Antifibrotic activity of Taraxacum officinale root in carbon tetrachloride-induced liver
damage in mice
Robert Domitrović, *,1 Hrvoje Jakovac,2 Željko Romić,3 Dario Rahelić, 4 Žarko Tadić5
1
Department of Chemistry and Biochemistry, School of Medicine, University of Rijeka,
Rijeka, Croatia
2
Department of Physiology and Immunology, School of Medicine, University of Rijeka,
Rijeka, Croatia
3
Department for Laboratory Diagnostics, Clinical Hospital Dubrava, Zagreb, Croatia
4
Department for Internal Diseases, Clinical Hospital Dubrava, Zagreb, Croatia
5
School of Medicine, University of Rijeka, Rijeka, Croatia
*
Corresponding author:
Robert Domitrović
Department of Chemistry and Biochemistry
School of Medicine, University of Rijeka
B. Branchetta 20, 51000 Rijeka, Croatia
Tel./fax.: +385 51 651 135
E-mail address: robertd@medri.hr
1
Abstract
Aim of the study: Dandelion (Taraxacum officinale) has been traditionally used in the
treatment of various liver disorders. The present study was aimed to assess the efficacy of
dandelion root water-ethanol extract (DWE) in carbon tetrachloride (CCl4)-induced hepatic
fibrosis.
Materials and Methods: The mice were treated with CCl4 dissolved in olive oil (20% v/v, 2
ml/kg) intraperitoneally (i.p.), twice a week for 4 weeks. DWE was administered i.p. once
daily for next 10 days, in doses of 200 and 600 mg/kg of body weight. The degree of hepatic
fibrosis was determined by hydroxyproline content and Mallory trichrome staining. Oxidative
stress was determined by measuring hepatic superoxide dismutase (Cu/Zn SOD) activity. The
expression and specific tissue distribution of glial fibrillary acidic protein (GFAP), alphasmooth muscle actin (α-SMA), and metallothionein (MT) I/II in the liver were determined by
immunohistochemistry.
Results: Hepatic Cu/Zn SOD activity has been decreased in intoxicated mice and normalized
in DWE treated groups. MT I/II immunopositivity was strongly reduced in the CCl4 group.
DWE treatment successfully decreased hepatic fibrinous deposits, restored histological
architecture, and modulate the expression of GFAP and α-SMA. Concomitantly, MT I/II
expression increased in the DWE treated groups.
Conclusions: Our results suggest the therapeutic effect of DWE on CCl4-induced liver fibrosis
by the inactivation of hepatic stellate cells and the enhancement of hepatic regenerative
capabilities. The present results provide scientific evidence to substantiate the traditional use
of Taraxacum officinale root in hepatic disorders.
Keywords: Carbon tetrachloride; Liver fibrosis; Dandelion (Taraxacum officinale);
Metallothionein; Glial fibrillary acidic protein; α-Smooth muscle actin.
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1. Introduction
Carbon tetrachloride is one of the most widely used models to study hepatic damage which
lead to progressive hepatic fibrosis and finally to cirrhosis (Natarajan et al., 2006). Fibrosis
can be considered as an excessive accumulation of connective tissue in parenchymal organs.
In the liver, fibrosis represents a very frequent event which follows chronic insult of sufficient
intensity to trigger a "wound healing''-like reaction (Poli, 2000). Activated portal fibroblasts,
myofibroblasts of bone marrow origin, and particularly hepatic stellate cells (HSCs), have
been identified as a major collagen-producing cells in the injured liver, playing a role in
fibrogenesis (Bataller and Brener, 2005). These cells are activated by fibrogenic cytokines
such as TGF-beta1, angiotensin II, and leptin. After liver injury, HSCs become activated,
converting themselves into a myofibroblast-like cells (Moreira, 2007).
The genus Taraxacum Wiggers, family Asteraceae, subfamily Cichorioideae, tribe Lactuceae,
commonly known as dandelion, includes approximately 30–57 varieties with many
microspecies, divided into nine sections (Hegi, 1987). Plants of the genus Taraxacum are
widely distributed in the warmer temperate zones of the Northern Hemisphere and have long
been used as medicinal herbs. Traditionally, root and herb from Taraxacum officinale Weber
ex Wiggers have been used for the treatment of various ailments, including liver and
gallbladder disorders (Schutz et al., 2006). Phytochemical investigations of dandelion root
showed the presence of various classes of natural compounds, phenolics, sesquiterpenes,
triterpenes, and phytosterols (Schütz et al., 2005; Schütz et al., 2006). Several healthbeneficial effects, including diuretic, laxative, cholagogue, anti-rheumatic, anti-inflammatory,
choleretic, anti-carcinogenic and hypoglycemic activities, have been attributed to the use of
dandelion extracts or the plant itself (for review see Schütz et al., 2006). One of the major
constituents of dandelion root, inulin, has been shown to exert hepatoprotective activity in
xenobiotics-induced liver injuries (Sugatani et al., 2006). The toxicity of dandelion was found
3
to be low, due to absence of any significant toxins. In mice, herb and root extracts
adiministered intraperitoneally showed the median lethal dose (LD50) of 28.8 and 36.6 g/kg
body weight, respectively (Rácz-Kotilla et al., 1974).
To our knowledge, to date no reports have recorded the therapeutic activity of Taraxacum
officinale in liver fibrosis. Therefore, the aim of this research was to investigate the effect of
dandelion root water-ethanolic extract (DWE) in CCl4-induced liver fibrosis and the possible
mechanism of the hepatoprotective activity.
2. Materials and methods
2.1. Materials
Dandelion root water-ethanol tincture 4:1 (12% v/v ethanol in water) was purchased from
Alternative Health & Herbs Remedies, Albany, OR, USA. Carbon tetrachloride (CCl4), olive
oil, hydroxyproline, 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-s-triazine
(TPTZ), FeCl3 · 6H2O, gallic acid, chlorogenic acid, rutin, and Trolox were obtained from
Sigma Chemical Co., St. Louis, MO, USA. Folin-Ciocalteus's phenol reagent and sodium
carbonate were from Merck (Damstadt, Germany). All other chemicals and solvents were of
the highest grade commercially available.
2.2. Animals
Male BALB/c mice from our breeding colony, 2-3 months old, were divided into 5 groups
with 5 animals per group. Mice were fed a standard rodent diet (pellet, type 4RF21 GLP,
Mucedola, Italy) containing 19.4% protein, 5.5% fiber, 11.1% water, 54.6% carbohydrates,
6.7% ash, and 2.6% by weight of lipids (native soya oil) to prevent essential fatty acid
deficiency. Total energy of the diet was 16.4 MJ/kg. The animals were maintained at 12 h
light/dark cycle, at constant temperature (20±1°C) and humidity (50±5%). All experimental
procedures were approved by the Ethical Committee of the Medical Faculty, University of
Rijeka.
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2.3. Experimental Design
Group I animals were the control untreated group, group II animals were treated with CCl4
only (the CCl4 group), groups III and IV were CCl4 and DWE treated mice. Mice were given
CCl4 dissolved in olive oil (20% v/v, 2 ml/kg) intraperitoneally (i.p.) twice a week for 4
weeks. DWE dissolved in saline was administered i.p. at dose of 200 and 600 mg/kg daily
(2% of dry weight after heating at 110°C) for next 10 days to the groups III and IV,
respectively. The control and CCl4 groups received saline instead of DWE. The toxicity of
DWE was tested on separate group which received DWE only (600 mg/kg) for 10 days (group
V). Animals were sacrificed 24 hours after the last dose of DWE or saline by cervical
dislocation. The blood was taken from orbital sinus of ether anesthetized mice. The abdomen
of sacrificed animals was cut open and the liver was removed. The liver samples were used to
assess the antioxidative status and hydroxyproline content or was preserved in a buffered
formalin solution to obtain the histological sections.
2.4. Antioxidant characterization of DWE
2.4.1. Total antioxidant activity (FRAP assay)
Total antioxidant activity is measured by Ferric Reducing Antioxidant Power (FRAP) assay
of Benzie and Strain (1999). (1996). The fresh working solution was prepared by mixing 25
ml 300 mM acetate buffer, pH 3.6, 2.5 ml 10 mM TPTZ solution in 40 mM HCl, and 2.5 ml
20 mM FeCl3 · 6H2O solution. The temperature of the solution was raised to 37°C before
using. The plant extract samples (150 μL) were allowed to react with 2850 μl of the FRAP
solution for 30 min in the dark condition. Readings of the colored product (ferrous
tripyridyltriazine complex) were taken at 593 nm. Results are expressed in μM Fe (II)/g dry
weight.
2.4.2. Analysis of total polyphenols
5
The total phenolic contents of the extract was determined using the Folin–Denis method
(Swain and Hills, 1959). A gallic acid calibration curve ranging from 6.25 to 200 μg/ml was
prepared and the results determined from the regression equation of the calibration curve were
expressed as mg gallic acid equivalents (GAE)/g of dry plant material. DWE (0.5 ml) was
mixed with 10-fold-diluted Folin-Ciocalteu phenol reagent (0.5 ml). Sodium carbonate
solution (1 ml, 35%, w/v) was added to the mixture, which was shaken thoroughly and diluted
to 3 ml by adding water. The mixture is allowed to stand for 30 min and the blue color formed
was measured spectrophotometically at 750 nm (Cary 100, Varian, Mulgrave, Australia).
2.4.3. DPPH radical scavenging assay
The method used to determine the free radical scavenging activity was adapted from that
described by Blois (1958). 0.1 ml samples of DWE in methanol with concentrations (10, 20,
50, 100, 300, and 600 μg/ml) or solvent itself (as a negative control) were mixed with 0.9 ml
of DPPH 100 μM solution in methanol. After the reaction was carried out in dark at room
temperature for 30 min the absorbance was measured at the wavelength 517 nm on a Cary
100 spectrophotometer. Trolox was used as a reference compound. The scavenging activity
against DPPH free radicals, expresed as a 50% scavenging concentration (SC50), was
determined from a graph in which concentration and reduction activity were plotted against
each other.
2.5. HPLC analysis
The sample identification of chlorogenic acid and rutin was performed by thin-layer
chromatography, according to the European Pharmacopoeia (2009) (data not shown). The
fingerprint of DWE was achieved using HPLC. The extract was dried using Laborota 4002
Control rotary evaporator (Heidolph Instruments GmbH & Co KG, Schwabach, Germany) at
40°C and the residue was dissolved in 1 ml of methanol. The HPLC analysis was carried out
using a Prostar/Dynamax System PS-210 Pump and an automated injection system (Prostar
6
410 Autosampler, Varian, Palo Alto, CA, USA). The column employed for HPLC
determination was a Beta-Basic C18 column (4.6 ×150 mm; Thermo Electron Corporation,
San Jose, CA, USA) with column temperature set at 25°C. The mobile phase was linear
gradient of aqueous acetic acid 2%:methanol (0 min, 100:0; 17 min, 100:0; 7 min, 25:75 0; 5
min 0:100) at 0.7 ml/min. A photodiode array detector (Prostar 335 PDA Detector, Varian)
was set at 280 and 350 nm. The loading volume was 15 μl. For calibration appropriate
volumes of standard stock solutions (100 μg/ml) were diluted with methanol, and five
concentration levels (5, 10, 20, 50, and 100 μg/ml) were analysed. The peak retention time of
the reference standard was used to identify the peak of sample constituent for fingerprint
analysis. Compound quantification was performed using a calibration curve of the standard
compound.
2.6. Hepatotoxicity
Serum levels of ALT, AST, and ALP (Herbos Diagnostic, Sisak, Croatia) as markers of
hepatic damage, were measured by using a Bio-Tek EL808 Ultra Microplate Reader (BioTek
Instruments, Winooski, VT, USA) according to manufacturer’s instructions.
2.7. Determination of Cu/Zn SOD activity
Cu/Zn SOD activity was measured spectrophotometrically using Superoxide Dismutase Assay
Kit (Cayman Chemical, Ann Arbor, MI, USA), according to manufacturer’s instructions, as
described previously. Briefly, livers were homogenized in 50 mM phosphate buffer saline
(PBS), pH 7.4. The homogenate was subjected to centrifugation at 12000g for 15 min, at 4°C.
Cu/Zn SOD activity in homogenate supernatants was assessed by the degree of suppression of
xanthine reduction in the presence of NADH and xanthine oxidase. The increase of
absorbance was monitored spectrophotometrically at 450 nm (Bio-Tek EL808 Ultra
Microplate Reader). Protein content in supernatants was estimated by Bradford's method
(Bradford, 1976).
7
2.8. Hepatic hydroxyproline
The tissue sample (50-60 mg) was hydrolyzed in 4 ml 6 M HCl at 110°C for 24 h. After being
filtered through a 0.45-μm filter, 2 ml of samples was extracted and analyzed by Stagemann's
colorimetric method, as modified by Bergman and Loxley (1963), as described previously
(Domitrović et al., 2009). Briefly, sample neutralization was obtained with 10 M NaOH and 3
M HCl. To a 200 μl of the above solution, 400 μl of isopropanol in citrate-acetate-buffered
Chloramine T were added. After 4 min, 2.5 ml of Ehrlich reagent was added. Tubes were
wrapped in aluminum foil and incubated for 25 min in a water-bath at 60°C, tap water cooled,
and the absorbance of each sample was measured spectrophotometrically at 550 nm (Cary
100, Varian, Mulgrave, Australia).
2.9. Histopathology
Liver specimens were fixed in 4% phosphate buffered formalin, embedded in paraffin and cut
into 4 μm thick sections. Sections for histopathological examination were stained with
hematoxylin and eosin and Mallory trichrome stain using standard procedure.
2.10. Immunohistochemical determination of GFAP, α-SMA, and MT I/II
Immunohistochemical studies were performed on paraffin embedded liver tissues using
mouse monoclonal anti-MT I+II antibody diluted 1:50 (clone E9; DakoCytomation,
Carpinteria, CA, USA), mouse monoclonal anti-GFAP antibody diluted 1:100 (clone 1B4;
BD Pharmingen, San Diego, CA, USA), and mouse monoclonal antibody to α-SMA diluted
1:100 (SPM332; Abcam, Cambridge, UK), employing DAKO EnVision+ System,
Peroxidase/DAB kit (DAKO Corporation, Carpinteria, CA, USA), as described previously
(Domitrović et al., 2009). Briefly, slides were incubated with peroxidase block. After
washing, monoclonal antibodies diluted in phosphate buffered saline supplemented with
bovine serum albumin were added to tissue samples and incubated overnight at 4°C in a
humid environment, followed by incubation with peroxidase labeled polymer conjugated to
8
secundary antibodies containing carrier protein linked to Fc fragments to prevent nonspecific
binding. The immunoreaction product was visualized by adding substrate-chromogen
diaminobenzidine (DAB) solution, resulting with brownish coloration at antigen sites. Tissues
were counterstained with hematoxylin, dehydrated in gradient of alcohol and mounted with
mounting medium. The intensity of staining was graded as weak, moderate, and intense. The
specificity of the reaction was confirmed by substitution of primary antibodies with irrelevant
immunoglobulins of matched isotype, used in the same conditions and dilutions as primary
antibodies. Stained slides were analyzed by light microscopy (Olympus BX51, Tokyo, Japan).
2.11. Statistical Analysis
Data were analyzed using StatSoft STATISTICA version 7.1 software. Data were analyzed by
one-way analysis of variance (ANOVA), followed by Dunnett's post hoc test. Values in the
text are means ± standard deviation (SD). Differences with p < 0.05 were considered to be
statistically significant.
3. Results
3.1. Antioxidant characterization of the extract
Total polyphenols and antioxidant activity obtained using DPPH and FRAP assays were
measured three times to test the reproducibility of the assays. The content of total polyphenols
in DWE was relatively low, 3.11±0.14 mg gallic acid equivalent/g of dry weight. The FRAP
value of DWE was 17.9±1.5 mM Fe(II) equivalent/g of dry weight. The DPPH radical
scavenging activity of DWE was also very modest, with SC50 value of 540.4±28.8 μg/ml.
3.2. HPLC fingerprint of the extract
At present, there is no report on the appropriate marker and HPLC fingerprint pattern of
Taraxacum officinale root extract. Between tested substances, chlorogenic acid and rutin, only
chlorogenic acid was identified (the peak retention time 14.06 min), in amount of 1.2±0.05
9
mg/g of dry weight (the mean of three determinations with standard deviation) (Fig. 1). Thus,
chlorogenic acid represents 38.6% of the total phenolics in DWE.
3.3. Hepatotoxicity
As shown in Table 1, body weight in the CCl4 group increased significantly when compared
to control mice, which was attenuated by DWE treatment (Table 1). Similarly, liver
enlargement in the CCl4 group was significantly reduced in the groups receiving DWE
therapy. DWE alone did not affected either body or liver weight compared to the control
group.
Liver damage has been determined by mice weight, liver enzymes, oxidative status, and
hydroxyproline content. One of the most sensitive indicators of hepatocyte injury is the
release of intracellular enzymes, such as transaminases and serum alkaline phosphatase in the
circulation. Ten days after discontinuation of the toxin exposure, serum AST, ALT, and ALP
activities were slightly but significantly increased in the CCl4 group (Table 2). The relative
liver weight increased in the CCl4 group compared to control mice. In the group receiving 600
mg/kg of DWE, AST and ALT activities were still increased, however, ALP activity was
normalized. On the other hand, the extract alone did not affected the serum markers.
3.4. Cu/Zn SOD activity
The hepatic activity of Cu/Zn SOD has been significantly decreased in the CCl4 group (Table
2). DWE treatment, on the other hand, restored Cu/Zn SOD activity up to the control values.
The extract alone did not shown significant effect on the enzyme activity.
3.5. Hepatic hydroxyproline
The liver hydroxyproline content was fourfold higher in the CCl4 group than in controls
(Table 2). DWE therapy significantly decreased hepatic hydroxyproline level, which was
returned to normal values in the group receiving 600 mg/kg of DWE. In the DWE only treated
group hydroxyproline level was similar to controls.
10
3.6. Histopathology and hepatic collagen
Fig. 2A and 3A show the liver sections from control mice with regular hepatic architecture
and collagen traces visible in vascular walls. The liver speciments from the CCl4 group
showed pronounced microvesicular steatosis, with remnants of degenerated and balooned
hepatocytes containing acidophilic hyaline inclusions (Fig. 2B). Mononuclear inflammatory
cell infiltration in these areas was also present. Hepatocytes in vicinity to the scaring tissue,
particularly those located immediately alongside the fibrotic border, showed extensive fibrosis
with multiple fibrotic nodules, predominantly in the periportal areas, with marked pericellular
collagen deposition (Fig. 3B). The livers of mice receiving 200 mg/kg of DWE did not show
any significant lesions within hepatic parenchyma (Fig. 2C), whereas a discrete pericellular
fibrosis and microvesicular steatosis were still present (Fig. 3C). The livers of mice treated
with 600 mg/kg of DWE showed maintained histoarchitecture, almost similar to control
animals, with only weak signs of microvesicular steatosis in periportal and perilobular
hepatocytes (Fig. 2D and 3D). Mice treated with the extract only showed regular hepatic
architecture similar to control mice (Fig. 2E and 3E).
3.7. Tissue expression of GFAP, α-SMA, and MT I/II
In the livers of control animals, α-SMA immunopositivity was restricted to smooth
musculature belonging to vascular endothel, while other liver cells remained negative (Fig.
4A). CCl4 strongly induced perisinusoidal α-SMA expression through affected lobuli,
connected between themselves with thin, "bridging" immunopositivity (Fig. 4B). The livers of
mice receiving DWE 200 and 600 mg/kg showed only sporadic α-SMA immunopositivity
(Fig. 4C and 4D). Mice treated with the extract alone shown no signs of hepatic collagen
deposition (Fig. 4E).
Immunohistochemical staining using monoclonal anti-GFAP antibody showed thin, irregular
immunopositive bands lining the hepatic sinusoids of control animals (Fig. 5A). In the CCl4
11
group, strong GFAP expression was limited to the scare areas (Fig. 5B). Treatment with DWE
200 mg/kg decreased GFAP expression in fibrotic areas, but induced sporadic assembling of
GFAP positive cells in surrounding tissue (Fig. 5C). Similarly, the livers of mice receiving
DWE 600 mg/kg showed a few immunopositive cells in perisinusoidal space (Fig. 5D). The
livers of mice treated with DWE only had staining pattern similar to control animals, with
GFAP immunopositive HSCs (Fig. 5E).
In the livers of control animals, moderate MT I/II immunopositivity of perilobular
hepatocytes was present mostly in the cytoplasm, with a few in the nucleus (Fig. 6A). In the
CCl4 group, MT I/II immunopositivity was sporadic and restricted to fibrous lessions, while
in the remnant liver tissue was not detected (Fig. 6B). Concomitantly with the
histoarchitecture improvement, MT I/II immunopositivity significantly increased in the livers
of animals treated with DWE 200 mg/kg (Fig. 6C) and 600 mg/kg (Fig. 6D). Mice receiving
DWE only showed MT I/II expression similar to controls (Fig. 6E).
4. Discussion
Liver fibrosis represents the excessive accumulation of extracellular matrix (ECM) proteins
including collagen that occurs in most types of chronic liver diseases. Advanced liver fibrosis
results in cirrhosis, liver failure, and portal hypertension and often requires liver
transplantation (Bataller and Brenner, 2005). Oxidative stress has been implicated in the
etiology of chronic liver disease and liver fibrosis, often in association with decreased
antioxidant defenses (Parola and Robino, 2001). In the present study, increased hepatic
collagen deposition in the CCl4 group has indicated the development of liver fibrosis. The
DWE therapy has induced withdrawal of collagen deposits in necrotic areas and the reversion
of hepatic fibrosis. The elevation in Cu/Zn SOD activity in DWE treated groups, compared to
the CCl4 group, suggest that the hepatoprotective effect of DWE could be partially attributed
to its antioxidant activity. Most recent study suggests that polyphenolic chlorogenic acid,
12
which has been identified in DWE, has inhibitory potential on CCl4-induced liver fibrosis in
rats by inactivating HSCs (Shi et al., 2009). However, medicinal plants typically contain
several different chemical compounds that may act individually, additively or in synergy to
improve health (Gurib-Fakim, 2006). Therefore, it is difficult to determine the contribution of
individual compounds to the pharmacological effectiveness of complex therapeutics.
The accumulation of ECM observed in fibrosis and cirrhosis is considered to be a result of
activation of fibrogenic cell types, such as HSCs and portal fibroblasts, which acquire a
myofibroblastic phenotype (Guyot et al., 2006). During pathological conditions, activated
HSC increase the production of ECM proteins and remodel the sinusoidal wall and necrotic
areas (Burt 1999; Jin et al., 2002). However, the mechanisms of hepatic fibrogenesis are not
yet fully understood. In particular, the role of HSCs remains unclear. The maintenance of
HSC morphology might be one of the factors playing a role in the prevention or slowing
down of liver fibrosis. Buniatian (2003) has observed different patterns of activation of HSC
in the ellagic acid treated hepatic cell cultures, which might correspond to distinct activities of
these cells, which, in turn, might lead to different outcomes of liver fibrosis. It has been
shown that HSCs have ability to migrate in vitro (Marra et al., 1999) and in vivo to the fibrotic
lesions and take part in the repair of injured liver tissue (Kinnman et al., 2000). In their
quiescent state, HSCs express various cellular markers, such as GFAP (Neubauer et al.,
1996). Upon activation, HSCs which acquire myofibroblast phenotype increase the levels of
desmin and α-SMA whereas GFAP expression decreases (Campbell et al., 2005). In this
study, deposition of collagen in the liver of CCl4-intoxicated mice, associated with increased
α-SMA and decreased GFAP perisinusoidal immunopositivity, indicate engagement of HSCs
in development of liver fibrosis. Furthermore, the resolution of fibrotic scares was
accompanied by HSCs deactivation, as indicated by decreased α-SMA immunopositivity.
13
Metallothioneins (MTs) constitute a family of low molecular weight, cysteine-rich
metalloproteins involved in cytoprotection during pathology. Increased MT synthesis in
response to oxidative stress in both the cytosol and nucleus has been reported previously (Sato
and Bremner, 1993; Levadoux-Martin et al., 2001). The biological function of MT remains
unsettled, however it is known that the induction of MT synthesis can protect animals from
hepatotoxicity induced by heavy metals and various toxins including CCl4, but also may play
a role in cell proliferation and liver regeneration (Cherian and Kang, 2006). Previously, we
have shown up-regulation of MT I/II expression and the enchantment of hepatic regenerative
capability in acute and chronic CCl4 hepatotoxicity by luteolin (Domitrović et al., 2008;
Domitrović et al., 2009). The mechanisms by which MT participates in the pathology and the
recovery of liver fibrosis are still largely unknown. Jiang and Kang (2004) have shown that
mice which have developed a reversible liver fibrosis upon removal of CCl4 had a high level
of hepatic MTs, but mice which developed an irreversible fibrosis had low expression of
MTs. Therefore, hepatic MT expression in pathological conditions could reflect the severity
of chronic liver damage (Carrera et al., 2003). MT I/II protein down-regulation in the CCl4
group, compared to controls, suggest the suppression of hepatic regenerative response and the
progression toward irreversible fibrosis. On the other hand, the positive correlation between
MT I/II expression and liver tissue repairment in the DWE treated groups suggest its
involvement in the liver regeneration.
In conclusion, the present study provides original evidence that the Taraxacum officinale
water-ethanolic root extract elicits the therapeutic effect on hepatic fibrosis. The DWE
treatment has promoted the complete regression of fibrosis and the enchantment of hepatic
regenerative capabilities. The results obtained from this study strongly suggest the therapeutic
potential of DWE in patients with liver fibrosis.
Acknowledgments
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This research was supported by grants from Ministry of Science, Education and Sport,
Republic of Croatia (project No. 062-0000000-3554). The authors thank Hrvoje Križan,
Ljubica Črnac, and Jadranka Eškinja for technical support and Orjen Petković for his help in
performing HPLC chromatography.
Conflict of interest
The authors declare that there are no conflicts of interest.
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Figure captions:
Fig. 1. The HPLC fingerprint of DWE, representative of three independent experiments.
Column, Beta-Basic C18; mobile phase, linear gradient of aqueous acetic acid 2%:methanol
(0 min, 100:0; 17 min, 100:0; 7 min, 25:75 0; 5 min 0:100); flow rate, 0.7 ml/min; injection
volume, 15 μl; detection, 350 nm. The peak at retention time of 14.06 min identified
chlorogenic acid.
Fig. 2. The effect of CCl4 on liver histology. (A) The livers from control mice showed normal
hepatic architecture. (B) The CCl4 group showed severe hepatic lesions, degenerated and
balooned/necrotic hepatocytes with cellular hyaline inclusions (arrow). (C) Hepatic lesions
and hyaline deposits in mice receiving 200 mg/kg DWE were completely removed although
the hepatic architecture was not restored. (D) The livers of mice receiving 600 mg/kg of DWE
showed fully recovered hepatic architecture. (E) Mice treated with 600 mg/kg of DWE only
showed regular hepatic architecture. Original magnification 100x. H&E stain.
Photomicrographs are representative of two experiments with five mice per group.
Fig. 3. Detection of collagen in the livers. (A) A normal liver section from control mice
showing collagen only in vascular walls. (B) The CCl4 group has developed extensive fibrosis
in the periportal areas, with degenerated, balooned hepatocytes and cellular inclusions in
fibrotic scares. Collagen fibers of the connective tissues are identified by their blue color.
Mice receiving 200 mg/kg (C) and 600 mg/kg (D) of DWE did not show significant signs of
fibrosis. (E) The livers of mice treated with 600 mg/kg DWE only were similar to controls.
Original magnification 100x, insets 1000x. Mallory trichrome stain. Photomicrographs are
representative of two experiments with five mice per group.
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Fig. 4. The expression and specific tissue distribution of α-SMA. (A) In the livers of control
animals, α-SMA immunopositivity was restricted to vascular smooth musculature (arrow),
while other liver cells remain negative. (B) In mice receiving α-SMA–containing
myofibroblasts were strongly and diffusely stained in affected lobuli, connected between
themselves with thin, "bridging" immunopositivity (arrowhead). (C) α-SMA
immunopositivity was significantly reduced in the livers of mice receiving 200 mg/kg and (D)
mice treated with 600 mg/kg of DWE showed only sporadic α-SMA immunopositivity in the
hepatocytes in vicinity to fibrotic lesions (arrowhead). (E) Mice treated with 600 mg/kg DWE
only showed staining pattern similar to control animals (arrow). Original magnification 400x.
Immunohistochemical stain for α-SMA. Photomicrographs are representative of two
experiments with five mice per group.
Fig. 5. The expression and specific tissue distribution of GFAP. (A) The liver sections from
control mice showed thin GFAP immunopositive bands lining the hepatic sinusoids (arrow).
(B) In the livers of mice receiving CCl4 moderate GFAP expression was limited to the scare
areas (arrowhead). Mice receiving 200 mg/kg (C) and 600 mg/kg DWE (D) had only sporadic
GFAP expression in surrounding tissue (arrowhead). (E) The livers of mice receiving 600
mg/kg DWE showed staining pattern similar to the control animals (arrow). Original
magnification 1000x. Immunohistochemical stain for GFAP. Photomicrographs are
representative of two experiments with five mice per group.
Fig. 6. The expression and specific tissue distribution of MT I/II. (A) The livers from control
mice showed weak perilobular MT I/II immunopositivity. (B) In the CCl4 group MT I/II
immunopositivity was only sporadic and restricted to the fibrotic scares. (C) Mice receiving
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200 mg/kg DWE had weak cytoplasmic MT I/II expression. (D) The livers of mice receiving
600 mg/kg of DWE showed moderate MT I/II immunopositivity. (E) The livers of mice
receiving 600 mg/kg DWE were negative for MT I/II. Original magnification 100x, insets
1000x. Immunohistochemical stain for MT I/II. Photomicrographs are representative of two
experiments with five mice per group. (F) The bar graph shows the extent of MT I/II hepatic
expression (mean ± SD, n=5).
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