

-tu

bulin

0.35

NFA

T3/α

-tu

bulin

(b)

Figure 3: The expressions of NFAT3 and phosphorylated-GATA4 by Western blotting analysis (a) and quantitative analysis (b) in the heartof CCl4-treated rats. The individual severity rates in rats were expressed as mean ± SE, n = 8, ∗P < 0.05 as compared with control group,and #P < 0.05 as compared with CCl4-treated group.

4. Discussion

Numerous reports center on the involvement of IL-6 andthe related cytokines in cardiac hypertrophy [7] as aninducer of downstream pathways. IL-6 is a typical cytokinewhich was found to have a potent hypertrophic effect oncardiomyocytes [27], as the overexpression of this cytokinehas been linked to hypertrophic myocardium injury [28]. Inthe present study, our data showed that the expressions ofIL-6 increased in CCl4-induced cirrhosis rats detected withoccurrence of cardiac hypertrophy, which suggests that thecirrhosis-associated cardiac hypertrophy may be related withthe IL-6 signaling pathway in the CCl4-treated rats.

IL-6 is involved in multiple intracellular signaling path-ways, particularly the MEK5-ERK5 pathway [29–32], whichplays a critical role in the induction of eccentric cardiachypertrophy that can progress to dilated cardiomyopathyand sudden death [33, 34], and the JAK-STAT3 pathway,which promotes the increase of cell dimensions [35–37].Since the experiments suggest a relationship between

CCl4-induced cirrhosis-associated cardiac hypertrophy andIL-6, we decided to analyze the mechanism concerning OGEand silymarin and how it may inhibit cardiac hypertrophythrough the inhibition of IL-6 extracellular signals. Westernblotting analysis shows that the expressions of IL-6, MEK5,ERK5, and p-ERK5 were increased in the CCl4-treatedgroups as compared to the control (Figure 4) and werepartially restored to control levels when cotreated with OGEor silymarin. Moreover, the expressions of p-JAK and STAT3were increased in the CCl4-treated group (Figure 5) andrestored by OGE or silymarin cotreatment, as in the abovegene expressions. Taken together, these findings indicate thatboth the JAK-STAT3 and the MEK5-ERK5 pathways relatedgenes were overexpressed by IL-6 expression in responseto CCl4-induced cirrhosis-associated cardiac hypertrophy(Figure 6), which confirms the importance of the two path-ways and also demonstrates that their overexpression may bereversed by OGE or silymarin treatment thus lowering livercirrhosis and reducing the chance of cardiac hypertrophy.An interesting note is that silymarin, which has rarely been

RETRACTED

6 Evidence-Based Complementary and Alternative Medicine

25 kDa

57 kDa

54 kDa

115 kDa

123 kDa

IL-6

MEK5

ERK5

p-ERK5

α-tubulin

CCl4+0.02 g/kg

OGE

CCl4+ CCl4+0.02 g/kg

OGE0.04 g/kg

silymarinCCl4Control

(a)

0.02 g/kg 0.02 g/kg

OGE

0.04 g/kg

OGE silymarin

Control CCl4+ CCl4+ CCl4+ CCl4+

0.02 g/kg 0.02 g/kg

OGE

0.04 g/kg

OGE silymarin

Control CCl4+ CCl4+ CCl4+ CCl4+0.02 g/kg 0.02 g/kg

OGE

0.04 g/kg

OGE silymarin

Control CCl4+ CCl4+ CCl4+ CCl4+

0.02 g/kg 0.02 g/kg

OGE

0.04 g/kg

OGE silymarin

Control CCl4+ CCl4+ CCl4+ CCl4+

0.4

0.3

0.2

0.1

0

0.16

0.14

0.12

0.1

0.08

0.06

0.04

0.02

0

0.16

0.14

0.12

0.1

0.08

0.06

0.04

0.02

0

0.5

0.4

0.3

0.2

0.1

0

∗∗

∗

∗

#

#

#

##

##

#

ME

K5/α

-tu

bulin

ER

K5/α

-tu

bulin

pER

K5/α

-tu

bulin

IL−6

/α-t

ubu

lin

(b)

Figure 4: The expressions of IL6 and its downstream signaling proteins MEK5, ERK5, and phosphorylated-ERK5 by Western blottinganalysis (a) and quantitative analysis (b) in the heart of CCl4-treated rats. The individual severity rates in rats were expressed as mean ± SE,n = 8, ∗P < 0.05 as compared with control group, and #P < 0.05 as compared with CCl4-treated group.

RETRACTED

Evidence-Based Complementary and Alternative Medicine 7

131 kDa

92 kDa

57 kDa

p-JAK

STAT3

α-tubulin

CCl4+0.02 g/kg

OGE

CCl4+ CCl4+0.02 g/kg

OGE0.04 g/kg

silymarinCCl4Control

(a)

0.25

0.3

0.2

0.15

0.1

0.05

0.25

0.2

0.15

0.1

0.05

00

∗

∗

#

#

#

# STA

T3/α

-tu

bulin

CCl4 CCl4+

0.02 g/kg

CCl4+

0.02 g/kg

OGE

CCl4+

0.04 g/kg

OGEsilymarin

ControlCCl4 CCl4+

0.02 g/kg

CCl4+

0.02 g/kg

OGE

CCl4+

0.04 g/kg

OGEsilymarin

Control

p-JA

K/α

-tu

bulin

(b)

Figure 5: The expressions of JAK-Stat3 pathway by Western blotting analysis (a) and quantitative analysis (b) in the heart of CCl4-treatedrats. The individual severity rates in rats were expressed as mean ± SE, n = 8. ∗P < 0.05 as compared with control group. #P < 0.05 ascompared with CCl4-treated group.

CCl4

Liver cirrhosis

Normal heart Cardiac hypertrophy

IL-6-MEK5-ERK5

IL6-JAK2-STAT1/3

GATA4

NAT3

Fetal gene BNP

Overexpression and activiation of thepathological cardiac hypertrophy markers

OGE and silymarin

Figure 6: The summary of the mechanism of CCl4-induced cirrhosis-associated cardiac hypertrophy. Our data demonstrated that Ocimumgratissimum and silymarin extracts attenuate cardiac cells from CCl4 induced damage possibly by lowering liver cirrhosis which reduces thechance of cardiac hypertrophy maybe via inhibiting IL-6 signaling pathway activation.

RETRACTED

8 Evidence-Based Complementary and Alternative Medicine

demonstrated to treat cardiac hypertrophy [38], suggeststhat some common elements between herbal preparations,such as their antioxidant properties, may be responsible fortreatment against liver cirrhosis-induced cardiac damage.

Cardiac hypertrophy can be classified as physiologicaland pathological hypertrophy [7], with the physiologicalbeing a natural bodily response to maturation, pregnancy,and exercise, and the pathological being a response topathological stress signals, such as inflammation, cardiacinjury, or exposure to toxicity. In our study, we found thatmany genes was responded to cardiac hypertrophy by CCl4induction, including MEK5, ERK5, JAK2, STAT3, NFAT3,GATA4, and fetal gene BNP, which are used as a pathologicalmarker [39–42] (Figure 6). Since pathological hypertrophyis also associated with observable loss of tissue integrity,which we also found in CCl4-treated rats, this suggests thatCCl4 induced cirrhosis-associated cardiac hypertrophy maybelong to pathological hypertrophy and can also be exploredfurther as a pathological model.

There is a peculiar phenomenon that a 0.02 g/kg BWdoseof OGE had a significant inhibition effect on CCl4-inducedcardiac hypertrophy and on the related gene expressions thana 0.04 g/kg BW dose. A possible explanation suggests thatthe saturation of the higher dose could have lowered theeffectiveness of the treatment.

5. Conclusions

In summary, CCl4-induced cirrhotic cardiac damage canoccur through the IL-6 signaling pathway which leadsto eventual cardiac hypertrophy. OGE and silymarin canprotect cardiac cells from CCl4-induced damage possiblyby inhibiting the expression of the IL-6 signaling pathwayrelated genes. Moreover, we also found in further researchthat CCl4 induced cardiac damage can induce the FASLsignaling pathway and the TGF-β signaling pathway, whichmay lead to cell apoptosis and eventual cardiac fibrosis(pending publication). It seems that multiple mechanismsare involved in the CCl4 induced cardiac damage. However,in the present study, we suggest that OGE and silymarin inthe form of herbal supplements are a viable option for theprotection of cardiac tissues against cirrhosis-related cardiachypertrophy.

Acknowledgments

The authors thank Dr. Edwin L. Cooper for reading the paperand making suggestions. This work was supported by grantsfrom the National Science Council, China (NSC 98-2320-B-039-042-MY3), by the Taiwan Department of Health ClinicalTrial and Research Center of Excellence (DOH101-TD-B-111-004) and in part by the (CMU97-203 and CMU98-asia-12), Taiwan.

References

[1] E. Theocharidou, A. Krag, F. Bendtsen, S. Moller, and A. K.Burroughs, “Cardiac dysfunction in cirrhosis—does adrenalfunction play a role? A hypothesis,” Liver International, vol. 32,no. 9, pp. 1327–1332, 2012.

[2] H. J. Kowalski and W. H. Abelmann, “The cardiac output atrest in Laennec’s cirrhosis,” The Journal of Clinical Investiga-tion, vol. 32, no. 10, pp. 1025–1033, 1953.

[3] L. Gould, M. Shariff, M. Zahir, and M. Di Lieto, “Cardiachemodynamics in alcoholic patients with chronic liver diseaseand a presystolic gallop,” Journal of Clinical Investigation, vol.48, no. 5, pp. 860–868, 1969.

[4] H. Kelbaek, J. Eriksen, I. Brynjolf et al., “Cardiac performancein patients with asymptomatic alcoholic cirrhosis of the liver,”American Journal of Cardiology, vol. 54, no. 7, pp. 852–855,1984.

[5] T. Timoh, M. A. Protano, G. Wagman, M. Bloom, and T. J.Vittorio, “A perspective on cirrhotic cardiomyopathy,” Trans-plantation Proceedings, vol. 43, no. 5, pp. 1649–1653, 2011.

[6] A. Helmy, R. Jalan, D. E. Newby, P. C. Hayes, and D. J. Webb,“Role of angiotensin II in regulation of basal and sympa-thetically stimulated vascular tone in early and advancedcirrhosis,” Gastroenterology, vol. 118, no. 3, pp. 565–572, 2000.

[7] A. Rohini, N. Agrawal, C. N. Koyani, and R. Singh, “Moleculartargets and regulators of cardiac hypertrophy,” Pharmacologi-cal Research, vol. 61, no. 4, pp. 269–280, 2010.

[8] R. Skoumal, M. Toth, R. Serpi et al., “Parthenolide inhibitsSTAT3 signaling and attenuates angiotensin II-induced leftventricular hypertrophy via modulation of fibroblast activity,”Journal of Molecular and Cellular Cardiology, vol. 50, no. 4, pp.634–641, 2011.

[9] H. Chung, D. P. Hong, H. J. Kim et al., “Differential geneexpression profiles in the steatosis/fibrosis model of rat liverby chronic administration of carbon tetrachloride,” Toxicologyand Applied Pharmacology, vol. 208, no. 3, pp. 242–254, 2005.

[10] J. H. Zavecz and H. D. Battarbee, “The role of lipophilicbile acids in the development of cirrhotic cardiomyopathy,”Cardiovascular Toxicology, vol. 10, no. 2, pp. 117–129, 2010.

[11] G. Ceolotto, I. Papparella, A. Sticca et al., “An abnormalgene expression of the β-adrenergic system contributes to thepathogenesis of cardiomyopathy in cirrhotic rats,”Hepatology,vol. 48, no. 6, pp. 1913–1923, 2008.

[12] A. Valenzuela and A. Garrido, “Biochemical bases of thepharmacological action of the flavonoid silymarin and of itsstructural isomer silibinin,” Biological Research, vol. 27, no. 2,pp. 105–112, 1994.

[13] J. Y. Liu, M. J. Lee, H. M. Chen et al., “Ocimum gratissimumaqueous extract protects H9c2 myocardiac cells from H2O2 -Induced cell apoptosis through Akt signalling,” Evidence-basedComplementary and Alternative Medicine, vol. 2011, Article ID578060, 8 pages, 2011.

[14] H. M. Chen, M. J. Lee, C. Y. Kuo, P. L. Tsai, J. Y. Liu, and S. H.Kao, “Ocimum gratissimum aqueous extract induces apoptoticsignalling in lung adenocarcinoma cell A549,” Evidence-BasedComplementary and Alternative Medicine, vol. 2011, Article ID739093, 7 pages, 2011.

[15] C. C. Chiu, C. Y. Huang, T. Y. Chen et al., “Beneficial effectsof Ocimum gratissimum aqueous extract on rats with CCl4-induced acute liver injury,” Evidence-Based Complementaryand AlternativeMedicine, vol. 2012, Article ID 736752, 9 pages,2012.

[16] P. Nangia-Makker, L. Tait, M. P. V. Shekhar et al., “Inhibitionof breast tumor growth and angiogenesis by a medicinal herb:Ocimum gratissimum,” International Journal of Cancer, vol.121, no. 4, pp. 884–894, 2007.

[17] N. K. Ayisi and C. Nyadedzor, “Comparative in vitro effe-cts of AZT and extracts of Ocimum gratissimum, Ficus

RETRACTED

Evidence-Based Complementary and Alternative Medicine 9

polita, Clausena anisata, Alchornea cordifolia, and Elaeophorbiadrupifera against HIV-1 and HIV-2 infections,” AntiviralResearch, vol. 58, no. 1, pp. 25–33, 2003.

[18] J. C. Aguiyi, C. I. Obi, S. S. Gang, and A. C. Igweh,“Hypoglycaemic activity of Ocimum gratissimum in rats,”Fitoterapia, vol. 71, no. 4, pp. 444–446, 2000.

[19] C. K. Atal, M. L. Sharma, A. Kaul, and A. Khajuria,“Immunomodulating agents of plant origin. I. Preliminaryscreening,” Journal of Ethnopharmacology, vol. 18, no. 2, pp.133–141, 1986.

[20] P. Chaturvedi, S. George, and A. John, “Preventive andprotective effects of wild basil in ethanol-induced liver toxicityin rats,” British Journal of Biomedical Science, vol. 64, no. 1, pp.10–12, 2007.

[21] S. George and P. Chaturvedi, “A comparative study of theantioxidant properties of two different species of ocimum ofsouthern africa on alcohol-induced oxidative stress,” Journalof Medicinal Food, vol. 12, no. 5, pp. 1154–1158, 2009.

[22] M. Mourelle, P. Muriel, L. Favari, and T. Franco, “Preventionof CCl4-induced liver cirrhosis by silymarin,” Fundamentaland Clinical Pharmacology, vol. 3, no. 3, pp. 183–191, 1989.

[23] R. Hernandez-Muoz, M. Dıaz-Muoz, J. A. Suarez-Cuencaet al., “Adenosine reverses a preestablished CCl4-inducedmicronodular cirrhosis through enhancing collagenolyticactivity and stimulating hepatocyte cell proliferation in rats,”Hepatology, vol. 34, no. 4, pp. 677–687, 2001.

[24] P. Muriel and M. Mourelle, “Prevention by silymarin ofmembrane alterations in acute CCl4 liver damage,” Journal ofApplied Toxicology, vol. 10, no. 4, pp. 275–279, 1990.

[25] P. Muriel, T. Garciapina, V. Perez-Alvarez, and M. Mourelle,“Silymarin protects against paracetamol-induced lipid perox-idation and liver damage,” Journal of Applied Toxicology, vol.12, no. 6, pp. 439–442, 1992.

[26] U. K. Laemmli, “Cleavage of structural proteins during theassembly of the head of bacteriophage T4,” Nature, vol. 227,no. 5259, pp. 680–685, 1970.

[27] T. Kanda and T. Takahashi, “Interleukin-6 and cardiovasculardiseases,” Japanese Heart Journal, vol. 45, no. 2, pp. 183–193,2004.

[28] K. Kaneko, T. Kanda, T. Yokoyama et al., “Expression ofinterleukin-6 in the ventricles and coronary arteries of patientswith myocardial infarction,” Research Communications inMolecular Pathology and Pharmacology, vol. 97, no. 1, pp. 3–12, 1997.

[29] H. Hirota, K. Yoshida, T. Kishimoto, and T. Taga, “Continuousactivation of gp130, a signal-transducing receptor compo-nent for interleukin 6-related cytokines, causes myocardialhypertrophy in mice,” Proceedings of the National Academy ofSciences of the United States of America, vol. 92, no. 11, pp.4862–4866, 1995.

[30] T. Hirano, “Interleukin 6 and its receptor: ten years later,”International Reviews of Immunology, vol. 16, no. 3-4, pp. 249–284, 1998.

[31] H. Kodama, K. Fukuda, J. Pan et al., “Leukemia inhibitoryfactor, a potent cardiac hypertrophic cytokine, activates theJAK/STAT pathway in rat cardiomyocytes,” Circulation Re-search, vol. 81, no. 5, pp. 656–663, 1997.

[32] A. Ogata, N. Nishimoto, and K. Yoshizaki, “Advances ininterleukin-6 therapy,” Rinsho Byori, vol. 47, no. 4, pp. 321–326, 1999 (Japanese).

[33] R. L. Nicol, N. Frey, G. Pearson, M. Cobb, J. Richardson,and E. N. Olson, “Activated MEK5 induces serial assemblyof sarcomeres and eccentric cardiac hypertrophy,” EMBOJournal, vol. 20, no. 11, pp. 2757–2767, 2001.

[34] S. J. Cameron, S. Itoh, C. P. Baines et al., “Activation of bigMAP kinase 1 (BMK1/ERK5) inhibits cardiac injury aftermyocardial ischemia and reperfusion,” FEBS Letters, vol. 566,no. 1–3, pp. 255–260, 2004.

[35] J. J. Jacoby, A. Kalinowski, M. G. Liu et al., “Cardiomyocyte-restricted knockout of STAT3 results in higher sensitivityto inflammation, cardiac fibrosis, and heart failure withadvanced age,” Proceedings of the National Academy of Sciencesof the United States of America, vol. 100, no. 22, pp. 12929–12934, 2003.

[36] P. Fischer and D. Hilfiker-Kleiner, “Survival pathways inhypertrophy and heart failure: the gp130-STAT3 axis,” BasicResearch in Cardiology, vol. 102, no. 4, pp. 279–297, 2007.

[37] K. Boengler, D. Hilfiker-Kleiner, H. Drexler, G. Heusch, and R.Schulz, “The myocardial JAK/STAT pathway: from protectionto failure,” Pharmacology and Therapeutics, vol. 120, no. 2, pp.172–185, 2008.

[38] W. Ai, Y. Zhang, Q. Z. Tang et al., “Silibinin attenuates cardiachypertrophy and fibrosis through blocking EGFR-dependentsignaling,” Journal of Cellular Biochemistry, vol. 110, no. 5, pp.1111–1122, 2010.

[39] S. W. Kong, N. Bodyak, P. Yue et al., “Genetic expressionprofiles during physiological and pathological cardiac hyper-trophy and heart failure in rats,” Physiological Genomics, vol.21, pp. 34–42, 2005.

[40] J. R. McMullen and G. L. Jennings, “Differences betweenpathological and physiological cardiac hypertrophy: noveltherapeutic strategies to treat heart failure,” Clinical andExperimental Pharmacology and Physiology, vol. 34, no. 4, pp.255–262, 2007.

[41] V. Beisvag, O. J. Kemi, I. Arbo et al., “Pathological andphysiological hypertrophies are regulated by distinct geneprograms,” European Journal of Cardiovascular Prevention andRehabilitation, vol. 16, no. 6, pp. 690–697, 2009.

[42] Y. J. Weng, W. W. Kuo, C. H. Kuo et al., “BNIP3 inducesIL6 and calcineurin/NFAT3 hypertrophic-related pathways inH9c2 cardiomyoblast cells,” Molecular and Cellular Biochem-istry, vol. 345, no. 1-2, pp. 241–247, 2010.