Scholarly article on topic 'Ameliorative effects of tannic acid on carbon tetrachloride-induced liver fibrosis in vivo and in vitro'

Ameliorative effects of tannic acid on carbon tetrachloride-induced liver fibrosis in vivo and in vitro Academic research paper on "Clinical medicine"

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Abstract of research paper on Clinical medicine, author of scientific article — Xi Chu, Hua Wang, Yan-min Jiang, Yuan-yuan Zhang, Yi-fan Bao, et al.

Abstract We investigated the ameliorative effects and potential mechanisms of tannic acid (TA) in carbon tetrachloride (CCl4)-intoxicated mice and hepatic stellate cells (HSCs). Liver fibrosis was observed in CCl4 (800 ml/kg)-induced mice, and high viability was observed in CCl4 (10 mM)-intoxicated HSCs. Pre-treatment of mice with TA (25 or 50 g/kg/day) significantly ameliorated hepatic morphology and coefficient 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-1β (IL-1β), tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), 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 μM, 0.1 μM or 1 μM) decreased the TIMP-1/MMP-1 ratio and reduced the viability of HSCs. These results indicated that TA exerts significant liver-protective effects in mice with CCl4-induced liver fibrosis. The potential mechanism may rely on the inhibition of collagen accumulation, oxidative stress, inflammation and apoptosis.

Academic research paper on topic "Ameliorative effects of tannic acid on carbon tetrachloride-induced liver fibrosis in vivo and in vitro"

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Ameliorative effects of tannic acid on carbon tetrachloride-induced liver fibrosis 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 ofHebei Medical University, No. 12, Jiankang Road, Shijiazhuang, 050011, Hebei, China b Hebei Medical University, No. 361, East Zhongshan Road, Shijiazhuang, 050017, Hebei, China

c 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 ofHebei, 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

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ARTICLE INFO

Article history:

Received 13 July 2015

Received in revised form

23 November 2015

Accepted 1 December 2015

Available online 15 December 2015

Keywords:

Tannic acid

Anti-fibrosis

Anti-oxidation

Anti-Inflammation

Anti-Apoptosis

ABSTRACT

We investigated the ameliorative effects and potential mechanisms of tannic acid (TA) in carbon tetrachloride (CCl4)-intoxicated mice and hepatic stellate cells (HSCs). Liver fibrosis was observed in CCl4 (800 ml/kg)-induced mice, and high viability was observed in CCl4 (10 mM)-intoxicated HSCs. Pre-treatment of mice with TA (25 or 50 g/kg/day) significantly ameliorated hepatic morphology and coefficient values and reduced the activities of aspartate aminotransferase (AST) and alanine aminotrans-ferase (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-1 b (IL-1 b), tumor necrosis factor-a (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 significant liver-protective effects in mice with CCl4-induced liver fibrosis. The potential mechanism may rely on the inhibition of collagen accumulation, oxidative stress, inflammation 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/).

1. Introduction

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

* 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, fibronectins 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-inflammatory cytokines (5). Inflammation plays an important role in the stimulation of HSCs and hepatic fibrosis (6). These liver disorders are associated with a significant 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 fibrosis.

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/jophs.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/).

herbs, green tea and red wine (4). This compound possesses strong antioxidant, astringent, antiviral and antibacterial properties (9—11), 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), influencing 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 CCl4-induced hepatic fibrogenesis 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). TAand 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 fixed 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 confirm 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 fluoride (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

A H&E staining

Fig. 1. A: Representative microscopic photographs were observed by H&E staining; B: Representative microscopic photographs were observed by Masson's trichrome staining. Original magnification 400x, 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.

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 fields at a 200 x magnification to determine the area of fibrosis (% positive area = stained area/total area of the tissue x 100). IL-1 b, 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 20—100 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-[4,5-dimethylthiazol-2-ly]-2,5-diphenyltetrazolium bromide (MTT) (Sigma—Aldrich, 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 x 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 for-mazan 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 significant differences were identified using one-way analysis of variance (ANOVA) and Tukey's multiple comparison tests. Differences were considered statistically significant at P < 0.05. The Statistical

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 coefficients 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 x 100. Our results demonstrated that the relative liver weight increased significantly 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 significantly 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 significantly 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 fibrous tissue (black triangles). After treatment with TA, the liver tissues had regained a normal structure and the degree of fibrous tissue proliferation was reduced (Fig. 1A).

3.2. Attenuation of hepatic fibrosis by TA

To observe the anti-fibrosis effects of TA and explore the underlying mechanisms, we first 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 immunohisto-chemistry and in HSCs using western blotting respectively.

3.2.1. Masson staining

Masson staining is typically used to observe the localization of collagen fibers in liver tissues. As shown in Fig. 1B, the livers of mice that were administered CCl4 showed typical characteristics of fibrosis (black triangles), and the fibrotic tissue area increased approximately 29-fold compared with the CONT group (Fig. 1C, P < 0.01). After treatment with TA, the fibrotic areas were significantly 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) LW(g) LW/BW (g/g) ALT (UI/L) AST (UI/L)

CONT 31.15 ± 1.28 1.21 ± 0.05 3.91 ± 0.11 38.04 ± 2.03 117.07 ± 6.53

CCl4 30.95 ± 0.65 2.03 ± 0.11 6.52 ± 0.41## 107.57 ± 5.62## 317.69 ± 14.13##

L-TA 32.83 ± 1.72 1.81 ± 0.10 5.52 ± 0.13* 71.95 ± 3.33** 156.27 ± 9.81**

H-TA 32.59 ± 1.61 1.71 ± 0.09 5.26 ± 0.13** 68.01 ± 3.54** 141.75 ± 8.39**

SIL 32.35 ± 1.12 1.80 ± 0.08 5.56 ± 0.14* 92.91 ± 4.03* 183.91 ± 6.74**

Data are presented as mean ± SEM. **P < 0.01 vs. CONT group. *P < 0.05, **P < 0.01 vs. CCl4 group.

Fig. 2. A—C: Representative microscopic photographs of MMP-9, MMP-1 and TIMP-1 in liver observed by immunohistochemistry. Original magnification 400x, the bars represent 50 mm. The triangles indicate positive expression of MMP-9, MMP-1 and TIMP-1. 2D—F: the percentage positive area of MMP-9, MMP-1 and TIMP-1 in each group was calculated. 2G—H: 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.

Table 2

Effects of TA on CCl4-intoxicated HSCs viability.

Groups A490 Cell viability (%)

CONT 0.26 t 0.04 —

CCl4 0.48 0.05 100

L-TA 0.31 0.04 70.71 H 10.32*

H-TA 0.29 - 0.03 66.67 t 9.89*

H-TA 0.23 0.05 51.62 t 10.44**

SIL 0.21 0.04 43.01 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 significantly in comparison to that observed in the CONT group (P < 0.01). However, this modulation effect was rescued by TA treatment in a dose-dependent 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 fibrosis. The expression of MMP-9 in CCl4 group was significantly decreased compared with the CONT group (P < 0.01). After pretreatment with TA (low-dose, middle dose and

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 significantly increased the T1MP-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) significantly reduced the T1MP-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 fibrosis. The results showed that CCl4 could increase HSC viability, promote cell proliferation and eventually lead to fibrosis. 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 fibrosis induced by CCl4 significantly 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 significantly 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 inflammation by TA

The overexpression of proinflammatory cytokines may play a role in accelerating the development of hepatic fibrosis. 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 < 0.01 vs. CONT group. *P < 0.05, **P < 0.01 vs. CCl4 group.

Fig. 4. Representative microscopic photographs of IL-1 b and TNF-a in liver were observed by immunohistochemistry. Original magnification 400 x, the bars represent 50 mm. The triangles indicate the positive expression. The percentage positive area of IL-1 b 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 inflammatory reactions, inevitably leading to fibrosis. The CCl4 group exhibited elevated expression of the IL-1 b and TNF-a proteins. In contrast, after treatment by TA, the expression levels of the IL-1b and TNF-a proteins were significantly decreased compared with the levels observed in the CCl4 group (Fig. 4, P < 0.01, respectively).

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).

Compared to the CCl4 group, TA reduced the expression of TGF-b, c-fos 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. 6A—F). 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).

4. Discussion

Liver fibrosis occurs due to a dynamic wound-healing response to hepatocellular damage that is associated with a high risk of significant morbidity and mortality (19). Thus, investigations that aim to identify effective methods for suppressing liver fibrosis 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-fibrotic 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 significantly after the administration of CCl4. However, in the TA treatment groups, these activities were significantly decreased, suggesting that TA prevents liver damage. This finding is

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 fibrogenesis 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 significantly 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 inflammatory 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, inflammation and liver fibrosis. 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 significantly reduced CCl4-induced hepatic fibrosis in mice due to the antioxi-dant activities of polyphenols.

The release of proinflammatory cytokines TNF-a and 1L-1 b by kuffer cells and stimulated multiplication of hepatic stellate cells is one of the manifestations of liver fibrosis (25). Inflammation contributes to a number of pathological events and is further evident in the current study based on the significant increases in TNF-a and 1L-

1 b 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 fibrosis, TNF-a is not only the mediator of inflammation 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-a-converting 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 proin-flammatory cytokines (such as TNF-a, IL-6, IL-8) and then induced fibrotic changes (25). Co-treatment with TA can significantly decrease the levels of inflammatory factors. This activity suggests that TA exerts a protective effect on liver function by inhibiting inflammatory responses via the reduction of inflammatory factors.

Endothelial dysfunction, as described by Panza and Schiffrin (18), is interpreted as a series of events that include vasoconstriction and the pro-inflammatory response (29). These results indicated that CCl4 could induce hepatic fibrosis 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 fibrosis in association with its ability to promote the proliferation of fibroblasts and the synthesis of ECM (30). This stimulatory effect is mediated by ATR-1 (31). Treatment with TA resulted in a significant dose-dependent reduction of ATR-1 expression (Fig. 5C), and this result demonstrated that the TA-mediated down-regulation of ATR-1 expression is a hepatic protection pathway.

Fig. 6. A—C: Representative microscopic photographs of TGF-b, c-fos and c-jun in hepatic observed by immunohistochemistry. Original magnification 400x, the bars represent 50 mm. The triangles indicate the positive expression. D—F: 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.

Apoptosis and necrosis contribute to the process of liver fibrosis (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 identified 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 fibrosis. These findings indicate a new pharmacological use for TA that may be attributed at least in part to the inhibition of lipid peroxidation, the suppression of inflammatory reactions, the induction of apoptosis in hepato-cytes and the inhibition of HSC activation.

Conflicts of interest

The authors declare that they have no conflicts 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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