|Year : 2014 | Volume
| Issue : 2 | Page : 216-221
Anti-fibrosis effects of Huisheng oral solution in CCl 4 -induced hepatic fibrosis in rat
Wenting Li1, Yuanbo Wu2, Chuanlong Zhu1, Zheng Wang3, Rentao Gao1, Quan Wu4
1 Department of Infectious Disease, Anhui Provincial Hospital, Hefei, China
2 Department of Neurology, Anhui Provincial Hospital, Hefei, China
3 Department of Respiratory and Critical Medicine, Zhengzhou University, China
4 Central Lab, Anhui Provincial Hospital, Hefei, China
|Date of Submission||11-Dec-2013|
|Date of Decision||04-Jan-2014|
|Date of Acceptance||04-Feb-2014|
|Date of Web Publication||24-Mar-2014|
Department of Infectious Disease, Anhui Provincial Hospital, Hefei
Department of Infectious Disease, Anhui Provincial Hospital, Hefei
Source of Support: None, Conflict of Interest: None
Aim: Some gradient of Huisheng oral solution (HOS) has been reported to have anti-fibrosis activity. This study was designed to investigate whether HOS could inhibit liver fibrosis and to elucidate its molecular mechanism of action.
Materials and Methods: Hepatic fibrosis model in rat was induced by subcutaneous injection of CCl 4 . Rats in the treatment group were administrated with HOS intragastrically. Hematoxylin and eosin (H and E) staining and Masson's trichrome staining were used to examine the changes in liver pathology. Levels of ALT, AST, LDH, hyaluronic acid (HA) and laminin (LN) in serum and hydroxyproline (Hyp) in liver were detected by biochemical examination and radioimmunoassay, respectively. The expression and distribution of Smad3, TGF-β1, α-SMA and TIMP-1 were observed and the active TGF-β1 was tested.
Results: Our data demonstrated that HOS alleviated CCl 4 -induced collagen deposition in liver tissue, improved liver condition and liver function in rats. HOS also significantly reduced the expression and distribution of Smad3, TGF-β1, α-SMA and TIMP-1 as well as decreased active TGF-β1.
Conclusions: This study revealed that HOS attenuates the development of liver fibrosis through suppressing the TGF-β1 pathway. It provides us a new approach to treatment of liver fibrosis.
Keywords: Huisheng oral solution, liver fibrosis, TGF-β1
|How to cite this article:|
Li W, Wu Y, Zhu C, Wang Z, Gao R, Wu Q. Anti-fibrosis effects of Huisheng oral solution in CCl 4 -induced hepatic fibrosis in rat. Indian J Pharmacol 2014;46:216-21
|How to cite this URL:|
Li W, Wu Y, Zhu C, Wang Z, Gao R, Wu Q. Anti-fibrosis effects of Huisheng oral solution in CCl 4 -induced hepatic fibrosis in rat. Indian J Pharmacol [serial online] 2014 [cited 2020 May 25];46:216-21. Available from: http://www.ijp-online.com/text.asp?2014/46/2/216/129323
| » Introduction|| |
Liver fibrosis has been considered to be the wound-healing response of liver to various toxic stimuli, including hepatitis, alcohol and immune compounds. Liver fibrosis is characterized by an excessive deposition of extra cellular matrix (ECM). Hepatic stellate cells (HSCs) have been regarded as the main source of ECM. Inhibition of the activation and function of HSCs has become the most important treatment strategy for liver fibrosis. 
Liver fibrosis appears in multiple stages and is regulated by many inflammatory mediators and cytokines. Among various factors involved in liver fibrosis, TGF-β1 is considered to be the most important. The TGF-β1-mediated signaling pathway plays key roles in the progress of liver fibrosis including cell development, HSC activation, collagen deposition and ECM remodeling.  It has been widely accepted that TGF-β1 initiates liver fibrosis by regulating the Smad-mediated signaling pathway, especially Smad3. Most importantly, TGF-β1 could be secreted by Kuffer cells and HSCs as well as their cell line, HSC-T6 cells. It was reported that HSCs could generate TGF-β1 leading to the production of ECM and releasing of chemokines and cytokines involved in liver fibrosis.  Anti-TGF-β1 has the ability of attenuating the progress of liver fibrosis by decreasing the expression of tissue inhibitors of metalloproteinase-1 (TIMP-1), α-SMA and collagen I. 
Huisheng oral solution was developed from a classical recipe called Huazheng Huisheng Dan which has been widely used in China for at least 1000 years. Huisheng oral solution contains ginseng, Rhizoma cyperi, angelica, motherwort, rhizome sparganii, trogopterus dung, turtle shell, frankincense, saffron, Ligusticum wallichii, peach kernel, rhubarb, leech, clove and ferula.  These ingredients have been reported to have many pharmaceutical activities including anti-fibrosis, anti-tumor, anti-coagulant and so on. , However, there is little known about anti-fibrosis effects of Huisheng oral solution. This study was designed to investigate the anti-fibrosis activity of Huisheng oral solution in rats induced by CCl 4.
| » Materials and Methods|| |
CCl 4 (Wuhan Yafa Biological Technology Co., Ltd) was diluted by 40% (v/v) in olive oil before use. Enzyme-linked immunosorbent assay (ELISA) kits for determining serum alanine transaminase (ALT), aspartate transaminase (AST) and lactic dehygrogenase (LDH) were obtained from Wuhan Boster Biological Technology Co. Ltd (Wuhan, China). ELISA kit for TGF -β1 was bought from R and D system (USA). Kits for serum hyaluronic acid (HA) and laminin (LN) were bought from Senxiong Company (Shanghai, China). Kit for liver hydroxyproline (Hyp) was provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Rabbit polyclonal antibodies against α-SMA and β-actin and anti-bodies of rabbit IgG conjugated with horseradish peroxidase (HRP) were obtained from Boster (Wuhan, China). Huisheng oral solution was provided by the Chengdu Di'ao group Tianfu Pharmaceutical Co., Ltd.
Male Wistar rats (Experimental Animal Center of Anhui Medical University, Hefei, China) weighing 200-300 g were included in this study. All animal procedures were performed under the guidelines set by Anhui Medical University Animal Care and Use Committee.
They were randomly divided into three groups (n = 20-25 rats per group). Rats in the model group were injected subcutaneously with CCl 4 at a dose of 3 ml/kg twice a week for 8 weeks. At the same time, rats in the treatment group were intragastrically given Huisheng oral solution at a dose of 2 ml/100 g body weight twice daily. Meanwhile, rats in the control group were treated with normal saline instead. At the end of the experiment, rats were anesthetized with 10% chloral hydrate and sacrificed. Serum samples were collected from each rats and stored at -80C. Livers were harvested at 24 h after the last injection for three purposes: (1) fixed in 10% formalin for histological examinations, (2) preserved at -80C for Hyp kits and (3) homogenized in TRIZOL for RNA isolation.
The serum levels of ALT, AST and LDH in rats were determined by ELISA kits and the levels of HA and LN in serum were detected by radioimmunoassay.
Hepatic Concentration of Active TGF-β1
The hepatic concentration of TGF-β1 was detected by ELISA kits according to the manufacturer's instructions. Liver tissues were homogenenized in extraction buffer. Active TGF-β1 was presented as percentage of total TGF-β.
Liver tissues were fixed in 10% formalin, embedded in paraffin and sectioned at 5 μm thickness. Changes in liver pathology and collagen deposition were observed by hematoxylin-eosin (H and E) staining and Masson's trichrome staining, respectively. The scores of hepatic fibrosis grading were blindly determined by two independent pathologists according to the score system described by Chevallier. 
Immunohistochemical staining was performed on paraffin-embedded liver tissue sections of 5-μm thickness, which were deparaffinized, treated with 0.3% endogenous peroxidase blocking solution for 20 minutes. The sections were treated sequentially with 3% hydrogen peroxidase in methanol for 10 min at room temperature and washed with phosphate-buffered saline for 5 min three times to block endogenous peroxidase activity. The liver sections were then incubated with a rat anti-Smad3 antibody at a dilution of 1:200 for 1.5 h at room temperature and then incubated with HRP-labeled goat-anti-rabbit secondary antibodies (diluted to 1:200). Samples were analyzed by confocal microscopy using 40 × magnification (Olympus, Tokyo, Japan). The expressions of TGF-β1, α-SMA and TIMP-1 in liver tissue were analyzed by the same method and were measured by a PI. Positive index (PI) = mean optical density × positive area percentage.
The level of Hyp in liver tissue was determined by a spectrophotometric method according to the kit's instruction. The level of Hyp was expressed as Hyp (μg)/protein (mg).
Reverse Transcription-Polymerase Chain Reaction) and Quantitative Real-time PCR0
Total mRNA was extracted from the liver tissue of rats as described by the manufacturer. Single-strand cDNA was synthesized from 1 μg of total RNA by reverse transcription according to the instructions (Toyobo, Japan). RT-PCR and qPCR were performed as described previously. 
The primer was as follows:
- Smad3 (Invitrogen, Shanghai):
Forward: 5′- TGATCCC TCCAATTCAGAGC-3′,
Reverse: 5′- GTTGGGAGACTGGACGAAAA-3′; 
-GAPDH (Toyobo, Japan):
Forward: 5′- ACCACAGTCCATGCCATCAC-3′,
Reverse: 5′- TCCACCACCCTGTTGCTGTA-3′.
- TIMP-1 (Invitrogen, Shanghai):
Forward: 5′- TTTGCATCTCTG GCCTCTG-3′,
Reverse: 5′- AATGACTGTCACTCTCCAG-3′;
-α-SMA (Invitrogen, Shanghai):
Forward: 5′- GATCACCATCGGGAATGAACGC-3′,
Reverse: 5′- CTTAGAAGCATTTGCGGTGGAC-3′,
- TGF-β1 (Invitrogen, Shanghai):
Forward: 5′-TGAGTGGCTGTCTTTTGACG -3′,
Reverse: 5′- ACTTCCAACCCAGGTCCTTC-3′. 
Western Blot Assays
The total protein from liver tissue was extracted according to the instructions provided in the kits (Yafa, Wuhan). The proteins were separated by a 10% SDS-PAGE gel and then transferred onto nitrocellulose membranes (Pierce, Rockfors, USA). After incubation with 10% nonfat milk, the membranes were probed with the polyclonal rabbit anti-rat Smad3 (TIMP-1, TGF-β1, α-SMA) antibody (1:400) overnight at 4°C. After washing for 2×3 min, membranes were incubated with HRP-labeled goat-anti-rabbit secondary antibodies for 1 h at room temperature and colored by ESL. The membranes were scanned for the relative value of protein expression in gray scale by Image-Pro plus software 6.0 (Media Cybernetics, Silver Spring, USA). The relative expression was measured according to the reference blots of β-actin.
Values are expressed as mean ± SD. Statistical analyses were performed using an independent t test and one-way analysis of variance (ANOVA). All statistical analyses were performed using SPSS v19.0 statistical software. Results with P < 0.05 were considered to be statistically significant.
| » Results|| |
Effects of Huisheng Oral Solution on CCl 4 -induced Hepatic Fibrosis in Rats
As shown in [Table 1], serum levels of HA and LN levels in rats were significantly increased in the model group compared with the control group, but they were markedly decreased in the treatment group compared with the model group (P < 0.05, respectively). Moreover, higher serum levels of ALT, AST and LDH were observed in the model group compared with the control group, while Huisheng oral solution obviously decreased all the above parameters compared with the model group (P < 0.05, respectively).
|Table 1: |
Serum levels of alanine aminotransferase, aspartate tranfaminase, lactic dehydrogenase, hyaluronic acid and laminin and hydroxyproline content in liver tissue
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At the end of the experiment, liver tissue samples from control rats showed normal lobular architecture with central veins and radiating hepatic cords. Liver tissue sections from the model group showed inflammatory cell infiltration, steatosis and fibrosis. In contrast, Huisheng oral solution treatment remarkably ameliorated the adipose degeneration of hepatocytes and reduced the immigration of inflammatory cells compared with the model group [Figure 1].
|Figure 1: Liver pathology. The changes in liver pathology were examined by hematoxylin-eosin (H&E) (A1, A2, A3) and Masson′s trichrome staining (B1, B2, B3), respectively. Liver samples from control rats (A1, B1) showed normal liver architecture, few inflammatory cell infiltration and little collagen fibers. Samples from model groups (A3, B3) showed lots of inflammatory cells as well as much collagen fibers compared with the control group, while the Huisheng oral solution treatment markedly improved the liver pathology of rats (A2, B2) compared with the model group|
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In addition, as shown by Masson, there were more collagen fibers in the model group compared with the normal group, extending into the hepatic lobules separating them completely. The collagen deposition was sharply decreased by Huisheng oral solution compared with the model group and no fibrotic septae could be observed [Figure 1].
Collagen content was also detected the measurement of Hyp level in liver tissue. As shown in [Table 1], the mean Hyp level in the model group was significantly higher than the normal group, but it was markedly decreased in the Huisheng oral solution-treated group.
Effects of Huisheng Oral Solution on the Expression and Distribution of Smad3, TGFβ1, α-SMA and TIMP-1 in Liver Tissue
Immunohistochemical analysis was used to detect the expression and distribution of Smad3, TGFβ1, α-SMA and TIMP-1 in liver tissue. As revealed in [Figure 2], there were few Smad3-positive regions in the control group (A1, PI = 0.0067 ± 0.006). In contrast, the expression of Smad3 was significantly increased in the model group, and Smad3-positive regions can be seen around the periportal fibrotic band areas, central vein and fibrous septa (A3, P1 = 0.0563 ± 0.007, P < 0.05), whereas they were sharply down-regulated in the Huisheng oral solution-treated group (A2, PI = 0.0325 ± 0.004, P < 0.05). The expression of TGF-β1, α-SMA and TIMP-1 was consistent with Smad3. (TGF-β1, 0.0055 ± 0.007 vs 0.0545 ± 0.006 vs 0.0344 ± 0.005; α-SMA, 0.0067 ± 0.008 vs 0.0643 ± 0.009 vs 0.0432 ± 0.010; TIMP-1, 0.0023 ± 0.009 vs 0.0531 ± 0.011 vs 0.0441 ± 0.010; P < 0.05, respectively).
|Figure 2: Expressions of Smad3 (A1-3), TGF-β1 (B1-3), α-SMA (C1-3), TIMP-1 (D1-3) in liver tissue (×200). Lane 1, rats in control group; Lane 2: rats in the treatment group (Huisheng oral solution); Lane 3: rats in model group (normal saline+CCl4). Considerable expression of Smad3 protein was observed among the periportal fibrotic areas, central vein, and fibrous septa in model group (A3) compared with normal group (A1). Huisheng oral solution treatment reduced the expression of Smad3 compared with the model group (A2). The expression of TGF-β1, α-SMA and TIMP-1 was consistent with Smad3|
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Effects of Huisheng Oral Solution on the Expression of Smad3, TGFβ1, α-SMA and TIMP-1 in Liver Tissue
To further investigate the molecular mechanism involved in anti-fibrosis effects of Huisheng oral solution, we tested the mRNA and protein expressions of Smad3, TGFβ1, α-SMA and TIMP-1 in liver tissue by RT-PCR and western blot, respectively.
As shown in [Figure 3], the mRNA and protein expressions of Smad3, TGFβ1, α-SMA and TIMP-1 in liver tissue were significantly increased in the model group compared with the control group (P < 0.05, respectively). However, Huisheng oral solution apparently decreased all the above parameters compared with the model group (P < 0.05, respectively). Therefore, Huisheng oral solution could significantly reduce the expression of α-SMA which may be associated with decreased TGFβ1 activation.
|Figure 3: Huisheng oral solution decreased the mRNA (A1, A2) and protein (B1, B2) expressions of Smad3, TGF-β1, α-SMA and TIMP-1 in liver tissue. The mRNA and protein levels of Smad3, TGF-β1, α-SMA and TIMP-1 in liver tissue were showed by qPCR (A1) and western blot (B1), respectively. Their relative expressions were respectively normalized to those of GADPH (A2) and β-actin (B2), respectively. *P<0.05 compared with the control group; *P<0.05 compared with the model group.|
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Active TGFβ1 in Liver Tissue
As shown in [Figure 4], CCl 4 treatment increased the concentration of active TGFβ1 in liver tissue in comparison to the control group, while it was markedly reduced in the Huisheng oral solution-treated group.
|Figure 4: Effects of Huisheng oral solution on active TGF-β1. Active TGF-β1 was detected by ELISA kits and expressed as percentage of total TGF-β. A higher percentage of active TGF-β1 was found in model group than in control group, while it was significantly decreased by Huisheng oral solution. *P<0.05 compared with the control group; *P<0.05 compared with the model group|
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| » Discussion|| |
Liver fibrosis represents a wound-healing response of liver to many liver injures including viral hepatitis, alcohol, copper, drug and immune compounds. Liver fibrosis was considered to be the imbalance of deposition and resolution of ECM in liver. Under physiological conditions, ECM could be degraded by matrix metalloproteinases (MMPs) immediately. Due to the hepatic injury initiated by the compounds, HSCs were activated and transformed into myofibroblast cells to synthesize ECM in liver.  In this process, ECM deposition excesses the resolution of ECM leading to the excessive deposition of ECM proteins in liver.  In addition, activated HSCs could produce many cytokines and ECM in liver, thus further boosting inflammatory process and activating HSCs.  Moreover, activated HSCs could secretly issue an inhibitor of MMP type I (TIMP-1), which could inhibit the activity of MMP, deteriorating the deposition of ECM in liver. 
Accumulated documents demonstrated that liver fibrosis was a chronic inflammation-associated disease, which involved the infiltration of many inflammatory cells and interplay of many cytokines and signal molecules.  Many studies suggested that reducing the release of cytokines and infiltration of inflammatory cells could prevent and reverse liver fibrosis. 
Increasing evidences demonstrated that the TGF-β1/Smad3 signaling pathway plays a pivotal role in the process of fibrosis, such as lung fibrosis, liver fibrosis and so on.  TGF-β1 could be secreted by active HSCs and their cell line, HSC-T6 cells. TGF-β1 could induce the activity of many signaling pathways such as MAPK and Wnt through a Smad3-mediated signaling cascade. These cytokines have been demonstrated to play important roles in liver fibrosis. TGF-β1 also regulates many biological functions including activating HSCs, up-regulating TIMP-1 and reducing the activation of MMPs which leads to the deterioration of liver fibrosis.  Recently, some compounds from Huisheng oral solution have been reported to have numerous pharmaceutical activities through inhibiting TGF-β1 including anti-fibrosis, anti-tumor.  However, there is little known about anti-fibrosis effects of Huisheng oral solution.
The hepatic fibrosis model induced by CCl 4 injection has long been used in scientific research. Thanks to this model, therapeutic effects against liver fibrosis have been greatly improved.  In this study, our data showed that the serum levels of ALT, AST and LDH in rats were significantly reduced by Huisheng oral solution compared to the model group. Histological examination also demonstrated a large number of inflammatory cells infiltrated into the intralobular and interlobular regions, and there were more collagen fibers in CCl 4 -treated rats compared with normal rats. In contrast, Huisheng oral solution treatment remarkably reduced the immigration of inflammatory cells and the deposition of collagen fibers compared with rats in the model group. In addition, Huisheng oral solution also reduced the expression and distribution of TGF-β1 in liver tissue, indicating that Huisheng oral solution attenuated the development of liver fibrosis which may be associated with its inhibitory effects on inflammation by down-regulating the TGF-β1-induced signaling pathway.
Levels of HA and LN in serum and Hyp in liver are the important indices reflecting the degree of liver fibrosis. In this study, CCl 4 -treated rats showed higher levels of HA, LN and Hyp while they were markedly decreased by Huisheng oral solution treatment, suggesting that Huisheng oral solution treatment could attenuate CCl 4 -induced liver fibrosis.
Activated HSCs are the main source of ECM in liver, and α-SMA has also been known as a specific marker of HSCs activation. Our studies indicated that considerable protein expression of α-SMA and TGF-β1 was observed in rat liver in model group compared with control group, while it was significantly reduced by Huisheng oral solution suggesting that anti-fibrosis effects of Huisheng oral solution were related to the inhibition of TGF-β1.
In recent years, TGF-β1 has been reported to play a key role in HSCs activation and liver fibrosis. Its molecular mechanism involved the process of epithelial to mesenchymal transition transition (EMT).  TGF-β1 was considered to be the most stimulus to EMT.  This process was characterized by an increase in the expression of α-SMA. Our data showed that Huisheng oral solution significantly reduced the expression of α-SMA and level of TGF-β1, suggesting that anti-fibrosis effects of Huisheng oral solution are associated with inhibition of TGF-β1-induced EMT.
In addition, it was observed that Huisheng oral solution reduced the gene and protein expressions of α-SMA, Smad3, TGF-β1 and TIMP-1 compared with the model group. These data were consistent with previous results,  suggesting that inhibition of TGF-β1 by Huisheng oral solution leads to the suppression of HSCs activation and liver fibrosis.
In conclusion, we showed that Huisheng oral solution significantly inhibited CCl 4 -induced liver fibrosis, and its anti-fibrosis maybe associated with its inhibitory effects on HSCs activation by down-regulating the TGF-β1/Smad3 signaling pathway. However, the molecular mechanism involved in anti-fibrosis effects of DKK-1 remains to be further explored.
| » Acknowledgments|| |
This work was supported by National Natural Science Foundation of China (No.81271713; No.81001046).
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]