International Immunopharmacology 17 (2013) 543–547
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International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp
Preliminary report
The hepatoprotective effect of fraxetin on carbon tetrachloride induced
hepatic fibrosis by antioxidative activities in rats
Xiaowei Chen a,1, Xiaozhou Ying b,1, Weiwei Zhang a, Yongping Chen a, Chunwei Shi a,
Yijun Hou a, Youcai Zhang a,⁎
a
b
Department of Infectious Diseases, the First Affiliated Hospital of Wenzhou Medical College, China
Department of Orthopaedic Surgery, the Second Affiliated Hospital of Wenzhou Medical College, China
a r t i c l e
i n f o
Article history:
Received 29 April 2013
Received in revised form 6 August 2013
Accepted 8 August 2013
Available online 27 August 2013
Keywords:
Fraxetin
Carbon tetrachloride (CCl4)
Antifibrotic
Antioxidant
Rats
a b s t r a c t
The aim of the study was to investigate the potentially protective effects of fraxetin on carbon tetrachloride (CCl4)
induced oxidative stress and hepatic fibrosis in Sprague–Dawley rats. In this study, rats were divided into five
groups, including normal controls, model, silymarin as the positive control, fraxetin 20 mg/kg and fraxetin
50 mg/kg. After 8 weeks, activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST)
and total bilirubin (TBIL) were checked. The levels of protein carbonyls, thiobarbituric acid-reactive substances
(TBARS) and antioxidant enzymes such as catalase, SOD and glutathione peroxidase (GSH-Px) were determined
after fraxetin administration. The hydroxyproline levels and histopathologic examinations of hepatocyte fibrosis
were also determined. We found that fraxetin at doses of 20 and 50 mg/kg for 8 weeks significantly reduced the
levels of TBARS and protein carbonyls compared with CCl4 group. Fraxetin significantly increased the activities of
catalase, SOD and GSH-Px in the liver. We also found that fraxetin prevented CCl4 induced hepatic fibrosis by histological observations. These results indicate that fraxetin exhibits potent protective effects against CCl4 induced
oxidative stress and hepatic fibrosis.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
Hepatic fibrosis is a common pathological process resulted from various chronic hepatic injuries, which is characterized by an increase of
extracelluarmatrix (ECM) deposition in the Disse's space and the imbalance between synthesis and degradation of ECM. Accumulating evidence suggests that hepatic fibrosis is a reversible disease, therefore
an effective treatment would probably prevent or reverse the hepatic
fibrotic process [1]. In recent years, considerably clinical and experimental evidences show that oxidative stress caused by an imbalance between the oxidant and antioxidant systems of the body in favor of the
oxidants should be a major apoptotic stimulus in the different types of
acute and chronic liver injury and hepatic fibrosis [2]. CCl4 is a potent
hepatoxin producing centrilobular hepatic necrosis which is widely
used for animal models of liver fibrosis. Hepatic fibrosis induced by
CCl4 is associated with the exacerbation of lipid peroxidation and the
depletion of antioxidant status [3]. Accordingly, successful antioxidant
interventions may be a potential and effective therapeutic strategy for
prevention and treatment of hepatic fibrosis.
Coumarins comprised a group of phenolic compounds widely distributed in natural plants, such as citrus fruits, tomatoes, vegetables,
and green tea. Their popularity has increased because of the range of
pharmacological properties demonstrated, such as antithrombotic [4],
anti-inflammatory [5], antiviral [6], and antitumor properties [7].
Fraxetin (7,8-dihydroxy-6-methoxy coumarin), a coumarin derivative,
has been reported to possess antioxidative, anti-inflammatory and
neuroprotective effects [8–11]. Fraxetin exhibits its antioxidant effect
through increasing the level of GSH and reducing oxidative damage in
a drosophila melanogaster experimental model [8]. In addition, previous studies reported that some coumarin derivatives include fraxetin,
was able to protect neuroblastoma cells against toxic effects induced
by rotenone [10,11].
Because of the strong antioxidant activity of fraxetin, we hypothesised
that fraxetin administration might be useful for preventing various types
of oxidative damage induced by oxidative stress. Therefore, the aims of
this study were to evaluate the antioxidant and antifibrotic properties
of fraxetin in vivo.
2. Materials and methods
⁎ Corresponding author at: Department of Infectious Diseases, the First Affiliated
Hospital of Wenzhou Medical College, Nanbaixiang streets, Ouhai, Wenzhou 325000,
China. Tel.: +86 18858707266.
E-mail address: [email protected] (Y. Zhang).
1
Contributed equally to this work.
1567-5769/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.intimp.2013.08.006
2.1. Chemicals
Carbon tetrachloride (CCl4), Fraxetin (7,8-dihydroxy-6-methoxy coumarin),and silymarin were obtained from Sigma Chemicals Company.
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X. Chen et al. / International Immunopharmacology 17 (2013) 543–547
Corn oil was purchased from the local market, sealed and stored at room
temperature after high temperature sterilization.
2.2. Animals
Male Sprague–Dawley rats (150–200 g) were obtained from Wenzhou Medical College. The rats were housed under normal laboratory
conditions (21 ± 2°C, 12/12 h light-dark cycle) with free access to standard pellet diet and water ad libitum. All animal procedures were
conducted in accordance with the standards set forth in the guidelines
for the care and use of experimental animals by the Committee for the
Purpose of Control and Supervision of Experiments on Animals and
the National Institutes of Health. The study protocol was approved by
the Animal Ethics Committee of Wenzhou Medical Collage.
2.3. Treatment
The animals were randomly divided into 5 groups of 7. Group I
served as the normal control and was orally administered distilled
water daily with intraperitoneally (i.p.) administered corn oil (1 ml/kg
body weight), which twice per week for 8 weeks. To induce oxidative
stress and hepatic fibrosis, animals of Groups II, III, IV and V were i.p. administered 2 ml/kg body weight of CCl4 (20% in corn oil) twice per week
for eight weeks. Group II served as the CCl4 control and was orally administered distilled water daily. Group III served as the positive control
and was orally administered silymarin (200 mg/kg) daily for 8 weeks.
Silymarin is a group of flavones extracted from Silybum marianum L.
and is a strong antioxidant. Many studies showed that silymarin was
an effective agent for CCl4 induced liver injury and hepatic fibrosis
[12–15]. Groups IV and V were orally administered fraxetin powder
dissolved in distilled water at doses of 20 and 50 mg/kg, respectively,
daily for 8 weeks.
Table 1
Effect of fraxetin on serum concentrations of ALT, AST and TBIL.
Groups
ALT (U/L)
AST (U/L)
TBIL (mmol/L)
Normal
CCl4 control
Silymarin 200 mg/kg
+CCl4
Fraxetin 20 mg/kg
+ CCl4
Fraxetin 50 mg/kg
+ CCl4
47.3 ± 15.2
450.6 ± 80.4#
152.4 ± 27.3⁎
66.2 ± 16.2
470.8 ± 75.9#
193.2 ± 55.7⁎
5.5 ± 2.1
27.4 ± 7.2#
8.56 ± 3.7⁎
225.7 ± 32.6⁎
246.3 ± 52.2⁎
12.5 ± 6.2⁎
176.4 ± 47.8⁎
185.8 ± 32.9⁎
7.82 ± 3.2⁎
Rats were seven in each group. Each value represents the mean ± SD. Significance was
determined by one-way ANOVA.
#
P b 0.05 as compared with Igroup.
⁎ P b 0.05 as compared with II group.
2.7. Measurement of hydroxyproline levels
The hydroxyproline levels in the livers were determined by a modified version of the previous method [18]. The liver samples were
weighed and completely hydrolysed in 6 M HCl. After hydrolysis, the
samples were derivatized using a chloramine T solution and Ehrlich's
2.4. Serum biochemical analysis
The rats were anesthetized with urethane (1.2 g/kg, intraperitoneally (i.p.)), the blood samples from abdominal aorta were drawn into heparinized injectors, and centrifuged at 3000 rpm at 4 °C for 15 min.
Serum ALT, AST and TBIL levels were measured by the first Affiliated
Hospital, Wenzhou Medical College (Wenzhou, China). After finishing
with the blood collection, the experimental animals were sacrificed,
liver samples were dissected and washed with ice-cold saline, then
they were immediately stored at −80°Cfor further analysis. The largest
right lobe of each liver was excised and fixed in a 10% formalin solution
for histopathologic analyses.
2.5. Measurement of lipid peroxidation and protein carbonyls
The quantitative measurement of lipid peroxidation was performed
by measuring the concentration of TBARS in the liver according to the
method reported by Tsaiet et al. [16]. In brief, samples were mixed
with a TBA reagent consisting of 0.375% TBA and 15% trichloroacetic
acid in 0.25 N hydrochloric acid.The supernatant was collected, and its
absorbance was measured at 535 nm with an ELISA plate reader. Oxidative damage to proteins was quantified by the carbonyl protein assay as
described previously [17]. The absorbance was measured at 370 nm
with an ELISA plate reader.
2.6. Measurement of SOD, catalase and GSH-Px levels
Liver homogenates were prepared in cold Tris–HCl (5 mmol/L, containing 2 mmol/L EDTA, pH 7.4) using a homogeniser. The unbroken
cells and cell debris were removed by centrifugation at 10,000 g for
10 min at 4°C. The supernatant was used immediately for the SOD, catalase and GSH-Px assays according to the protocols of commercially
available kits (Jiancheng Bioengineering Institute, Nanjing, China).
Fig. 1. Effects of fraxetin on liver TBARS (A) and protein carbonyls (B) in CCl4 intoxicated
rats. Values are the mean ± SD for 7 rats; #P b 0.05 as compared with normal group;
*P b 0.05 as compared with CCl4 treated control group.
X. Chen et al. / International Immunopharmacology 17 (2013) 543–547
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reagent, and the optical density was measured at 558 nm. A standard
calibration curve was prepared using trans-4-hydroxy-L-proline.
2.8. Liver histopathology
Liver samples were fixed in 10% buffered formaldehyde solution,
processed by the paraffin slice technique. For H&E staining, sections
were stained with hematoxylin for 3 min, washed, and stained with
0.5% eosin for an additional 3 min. The degree of liver damage was examined blindly by a special pathologist under a light Olympus microscope (Olympus, Hamburg, Germany).
2.9. Statistical analysis
Data bars represent the means ± SD (standard deviations) for at
least three independent experiments in all cases. One-way Analysis of
Variance (one-way ANOVA) was used to compare the differences in
means of more than two groups, followed by the Dunnett multiple
comparison tests to determine significant differences among the pairs.
P value of 0.05 or less was considered statistically significant.
3. Results
3.1. Effects of fraxetin on serum concentrations of ALT, AST and TBIL
As shown in Table 1, compared with group I, serum concentrations
of ALT, AST and TBIL were significantly increased in group II
(p b 0.05). Treatment with fraxetin at 20–50 mg/kg or silymarin significantly attenuated the increase of ALT, AST and TBIL.
3.2. Effects of fraxetin on lipid peroxidation and protein carbonyls
We found CCl4 induced toxicity caused a significant increase in
liver TBARS levels and protein carbonyl compared to the control
group (p b 0.05) (Fig. 1A, B). However, treatment with fraxetin at
20–50 mg/kg significantly reversed these changes. Similar results
were also found after the administration of 200 mg/kg of silymarin.
3.3. Effect of fraxetin on CCl4 induced changes in the levels of hepatic antioxidant enzyme activities
In the present study, CCl4 treatment significantly decreased hepatic
SOD, CAT and GSH-Px activities compared to the control group
(p b 0.05). By contrast, the decreased hepatic SOD, CAT and GSH-Px
Fig. 2. Effects of fraxetin on liver antioxidant enzymes in CCl4 intoxicated rats. (A) SOD;
(B) catalase and (C) GSH-Px. Values are the mean ± SD for 7 rats; #P b 0.05 as compared
with normal group;*P b 0.05 as compared with CCl4-treated control group.
Fig. 3. Effects of fraxetin on hydroxyproline in CCl4 intoxicated rats. Values are the
mean ± SD for 7 rats; #P b 0.05 as compared with normal group; *P b 0.05 as compared
with CCl4 treated control group.
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X. Chen et al. / International Immunopharmacology 17 (2013) 543–547
activities were significantly elevated in the fraxetin and silymarin
groups (Fig. 2A, B, C).
3.4. Effect of fraxetin on hydroxyproline levels
The levels of hydroxyproline in CCl4 treated group were significantly
higher than those in the control group (p b 0.05) (Fig. 3). However,
treatment with fraxetin at 20–50 mg/kg significantly inhibited the elevation in hydroxyproline levels following CCl4 administration. A similar
result was found in animals which were treated with silymarin.
3.5. Effects of fraxetin on histopathologic characteristics
H&E staining for the control group showed normal architecture,
whereas the CCl4 treated group exhibited fatty degeneration, necrosis and inflammation of hepatocytes. Treatment with fraxetin at
20–50 mg/kg or 200 mg/kg of silymarin, however, markedly improved the hepatic morphology and architecture, with less pseudo
lobules and inflammatory cell infiltration compared with CCl4 treated group (Fig. 4).
4. Discussion
Chronic liver diseases constitute a global connection, and the medical treatments for these diseases are usually difficult to handle and have
limited efficacy. Therefore, there has been considerable interest in finding new medicines for the treatment of liver diseases [19]. In an attempt
to model the liver fibrosis process, CCl4 has been used to experimentally
induce liver fibrosis in rodents widely [3,13,16,20].
As a result of the hepatic injury induced by CCl4, the altered permeability of the membrane causes the enzymes from the cells to be released into circulation [21], which damages the hepatic cells, as shown
by the abnormally high level of serum hepatospecific enzymes. In this
study, the serum ALT, AST, and TBIL levels increased markedly after
the CCl4 administration, but these increases were attenuated by the
treatment of fraxetin. In parallel with the alteration of liver function
markers, these phenomena were also confirmed by histological
observation.
The lipid peroxidation is one of the major outcomes of a free radicalmediated injury that directly damages membranes and generates a
number of secondary products in vivo. It is not only as the standard of
Fig. 4. Histopathological changes of fibrosis occurred in CCl4 intoxicated rats and prevention by the treatment with fraxetin (hematoxylin and eosin staining).(A) Normal control; (B) CCl4
control; (C) silymarin 200 mg/kg + CCl4; (D) fraxetin 20 mg/kg + CCl4; (E) fraxetin 50 mg/kg + CCl4.
X. Chen et al. / International Immunopharmacology 17 (2013) 543–547
liver fibrosis, but could also directly induce liver fibrosis [22,23]. The
measurement of TBARS is a well-established method for screening and
monitoring lipid peroxidation. Protein carbonyl groups are an important biomarker of protein oxidation, and the accumulation of protein
carbonyls has been observed in chronic liver diseases [24]. In our
study, fraxetin significantly reversed the elevation of liver TBARS and
protein carbonyls levels. Our results are consistent with previous reports that fraxetin showed scavenging activity against reactive oxygen
species [25] and inhibits lipid peroxidation in rat brain [26,27],
suggesting that the free radicals which were released in the liver were
effectively scavenged by fraxetin.
An increase in hydroxyproline levels in the liver indicates enhanced
hepatic fibrosis, which is associated with the exacerbation of lipid peroxidation and the depletion of antioxidant status after treatment with
CCl4 [18]. We found fraxetin or silymarin significantly inhibited the
elevation in hydroxyproline levels following CCl4 administration, suggesting that fraxetin has the ability to protect against CCl4 induced hepatic fibrosis in rats.
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5. Conclusions
The present study showed that fraxetin exerted beneficially
hepatoprotective effects on oxidative damage induced by CCl4, mainly
enhancing antioxidant capacity of liver organizations; reduce the level
of lipid peroxidation and protein carbonyls induced by CCl4. Further
studies are required to define the exact mechanism underlying the
anti-oxidative effects of fraxetin.
Acknowledgments
[15]
[16]
[17]
[18]
[19]
This work was supported by grants from the Hainan Provincial
Health Department (2011-34). The authors declare no conflicts of
interest.
[20]
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