Herbal Supplement Ameliorates Cardiac Hypertrophy in Rats with Ccl4-Induced Liver Cirrhosis

Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2017, Article ID 5276749, 1 page https://doi.org/10.1155/2017/5276749

Retraction Retracted: Herbal Supplement Ameliorates Cardiac Hypertrophy in 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 the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Evidence-Based Complementary and Alternative Medicine has retracted the article titled “Herbal Supplement Amelio- ratesCardiacHypertrophyinRatswithCCl4-Induced Liver Cirrhosis” [1]. The article was found to contain duplicated images in Figure 4(a), in which the IL6 blots are identical to the p-ERK5 blots. The authors provided a corrected figure, with replacements for p-ERK5, which is available as Supplementary Materials. However, they could not provide the underlying blots.

References

[1] P.-C. Li, Y.-W. Chiu, Y.-M. Lin et al., “Herbal supplement ameliorates cardiac hypertrophy in rats with CCl4-induced liver cirrhosis,” Evidence-Based Complementary and Alternative Medicine, vol. 2012, Article ID 139045, 9 pages, 2012. Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2012, Article ID 139045, 9 pages doi:10.1155/2012/139045

Research Article Herbal Supplement Ameliorates Cardiac Hypertrophy in Rats with CCl4-Induced Liver Cirrhosis

Ping-Chun Li,1, 2 Yung-Wei Chiu,3, 4 Yueh-Min Lin,5 Cecilia Hsuan Day,6 Guang-Yuh Hwang,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, Taiwan 2Department of Life Science, Tunghai University, Taichung 40704, Taiwan 3Emergency Department and Center of Hyperbaric Oxygen Therapy, Tungs’ Taichung MetroHarbor Hospital, Taichung 43503, Taiwan 4Institute of Medicine, Chung Shan Medical University, Taichung 40201, Taiwan 5Department of Pathology, Changhua Christian Hospital, Changhua 50006, Taiwan 6Department of Nursing, MeiHo University, Pingtung 91202, Taiwan 7Division of Cardiology, China Medical University Hospital, Taichung 40402, Taiwan 8Graduate Institute of Chinese Medical Science, China Medical University, Taichung 40402, Taiwan 9Department of Healthcare Administration, Asia University, Taichung 41354, Taiwan 10Graduate Institute of Basic Medical Science, China Medical University, Taichung 40402, Taiwan 11Department of Biotechnology, Asia University, Taichung 41354, Taiwan 12Center for Molecular Medicine, China Medical University Hospital, Taichung 40402, Taiwan 13Graduate Institute of Cancer Biology, China Medical University, Taichung 40402, Taiwan 14Department of Health and Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan

Correspondence should be addressed to Jer-Yuh Liu, [email protected] and Chih-Yang Huang, [email protected]

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 (CCl 4) induced liver cirrhosis model to test the molecular mechanism of action involved in cirrhosis-associated cardiac hypertrophy and the eﬀectiveness of Ocimum gratissimum extract (OGE) and silymarin against cardiac hypertrophy. 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 related genes NFAT, TAGA4, and NBP, and the interleukin-6 (IL-6) signaling pathway related genes MEK5, ERK5, JAK, and STAT3. OGE or silymarin co-treatment attenuated CCl4-induced cardiac abnormalities, and lowered expression of genes which were elevated by 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 the expressions of IL-6 signaling pathway related genes. We conclude that treatment of cirrhosis using herbal supplements is a viable optionRETRACTED for protecting cardiac tissues against cirrhosis-related cardiac hypertrophy.

1. Introduction patients with cirrhosis and potential clinical implications have long been known [5], little is understood regarding Patients with advanced cirrhosis have consistently been the molecular mechanism of action involved in cirrhosis- diagnosed with cardiac dysfunction under the condition associated alteration in cardiac structure and function, of hyperdynamic circulation [1]. Increased cardiac output especially cardiac hypertrophy. and reduced systemic vascular resistance are both signs Cirrhosis is known as a possible cause of portal vein of this condition [2–4]. Although cardiac dysfunction in constriction which may induce the activation of vasopressin, 2 Evidence-Based Complementary and Alternative Medicine

angiotensin II (Ang II), and the sympathetic nervous system intraperitoneally with CCl4 (8% CCl4/corn oil, 1 mL/kg body [6]. Cardiac hypertrophy is induced by such direct mechan- weight (BW) twice a week, Monday and Thursday) for 8 ical wall stress as well as paracrine/autocrine factors such as weeks, as described by Hernandez-Muoz´ et al. [23], with Ang II, which in turn activates specific signaling pathways, some modifications. At the same time, the rats were treated for instance, mitogen-activated protein kinases (MAPKs) with various dosages of OGE (0–0.04 g/kg BW), or silymarin and calcineurin. These can cause cardiac hypertrophy and orally (0.2 g/kg BW, four times a week, Tuesday, Wednesday, increase of related gene expressions, such as proto-oncogenes Friday, and Saturday) [24, 25]. The control rats were treated c-Fos and c-JUN, genes which encode atrial natriuretic with corn oil (1 mL/kg BW) and fed a normal diet. At the peptide (ANP) and B-type natriuretic peptide (BNP), and end of the experiment, blood and heart were immediately structural genes β-myosin heavy chain (β-MHC) and skeletal obtained after the animals were sacrificed. α-actin [7]. Ang II is associated with increased plasma levels of proinflammatory cytokines such as interleukin-6 (IL-6) 2.3. Histological Examinations. The heart was fixed in 10% [8], which is an effective stimulator of the Janus kinase/signal formalin, processed using routine histology procedures, transducer and activator of transcription (JAK/STAT) path- embedded in paraffin, cut in 5 μm sections, and mounted on way in cardiac hypertrophy [7]. However, the role of a slide. The samples were stained with hematoxylin and eosin these protein markers and transcriptional factors in cardiac for histopathological examination. hypertrophy and remodeling in vivo has not been examined in cirrhosis-associated hypertrophy. 2.4. Preparation of Tissue Extract. Allprocedureswereper- ◦ Carbon tetrachloride (CCl4) is frequently used to induce formed at 4 C. The heart samples were lysed by 30 strokes experimental cirrhosis in rats [9]. This model has recently using a Kontes homogenizer at a ratio of 100 mg tissue/1 been used to investigate the role of lipophilic bile acids mL lysis buffer. The lysis buffer consisted of 50 mM Tris- and examine cardiac gene expression profiles in cirrhotic HCl (pH 7.4), 2 mM EDTA, 2 mM EGTA, 150 mM NaCl, cardiomyopathy [10, 11]. Silymarin, a standardized extract 1mMPMSF,10μg/mL leupeptin, 1 mM sodium orthovana- of the milk thistle (Silybum marianum L. Gaertner), contains date, 1% (v/v) 2-mercaptoethanol, 1% (v/v) Nonidet P40, three biochemicals: silybin, silydianin, and silychristin and and 0.3% sodium deoxycholate. These homogenates were has a long tradition as a herbal remedy [12]. Ocimum centrifuged at 100,000 g for 1 h at 4◦C. The supernatant was gratissimum extract(OGE),acommonlyusedherbinfolk stored at −70◦C for Western blot assay. medicine, is rich in antioxidants and possesses many therapeutic functions [13–21]. Both herbal extracts have been 2.5. Electrophoresis and Western Blot. Tissue extract sam- shown using the CCl4 model to inhibit liver cirrhosis [22]. ples were prepared as described above. Sodium do deco Therefore the motive for this experiment is to use the CCl4- sulfate-polyacrylamide gel electrophoresis is carried out as induced liver cirrhosis model to understand the molecular described by Laemmli [26] using 10% polyacrylamide gels. mechanism of action involved in cirrhosis-associated cardiac After samples are electrophoresed at 140 V for 3.5 h, the hypertrophy, as well as to test effectiveness of silymarin and gels are equilibrated for 15 min in 25 mM Tris-HCl, pH OGE against cardiac damage and hypertrophy. 8.3, containing 192 mM glycine and 20% (v/v) methanol. Electrophoresed proteins are transferred to nitrocellulose 2. Materials and Methods paper (Amersham, Hybond-C Extra Supported, 0.45 Micro) using Hoefer Scientific Instruments Transpher Units at 2.1. Preparation of OGE. Leaves of Ocimum gratissimum 100 mA for 14 h. The nitrocellulose paper was incubated were harvested and washed with distilled water followed at room temperature for 2 h in blocking buffer containing by homogenization with distilled water using polytron. 100 mM Tris-HCl, pH 7.5, 0.9% (w/v) NaCl, 0.1% (v/v) The homogenate was incubated at 95◦Cfor1hour(h) Tween 20, and 3% (v/v) fetal bovine serum. Antibodies and then filtered through two layers of gauze. The filtrate BNP, phospho-GATA binding protein 4 (p-GATA4), nuclear was centrifuged at 20,000 g, 4◦C for 15 minutes (min) factor of activated T cells (NFAT), mitogen-activated protein to remove insoluble pellets and the supernatant (OGE) kinase kinase 5 (MEK5), extracellular signal-regulated pro- was thereafter collected, lyophilized, and stored at −20◦C tein kinase 5 (ERK5), phospho-extracellular signal-regulated until use. The final extract (OGE) was composed of 11.1% protein kinase 5 (p-ERK5), phospho-Janus kinase (p-JAK), polyphenol (including 0.03% catechins, 0.27% caffeic acid, signal transducer and activator of transcription 3 (STAT3), 0.37% epicatechin, and 3.27% rutin). α-tubulin purchased from Santa Cruz Biotechnology, Inc. RETRACTED(CA, USA), and IL-6 purchased from Abcam Inc. (MA, USA) 2.2. Animals and Treatment. Forty male wistar rats weighing are diluted to 1 : 2000 in antibody binding buffer containing 200–240 g were purchased from the National Animal Center 100 mM Tris-HCl, pH 7.5, 0.9% (w/v) NaCl, 0.1% (v/v) and housed in conventional cages with free access to water Tween 20, and 1% (v/v) fetal bovine serum. Incubations were and rodent chow at 20–22◦C with a 12-hour light-dark cycle. performed at room temperature for 3.5 h. The immunoblots All procedures involving laboratory animal use were in accor- were washed three times in 50 mL blotting buffer for dance with the guidelines of the Instituted Animal Care and 10 min and then immersed for 1 h in the second antibody Use Committee of Chung Shan Medical University (IACUC, solution containing horseradish peroxidase goat anti-rabbit CSMU) for the care and use of laboratory animals. The rats or anti-mouse IgG (Promega, WI, USA), which were diluted were divided evenly into five groups of 8 rats and treated in binding buffer to 1000-fold, for various antibodies. Evidence-Based Complementary and Alternative Medicine 3

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

Aa BCDE (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 ± 0.030 a Group A is given olive oil and water, Group B is given CCl4 and water, Group C is given CCl4 and0.02g/kgofOGE,GroupDisgivenCCl4 and 0.04 g/kg of OGE, 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: whole heart 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 BCDE (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 ± 0.01 a Group A is given olive oil and water, Group B is given CCl4 and water, Group C is given CCl4 and0.02g/kgofOGE,GroupDisgivenCCl4 and 0.04 g/kg of ∗ OGE,andGroupEisgivenCCl4 and 0.2/kg g of silymarin. The individual severity rates in rats were expressed as mean ± SE. LV: left ventricle. Significant differences from Group A, P < 0.05. #Significant differences from Group B, P < 0.05.

After washing with blocking buffer, the membrane was 3.2. Changes in Diameter and Thickness and Histological visualized using chemiluminescence (Amersham Pharmacia Structure of Left Heart Ventricle of CCl4-Induced Cirrhosis- Biotech, Piscataway, NJ, USA). Associated Cardiac Hypertrophy. The left ventricle diameter of the CCl4-treated group was significantly larger and 2.6. Statistical Analysis. The experimental results are expre- the walls were moderately thicker than the control group ssed as the mean ± SE. Data were assessed using analysis (Figure 1 upper panel and Table 2), but a change of that of variance (ANOVA) followed by a Student-Newman-Keuls scale in ventricle diameter was not present in the OGE and correction to adjust the significance level to avoid a type I silymarin cotreated groups. error. Student’s t-test was used in the comparison between The left most picture in Figure 1 (lower panel) shows groups. A P value less than 0.05 was considered statistically the appearance of a normal heart: one with a unified tissue different. pattern. However, hearts treated with CCl4 had clearly lost its tissue integrity, but such a change was clearly not observed in 3. Results groups cotreated with 0.02 g/kg BW OGE and silymarin.

3.1. Changes in Heart Weight of CCl4-Induced Cirrhosis- 3.3. The Expression of Cardiac Hypertrophy Related Genes in Associated Cardiac Hypertrophy. Throughout the experi- the Heart of CCl4-Treated Rats. The expression of cardiac mental period of 8 weeks, there was no difference in body hypertrophy related genes, such as BNP, p-GATA4, and weight of rats within the 5 groups. At the end of the NFAT4, were also tested [7]. Their figures were increased in experiments when rat livers were measured, liver fibrosis the CCl4-treated group as compared to the control group was observed in the CCl4-treated group, as compared to (Figures 2 and 3). In the groups cotreated with 0.02 g/kg the control group which was given olive oil. And for the BW OGE or silymarin, the expression of BNP, p-GATA4, groups treated with OGE or silymarin, a protective effect was and NFAT returned to control level. The results of the observed: liver fibrosis was significantly ameliorated com- 0.04 g/kg BW OGE-treated group were consistent with the pared to the CClRETRACTED4-treated group (data pending publication). above figures, in that their expressions were decreased, but In comparison, Table 1 shows that the whole heart weight not back to control levels. (WHW), left ventricle weight (LVW), and their ratio to the body weight of the CCl4-treated group were significantly 3.4. The Expression of IL-6 Signaling Pathway Related Genes higher than the control group. For groups treated with in the Heart of CCl4-Treated Rats. We wanted to test for IL-6 0.02 g/kg BW OGE and treated with 0.2 g/kg BW silymarin, signaling pathways because studies have shown that cardiac weights of the heart remained equal to the control group. hypertrophy can be attributed to IL-6 related cytokines However, for the group treated with 0.04 g/kg BW OGE, the [7]. Western blotting analysis shows that the expressions weight values had a less significant decrease compared to the of IL-6, MEK5, ERK5, and p-ERK5 were increased in CCl4-treated only group. the CCl4-treated group as compared to the control group 4 Evidence-Based Complementary and Alternative Medicine

Control CCl4 CCl4 + 0.02 g/kg CCl4 + 0.04 g/kg CCl4 + 0.2 g/kg OGE OGE silymarin

LV RV LV RV RV LV RV RV LV LV

000010 10 (cm) 10 10 0 10

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.

CCl4+ CCl4+ CCl4+ 0.02 g/kg 0.04 g/kg 0.02 g/kg CCl Control 4 OGE OGE silymarin

BNP 36 kDa

α-tubulin 57 kDa

(a) ∗ 0.12 0.1 0.08 # 0.06 # 0.04 BNP/ α -tubulin 0.02 0 Control CCl4 CCl4+ CCl4+CCl4+ 0.02 g/kg 0.04 g/kg 0.02 g/kg silymarin OGE OGE (b)

Figure 2: The expressions of BNP by Western blotting analysis (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. #P<0.05 as compared with the CCl4-treatedRETRACTED group.

(Figure 4). In the groups cotreated with 0.02 g/kg BW OGE expressions were increased in the CCl4-treated group as com- or silymarin, the expression of IL-6, MEK5, ERK5, and p- pared to the control group (Figure 5). In the groups cotreated ERK5 returned to control level. The expressions were also with 0.02 g/kg BW OGE or silymarin, the expressions of p- lowered in the 0.04 g/kg BW OGE-treated group, but not JAK and STAT3 returned to control levels, except for the back to the levels of the control group. 0.04 g/kg BW OGE group, which were lowered but not back The expressions of other IL-6 signaling pathway genes, p- to the control levels. This result is consistent with the data JAK and STAT3, were tested, the data shows that both their above. Evidence-Based Complementary and Alternative Medicine 5

CCl4+ CCl4+CCl4+ 0.02 g/kg 0.04 g/kg 0.02 g/kg Control CCl 4 OGE OGE silymarin

p-GATA4 92 kDa

NFAT3 120 kDa

α-tubulin 57 kDa

(a) ∗ 0.35

0.3 ∗ 0.3

0.25 0.25

0.2 0.2

0.15 0.15 # #

# α -tubulin NFAT3/ # 0.1 0.1 α -tubulin p-GATA4/

0.05 0.05

0 0 Control CCl4 CCl4+ CCl4+ CCl4+ Control CCl4 CCl4+ CCl4+ CCl4+ 0.02 g/kg 0.04 g/kg 0.02 g/kg 0.02 g/kg 0.04 g/kg 0.02 g/kg OGE OGE silymarin OGE OGE silymarin (b)

Figure 3: The expressions of NFAT3 and phosphorylated-GATA4 by Western blotting analysis (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.

4. Discussion CCl4-induced cirrhosis-associated cardiac hypertrophy and IL-6, we decided to analyze the mechanism concerning OGE Numerous reports center on the involvement of IL-6 and and silymarin and how it may inhibit cardiac hypertrophy the related cytokines in cardiac hypertrophy [7]asan through the inhibition of IL-6 extracellular signals. Western inducer of downstream pathways. IL-6 is a typical cytokine blotting analysis shows that the expressions of IL-6, MEK5, ff which was found to have a potent hypertrophic e ect on ERK5, and p-ERK5 were increased in the CCl4-treated cardiomyocytes [27], as the overexpression of this cytokine groups as compared to the control (Figure 4)andwere has been linked to hypertrophic myocardium injury [28]. In partially restored to control levels when cotreated with OGE the present study, our data showed that the expressions of or silymarin. Moreover, the expressions of p-JAK and STAT3 IL-6 increased in CCl4-induced cirrhosis rats detected with were increased in the CCl4-treated group (Figure 5)and occurrence ofRETRACTED cardiac hypertrophy, which suggests that the restored by OGE or silymarin cotreatment, as in the above cirrhosis-associated cardiac hypertrophy may be related with gene expressions. Taken together, these findings indicate that the IL-6 signaling pathway in the CCl4-treated rats. both the JAK-STAT3 and the MEK5-ERK5 pathways related IL-6 is involved in multiple intracellular signaling path- genes were overexpressed by IL-6 expression in response ways, particularly the MEK5-ERK5 pathway [29–32], which to CCl4-induced cirrhosis-associated cardiac hypertrophy plays a critical role in the induction of eccentric cardiac (Figure 6), which confirms the importance of the two path- hypertrophy that can progress to dilated cardiomyopathy ways and also demonstrates that their overexpression may be and sudden death [33, 34], and the JAK-STAT3 pathway, reversed by OGE or silymarin treatment thus lowering liver which promotes the increase of cell dimensions [35–37]. cirrhosis and reducing the chance of cardiac hypertrophy. Since the experiments suggest a relationship between An interesting note is that silymarin, which has rarely been 6 Evidence-Based Complementary and Alternative Medicine

CCl4+ CCl4+ CCl4+ 0.02 g/kg 0.04 g/kg 0.02 g/kg Control CCl 4 OGE OGE silymarin

IL-6 25 kDa

MEK5 54 kDa

ERK5 123 kDa

p-ERK5 115 kDa

α-tubulin 57 kDa

(a)

∗ 0.4 ∗ 0.16

0.14 0.3 0.12

0.1 0.2

6/ α -tubulin 0.08 − IL

MEK5/ α -tubulin 0.06 # # 0.1 0.04 # #

0.02

0 0 Control CCl4+ CCl4+CCl4+ CCl4+ Control CCl4+CCl4+CCl4+CCl4+ 0.02 g/kg 0.04 g/kg 0.02 g/kg 0.02 g/kg0.04 g/kg 0.02 g/kg OGE OGE silymarin OGE OGE silymarin

0.5 ∗ 0.16 ∗

0.14 0.4

0.12

0.3 0.1

0.08

ERK5/ α -tubulin 0.2

pERK5/ α -tubulin 0.06

# # # 0.04 # RETRACTED0.1 0.02

0 0 Control CCl4+ CCl4+ CCl4+ CCl4+ Control CCl4+CCl4+CCl4+CCl4+ 0.02 g/kg 0.04 g/kg 0.02 g/kg 0.02 g/kg0.04 g/kg 0.02 g/kg OGE OGE silymarin OGE OGE silymarin (b)

Figure 4: The expressions of IL6 and its downstream signaling proteins MEK5, ERK5, and phosphorylated-ERK5 by Western blotting analysis (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. Evidence-Based Complementary and Alternative Medicine 7

CCl4+ CCl4+CCl4+ 0.02 g/kg 0.04 g/kg 0.02 g/kg Control CCl4 OGE OGE silymarin

p-JAK 131 kDa

STAT3 92 kDa

α-tubulin 57 kDa

(a)

∗

0.3 0.25 ∗

0.25 0.2

0.2 0.15 0.15 #

# α -tubulin STAT3/ 0.1 α -tubulin p-JAK/ 0.1 # # 0.05 0.05

0 0 Control CCl4 CCl4+ CCl4+ CCl4+ Control CCl4 CCl4+ CCl4+ CCl4+ 0.02 g/kg 0.02 g/kg 0.04 g/kg 0.02 g/kg 0.04 g/kg 0.02 g/kg silymarin silymarin OGE OGE OGE OGE (b)

Figure 5: The expressions of JAK-Stat3 pathway by Western blotting analysis (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. #P<0.05 as compared with CCl4-treated group.

CCl4 OGE and silymarin

Liver cirrhosis

Normal heart Cardiac hypertrophy

IL-6-MEK5-ERK5 RETRACTEDIL6-JAK2-STAT1/3 GATA4 NAT3 Fetal gene BNP

Overexpression and activiation of the pathological cardiac hypertrophy markers

Figure 6: The summary of the mechanism of CCl4-induced cirrhosis-associated cardiac hypertrophy. Our data demonstrated that Ocimum gratissimum and silymarin extracts attenuate cardiac cells from CCl4 induced damage possibly by lowering liver cirrhosis which reduces the chance of cardiac hypertrophy maybe via inhibiting IL-6 signaling pathway activation. 8 Evidence-Based Complementary and Alternative Medicine demonstrated to treat cardiac hypertrophy [38], suggests [2] H. J. Kowalski and W. H. Abelmann, “The cardiac output at that some common elements between herbal preparations, rest in Laennec’s cirrhosis,” The Journal of Clinical Investiga- such as their antioxidant properties, may be responsible for tion, vol. 32, no. 10, pp. 1025–1033, 1953. treatment against liver cirrhosis-induced cardiac damage. [3]L.Gould,M.Shariff, M. Zahir, and M. Di Lieto, “Cardiac Cardiac hypertrophy can be classified as physiological hemodynamics in alcoholic patients with chronic liver disease and pathological hypertrophy [7], with the physiological and a presystolic gallop,” Journal of Clinical Investigation, vol. being a natural bodily response to maturation, pregnancy, 48, no. 5, pp. 860–868, 1969. and exercise, and the pathological being a response to [4] H. Kelbaek, J. Eriksen, I. Brynjolf et al., “Cardiac performance pathological stress signals, such as inflammation, cardiac in patients with asymptomatic alcoholic cirrhosis of the liver,” American Journal of Cardiology, vol. 54, no. 7, pp. 852–855, injury, or exposure to toxicity. In our study, we found that 1984. many genes was responded to cardiac hypertrophy by CCl 4 [5]T.Timoh,M.A.Protano,G.Wagman,M.Bloom,andT.J. induction, including MEK5, ERK5, JAK2, STAT3, NFAT3, Vittorio, “A perspective on cirrhotic cardiomyopathy,” Trans- GATA4, and fetal gene BNP, which are used as a pathological plantation Proceedings, vol. 43, no. 5, pp. 1649–1653, 2011. marker [39–42](Figure 6). Since pathological hypertrophy [6]A.Helmy,R.Jalan,D.E.Newby,P.C.Hayes,andD.J.Webb, is also associated with observable loss of tissue integrity, “Role of angiotensin II in regulation of basal and sympa- which we also found in CCl4-treated rats, this suggests that thetically stimulated vascular tone in early and advanced CCl4 induced cirrhosis-associated cardiac hypertrophy may cirrhosis,” Gastroenterology, vol. 118, no. 3, pp. 565–572, 2000. belong to pathological hypertrophy and can also be explored [7] A. Rohini, N. Agrawal, C. N. Koyani, and R. Singh, “Molecular further as a pathological model. targets and regulators of cardiac hypertrophy,” Pharmacologi- There is a peculiar phenomenon that a 0.02 g/kg BW dose cal Research, vol. 61, no. 4, pp. 269–280, 2010. of OGE had a significant inhibition effect on CCl4-induced [8]R.Skoumal,M.Toth,´ R. Serpi et al., “Parthenolide inhibits cardiac hypertrophy and on the related gene expressions than STAT3 signaling and attenuates angiotensin II-induced left a 0.04 g/kg BW dose. A possible explanation suggests that ventricular hypertrophy via modulation of fibroblast activity,” the saturation of the higher dose could have lowered the Journal of Molecular and Cellular Cardiology, vol. 50, no. 4, pp. effectiveness of the treatment. 634–641, 2011. [9]H.Chung,D.P.Hong,H.J.Kimetal.,“Differential gene 5. 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