Journal of Pharmacological Sciences 130 (2016) 15e23

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Ameliorative effects of tannic acid on carbon tetrachloride-induced

liver brosis in vivo and in vitro
Xi Chu a, *, Hua Wang b, Yan-min Jiang c, Yuan-yuan Zhang d, e, Yi-fan Bao f,
Xuan Zhang d, e, Jian-ping Zhang d, e, Hui Guo d, Fan Yang d, Yan-chao Luan g,
Yong-sheng Dong h
a

Department of Pharmacy, The Fourth Hospital of Hebei Medical University, No. 12, Jiankang Road, Shijiazhuang, 050011, Hebei, China
Hebei Medical University, No. 361, East Zhongshan Road, Shijiazhuang, 050017, Hebei, China
Department of Respiration, The Third Hospital of Shijiazhuang, No. 15, South Sports Street, Shijiazhuang, 050011, Hebei, China
d
Hebei University of Chinese Medicine, No. 3, Xingyuan Road, Shijiazhuang, 050200, Hebei, China
e
Hebei Key Laboratory of Integrative Medicine on Liver-Kidney Patterns, China
f
Key Laboratory of Drug Metabolism & Pharmacokinetics, China Pharmaceutical University, No. 1, Shennong Road of the Central Door, Nanjing, 210038,
Jiangsu, China
g
Chest Hospital of Hebei, No. 372, North Shengli Street, Shijiazhuang, 050041, Hebei, China
h
Intensive Care Unit, Air Force General Hospital, No. 30, Fucheng Road, Haidian, 100142, Beijing, China
b
c

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 13 July 2015
Received in revised form
23 November 2015
Accepted 1 December 2015
Available online 15 December 2015

We investigated the ameliorative effects and potential mechanisms of tannic acid (TA) in carbon tetrachloride (CCl4)-intoxicated mice and hepatic stellate cells (HSCs). Liver brosis was observed in CCl4
(800 ml/kg)-induced mice, and high viability was observed in CCl4 (10 mM)-intoxicated HSCs. Pretreatment of mice with TA (25 or 50 g/kg/day) signicantly ameliorated hepatic morphology and coefcient values and reduced the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), the concentrations of malondialdehyde (MDA) and serum levels of endothelin-1 (ET-1). In
addition, TA increased the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and endothelial nitric oxide synthase (eNOS) and the serum level of NO. Moreover, TA
reduced the expression of angiotensin II receptor-1 (ATR-1), interleukin-1b (IL-1b), tumor necrosis factora (TNF-a), transforming growth factor-b (TGF-b), caspase-3, c-fos, c-jun, the ratio of Bax/bcl-2, tissue
inhibitor of metalloproteinase-1 (TIMP-1) and TA increased matrix metal proteinase-9 (MMP-9), matrix
metalloproteinase-1 (MMP-1). Furthermore, TA (0.01 mM, 0.1 mM or 1 mM) decreased the TIMP-1/MMP-1
ratio and reduced the viability of HSCs. These results indicated that TA exerts signicant liver-protective
effects in mice with CCl4-induced liver brosis. The potential mechanism may rely on the inhibition of
collagen accumulation, oxidative stress, inammation and apoptosis.
2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords:
Tannic acid
Anti-brosis
Anti-oxidation
Anti-Inammation
Anti-Apoptosis

1. Introduction
A continuous brogenic stimulus, such as the physiological
response of chronically activated wound healing, can lead to hepatic brosis and even cirrhosis (1, 2). Hepatic brosis is characterized by reversible scarring and the massive deposition of
extracellular matrix (ECM) in the liver (3). During brogenesis,

* Corresponding author. Department of Pharmacy, The Fourth Hospital of Hebei

Medical University, 12, Jiankang Road, Shijiazhuang 050011, Hebei, China. Tel./
fax: 86 311 89926911.
E-mail address: chux2014@126.com (X. Chu).
Peer review under responsibility of Japanese Pharmacological Society.

HSCs are activated and excessive ECM components, such as hyaluronic acid, bronectins and collagens, accumulate (4). In addition,
activated HSCs exhibit a strong proliferative activity by expressing
excessive collagen I and a-smooth muscle actin and secreting
chemotactic cytokines and pro-inammatory cytokines (5).
Inammation plays an important role in the stimulation of HSCs
and hepatic brosis (6). These liver disorders are associated with a
signicant mortality risk; over 100 million people are affected by
this disease worldwide (7, 8), and cirrhosis is the leading cause of
liver disease-related mortality worldwide. Therefore, it is vital to
develop therapeutic strategies for inhibiting liver brosis.
Tannic acid (TA), which is a component of traditional Chinese
medicines (TCMs) (Fig. 1D), widely exists in cereals, legumes, fruits,

http://dx.doi.org/10.1016/j.jphs.2015.12.002
1347-8613/ 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

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X. Chu et al. / Journal of Pharmacological Sciences 130 (2016) 15e23

herbs, green tea and red wine (4). This compound possesses strong
antioxidant, astringent, antiviral and antibacterial properties
(9e11), reduces serum cholesterol and triglycerides, and suppresses lipogenesis (12). As a positive control, silymarin (SIL) is a
traditional herbal medicine, and previous pharmacological studies
demonstrated that SIL has a positive effect on the function of liver
cells (13), inuencing the regenerative capacity of these cells via
antioxidant and protein-restoring activities. Carbon tetrachloride
(CCl4) induces hepatotoxicity via activated hepatic cytochrome
P450 concomitant with lipid peroxidation and eventually leads to
necrosis (14, 15). In this study, we studied the effect of TA on CCl4induced hepatic brogenesis in vivo and on HSCs in vitro.
2. Material and methods
2.1. Animals
Fifty male Kunming mice (20 2 g) were obtained from the
Laboratory Animal Center of Hebei Medical University. All animal
handing procedures were performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory
Animals, and all experiments were endorsed by the Animal Ethics
and Use Committee of Hebei Science and Technical Bureau in China.
After one week of acclimation, the mice were weighed and randomly
divided into the control (CONT, saline, intraperitoneal (i.p.)), CCl4
(800 ml/kg, 3 times per week, i.p.), CCl4 low-dose TA (L-TA, 25 mg/
kg/day, intragastric (i.g.)), CCl4 high-dose TA (H-TA, 50 mg/kg/day,
i.g.) and CCl4 SIL (200 mg/kg/day, i.p., positive control) groups
(n 10). TA and SIL were supplied by Sigma Chemicals (St. Louis, MO,
USA). At the end of the 12th experimental week, all of the mice were

weighed and anesthetized with sodium pentobarbital (50 mg/kg).

The serum was separated for biochemical analysis, and the liver
samples were quickly excised and snap frozen in liquid nitrogen or
xed in a 4% paraformaldehyde solution.
2.2. HSC culture
HSCs were purchased from (Baili Biotechnology Co., Ltd,
Shanghai, China) and the purity is more than 98%. HSCs were
assessed via Trypan Blue exclusion to conrm that the cell viability
was 95%. The HSCs were cultured according to a previous study
(16). To detect the cytotoxic effects of TA on HSCs, the experiments
were divided into six groups: the control (CONT), CCl4 (10 mM),
CCl4 low-dose TA (L-TA, 0.01 mM), CCl4 middle-dose TA (M-TA,
0.1 mM), CCl4 high-dose TA (H-TA, 1 mM) and CCl4 SIL (10 mg/ml)
groups. Cells in the CCl4 group and CONT group were cultured in
DMEM medium, and the cells in the other groups were cultured
with TA or SIL. After 24 h, cells were exposed to CCl4 for 60 min.
Then, we removed the culture medium, washed the cells using
phosphate-buffered saline (PBS) and lysed the cells in 50 mmol/l
Na3VO4, 1 mmol/l phenylmethanesulfonyl uoride (PMSF) and
0.1 mmol/l aprotinin. The samples were centrifuged (15,000 g,
30 min, 4  C) to remove cell debris, and protein concentrations
were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., California, USA).
2.3. Biochemical analysis
The blood samples were centrifuged at 3000 g for 15 min, and
serum levels of aspartate aminotransferase (AST) and alanine

Fig. 1. A: Representative microscopic photographs were observed by H&E staining; B: Representative microscopic photographs were observed by Masson's trichrome staining.
Original magnication 400, the bars represent 50 mm. C: Masson's trichrome staining in each group was calculated. Data are presented as mean SEM. ##P < 0.01 vs. CONT group.
*
P < 0.05, **P < 0.01 vs. CCl4 group. D: Chemical structure of TA.

X. Chu et al. / Journal of Pharmacological Sciences 130 (2016) 15e23

aminotransferase (ALT) were detected using an Autodry Chemistry

Analyzer (AU400; Olympus, Tokyo, Japan). The serum activities of
ET-1 and NO were assayed using ELISA kits (Jian Cheng Biological
Engineering Institute, Nanjing, China). The contents of MDA and the
activities of SOD, GSH-Px and CAT in hepatic tissues were estimated
according to the manuals provided with the assay kits (Jian Cheng
Biological Engineering Institute, Nanjing, China), and the procedures were described previously (17).
2.4. Histopathology analysis
Hepatic tissue samples were stained with hematoxylin and
eosin (H&E) for histopathological studies or with Masson staining
reagents for hepatic collagen deposition, according to routine
staining steps that were described previously (18). We randomly
selected 50 elds at a 200 magnication to determine the area of
brosis (% positive area stained area/total area of the
tissue  100). IL-1b, TNF-a, TGF-b, c-fos, c-jun, MMP-9, TIMP-1 and
MMP-1 were analyzed via immunohistochemistry, which was
performed according to previously described manufacturer protocols (18).
2.5. Western blotting analysis
A total of 20e100 mg of protein obtained from liver homogenate
fractions or HSCs lysates were used for analysis. Western blotting
was used to detect the expression of Bax, bcl-2, angiotensin II
receptor-1 (ATR-1), caspase-3, endothelial nitric oxide synthase
(eNOS), in liver tissues and expressions of TIMP-1, MMP-1 and
MMP-9 in HSCs. The Quantity One software (Bio-Rad, USA) was
used to scan the blots and quantify the bands.
2.6. MTT assay for the analysis of HSC viability
The cell proliferation assay was performed using the 3-[4,5dimethylthiazol-2-ly]-2,5-diphenyltetrazolium bromide (MTT)
(SigmaeAldrich, St. Louis, MO, USA) colorimetric assay, which is
based on the reduction of the tetrazolium salt. The HSCs were
seeded in a 96-well plate (4  103 cells/well) and incubated with TA
and SIL for 24 h. Then, we added the MTT working solution (10 ml/
well, 5 mg/ml in PBS) and incubated the samples for 4 h at 37  C.
The medium were removed and the HSCs was dissolved using a
microplate reader. The cell viability was calculated as the percentage of MTT reduction, the absorption (A) of solubilized formazan was measured at the wavelength of 490 nm by ELISA plate
reader and the absorbance of the CCl4 cells was set as 100%.
2.7. Statistical analysis
Data are expressed as the mean SME. Statistically signicant
differences were identied using one-way analysis of variance
(ANOVA) and Tukey's multiple comparison tests. Differences were
considered statistically signicant at P < 0.05. The Statistical

17

Package for Social Sciences (SPSS, 16.0) software was used for the
analyses.
3. Results
3.1. Amelioration of morphological changes by TA
3.1.1. Liver coefcients and liver function changes in mice
The body weights and liver weights of the mice were measured,
and the relative liver weight was calculated as liver weight/body
weight  100. Our results demonstrated that the relative liver
weight increased signicantly in the CCl4 group compared to the
CONT group (P < 0.01, Table 1). The results also demonstrated that
the extent of liver injury was signicantly reduced in the L-TA and
H-TA groups (P < 0.01).
A biochemical analysis of the activities of ALT and AST in the
serum was performed to verify the role of TA in the protection of
livers treated with CCl4. As shown in Table 1, the activities of serum
ALT and AST in the CCl4 group (107.57 IU/L and 317.69 IU/L,
respectively) increased approximately 3-fold compared with the
activities observed in the CONT group (38.04 IU/L and 117.07 IU/L,
respectively). However, the activities of serum ALT and AST were
signicantly reduced by L-TA (71.95 IU/L and 156.27 IU/L, respectively) and H-TA (68.01 IU/L and 141.75 IU/L, respectively, both
P < 0.01).
3.1.2. Histological changes in mice
A histological examination was performed using H&E staining to
visualize the extent of the liver damage induced by CCl4. The liver
tissues in the control group revealed a normal architecture,
whereas the liver tissues in the CCl4 group showed the destruction
of the structure and the proliferation of brous tissue (black triangles). After treatment with TA, the liver tissues had regained a
normal structure and the degree of brous tissue proliferation was
reduced (Fig. 1A).
3.2. Attenuation of hepatic brosis by TA
To observe the anti-brosis effects of TA and explore the underlying mechanisms, we rst measured the collagen distribution
in mice using Masson staining. We also detected the expression
levels of MMP-9, TIMP-1 and MMP-1 in mice using immunohistochemistry and in HSCs using western blotting respectively.
3.2.1. Masson staining
Masson staining is typically used to observe the localization of
collagen bers in liver tissues. As shown in Fig. 1B, the livers of mice
that were administered CCl4 showed typical characteristics of
brosis (black triangles), and the brotic tissue area increased
approximately 29-fold compared with the CONT group (Fig. 1C,
P < 0.01). After treatment with TA, the brotic areas were signicantly reduced in a dose-dependent manner.

Table 1
Effects of TA on liver weight and the activities of ALT and AST in serum of CCl4-intoxicated mice.
Groups

BW (g)

CONT
CCl4
L-TA
H-TA
SIL

31.15
30.95
32.83
32.59
32.35

LW (g)
1.28
0.65
1.72
1.61
1.12

Data are presented as mean SEM.

##

1.21
2.03
1.81
1.71
1.80

LW/BW (g/g)
0.05
0.11
0.10
0.09
0.08

3.91
6.52
5.52
5.26
5.56

0.11
0.41##
0.13*
0.13**
0.14*

P < 0.01 vs. CONT group. *P < 0.05, **P < 0.01 vs. CCl4 group.

ALT (UI/L)
38.04
107.57
71.95
68.01
92.91

2.03
5.62##
3.33**
3.54**
4.03*

AST (UI/L)
117.07
317.69
156.27
141.75
183.91

6.53
14.13##
9.81**
8.39**
6.74**

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X. Chu et al. / Journal of Pharmacological Sciences 130 (2016) 15e23

Fig. 2. AeC: Representative microscopic photographs of MMP-9, MMP-1 and TIMP-1 in liver observed by immunohistochemistry. Original magnication 400, the bars represent
50 mm. The triangles indicate positive expression of MMP-9, MMP-1 and TIMP-1. 2DeF: the percentage positive area of MMP-9, MMP-1 and TIMP-1 in each group was calculated.
2GeH: expression levels of and the ratio of MMP-9 and TIMP-1/MMP-1 in HSCs observed by western blotting, the value of MMP-9 and ratio of TIMP-1/MMP-1 in each group was
calculated. Data are presented as mean SEM. ##P < 0.01 vs. CONT group. **P < 0.01 vs. CCl4 group.

X. Chu et al. / Journal of Pharmacological Sciences 130 (2016) 15e23

Table 2
Effects of TA on CCl4-intoxicated HSCs viability.
Groups

A490

CONT
CCl4
L-TA
H-TA
H-TA
SIL

0.26
0.48
0.31
0.29
0.23
0.21

Cell viability (%)

0.04
0.05
0.04
0.03
0.05
0.04

e
100
70.71
66.67
51.62
43.01

10.32*
9.89*
10.44**
6.99**

The value of cell viability in CCl4 group was standardized as 100%. Data are presented as mean SEM. *P < 0.05, **P < 0.01 vs. CCl4 group.

3.2.2. Immunohistochemistry analysis of MMP-9, TIMP-1 and

MMP-1 in mice
MMP-9 has the ability to denature and degrade collagens in
ECM. In the CCl4 group, the expression of MMP-9 and MMP-1 was
decreased, but the TIMP-1 was increased signicantly in comparison to that observed in the CONT group (P < 0.01). However, this
modulation effect was rescued by TA treatment in a dosedependent manner. Compared with the CCl4 group, in the L-TA
group and the H-TA group, the expression levels of MMP-9
increased by approximately 5.2-fold and 8.7-fold, respectively
(Fig. 2A, D), the expression levels of MMP-1 increased by approximately 2.2-fold and 4.7-fold, respectively (Fig. 2B, E), and the
expression levels of TIMP-1 decreased by approximately 3.0-fold
and 6.4-fold, respectively (Fig. 2C, F).
3.2.3. Western blotting analysis of MMP-9, TIMP-1 and MMP-1 in
HSCs
The TIMP-1/MMP-1 ratio and expression of MMP-9 were used to
express the degree of liver brosis. The expression of MMP-9 in CCl4
group was signicantly decreased compared with the CONT group
(P < 0.01). After pretreatment with TA (low-dose, middle dose and

19

high-dose), the expression of MMP-9 was increased by 0.13-fold,

0.83-fold and 1.3-fold respectively (Fig. 2G). As shown in Fig. 2H,
CCl4 signicantly increased the TIMP-1/MMP-1 ratio by nearly 10fold in comparison to the CONT samples (P < 0.01). However, the
presence of TA (low-dose, middle dose and high-dose) signicantly
reduced the TIMP-1/MMP-1 ratio by 49.7%, 79.6% and 90.5%
respectively.
3.2.4. MTT analysis of HSC viability
The activation and proliferation of HSCs are closely related to the
pathogenesis and development of liver brosis. The results showed
that CCl4 could increase HSC viability, promote cell proliferation
and eventually lead to brosis. After treatment with TA, the cell
viability was reduced in a dose-dependent manner (Table 2).
3.3. Inhibition of oxidative stress by TA
CCl4 can trigger oxidative stress and lipid peroxidation and
cause the overproduction of free radicals, so we assessed the activities of GSH-Px, SOD, and CAT and the contents of MDA in the
livers using biochemical analysis methods. The liver brosis
induced by CCl4 signicantly reduced the liver GSH-Px, SOD and
CAT activities and increased the liver MDA content in comparison to
the CONT group (P < 0.01, Fig. 3). The results demonstrated that the
liver SOD, CAT and GSH-Px activities were signicantly increased by
TA treatment (P < 0.01 for both the L-TA and H-TA groups). In
contrast, liver MDA levels were markedly decreased by treatment
with TA.
3.4. Prevention of inammation by TA
The overexpression of proinammatory cytokines may play a
role in accelerating the development of hepatic brosis. The

Fig. 3. The activities of SOD, GSH-Px, CAT and the contents of MDA were measured in liver tissue homogenates. Data are presented as mean SEM.
*
P < 0.05, **P < 0.01 vs. CCl4 group.

##

P < 0.01 vs. CONT group.

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X. Chu et al. / Journal of Pharmacological Sciences 130 (2016) 15e23

Fig. 4. Representative microscopic photographs of IL-1b and TNF-a in liver were observed by immunohistochemistry. Original magnication 400, the bars represent 50 mm. The
triangles indicate the positive expression. The percentage positive area of IL-1b and TNF-a in each group was calculated. Data are presented as mean SEM. ##P < 0.01 vs. CONT
group. **P < 0.01 vs. CCl4 group.

immunohistochemical results showed that CCl4 could trigger inammatory reactions, inevitably leading to brosis. The CCl4 group
exhibited elevated expression of the IL-1b and TNF-a proteins. In
contrast, after treatment by TA, the expression levels of the IL-1b
and TNF-a proteins were signicantly decreased compared with
the levels observed in the CCl4 group (Fig. 4, P < 0.01, respectively).

Compared to the CCl4 group, TA reduced the expression of TGF-b, cfos and c-jun by approximately 2.4-fold, 1.3-fold and 0.7-fold,
respectively, at the low dose and by 7.5-fold, 8.2-fold and 7.0-fold,
respectively, at the high dose (Fig. 6AeF). Similarly, western blotting indicated that TA reduced the expression of caspase-3 and Bax/
bcl-2, which indicated that the anti-apoptosis abilities of the cells
increased (Fig. 6G, H).

3.5. Effects of TA on endothelial function and on ATR-1

In this study, we detected changes in ET-1 and NO in the serum
using biochemical analysis methods and assessed the protein
expression levels of eNOS and ATR-1 in livers via western blotting.
The results showed that CCl4 may lead to endothelial dysfunction,
as indicated by increased ET-1 and ATR-1 levels and decreased NO
and eNOS levels compared with the control (all P < 0.01). After
treatment with TA, the function of the endothelial cells was
ameliorated, potentially contributing to the ability of TA to increase
NO and eNOS levels and decrease ET-1 and ATR-1 levels (Fig. 5).
3.6. Suppression of apoptosis by TA
Immunohistochemical analysis showed that the TGF-b, c-fos
and c-jun protein levels were rapidly up-regulated in the CCl4
group in comparison to the CONT group (P < 0.01, respectively).

4. Discussion
Liver brosis occurs due to a dynamic wound-healing response
to hepatocellular damage that is associated with a high risk of
signicant morbidity and mortality (19). Thus, investigations that
aim to identify effective methods for suppressing liver brosis and
preventing the development of cirrhosis are urgently needed (20).
In this study, we used a model of chronic CCl4-induced liver injury
to study the potential molecular mechanisms that underlie the
anti-brotic activities of TA. The results of this study clearly
demonstrate that certain biochemical changes occur in the liver
and serum of mice after the chronic administration of CCl4 for 12
weeks (7). Among these changes, the levels of AST and ALT
increased signicantly after the administration of CCl4. However, in
the TA treatment groups, these activities were signicantly
decreased, suggesting that TA prevents liver damage. This nding is

X. Chu et al. / Journal of Pharmacological Sciences 130 (2016) 15e23

21

Fig. 5. A, B: Serum level of ET-1 and NO in the mice; 5C, D: Expression levels of ATR-1 and eNOS in hepatic tissues observed by western blotting. The data are presented as
mean SEM. ##P < 0.01 vs. CONT group. **P < 0.01 vs. CCl4 group.

consistent with the results of the histopathological examination

(Fig. 1A). Further investigations are currently underway to identify
the mechanism that underlies the protective effect of TA.
Liver brogenesis is accompanied by increased deposition of
ECM in the perisinusoidal and periportal spaces (21). MMPs are
produced by activated HSCs and catalyze proteolysis. In contrast,
TIMPs regulate ECM homeostasis by binding to a particular MMP
and preventing its activity (22). The administration of CCl4 stimulates HSCs to secrete TIMP-1 and decreases the activity of MMP-1
and MMP-9. In the TA-treated groups, the expression of TIMP-1
was signicantly decreased and the viability of HSCs was
reduced. Therefore, it is possible that TA regulates the ECM balance
via TIMP/MMP components and inhibits the activation of HSCs.
Oxidative stress is involved in the pathogenesis of hepatic injury
via the release of reactive oxygen species, and this type of stress
promotes the inammatory response and damages the cellular
membrane, leading to the release of enzymes associated with
hepatotoxicity (23). We studied the hepatoprotective mechanisms
of TA using markers of oxidative stress, inammation and liver
brosis. In this study, TA decreased lipid peroxides in the liver tissue, increased SOD, GSH-Px and CAT activity and decreased MDA
content in CCl4-treated mice (P < 0.01). Polyphenols, including TA,
have been considered to play a vital antioxidant role as dietary
antioxidants in the prevention of oxidative damage (24). The results
of this study revealed that the administration of TA signicantly
reduced CCl4-induced hepatic brosis in mice due to the antioxidant activities of polyphenols.
The release of proinammatory cytokines TNF-a and IL-1b by
kuffer cells and stimulated multiplication of hepatic stellate cells is
one of the manifestations of liver brosis (25). Inammation contributes to a number of pathological events and is further evident in
the current study based on the signicant increases in TNF-a and IL-

1b levels that occurred in the CCl4-treated group in comparison to

either the control or the TA co-treated group. In the process of liver
brosis, TNF-a is not only the mediator of inammation but is also
involved in the mechanism of repairing parenchyma by activating
the proliferation of hepatocytes (26). Under normal conditions,
TNF-a exists as a type II transmembrane protein; however, this
factor is released in a soluble form by the metalloprotease TNF-aconverting enzyme in response to cellular stress (27). Maintaining
TNF-a in a certain concentration range is important for cell protection; however, excessive amounts of this cytokine may cause cell
injury. Increased expression of TNF-a could stimulate the release of
cytokines from macrophages and induce phagocyte oxidative
metabolism (28), thus leading to cascade synthesis of proinammatory cytokines (such as TNF-a, IL-6, IL-8) and then induced
brotic changes (25). Co-treatment with TA can signicantly
decrease the levels of inammatory factors. This activity suggests
that TA exerts a protective effect on liver function by inhibiting
inammatory responses via the reduction of inammatory factors.
Endothelial dysfunction, as described by Panza and Schiffrin (18),
is interpreted as a series of events that include vasoconstriction and
the pro-inammatory response (29). These results indicated that
CCl4 could induce hepatic brosis and apoptosis primarily by interfering with endothelial function (increased hepatic ET-1 levels and
decreased serum levels of NO and eNOS). However, TA can increase
the level of NO, decrease hepatic levels of ET-1 and improve the
hepatic microcirculation. Previous studies suggested that Ang-II
induced brosis in association with its ability to promote the proliferation of broblasts and the synthesis of ECM (30). This stimulatory effect is mediated by ATR-1 (31). Treatment with TA resulted
in a signicant dose-dependent reduction of ATR-1 expression
(Fig. 5C), and this result demonstrated that the TA-mediated downregulation of ATR-1 expression is a hepatic protection pathway.

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X. Chu et al. / Journal of Pharmacological Sciences 130 (2016) 15e23

Fig. 6. AeC: Representative microscopic photographs of TGF-b, c-fos and c-jun in hepatic observed by immunohistochemistry. Original magnication 400, the bars represent
50 mm. The triangles indicate the positive expression. DeF: The percentage positive area of TGF-b, c-fos and c-jun in each group was calculated. G, H: Expression levels of caspase-3,
Bax and bcl-2 in hepatic tissues observed by western blotting, the value of TIMP-1/MMP-1 in each group was calculated. Data are presented as mean SEM. ##P < 0.01 vs. CONT
group. **P < 0.01 vs. CCl4 group.

X. Chu et al. / Journal of Pharmacological Sciences 130 (2016) 15e23

Apoptosis and necrosis contribute to the process of liver brosis

(32). A previous study indicated that CCl4 can induce hepatocellular
damage, which is characterized by necrotic cell death (33). To date,
TGF-b, c-fos, c-jun, caspase-3, Bax and bcl-2 have been identied as
markers of apoptosis (Fig. 6). All of our results showed that TA could
inhibit the occurrence and progression of apoptosis by regulating
the expression of the above-mentioned apoptosis factors.
In summary, the results of this study showed that TA could
effectively repair the injury and improve the pathological changes
that occur during CCl4-induced liver brosis. These ndings indicate a new pharmacological use for TA that may be attributed at
least in part to the inhibition of lipid peroxidation, the suppression
of inammatory reactions, the induction of apoptosis in hepatocytes and the inhibition of HSC activation.
Conicts of interest
The authors declare that they have no conicts of interest.
Acknowledgments
This work was supported by grants from the Research Foundation of the Education Bureau of Heibei Province (QN20131046) and
the National Natural Science Foundation of China (31401003) that
were awarded to XZ and by a grant from the Youth Foundation of
Hebei University of Chinese Medicine (QNZ2015003), which was
awarded to YZ.
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