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Protective effect of pilose antler peptide on carbon tetrachloride-induced hepatotoxicity in mice
Accepted Manuscript Title: Protective effect of pilose antler peptide on carbon tetrachloride-induced hepatotoxicity in mice Authors: Ma Chunhua, Long Hongyan PII: DOI: Reference:
S0141-8130(17)30594-9 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.03.024 BIOMAC 7190
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
16-2-2017 3-3-2017 4-3-2017
Please cite this article as: Ma Chunhua, Long Hongyan, Protective effect of pilose antler peptide on carbon tetrachloride-induced hepatotoxicity in mice, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2017.03.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Protective effect of pilose antler peptide on carbon tetrachloride-induced hepatotoxicity in mice Ma Chunhua, Long Hongyan* Central Laboratory, Nanjing Municipal Hospital of T.C.M, The Third Affiliated Hospital of Nanjing University of T.C.M, Nanjing, 210001,China. To whom correspondence should be addressed at [email protected] (H. Long)
Abstract The present study was designed to investigate the beneficial effects of pilose antler peptide (PAP) against carbon tetrachloride (CCl4) hepatotoxicity in mice, and explore the underlying mechanisms. In our results, animals were treated with either CCl4 and/or PAP for six consecutive weeks. The levels of inflammatory cytokines in serum and liver, hepatotoxicity markers in serum and histopathological evaluation were determined. PAP (50, 100 mg/kg) significantly inhibited the CCl4-incuded overproduction of inflammatory cytokines including tumor necrosis factor α (TNF-α), cytokines interleukin-1β (IL-1β), and interleukin-6 (IL-6) in serum and liver. Animals treated with PAP exhibited lower levels of alanine transaminase (ALT) and aspartate transaminase (AST), PAP administration alleviated CCl4 induced hepatic fibrosis as manifested in histopathological records. In fact, PAP prevented the activation of TLR/NF-κB pathway, by inhibiting the expression of TLR2, TLR4 MyD88, p-NF-κBp65 and p-IκBα. In addition, PAP also significantly reversed CCl4-induced alteration of TGF-β and p-samd-3 in liver tissue. In conclusion, PAP restored CCl4-induced hepatotoxicity via TLR/NF-κB and TGF-β/samd-3 pathways.
Keywords: Pilose antler peptide, Carbon tetrachloride hepatotoxicity, Inflammation.
Introduction Liver fibrosis is a major health concern which results in significant morbidity and mortality around the world [1]. The pathogenesis of liver fibrosis is complex and the deposition of extracellular matrix by activated hepatic stellate cells is associated [2]. Plenty of proinflammatory cytokines, chemokines, and molecules were produced during the activation of hepatic stellate cells, and inflammation, is a key event during the activation of hepatic stellate cells. Many inflammatory factors play key pathogenic roles in the activation of hepatic stellate cells, such as tumor necrosis factor α (TNF-α), cytokines interleukin-1β (IL-1β), and interleukin-6 (IL-6) [3]. Also, NF-κB, reactive oxygen species, lipid peroxidation products and multiple growth factors accelerate the stimulation of hepatic stellate cells, which together with accumulation of myofibroblasts indicate the core pathophysiologic convergence point of diverse signaling pathways ultimately resulting in liver fibrosis [4]. Several attention have been paid to link liver fibrosis to molecular targets or pathways critically involved in the process of fibrogenesis. Among these, molecular target such as transforming growth factor beta (TGF-β) is of great importance. TGF-β belongs to gene superfamily of growth factors and is one of the best characterized cytokine of this family with multiple influences on various target organs [5, 6]. It is ubiquitously distributed and has been recognized as one of most powerful pro-fibrogenic mediators. TGF-β promoted the activation of hepatic stellate cells and accumulation of extracellular matrix proteins which contributed to fibrosis [7]. The overproduction of TGF-β stimulates the synthesis and release of various components of extracellular matrix proteins, such as collagen and fibronectin. Also, the fibrogenic cytokine, TGF-β, is closely associated with cirrhosis development as it stimulated the production of extracellular matrix proteins and abolished matrix protein removal [8]. More importantly, the elevation of TGF-β has been observed in patients with liver fibrosis as well as in experimental models of liver fibrosis [9, 10]. Deer antlers, namely‘‘lu rong’’ in China, “nokyong’’ in Korea or “tokujo’’ in Japan, were widely used folk medicines in Asia. These soft growing tissues were applied in traditional Chinese medicine for strengthening kidney, nursing the blood treating neurosis and prolonging life. Deer antlers have been reported to exert a variety of properties, such as anti-inflammatory, anti-stress
and anti-aging effects in previous research. Pilose antler peptide (PAP: MW: 7200; amino acid residue: 68) is isolated from the deer antlers and has been shown beneficial effects on chronic inflammatory and oxidative damages [11, 12]. However, the hepatoprotective action of PAP on CCl4-induced liver injury haven’t been reported before. The present study was designed to
investigate the beneficial effects of PAP against carbon tetrachloride hepatotoxicity in mice.
Materials and Methods Reagents CCl4 purchased from Sigma (St. Louis, MO, USA). All primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The horseradish peroxidase-conjugated anti-rabbit antibody was supplied by Bioworld Technology Inc. (MN, USA). TNF-α, IL-6 and IL-1β enzyme-linked immuno-sorbent assay (ELISA) kits were supplied by Nanjing Key GEN Biotech. Co., Ltd. (Nanjing, China). AST and ALT kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China).
Preparation of PAP
Cornu Corvi nippon parvum were provided and authenticated by a pharmacognosist, Mingjian Qin, in China Pharmaceutical University. The PAP preparation as follow: 10g Cornu Corvi nippon parvum were powdered, soaked with 500 mL of Na2HPO4-NaOH (PH=12, 50 mmol/L
EDTA,0.5 mol/L NaCl) and refluxed two times for 4h, and then it was centrifuged at 4000 r/min, 20min, supernatant was added ethanol (-20 ℃) for 30min, the it was centrifuged to obtain residue, The residue was added trypsin (PH=9) for 4h (37℃) and then the PAP (PAP: MW: 7200; amino acid residue: 68) was obtained, the PAP powders were dissolved in sterilized distilled water before administration to the experimental animals.
Animals 50 male ICR mice (8–12 weeks old) were obtained from Jiangning Qinglongshan Animal Cultivation Farm (Nanjing, China). Water and food, supplied in special steel containers, were provided ad libitum. During the study, rats were kept in an air-conditioned room at 23 ± 2 ℃ with a 12-h light/dark cycle. All the experimental procedures were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Experimental Design The mice were randomly divided into five groups (n = 10): the control group, the CCl4 group, the CCl4 and PAP (50 mg/kg) group, the CCl4 and PAP (100 mg/kg) group,CCl4 and silymarin (100 mg/kg) group. Hepatic fibrosis was induced by subcutaneous injection of carbon tetrachloride (1:1 in olive oil) in rats at a dose of 3 ml/kg twice-weekly for consecutive 6 weeks. The rats in the control group were injected with an equal volume of olive oil without CCl4. When the hepatic fibrosis model was established and identified, the mice in the PAP groups intragastrically received 100, 200 mg/kg PAP and 100 mg/kg silymarin dissolved in normal saline daily for consecutive 4 weeks. Accordingly, mice in the control and model groups received an equal volume of normal saline.
Measurement of AST and ALT activities
The activities of AST and ALT in serum in mice were measured by corresponding commercial kits (Jiancheng Bioengineering Institute, Nanjing, China).
Cytokine measurements
The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in serum and liverin mice were determined on the basis of enzyme immunoassays by ELISA kit in accordance with the instruction of the manufacturers. Histopathological measurement
Liver tissues were excised from the left lobe of livers. The liver specimens were fixed with 10 % neutral formalin and embedded in paraffin blocks. Then the paraffin-embedded liver tissues were cut into 4-lm sections with a microtome (Leica, Nussloch, Germany) and stained with hematoxylin and eosin (H&E) (Sigma, Korea) for photomicroscopic assessment (2009 magnification). Liver inflammatory cell count based on a five point scoring system was performed to estimate the severity of leukocyte infiltration. The scoring system was: 0: no cells, 1: a few cells, 2: a ring of cells with 1 cell layer deep, 3: a ring of cells with 2–4 cell layers deep; and 4: a ring of cells with more than 4 cell layers deep. Masson stain was performed to confirm the CCl4-induced hepatic fibrosis evidenced by fiber extension and collagen accumulation. The scoring system hepatic fibrosis was: 0: no, 1-2: a few of fibrosis, 3-4: a large of fibrosis, 4-5: serious fibrosis; and 6: very serious fibrosis
Western blot analysis
The liver tissues in each group were chopped into small pieces and extracted by RIPA buffer (RIPA with 0.1% phenylmethylsulfonyl fluoride) on ice. Then, the samples were centrifuged at 12,000 rpm for 10 min at 4 ℃. A BCA protein assay kit was chose to measure the concentration of total protein of liver tissues lysates. The samples of liver tissues lysates were isolated through SDS-polyacrylamide gel electrophoresis and then transferred on the PVDF membranes. After incubated with the specific antibodies (1:1000) overnight at 4 ℃, the membranes were exposure into the secondary antibodies for 2 h. The blots were washed for 3 times by TBST and analysed for quantification. Statistical analysis All of the data in this experiment was presented as mean ± standard error. Divergence between groups were compared by one-way ANOVA with Tukey multiple comparison test. P < 0.05 was considered as significant.
Results Effect of PAP on serum biomarkers To detect liver fibrosis markers, the levels of aminotransferase (AST) and alanine aminotransferase (ALT) in serum were measured. As illustrated in Fig. 1, the serum levels of AST and ALT in CCl4-treated were significantly higher than those of the control group. PAP (50, 100 mg/kg) or silymarin (100 mg/kg) treatment group obviously reduced the serum AST and ALT as compared to the model group (Fig. 1).
Effects of PAP on inflammatory cytokines in serum and liver Inflammatory reaction is one of the major features in during CCl4-induced liver fibrosis. To evaluate whether PAP affect the inflammatory responses during liver damage, the levels of TNF-α, IL-6 and IL-1β in serum and liver were assessed. It was observed that there were significant increases of TNF-α, IL-6 and IL-1β in model group both in serum and liver. To the respective, the contents of TNF-α, IL-6 and IL-1β were effectively inhibited in PAP (50, 100 mg/kg) or silymarin (100 mg/kg) treated group compared with those in CCl4 group mice. (Fig.2).
Effect of PAP on liver histology As illustrated in Fig. 3, liver sections obtained from control group depicted normal architecture of hepatic central vein and surrounding hepatocytes. However, samples taken from CCl4-treated mice group associated ballooning degeneration and fatty change in the hepatocytes related to strands of fibrous connective tissue and severe inflammatory cells infiltration. Nevertheless, PAP (50, 100 mg/kg) or silymarin (100 mg/kg) treatment effectively reversed the hepatic architecture with mild ballooning degeneration and few inflammatory cells infiltration (Fig.3).
Effects of PAP on inflammation-related pathways in CCl4-induced liver As shown in Fig.4, the expressions of TLR2, TLR4 MyD88, p-NF-κBp65 and p-IκBα were significantly up-regulated in liver tissues after CCl4 stimulation. However, treatment with PAP (50, 100 mg/kg) or silymarin (100 mg/kg) obviously ameliorated these situations. Liver injury markedly up-regulated the expression of TGF-β and p-samd-3 in liver tissue. On the other hand,
treatment with PAP (50, 100 mg/kg) or silymarin (100 mg/kg) inhibited the protein levels of TGF-β and p-samd-3 compared with those in model group (Fig.4).
Discussion Liver fibrosis is a major health concern which results in significant morbidity and mortality around the world. Livers respond to injuries in a systematic manner, initially removing injured tissue through invading inflammatory cells and then synthesizing matrix proteins in an organized manner associated with tissue remodeling and regeneration. Attention has been focused on towards the emerging signaling pathways underlying fibrosis progression develop new therapies. Recent studies have shed the light on the inflammatory related pathways as promising therapeutic target for liver fibrosis. In our present study, we found PAP prevented the activation of TLR/NF-κB and inhibited the expression of TGF-β/samd-3 in liver tissue. CCl4 has been widely adopted for experimental induction of hepatic fibrosis [13]. CCl4 produced the overproduction of free radicals which caused lipid peroxidation of the hepatocellular membrane, followed by the release of inflammatory cytokines and hepatocellular damage [14]. In our present study, the hepatic fibrosis was evidenced by the elevation of serum ALT, AST, inflammatory cytokines, and damaged liver functions. PAP (50, 100 mg/kg) significantly attenuated these hepatic alterations demonstrating restored hepatocellular architecture and improved hepatocytes functions. One important etiology of liver fibrosis is excessive accumulation of ECM components especially collagen [15]. Chronic CCl4 treatment produced liver fibrosis and collagen deposition as manifested by Masson’s trichrome staining of collagen fibers. The present study manifested that PAP (50, 100 mg/kg) exhibited a potent antifibrotic activity which abolished CCl4-induced fibrogenesis. This was further confirmed by the significant reduction of liver collagen deposition observed in Masson’s stain. Inflammatory process, initiated by kupffer cell activation, is a main event involved in the fibrogenic process [16]. Upon liver damages, NF-κB and its downstream signaling are stimulated in both kupffer cells and HSCs. As a transcription factor, NF-κB up-regulates the release of multiple inflammatory cytokines including TNF-α, IL-6 and IL-1β. TNF-α, IL-6 and IL-1β further perpetuates the inflammatory cascades. In addition, NF-κB signaling contributes to the activation
and survival of HSCs [17]. The activation of NF-κB is controlled by the degradation of the IκBα . Consistent with previous studies, our study showed that CCl4 increased the concentration of TNF-α, IL-6 and IL-1β, and expression of p-NF-κB and p-IκBα, thus, worsening hepatic inflammation and fibrosis [18]. TGF-β is an important mediator during HSC activation and ECM accumulation which leads to liver fibrosis. TGF-β promoted the activation of hepatic stellate cells and accumulation of extracellular matrix proteins which contributed to fibrosis. The overproduction of TGF-β stimulates the synthesis and release of various components of extracellular matrix proteins, such as collagen and fibronectin. Upon activation, TGF-β binds to type I and type II serine/threonine kinase receptors, a heteromeric complex, which activate receptor-regulated Smads including Smad3 [19]. In fact, nuclear translocation of activated Smads is a fundamental process for manipulating TGF-β-dependent genes [20]. TGF-β receptors also interact with Smad-independent pathways and regulate Smad signaling as well as Smad independent TGF-β responses [21]. Studies have shown that TGF-β/Smad signaling pathway inactivation may serve as a promising therapeutic target against hepatic fibrosis. The present study manifested that increased expression of TGF-β and phospho-Smad3 were observed in CCl4-treated mice. PAP (50, 100 mg/kg) significantly inhibited phosphorylation of Smad3 and activation of TGF-β. Thus, our results demonstrated the hepatoprotective action of PAP against carbon tetrachloride hepatotoxicity in mice, and explored the possibly mechanisms, in conclusion. Further research are needed for more details in the future.
Conflict of interest All authors have no conflict of interest
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Figure 1 Effect of PAP on serum AST and ALT Values are expressed as mean ± SD. Compared with control: #P<0.01, #P<0.01; Compared with model: *p< 0.05, **p< 0.01. Figure 2 Effects of PAP on inflammatory cytokines in serum and liver Values are expressed as mean ± SD. Compared with control: #P<0.01, #P<0.01; Compared with model: *p< 0.05, **p< 0.01. Figure 3 Effect of PAP on liver HE stain(A,x200) and masson stain(B,x200) Figure 4 Effects of PAP on inflammation-related pathways in CCl4-induced liver Values are expressed as mean ± SD. Compared with control: #P<0.01, #P<0.01; Compared with model: *p< 0.05, **p< 0.01.
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S0141-8130(17)30594-9 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.03.024 BIOMAC 7190
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
16-2-2017 3-3-2017 4-3-2017
Please cite this article as: Ma Chunhua, Long Hongyan, Protective effect of pilose antler peptide on carbon tetrachloride-induced hepatotoxicity in mice, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2017.03.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Protective effect of pilose antler peptide on carbon tetrachloride-induced hepatotoxicity in mice Ma Chunhua, Long Hongyan* Central Laboratory, Nanjing Municipal Hospital of T.C.M, The Third Affiliated Hospital of Nanjing University of T.C.M, Nanjing, 210001,China. To whom correspondence should be addressed at [email protected] (H. Long)
Abstract The present study was designed to investigate the beneficial effects of pilose antler peptide (PAP) against carbon tetrachloride (CCl4) hepatotoxicity in mice, and explore the underlying mechanisms. In our results, animals were treated with either CCl4 and/or PAP for six consecutive weeks. The levels of inflammatory cytokines in serum and liver, hepatotoxicity markers in serum and histopathological evaluation were determined. PAP (50, 100 mg/kg) significantly inhibited the CCl4-incuded overproduction of inflammatory cytokines including tumor necrosis factor α (TNF-α), cytokines interleukin-1β (IL-1β), and interleukin-6 (IL-6) in serum and liver. Animals treated with PAP exhibited lower levels of alanine transaminase (ALT) and aspartate transaminase (AST), PAP administration alleviated CCl4 induced hepatic fibrosis as manifested in histopathological records. In fact, PAP prevented the activation of TLR/NF-κB pathway, by inhibiting the expression of TLR2, TLR4 MyD88, p-NF-κBp65 and p-IκBα. In addition, PAP also significantly reversed CCl4-induced alteration of TGF-β and p-samd-3 in liver tissue. In conclusion, PAP restored CCl4-induced hepatotoxicity via TLR/NF-κB and TGF-β/samd-3 pathways.
Keywords: Pilose antler peptide, Carbon tetrachloride hepatotoxicity, Inflammation.
Introduction Liver fibrosis is a major health concern which results in significant morbidity and mortality around the world [1]. The pathogenesis of liver fibrosis is complex and the deposition of extracellular matrix by activated hepatic stellate cells is associated [2]. Plenty of proinflammatory cytokines, chemokines, and molecules were produced during the activation of hepatic stellate cells, and inflammation, is a key event during the activation of hepatic stellate cells. Many inflammatory factors play key pathogenic roles in the activation of hepatic stellate cells, such as tumor necrosis factor α (TNF-α), cytokines interleukin-1β (IL-1β), and interleukin-6 (IL-6) [3]. Also, NF-κB, reactive oxygen species, lipid peroxidation products and multiple growth factors accelerate the stimulation of hepatic stellate cells, which together with accumulation of myofibroblasts indicate the core pathophysiologic convergence point of diverse signaling pathways ultimately resulting in liver fibrosis [4]. Several attention have been paid to link liver fibrosis to molecular targets or pathways critically involved in the process of fibrogenesis. Among these, molecular target such as transforming growth factor beta (TGF-β) is of great importance. TGF-β belongs to gene superfamily of growth factors and is one of the best characterized cytokine of this family with multiple influences on various target organs [5, 6]. It is ubiquitously distributed and has been recognized as one of most powerful pro-fibrogenic mediators. TGF-β promoted the activation of hepatic stellate cells and accumulation of extracellular matrix proteins which contributed to fibrosis [7]. The overproduction of TGF-β stimulates the synthesis and release of various components of extracellular matrix proteins, such as collagen and fibronectin. Also, the fibrogenic cytokine, TGF-β, is closely associated with cirrhosis development as it stimulated the production of extracellular matrix proteins and abolished matrix protein removal [8]. More importantly, the elevation of TGF-β has been observed in patients with liver fibrosis as well as in experimental models of liver fibrosis [9, 10]. Deer antlers, namely‘‘lu rong’’ in China, “nokyong’’ in Korea or “tokujo’’ in Japan, were widely used folk medicines in Asia. These soft growing tissues were applied in traditional Chinese medicine for strengthening kidney, nursing the blood treating neurosis and prolonging life. Deer antlers have been reported to exert a variety of properties, such as anti-inflammatory, anti-stress
and anti-aging effects in previous research. Pilose antler peptide (PAP: MW: 7200; amino acid residue: 68) is isolated from the deer antlers and has been shown beneficial effects on chronic inflammatory and oxidative damages [11, 12]. However, the hepatoprotective action of PAP on CCl4-induced liver injury haven’t been reported before. The present study was designed to
investigate the beneficial effects of PAP against carbon tetrachloride hepatotoxicity in mice.
Materials and Methods Reagents CCl4 purchased from Sigma (St. Louis, MO, USA). All primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The horseradish peroxidase-conjugated anti-rabbit antibody was supplied by Bioworld Technology Inc. (MN, USA). TNF-α, IL-6 and IL-1β enzyme-linked immuno-sorbent assay (ELISA) kits were supplied by Nanjing Key GEN Biotech. Co., Ltd. (Nanjing, China). AST and ALT kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China).
Preparation of PAP
Cornu Corvi nippon parvum were provided and authenticated by a pharmacognosist, Mingjian Qin, in China Pharmaceutical University. The PAP preparation as follow: 10g Cornu Corvi nippon parvum were powdered, soaked with 500 mL of Na2HPO4-NaOH (PH=12, 50 mmol/L
EDTA,0.5 mol/L NaCl) and refluxed two times for 4h, and then it was centrifuged at 4000 r/min, 20min, supernatant was added ethanol (-20 ℃) for 30min, the it was centrifuged to obtain residue, The residue was added trypsin (PH=9) for 4h (37℃) and then the PAP (PAP: MW: 7200; amino acid residue: 68) was obtained, the PAP powders were dissolved in sterilized distilled water before administration to the experimental animals.
Animals 50 male ICR mice (8–12 weeks old) were obtained from Jiangning Qinglongshan Animal Cultivation Farm (Nanjing, China). Water and food, supplied in special steel containers, were provided ad libitum. During the study, rats were kept in an air-conditioned room at 23 ± 2 ℃ with a 12-h light/dark cycle. All the experimental procedures were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Experimental Design The mice were randomly divided into five groups (n = 10): the control group, the CCl4 group, the CCl4 and PAP (50 mg/kg) group, the CCl4 and PAP (100 mg/kg) group,CCl4 and silymarin (100 mg/kg) group. Hepatic fibrosis was induced by subcutaneous injection of carbon tetrachloride (1:1 in olive oil) in rats at a dose of 3 ml/kg twice-weekly for consecutive 6 weeks. The rats in the control group were injected with an equal volume of olive oil without CCl4. When the hepatic fibrosis model was established and identified, the mice in the PAP groups intragastrically received 100, 200 mg/kg PAP and 100 mg/kg silymarin dissolved in normal saline daily for consecutive 4 weeks. Accordingly, mice in the control and model groups received an equal volume of normal saline.
Measurement of AST and ALT activities
The activities of AST and ALT in serum in mice were measured by corresponding commercial kits (Jiancheng Bioengineering Institute, Nanjing, China).
Cytokine measurements
The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in serum and liverin mice were determined on the basis of enzyme immunoassays by ELISA kit in accordance with the instruction of the manufacturers. Histopathological measurement
Liver tissues were excised from the left lobe of livers. The liver specimens were fixed with 10 % neutral formalin and embedded in paraffin blocks. Then the paraffin-embedded liver tissues were cut into 4-lm sections with a microtome (Leica, Nussloch, Germany) and stained with hematoxylin and eosin (H&E) (Sigma, Korea) for photomicroscopic assessment (2009 magnification). Liver inflammatory cell count based on a five point scoring system was performed to estimate the severity of leukocyte infiltration. The scoring system was: 0: no cells, 1: a few cells, 2: a ring of cells with 1 cell layer deep, 3: a ring of cells with 2–4 cell layers deep; and 4: a ring of cells with more than 4 cell layers deep. Masson stain was performed to confirm the CCl4-induced hepatic fibrosis evidenced by fiber extension and collagen accumulation. The scoring system hepatic fibrosis was: 0: no, 1-2: a few of fibrosis, 3-4: a large of fibrosis, 4-5: serious fibrosis; and 6: very serious fibrosis
Western blot analysis
The liver tissues in each group were chopped into small pieces and extracted by RIPA buffer (RIPA with 0.1% phenylmethylsulfonyl fluoride) on ice. Then, the samples were centrifuged at 12,000 rpm for 10 min at 4 ℃. A BCA protein assay kit was chose to measure the concentration of total protein of liver tissues lysates. The samples of liver tissues lysates were isolated through SDS-polyacrylamide gel electrophoresis and then transferred on the PVDF membranes. After incubated with the specific antibodies (1:1000) overnight at 4 ℃, the membranes were exposure into the secondary antibodies for 2 h. The blots were washed for 3 times by TBST and analysed for quantification. Statistical analysis All of the data in this experiment was presented as mean ± standard error. Divergence between groups were compared by one-way ANOVA with Tukey multiple comparison test. P < 0.05 was considered as significant.
Results Effect of PAP on serum biomarkers To detect liver fibrosis markers, the levels of aminotransferase (AST) and alanine aminotransferase (ALT) in serum were measured. As illustrated in Fig. 1, the serum levels of AST and ALT in CCl4-treated were significantly higher than those of the control group. PAP (50, 100 mg/kg) or silymarin (100 mg/kg) treatment group obviously reduced the serum AST and ALT as compared to the model group (Fig. 1).
Effects of PAP on inflammatory cytokines in serum and liver Inflammatory reaction is one of the major features in during CCl4-induced liver fibrosis. To evaluate whether PAP affect the inflammatory responses during liver damage, the levels of TNF-α, IL-6 and IL-1β in serum and liver were assessed. It was observed that there were significant increases of TNF-α, IL-6 and IL-1β in model group both in serum and liver. To the respective, the contents of TNF-α, IL-6 and IL-1β were effectively inhibited in PAP (50, 100 mg/kg) or silymarin (100 mg/kg) treated group compared with those in CCl4 group mice. (Fig.2).
Effect of PAP on liver histology As illustrated in Fig. 3, liver sections obtained from control group depicted normal architecture of hepatic central vein and surrounding hepatocytes. However, samples taken from CCl4-treated mice group associated ballooning degeneration and fatty change in the hepatocytes related to strands of fibrous connective tissue and severe inflammatory cells infiltration. Nevertheless, PAP (50, 100 mg/kg) or silymarin (100 mg/kg) treatment effectively reversed the hepatic architecture with mild ballooning degeneration and few inflammatory cells infiltration (Fig.3).
Effects of PAP on inflammation-related pathways in CCl4-induced liver As shown in Fig.4, the expressions of TLR2, TLR4 MyD88, p-NF-κBp65 and p-IκBα were significantly up-regulated in liver tissues after CCl4 stimulation. However, treatment with PAP (50, 100 mg/kg) or silymarin (100 mg/kg) obviously ameliorated these situations. Liver injury markedly up-regulated the expression of TGF-β and p-samd-3 in liver tissue. On the other hand,
treatment with PAP (50, 100 mg/kg) or silymarin (100 mg/kg) inhibited the protein levels of TGF-β and p-samd-3 compared with those in model group (Fig.4).
Discussion Liver fibrosis is a major health concern which results in significant morbidity and mortality around the world. Livers respond to injuries in a systematic manner, initially removing injured tissue through invading inflammatory cells and then synthesizing matrix proteins in an organized manner associated with tissue remodeling and regeneration. Attention has been focused on towards the emerging signaling pathways underlying fibrosis progression develop new therapies. Recent studies have shed the light on the inflammatory related pathways as promising therapeutic target for liver fibrosis. In our present study, we found PAP prevented the activation of TLR/NF-κB and inhibited the expression of TGF-β/samd-3 in liver tissue. CCl4 has been widely adopted for experimental induction of hepatic fibrosis [13]. CCl4 produced the overproduction of free radicals which caused lipid peroxidation of the hepatocellular membrane, followed by the release of inflammatory cytokines and hepatocellular damage [14]. In our present study, the hepatic fibrosis was evidenced by the elevation of serum ALT, AST, inflammatory cytokines, and damaged liver functions. PAP (50, 100 mg/kg) significantly attenuated these hepatic alterations demonstrating restored hepatocellular architecture and improved hepatocytes functions. One important etiology of liver fibrosis is excessive accumulation of ECM components especially collagen [15]. Chronic CCl4 treatment produced liver fibrosis and collagen deposition as manifested by Masson’s trichrome staining of collagen fibers. The present study manifested that PAP (50, 100 mg/kg) exhibited a potent antifibrotic activity which abolished CCl4-induced fibrogenesis. This was further confirmed by the significant reduction of liver collagen deposition observed in Masson’s stain. Inflammatory process, initiated by kupffer cell activation, is a main event involved in the fibrogenic process [16]. Upon liver damages, NF-κB and its downstream signaling are stimulated in both kupffer cells and HSCs. As a transcription factor, NF-κB up-regulates the release of multiple inflammatory cytokines including TNF-α, IL-6 and IL-1β. TNF-α, IL-6 and IL-1β further perpetuates the inflammatory cascades. In addition, NF-κB signaling contributes to the activation
and survival of HSCs [17]. The activation of NF-κB is controlled by the degradation of the IκBα . Consistent with previous studies, our study showed that CCl4 increased the concentration of TNF-α, IL-6 and IL-1β, and expression of p-NF-κB and p-IκBα, thus, worsening hepatic inflammation and fibrosis [18]. TGF-β is an important mediator during HSC activation and ECM accumulation which leads to liver fibrosis. TGF-β promoted the activation of hepatic stellate cells and accumulation of extracellular matrix proteins which contributed to fibrosis. The overproduction of TGF-β stimulates the synthesis and release of various components of extracellular matrix proteins, such as collagen and fibronectin. Upon activation, TGF-β binds to type I and type II serine/threonine kinase receptors, a heteromeric complex, which activate receptor-regulated Smads including Smad3 [19]. In fact, nuclear translocation of activated Smads is a fundamental process for manipulating TGF-β-dependent genes [20]. TGF-β receptors also interact with Smad-independent pathways and regulate Smad signaling as well as Smad independent TGF-β responses [21]. Studies have shown that TGF-β/Smad signaling pathway inactivation may serve as a promising therapeutic target against hepatic fibrosis. The present study manifested that increased expression of TGF-β and phospho-Smad3 were observed in CCl4-treated mice. PAP (50, 100 mg/kg) significantly inhibited phosphorylation of Smad3 and activation of TGF-β. Thus, our results demonstrated the hepatoprotective action of PAP against carbon tetrachloride hepatotoxicity in mice, and explored the possibly mechanisms, in conclusion. Further research are needed for more details in the future.
Conflict of interest All authors have no conflict of interest
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Figure 1 Effect of PAP on serum AST and ALT Values are expressed as mean ± SD. Compared with control: #P<0.01, #P<0.01; Compared with model: *p< 0.05, **p< 0.01. Figure 2 Effects of PAP on inflammatory cytokines in serum and liver Values are expressed as mean ± SD. Compared with control: #P<0.01, #P<0.01; Compared with model: *p< 0.05, **p< 0.01. Figure 3 Effect of PAP on liver HE stain(A,x200) and masson stain(B,x200) Figure 4 Effects of PAP on inflammation-related pathways in CCl4-induced liver Values are expressed as mean ± SD. Compared with control: #P<0.01, #P<0.01; Compared with model: *p< 0.05, **p< 0.01.
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