transforming-growth-factor-beta and Carbon-Tetrachloride-Poisoning

Sustained activation of hepatic stellate cells (HSCs) leads to hepatic fibrosis, which is characterized by excessive collagen production, and for which there is no available drug clinically. Despite tremendous progress, the cellular activities underlying HSC activation, especially the driving force in the perpetuation stage, are only partially understood. Recently, microRNA-21 (miR-21) has been found to be prevalently up-regulated during fibrogenesis in different tissues, although its detailed role needs to be further elucidated. In the present study, miR-21 expression was examined in human cirrhotic liver samples and in murine fibrotic livers induced by thioacetamide or carbon tetrachloride. A dramatic miR-21 increase was noted in activated HSCs. We further found that miR-21 maintained itself at constant high levels by using a microRNA-21/programmed cell death protein 4/activation protein-1 (miR-21/PDCD4/AP-1) feedback loop. Disrupting this loop with miR-21 antagomir or AP-1 inhibitors significantly suppressed fibrogenic activities in HSCs and ameliorated liver fibrosis. In contrast, reinforcing this loop with small interfering RNA (siRNA) against PDCD4 promoted fibrogenesis in HSCs. Further analysis indicated that the up-regulated miR-21 promoted the central transforming growth factor-β (TGF-β) signaling pathway underlying HSC activation. In summary, we suggest that the miR-21/PDCD4/AP-1 autoregulatory loop is one of the main driving forces for hepatic fibrosis progression. Targeting this aberrantly activated feedback loop may provide a new therapeutic strategy and facilitate drug discovery against hepatic fibrosis.

Topics: Adult; Aged; Animals; Apoptosis Regulatory Proteins; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Female; Hepatic Stellate Cells; Humans; Liver Cirrhosis, Experimental; Male; Mice; Mice, Inbred ICR; MicroRNAs; Middle Aged; RNA-Binding Proteins; Signal Transduction; Thioacetamide; Transcription Factor AP-1; Transforming Growth Factor beta

The in vivo antifibrotic effect of a fucoidan extract (FE) from Sargassum fluitans Borgesen was evaluated in a carbon tetrachloride-induced liver damage model in rats over twelve weeks. Chemical analysis showed the FE to contain carbohydrates, sulfates, uronic acids, protein, phenols, and to have a molecular weight of ~60 kDa. Physiological, biochemical, histological and genetic assays were done. Daily oral administration of FE (50 mg/kg) reduced liver enzymatic activity, liver infiltration of inflammatory cells, collagen fiber deposition and gene expression cytokines such as interleukin beta 1 (IL-β1), tumor necrosis factor alpha (TNF-α), transforming growth factor beta 1 (TGF-β1), Smad-3, Smad-2, collagen 1 alpha 1 (col1α1) and tissue inhibitor of metalloproteinase 1 (TIMP-1). It also increased RNA expression of Smad-7 and metalloproteinase 2 and 9 (MMP2 and MMP9). The fucoidan extract exhibited an antifibrotic effect mediated by the inhibiting TGF-β1/Smad pathway, as well as anti-inflammatory effects.

Topics: Animals; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Disease Models, Animal; Gene Expression Regulation; Humans; Liver; Liver Cirrhosis; Plant Extracts; Polysaccharides; Rats; Sargassum; Signal Transduction; Smad Proteins; Smad3 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

Schisandrin B (Sch B) and miR-101 family members play critical roles in the pathogenesis of liver fibrosis. However, the relationship between them has not been reported yet. Thus, this study aims to fill this research gap. Results showed that Sch B significantly upregulated the expression of miR-101-5p in HSC-T6 cells. Sch B also increased the expression of miR-101-5p by combined administration of TGF-β1 and Sch B. Using miR-101-5p inhibitor, we demonstrated that Sch B can target miR-101-5p through the TGF-β signalling pathway to regulate the proliferation and activation of HSC-T6 cells. A rat model of carbon tetrachloride-induced liver fibrosis was established, and results indicated that Sch B can attenuate liver fibrosis by upregulating the expression of miR-101-5p. In conclusion, Sch B can directly target miR-101 to suppress liver fibrosis. Sch B or miR-101-5p may be used as a therapeutic approach for the prevention and treatment of liver fibrosis.

Topics: Animals; Carbon Tetrachloride Poisoning; Cyclooctanes; Humans; Lignans; Liver Cirrhosis; Male; MicroRNAs; Polycyclic Compounds; Rats; Rats, Sprague-Dawley; Signal Transduction; Transforming Growth Factor beta

Non-alcoholic steatohepatitis (NASH) is characterized by lipotoxicity, inflammation and fibrosis, ultimately leading to end-stage liver disease. The molecular mechanisms promoting NASH are poorly understood, and treatment options are limited. Here, we demonstrate that hepatic expression of bone morphogenetic protein 8B (BMP8B), a member of the transforming growth factor beta (TGFβ)-BMP superfamily, increases proportionally to disease stage in people and animal models with NASH. BMP8B signals via both SMAD2/3 and SMAD1/5/9 branches of the TGFβ-BMP pathway in hepatic stellate cells (HSCs), promoting their proinflammatory phenotype. In vivo, the absence of BMP8B prevents HSC activation, reduces inflammation and affects the wound-healing responses, thereby limiting NASH progression. Evidence is featured in primary human 3D microtissues modelling NASH, when challenged with recombinant BMP8. Our data show that BMP8B is a major contributor to NASH progression. Owing to the near absence of BMP8B in healthy livers, inhibition of BMP8B may represent a promising new therapeutic avenue for NASH treatment.

Topics: Animals; Bone Morphogenetic Proteins; Carbon Tetrachloride Poisoning; Diet, High-Fat; Diet, Western; Hepatic Stellate Cells; Humans; Inflammation; Liver Regeneration; Mice; Mice, Inbred C57BL; Non-alcoholic Fatty Liver Disease; Recombinant Proteins; Smad Proteins; Transforming Growth Factor beta; Wound Healing

Liver exhibits a remarkable maintenance of functional homeostasis in the presence of a variety of damaging toxic factors. Tissue regeneration involves cell replenishment and extracellular matrix remodeling. Key regulator of homeostasis is the transforming growth factor-β (TGFβ) cytokine. To understand the role of TGFβ during liver regeneration, we used the single-dose carbon tetrachloride (CCl4) treatment in mice as a model of acute liver damage. We combined this with in vivo inhibition of the TGFβ pathway by a small molecule inhibitor, LY364947, which targets the TGFβ type I receptor kinase [activin receptor-like kinase 5 (ALK5)] in hepatocytes but not in activated stellate cells. Co-administration of LY364947 inhibitor and CCl4 toxic agent resulted in enhanced liver regeneration; cell proliferation (measured by PCNA, phosphorylated histone 3, p21) levels were increased in CCl4 + LY364947 versus CCl4-treated mice. Recovery of CCl4-metabolizing enzyme CYP2E1 expression in hepatocytes is enhanced 7 days after CCl4 intoxication in the mice that received also the TGFβ inhibitor. In summary, a small molecule inhibitor that blocks ALK5 downstream signaling and halts the cytostatic role of TGFβ pathway results in increased cell regeneration and improved liver function during acute liver damage. Thus, in vivo ALK5 modulation offers insight into the role of TGFβ, not only in matrix remodeling and fibrosis, but also in cell regeneration.

Topics: Animals; Biomarkers; Carbon Tetrachloride Poisoning; Hepatic Stellate Cells; Hepatocytes; Liver; Liver Regeneration; Male; Mice, Inbred C57BL; Protein Serine-Threonine Kinases; Pyrazoles; Pyrroles; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Here we evaluated the ability of L-theanine in preventing experimental hepatic cirrhosis and investigated the roles of nuclear factor-κB (NF-κB) activation as well as transforming growth factor β (TGF-β) and connective tissue growth factor (CTGF) regulation. Experimental hepatic cirrhosis was established by the administration of carbon tetrachloride (CCl4) to rats (0.4 g/kg, intraperitoneally, three times per week, for 8 weeks), and at the same time, adding L-theanine (8.0 mg/kg) to the drinking water. Rats had ad libitum access to water and food throughout the treatment period. CCl4 treatment promoted NF-κB activation and increased the expression of both TGF-β and CTGF. CCl4 increased the serum activities of alanine aminotransferase and γ-glutamyl transpeptidase and the degree of lipid peroxidation, and it also induced a decrease in the glutathione and glutathione disulfide ratio. L-Theanine prevented increased expression of NF-κB and down-regulated the pro-inflammatory (interleukin (IL)-1β and IL-6) and profibrotic (TGF-β and CTGF) cytokines. Furthermore, the levels of messenger RNA encoding these proteins decreased in agreement with the expression levels. L-Theanine promoted the expression of the anti-inflammatory cytokine IL-10 and the fibrolytic enzyme metalloproteinase-13. Liver hydroxyproline contents and histopathological analysis demonstrated the anti-fibrotic effect of l-theanine. In conclusion, L-theanine prevents CCl4-induced experimental hepatic cirrhosis in rats by blocking the main pro-inflammatory and pro-fibrogenic signals.

Topics: Alanine Transaminase; Animals; Antioxidants; Aspartate Aminotransferases; Carbon Tetrachloride Poisoning; Chemical and Drug Induced Liver Injury; Connective Tissue Growth Factor; Cytokines; Down-Regulation; Glutamates; Lipid Peroxidation; Liver Cirrhosis; Male; Matrix Metalloproteinase 13; NF-kappa B; Rats; Rats, Wistar; Transforming Growth Factor beta

Mesothelial cells (MCs) form a single layer of the mesothelium and cover the liver surface. A previous study demonstrated that, upon liver injury, MCs migrate inward from the liver surface and give rise to hepatic stellate cells (HSCs) in biliary fibrosis induced by bile duct ligation (BDL) or myofibroblasts in CCl4-induced fibrosis. The present study analyzed the role of transforming growth factor-β (TGF-β) signaling in mesothelial-mesenchymal transition (MMT) and the fate of MCs during liver fibrosis and its regression. Deletion of TGF-β type II receptor (Tgfbr2) gene in cultured MCs suppressed TGF-β-mediated myofibroblastic conversion. Conditional deletion of Tgfbr2 gene in MCs reduced the differentiation of MCs to HSCs and myofibroblasts in the BDL and CCl4 models, respectively, indicating that the direct TGF-β signaling in MCs is responsible to MMT. After BDL and CCl4 treatment, MC-derived HSCs and myofibroblasts were distributed near the liver surface and the thickness of collagen was increased in Glisson's capsule beneath the liver surface. Fluorescence-activated cell sorting analysis revealed that MC-derived HSCs and myofibroblasts store little vitamin A lipids and have fibrogenic phenotype in the fibrotic livers. MCs contributed to 1.4 and 2.0% of activated HSCs in the BDL and CCl4 models, respectively. During regression of CCl4-induced fibrosis, 20% of MC-derived myofibroblasts survived in the liver and deactivated to vitamin A-poor HSCs. Our data indicate that MCs participate in capsular fibrosis by supplying vitamin A-poor HSCs during a process of liver fibrosis and regression.

Topics: Animals; Bile Ducts; Carbon Tetrachloride Poisoning; Cell Differentiation; Cells, Cultured; Epithelial-Mesenchymal Transition; Epithelium; Fibroblasts; Hepatic Stellate Cells; Ligation; Liver; Liver Cirrhosis; Mice; Mice, Knockout; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Vitamin A Deficiency

NLRC5, as the largest member of NLRs family, has recently been identified as a critical regulator of immune responses through negatively regulating NF-κB which is associated with the development of hepatic fibrosis. However, the expression and potential roles of NLRC5 in hepatic fibrosis and its reversal are still to be defined.. C57BL/6 mice were treatment with carbon tetrachloride (CCl4) induce hepatic fibrosis and its reversal. In vitro, models of hepatic fibrosis and its reversal are established by the treatment with TGF-β and MDI. The expression of NLRC5 was determined by RT-PCR, Western blot and immunohistochemistry. Consequently, NLRC5 was overexpressed or knockdown by transfecting PEGFP-C2-NLRC5 or NLRC5-siRNA respectively in the reversal of hepatic fibrosis, and the expression of fibrogenic genes such as α-SMA and Col1α1 was quantified. The NF-κB activity was detected as well.. Immunohistochemistry, RT-PCR and Western blot analysis with liver tissues and primary HSCs showed that NLRC5 was highly expressed in hepatic fibrosis and correspondingly decreased in the reversal stage. The differential expression of NLRC5 was confirmed in vitro. Enforced NLRC5 expression increased the expression of α-SMA and Col1α1, and blockade of NLRC5 reduced the fibrotic response. While the opposite expression of phosphorylated NF-кB p65 and phospho-IκBα was found.. NLRC5 is differentially expressed in hepatic tissues and hepatic stellate cells during hepatic fibrosis and its reversal. All the data indicated that NLRC5 may play a crucial role in regulating the reversal of hepatic fibrosis through NF-κB signaling pathway.

Topics: Actins; Animals; Carbon Tetrachloride Poisoning; Collagen Type I; Collagen Type I, alpha 1 Chain; Gene Knockdown Techniques; Hepatic Stellate Cells; I-kappa B Proteins; Intracellular Signaling Peptides and Proteins; Liver Cirrhosis; Mice; Mice, Inbred C57BL; Phosphorylation; RNA, Small Interfering; Transcription Factor RelA; Transfection; Transforming Growth Factor beta

TGF-β, the most potent profibrogenic factor, acts by activating SMAD (mothers against decapentaplegic) transcription factors, which bind to SMAD-binding elements in target genes. Here, we show that the thyroid hormone triiodothyronine (T3), through binding to its nuclear receptors (TRs), is able to antagonize transcriptional activation by TGF-β/SMAD. This antagonism involves reduced phosphorylation of SMADs and a direct interaction of the receptors with SMAD3 and SMAD4 that is independent of T3-mediated transcriptional activity but requires residues in the receptor DNA binding domain. T3 reduces occupancy of SMAD-binding elements in response to TGF-β, reducing histone acetylation and inhibiting transcription. In agreement with this transcriptional cross-talk, T3 is able to antagonize fibrotic processes in vivo. Liver fibrosis induced by carbon tetrachloride is attenuated by thyroid hormone administration to mice, whereas aged TR knockout mice spontaneously accumulate collagen. Furthermore, skin fibrosis induced by bleomycin administration is also reduced by the thyroid hormones. These findings define an important function of the thyroid hormone receptors and suggest TR ligands could have beneficial effects to block the progression of fibrotic diseases.

Topics: Animals; Bleomycin; Carbon Tetrachloride Poisoning; Liver Cirrhosis; Mice; Mice, Knockout; Signal Transduction; Smad3 Protein; Smad4 Protein; Transforming Growth Factor beta; Triiodothyronine

Transforming growth factor beta (TGF-β) is crucial for transdifferentiation of hepatic stellate cells (HSCs) and the blunting of TGF-β signaling in HSCs can effectively prevent liver fibrosis. Krüppel-like factor 11 (KLF11) is an early response transcription factor that potentiates TGF-β/Smad signaling by suppressing the transcription of inhibitory Smad7. Using a mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis, we observed significant upregulation of KLF11 in the activated HSCs during liver fibrogenesis. Meanwhile, the downregulation of miR-30 was observed in the HSCs isolated from fibrotic liver. Adenovirus-mediated ectopic expression of miR-30 was under the control of smooth muscle α-actin promoter, showing that the increase in miR-30 in HSC greatly reduced CCl4-induced liver fibrosis. Subsequent investigations showed that miR-30 suppressed KLF11 expression in HSC and led to a significant upregulation of Smad7 in vivo. Mechanistic studies further confirmed that KLF11 was the direct target of miR-30, and revealed that miR-30 blunted the profibrogenic TGF-β signaling in HSC by suppressing KLF11 expression and thus enhanced the negative feedback loop of TGF-β signaling imposed by Smad7. Finally, we demonstrated that miR-30 facilitated the reversal of activated HSC to a quiescent state as indicated by the inhibition of proliferation and migration, the loss of activation markers, and the gain of quiescent HSC markers. In conclusion, our results define miR-30 as a crucial suppressor of TGF-β signaling in HSCs activation and provide useful insights into the mechanisms underlying liver fibrosis.

Topics: Animals; Carbon Tetrachloride Poisoning; Chemical and Drug Induced Liver Injury; Hepatic Stellate Cells; Liver Cirrhosis; Mice; MicroRNAs; Transforming Growth Factor beta

Mistletoe (Viscum coloratum (Kom.) Nakai) has long been categorized as a traditional herbal medicine in Asia. In addition to its application in cancer therapy, mistletoe has also been used in the treatment of chronic hepatic disorders in China. In the present study, we investigated the antifibrotic effect and mechanisms of action of mistletoe extracts in a rat model of carbon tetrachloride (CCl4)-induced hepatotoxicity.. An experimental model of hepatic fibrosis was established by intraperitoneal injection of rats with CCl4 for 8 weeks. Rats were subsequently treated with a mistletoe alkaloid fraction preparation via oral administration (120mg/kg daily for 8 weeks) or with distilled water as a control. Histopathological changes were observed by hematoxylin and eosin staining and Masson׳s trichrome staining. The expression of markers relevant to hepatic stellate cell (HSC) activation in the liver was assessed by real-time reverse transcription-polymerase chain reaction, immunohistochemistry and western blotting. The anti-fibrosis activity and mechanisms of action of mistletoe alkaloid fractions were further investigated in the HSC-T6 HSC line, following treatment with mistletoe alkaloid fractions (12mg/ml) for 48h.. Hepatic fibrosis decreased markedly in CCl4-treated animals following treatment with mistletoe alkaloid fractions, compared to controls. The mRNA levels of transforming growth factor-β1 (TGF-β1), procollagen I and tissue inhibitors of metalloproteinases (TIMPs) were significantly downregulated, by about 40%, 40% and 45%, respectively, in liver tissues from rats treated with mistletoe alkaloid fractions. Furthermore, significant downregulation of TGF-β1, TGF-β1 receptor, phosphorylated Smad 2 and alpha smooth muscle actin (α-SMA) proteins, by about 45%, 30% and 40%, respectively, was also observed in liver tissues from mistletoe alkaloid fractions-treated rats. In contrast, Smad 7 levels were significantly increased by about 30% in mistletoe alkaloid fractions-treated rats. Treatment of HSC-T6 cells with mistletoe alkaloid fractions significantly induced Smad 7 expression and inhibited the expression of α-SMA, TGFβ1, TGF-β1 receptor, Smad 2 and TIMP-1, in vitro.. We demonstrate that mistletoe alkaloid fractions decrease extracellular matrix accumulation by inhibiting HSC activation. Mechanistically, this may occur via inhibition of TGF-β1/Smad 2 and Smad 7 signal transduction, thereby blocking the synthesis of procollagen I and TIMP-1. These findings suggest that mistletoe alkaloid fractions may be a potential therapeutic agent for the treatment of hepatic fibrosis.

Topics: Alkaloids; Animals; Base Sequence; Carbon Tetrachloride Poisoning; Cell Line; DNA Primers; Hepatic Stellate Cells; Liver Cirrhosis; Male; Mistletoe; Plant Extracts; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Smad Proteins; Transforming Growth Factor beta

Liver fibrosis is a severe, life-threatening clinical condition resulting from nonresolving hepatitis of different origins. IL-17A is critical in inflammation, but its relation to liver fibrosis remains elusive. We find increased IL-17A expression in fibrotic livers from HBV-infected patients undergoing partial hepatectomy because of cirrhosis-related early-stage hepatocellular carcinoma in comparison with control nonfibrotic livers from uninfected patients with hepatic hemangioma. In fibrotic livers, IL-17A immunoreactivity localizes to the inflammatory infiltrate. In experimental carbon tetrachloride-induced liver fibrosis of IL-17RA-deficient mice, we observe reduced neutrophil influx, proinflammatory cytokines, hepatocellular necrosis, inflammation, and fibrosis as compared with control C57BL/6 mice. IL-17A is produced by neutrophils and T lymphocytes expressing the Th17 lineage-specific transcription factor Retinoic acid receptor-related orphan receptor γt. Furthermore, hepatic stellate cells (HSCs) isolated from naive C57BL/6 mice respond to IL-17A with increased IL-6, α-smooth muscle actin, collagen, and TGF-β mRNA expression, suggesting an IL-17A-driven fibrotic process. Pharmacologic ERK1/2 or p38 inhibition significantly attenuated IL-17A-induced HSC activation and collagen expression. In conclusion, IL-17A(+) Retinoic acid receptor-related orphan receptor γt(+) neutrophils and T cells are recruited into the injured liver driving a chronic, fibrotic hepatitis. IL-17A-dependent HSC activation may be critical for liver fibrosis. Thus, blockade of IL-17A could potentially benefit patients with chronic hepatitis and liver fibrosis.

Topics: Actins; Adult; Animals; Carbon Tetrachloride Poisoning; Carcinoma, Hepatocellular; Chemical and Drug Induced Liver Injury; Collagen; Cytokines; Female; Gene Expression Regulation; Hemangioma; Hepatectomy; Hepatic Stellate Cells; Hepatitis B, Chronic; Hepatitis, Animal; Humans; Interleukin-17; Liver Cirrhosis; Liver Neoplasms; Male; MAP Kinase Signaling System; Mice; Mice, Knockout; Middle Aged; Neutrophils; Nuclear Receptor Subfamily 1, Group F, Member 3; Protein Kinase Inhibitors; Receptors, Interleukin-17; Recombinant Proteins; Th17 Cells; Transforming Growth Factor beta

Liver fibrosis is characterized by the deposition and increased turnover of extracellular matrix. This process is controlled by matrix metalloproteinases (MMPs), whose expression and activity dynamically change during injury progression. MMP-19, one of the most widely expressed MMPs, is highly expressed in liver; however, its contribution to liver pathology is unknown. The aim of this study was to elucidate the role of MMP-19 during the development and resolution of fibrosis by comparing the response of MMP-19-deficient (MMP19KO) and wild-type mice upon chronic liver CCl(4)-intoxication. We show that loss of MMP-19 was beneficial during liver injury, as plasma ALT and AST levels, deposition of fibrillar collagen, and phosphorylation of SMAD3, a TGF-ß1 signaling molecule, were all significantly lower in MMP19KO mice. The ameliorated course of the disease in MMP19KO mice likely results from a slower rate of basement membrane destruction and ECM remodeling as the knockout mice maintained significantly higher levels of type IV collagen and lower expression and activation of MMP-2 after 4 weeks of CCl(4)-intoxication. Hastened liver regeneration in MMP19KO mice was associated with slightly higher IGF-1 mRNA expression, slightly increased phosphorylation of Akt kinase, decreased TGF-ß1 mRNA levels and significantly reduced SMAD3 phosphorylation. In addition, primary hepatocytes isolated from MMP19KO mice showed impaired responsiveness towards TGF-ß1 stimulation, resulting in lower expression of Snail1 and vimentin mRNA. Thus, MMP-19-deficiency improves the development of hepatic fibrosis through the diminished replacement of physiological extracellular matrix with fibrotic deposits in the beginning of the injury, leading to subsequent changes in TGF-ß and IGF-1 signaling pathways.

Topics: Animals; Carbon Tetrachloride Poisoning; Cell Proliferation; Chronic Disease; Disease Models, Animal; Disease Progression; Hepatocytes; Insulin-Like Growth Factor I; Liver Cirrhosis; Matrix Metalloproteinases, Secreted; Mice; Mice, Knockout; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta

Liver fibrosis is orchestrated by a complex network of signaling pathways regulating the deposition of extracellular matrix proteins during fibrogenesis. MicroRNAs (miRNAs) represent a family of small noncoding RNAs controlling translation and transcription of many genes. Recently, miRNAs have been suggested to crucially modulate cellular processes in the liver such as hepatocarcinogenesis. However, their role in liver fibrosis is not well understood. We systematically analyzed the regulation of miRNAs in a mouse model of carbon tetrachloride-induced hepatic fibrogenesis (CCl(4) ) by gene array analysis, which revealed a panel of miRNA that were specifically regulated in livers of mice undergoing hepatic fibrosis. Within those, all three members of the miR-29-family were significantly down-regulated in livers of CCl(4) -treated mice as well as in mice that underwent bile duct ligation. Specific regulation of miR-29 members in murine fibrosis models correlated with lower expression of miR-29 in livers from patients with advanced liver fibrosis. Moreover, patients with advanced liver cirrhosis showed significantly lower levels of miR-29a in their serum when compared with healthy controls or patients with early fibrosis. On a cellular level, down-regulation of miR-29 in murine hepatic stellate cells (HSCs) was mediated by transforming growth factor beta (TGF-β) as well as inflammatory signals, namely, lipopolysaccharide (LPS) and nuclear factor kappa B (NF-κB). Furthermore, overexpression of miR-29b in murine HSC resulted in down-regulation of collagen expression.. Our data indicate that miR-29 mediates the regulation of liver fibrosis and is part of a signaling nexus involving TGF-β- and NF-κB-dependent down-regulation of miR-29 family members in HSC with subsequent up-regulation of extracellular matrix genes. Thus they may represent targets for novel therapeutic strategies against hepatic fibrogenesis and also might evolve as biomarkers in the diagnosis of liver fibrosis.

Topics: Adult; Aged; Aged, 80 and over; Animals; Bile Ducts; Carbon Tetrachloride Poisoning; Disease Models, Animal; Down-Regulation; Extracellular Matrix; Female; Hepatic Stellate Cells; Humans; Ligation; Lipopolysaccharides; Liver Cirrhosis; Male; Mice; Mice, Inbred BALB C; MicroRNAs; Middle Aged; NF-kappa B; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) is considered to be a major factor contributing to liver fibrosis. We have previously shown that nuclear translocation of YB-1 antagonizes the TGF-β/Smad3 signaling in regulating collagen gene expression. More recently, we have demonstrated that the novel small compound HSc025 promotes nuclear translocation of YB-1, resulting in the improvement of skin and pulmonary fibrosis. Here, we presented evidence as to the mechanism by which HSc025 stimulates nuclear translocation of YB-1 and the pharmacological effects of HSc025 on a murine model of hepatic fibrosis. A proteomics approach and binding assays using HSc025-immobilized resin showed that HSc025 binds to the amino acid sequence within the C-tail region of YB-1. In addition, immunoprecipitation experiments and glutathione S-transferase pulldown assays identified poly(A)-binding protein (PABP) as one of the cytoplasmic anchor proteins of YB-1. HSc025 directly binds to YB-1 and interrupts its interaction with PABP, resulting in accelerated nuclear translocation of YB-1. Transfection of cells with PABP siRNA promoted nuclear translocation of YB-1 and subsequently inhibited basal and TGF-β-stimulated collagen gene expression. Moreover, HSc025 significantly suppressed collagen gene expression in cultured activated hepatic stellate cells. Oral administration of HSc025 to mice with carbon tetrachloride-induced hepatic fibrosis improved liver injury as well as the degree of hepatic fibrosis. Altogether, the results provide a novel insight into therapy for organ fibrosis using YB-1 modulators.

Topics: Active Transport, Cell Nucleus; Alkadienes; Animals; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Cell Nucleus; Collagen; DNA-Binding Proteins; Gene Expression Regulation; Humans; Liver Cirrhosis, Experimental; Mice; Nuclear Proteins; Poly(A)-Binding Proteins; Protein Binding; Protein Structure, Tertiary; Rats; Signal Transduction; Smad3 Protein; Transcription Factors; Transforming Growth Factor beta; Y-Box-Binding Protein 1

Activation of innate immunity (natural killer [NK] cell/interferon-γ [IFN-γ]) has been shown to play an important role in antiviral and antitumor defenses as well as antifibrogenesis. However, little is known about the regulation of innate immunity during chronic liver injury. Here, we compared the functions of NK cells in early and advanced liver fibrosis induced by a 2-week or a 10-week carbon tetrachloride (CCl(4) ) challenge, respectively. Injection of polyinosinic-polycytidylic acid (poly I:C) or IFN-γ induced NK cell activation and NK cell killing of hepatic stellate cells (HSCs) in the 2-week CCl(4) model. Such activation was diminished in the 10-week CCl(4) model. Consistent with these findings, the inhibitory effect of poly I:C and IFN-γ on liver fibrosis was markedly reduced in the 10-week versus the 2-week CCl(4) model. In vitro coculture experiments demonstrated that 4-day cultured (early activated) HSCs induce NK cell activation via an NK group 2 member D/retinoic acid-induced early gene 1-dependent mechanism. Such activation was reduced when cocultured with 8-day cultured (intermediately activated) HSCs due to the production of transforming growth factor-β (TGF-β) by HSCs. Moreover, early activated HSCs were sensitive, whereas intermediately activated HSCs were resistant to IFN-γ-mediated inhibition of cell proliferation, likely due to elevated expression of suppressor of cytokine signaling 1 (SOCS1). Disruption of the SOCS1 gene restored the IFN-γ inhibition of cell proliferation in intermediately activated HSCs. Production of retinol metabolites by HSCs contributed to SOCS1 induction and subsequently inhibited IFN-γ signaling and functioning, whereas production of TGF-β by HSCs inhibited NK cell function and cytotoxicity against HSCs.. The antifibrogenic effects of NK cell/IFN-γ are suppressed during advanced liver injury, which is likely due to increased production of TGF-β and expression of SOCS1 in intermediately activated HSCs.

Topics: Animals; Carbon Tetrachloride Poisoning; Hepatic Stellate Cells; Immunity, Innate; Interferon-gamma; Killer Cells, Natural; Liver Cirrhosis; Mice; Poly I-C; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling Proteins; Transforming Growth Factor beta; Vitamin A

Experimental liver fibrosis induced by carbon tetrachloride (CCl(4)) is associated with oxidative stress, lipid peroxidation, and inflammation. This work was focused on elucidating the anti-inflammatory and antioxidant effects of ethylenediaminetetraacetic acid (EDTA) in this model of hepatotoxicity.. Wistar male rats were treated with CCl(4) and EDTA (60, 120, or 240 mg/kg). Morphometric analyses were carried out in Masson's stained liver sections to determine fibrosis index. Coagulation tests prothrombin time (PT) and partial thromboplastin time (PTT) were also determined. Gene expression for transforming growth factor beta (TGF-beta1), alpha1(I) procollagen gene (alpha1 Col I), tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and superoxide dismutase (SOD) was monitored by real-time PCR. Antioxidant effect of EDTA was measured by its effects on lipid peroxidation; biological activity of ceruloplasmin (Cp), SOD, and catalase (Cat) were analyzed by zymography assays.. Animals with CCl(4)-hepatic injury that received EDTA showed a decrement in fibrosis (20%) and lipid peroxidation (22%). The mRNA expression for TNF-alpha (55%), TGF-beta1 (50%), IL-6 (52%), and alpha1 Col I (60%) was also decreased. This group of animals showed increased Cp (62%) and SOD (25%) biological activities. Coagulation blood tests, Cat activity, and gene expression for SOD were not modified by EDTA treatment.. This study demonstrates that EDTA treatment induces the activity of antioxidant enzymes, decreases lipid peroxidation, hepatic inflammation, and fibrosis in experimental liver fibrosis induced by CCl(4).

Topics: Animals; Anti-Inflammatory Agents; Anticoagulants; Antioxidants; Blotting, Western; Carbon Tetrachloride Poisoning; Catalase; Edetic Acid; Immunoenzyme Techniques; Inflammation; Interleukin-6; Lipid Peroxidation; Liver; Liver Cirrhosis; Male; Oxidative Stress; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Superoxide Dismutase; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Chronic liver injury and inflammation lead to hepatic fibrosis, cirrhosis, and liver failure. Embryonic and mesenchymal stem cells have been shown to reduce experimental liver fibrosis but have potential limitations, including the formation of dysplastic precursors, tumors, and profibrogenic cells. Other stem-like cells may reduce hepatic inflammation and fibrosis without tumor and profibrogenic cell formation. To test this hypothesis we transplanted human amnion epithelial cells (hAEC), isolated from term delivered placenta, into immunocompetent C57/BL6 mice at week 2 of a 4-week regimen of carbon tetrachloride (CCl₄) exposure to induce liver fibrosis. Two weeks following hAEC infusion, intact cells expressing the human-specific markers inner mitochondrial membrane protein and human leukocyte antigen-G were found in mouse liver without evidence of host rejection of the transplanted cells. Human albumin, known to be produced by hAEC, was detected in sera of hAEC-treated mice. Human DNA was detected in mouse liver and also spleen, lungs, and heart of some animals. Following hAEC transplantation, CCl₄-treated animals showed decreased serum ALT levels and reduced hepatocyte apoptosis, compared to controls. hAEC-treated mouse liver had lower TNF-α and IL-6 protein levels and higher IL-10 compared to animals given CCl₄ alone. Compared to CCl₄ controls, hAEC-treated mice showed fewer activated collagen-producing hepatic stellate cells and less fibrosis area and collagen content. Reduced hepatic TGF-β levels in conjunction with a twofold increase in the active form of the collagen-degrading enzyme matrix metalloproteinase-2 in hAEC-treated mice compared to CCl₄ controls may account for the reduction in fibrosis. hAEC transplantation into immunocompetent mice leads to cell engraftment, reduced hepatocyte apoptosis, and decreased hepatic inflammation and fibrosis.

Topics: Amnion; Animals; Apoptosis; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Cell Separation; Cell Transplantation; Epithelial Cells; Female; Humans; Immunohistochemistry; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Pregnancy; Transforming Growth Factor beta; Transplantation, Heterologous

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is an endogenous tetrapeptide which has antifibrogenic effects at physiological concentrations in various tissues. AcSDKP is produced locally in the liver, however, little is known about its biological effect in this organ. We hypothesize that basal levels of endogenous AcSDKP decrease during the development of liver fibrosis and preservation of basal AcSDKP attenuates liver fibrosis.. Endogenous levels of AcSDKP in the liver were measured by enzyme immunoassay after 2, 6, and 10 weeks of carbon tetrachloride (CCl(4))-induced liver fibrosis in rats. Subcutaneous osmotic pump infusion of vehicle or AcSDKP (800 microg/kg/day) was administered to CCl(4)-treated rats for 8 weeks to study the effect of exogenous AcSDKP on liver fibrosis. The effect of AcSDKP on profibrogenic properties of hepatic stellate cells was studied in vitro.. Endogenous AcSDKP was significantly decreased in the liver of CCl(4)-treated rats. Chronic AcSDKP infusion preserved basal levels of AcSDKP and reduced liver injury, inflammation, fibrosis, and profibrogenic transforming growth factor-beta signaling. This was demonstrated by decreased aminotransferase serum levels, CD45 positive cells, collagen accumulation, alpha-smooth muscle actin positivity, transforming growth factor-beta1, phosphorylated Smad2/3 protein, increased bone morphogenetic protein-7, and phosphorylated Smad1/5/8. Further, AcSDKP exerts antifibrogenic effects on hepatic stellate cells (HSCs) by downregulation of HSC activation in vitro.. Maintaining physiological levels of AcSDKP is critical in negatively regulating the development of fibrosis in chronic liver injury. Preservation of AcSDKP may be a useful therapeutic approach in the management of liver fibrosis.

Topics: Actins; Animals; Carbon Tetrachloride Poisoning; Collagen; Gene Expression; Hepatic Stellate Cells; In Vitro Techniques; Liver Cirrhosis, Experimental; Male; Oligopeptides; Rats; Rats, Sprague-Dawley; Signal Transduction; Transforming Growth Factor beta

N-acetylcysteine (NAC) is an antioxidant, a precursor of reduced glutathione, and an inhibitor of the profibrotic cytokine liver transforming growth factor-beta (TGF-beta). Carbon tetrachloride (CCl4) cirrhosis is characterized by oxidative stress and fibrosis. Therefore, the aim of this work was to study the effect of NAC on experimental cirrhosis.. CCl4 was chronically administered for 8 weeks along with 300 mg/kg of NAC orally once a day. Alkaline phosphatase, alanine aminotransferase, and gamma-glutamyltranspeptidase were measured in plasma. Hydroxyproline, glycogen, lipid peroxidation, glutathione were determined in liver samples by colorimetric methods. TGF-beta was evaluated by western blotting, and a histopathological analysis was performed.. Serum markers of liver damage increased by CCl4 intoxication (P<0.05), whereas cotreatment with NAC prevented these increases (P<0.05); glycogen was depleted in the cirrhotic group (P<0.05), but preserved by NAC (P<0.05). Lipid peroxidation increased and glutathione decreased by the administration of CCl4 (P<0.05), again NAC prevented both effects (P<0.05). Importantly, collagen increased by about seven-fold in the CCl4 group (P<0.05); administration of NAC preserved the normal levels of collagen (P<0.05). Biochemical determinations were corroborated by hematoxylin and eosin, and trichromic stains. Western blots revealed a four-fold increase in TGF-beta in the group receiving CCl4, NAC cotreatment abolished TGF-beta signal (P<0.05).. Our results strongly suggest that NAC prevents experimental cirrhosis by two mechanisms: by preventing oxidative stress and by downregulating the profibrogenic cytokine TGF-beta. As NAC is currently used in humans intoxicated with paracetamol, it can be tested in fibrotic or cirrhotic patients under controlled trials.

Topics: Acetylcysteine; Animals; Antioxidants; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Liver Cirrhosis, Experimental; Male; Oxidative Stress; Rats; Rats, Wistar; Transforming Growth Factor beta

Liver fibrosis is characterized by an excess of collagen fiber deposition, and it is known that Kupffer cells play an important role by immunomodulation of the toxic response. Methyl palmitate (MP) is an effective Kupffer cell inhibitor. The aim of this work was to evaluate the effect of MP on experimental liver fibrosis. Four groups were formed: the control group, which received the vehicles only; CCl(4) group (0.4 g kg(-1), i.p., three times a week, for eight weeks); CCl(4) plus MP (300 mg kg(-1), i.p., daily); and MP alone. Alanine aminotransferase was increased by CCl(4), and MP did not prevent this increase. Lipid peroxidation was increased markedly by CCl(4); again, MP was not able to prevent this effect. Fibrosis increased nearly 6-fold (measured as liver hydroxyproline content) in the CCl(4) group; MP preserved the normal content of collagen. These results were corroborated by histopathology. To elucidate the antifibrogenic mechanism of MP, we measured the production of TGF-beta; CCl(4) increased this cytokine several-fold, and MP abolished this increase. Collectively the present results indicate that MP possesses a strong antifibrogenic effect at least in the CCl(4) model of fibrosis. The antifibrotic effect of MP is probably associated with its ability to reduce TGF-beta content, maybe by immunomodulation of Kupffer cells functioning.

Topics: Animals; Biotransformation; Blotting, Western; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Collagen; Down-Regulation; Histocytochemistry; Lipid Peroxidation; Liver Cirrhosis; Male; Necrosis; Palmitates; Rats; Rats, Wistar; Transforming Growth Factor beta

Curcumin is a phytophenolic compound, which is highly efficacious for treating several inflammatory diseases. The aim of this study was to evaluate the efficacy of curcumin in preventing or reversing liver cirrhosis. A 4-week bile duct ligation (BDL) rat model was used to test the ability of curcumin (100 mg/kg, p.o., daily) to prevent cirrhosis. To reverse cirrhosis, CCl(4) was administered chronically for 3 months, and then it was withdrawn and curcumin administered for 2 months. Alanine aminotransferase, gamma-glutamyl transpeptidase, liver histopathology, bilirubin, glycogen, reduced and oxidized glutathione, and TGF-beta (mRNA and protein) levels were assessed. Curcumin preserved normal values of markers of liver damage in BDL rats. Fibrosis, assessed by measuring hydroxyproline levels and histopathology, increased nearly fivefold after BDL and this effect was partially but significantly prevented by curcumin. BDL increased transforming growth factor-beta (TGF-beta) levels (mRNA and proteins), while curcumin partially suppressed this mediator of fibrosis. Curcumin also partially reversed the fibrosis induced by CCl(4). Curcumin was effective in preventing and reversing cirrhosis, probably by its ability of reducing TGF-beta expression. These data suggest that curcumin might be an effective antifibrotic and fibrolitic drug in the treatment of chronic hepatic diseases.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antioxidants; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Cholestasis; Curcumin; Dose-Response Relationship, Drug; Drug Interactions; Liver Cirrhosis, Experimental; Male; Oxidative Stress; Rats; Rats, Wistar; Transforming Growth Factor beta

Arazyme is a novel protease produced by the HY-3 strain of Aranicola proteolyticus, which is a Gram-negative aerobic bacterium that has been isolated from the intestine of the spider Nephila clavata. This study focused on the hepatoprotective effect of Arazyme on carbon tetrachloride (CCl4)-induced acute hepatic injury in senescence marker protein 30 (SMP30) knock-out (KO) mice and SMP30 wild-type (WT) mice. WT mice and SMP30 KO mice were divided into eight groups as follows: (i) two negative control groups (G1, G5) which were treated with a single intraperitoneal (i.p.) olive oil injection. (ii) Two positive control groups (G2, G6) which received a single i.p. CCl4 (0.4mL/kg) injection. (iii) Two vitamin C-treated groups (G3, G7) which received a single oral administration of vitamin C (100mg/kg) and were injected with a single i.p. CCl4 (0.4mL/kg). (iv) Two Arazyme-treated groups (G4, G8) which received a single oral administration of Arazyme (500mg/kg) and were injected with a single i.p. CCl4 (0.4mL/kg). Through present study, we could find that Arazyme-treated groups showed decreased degree of liver injury, increased expression of SMP30, decreased expression of phospho-Smad3 (p-Smad3), elevated expression of antioxidant proteins including sorbitol dehydrogenase, dihydropteridine reductase (DHPR), dehydrofolate reductase (DHFR), NADH dehydrogenase, glutathione S-transferase kappa 1 (GSTK1) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) compared with non-Arazyme-treated groups. Therefore, it is concluded that Arazyme plays a significant role in protecting injured hepatocytes by increasing the expression of SMP30, inhibiting the transforming growth factor-beta (TGF-beta)/Smad pathway and elevating the expression of antioxidant proteins.

Topics: Animals; Antioxidants; Ascorbic Acid; Calcium-Binding Proteins; Carbon Tetrachloride Poisoning; Chemical and Drug Induced Liver Injury; Chemoprevention; Disease Models, Animal; Female; Fluorescent Antibody Technique, Indirect; Immunoenzyme Techniques; Intracellular Signaling Peptides and Proteins; Liver; Male; Metalloproteases; Mice; Mice, Knockout; Oxidoreductases; Proteomics; Serratia; Smad3 Protein; Specific Pathogen-Free Organisms; Transforming Growth Factor beta; Up-Regulation

Acute liver injury induced by administration of carbon tetrachloride (CCl4) was shown to be a model of wound-repair in rat liver. Albumin gene expression was significantly reduced at 24 h post injection with CCl4, but recovered at 48 h. We also observed significant and transient expression of bone morphogenic protein-2 (BMP-2) at 6-24 h post treatment. This expression was also shown with depletion of Kupffer cell by GdCl3, and immunostaining with anti-BMP-2 antibody showed BMP-2-producing cells interspersed in intralobular spaces of injured liver. These observations suggest that BMP-2 secreted from oval-like cells plays important roles in the wound healing response of injured liver.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Carbon Tetrachloride Poisoning; Chemical and Drug Induced Liver Injury; Guanidine; Hepatocytes; Kupffer Cells; Lipopolysaccharide Receptors; Liver Diseases; Liver Regeneration; Male; Rats; Rats, Wistar; Transforming Growth Factor beta

Y-box protein-1 (YB-1) is a known negative regulator of collagen (Col) expression by two different mechanisms, acting directly through binding to an interferon-gamma response element within the col1A2 promoter and/or by physically interacting with p300/Smad3, thereby abrogating the stimulatory effect of transforming growth factor-beta (TGF-beta). Here, we report that YB-1 activation via the Jak1 signaling pathway is required and sufficient to confer interferon-gamma-dependent activation of the smad7 gene. By binding to a bona fide recognition site within the smad7 promoter, YB-1 up-regulates smad7 transcription, which was additively enhanced by autoinhibitory TGF-beta signaling. Importantly, the anti-TGF-beta effect was not only supplied by induced Smad7 expression but was recapitulated in the context of the col1A2 promoter, where YB-1 overexpression abolished the trans-stimulatory TGF-beta effect in a dominant fashion. In conclusion, YB-1 is the main target of interferon-gamma signaling via Jak1 that exerts antifibrotic action by both interference with TGF-beta signaling and direct down-regulation of collagen expression.

Topics: Animals; Carbon Tetrachloride Poisoning; Cell Line; Collagen; Fibrosis; Gene Expression Regulation; Interferon-gamma; Liver Cirrhosis, Experimental; Rats; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta; Y-Box-Binding Protein 1

Smad family proteins Smad2 and Smad3 are activated by transforming growth factor beta (TGF-beta)/activin/nodal receptors and mediate transcriptional regulation. Although differential functional roles of Smad2 and Smad3 are apparent in mammalian development, the relative functional roles of Smad2 and Smad3 in postnatal systems remain unclear. We used Cre/loxP-mediated gene targeting for hepatocyte-specific deletion of Smad2 (S2HeKO) in adult mice and generated hepatocyte-selective Smad2/Smad3 double knockouts by intercrossing AlbCre/Smad2(f/f) (S2HeKO) and Smad3-deficient Smad3ex8/ex8 (S3KO) mice. All strains were viable and had normal adult liver. However, necrogenic CCL4-induced hepatocyte proliferation was significantly increased in S2HeKO compared to Ctrl and S3KO livers, and transplanted S2HeKO hepatocytes repopulated recipient liver at dramatically increased rates compared to Ctrl hepatocytes in vivo. Using primary hepatocytes, we found that TGF-beta-induced G1 arrest, apoptosis, and epithelial-to-mesenchymal transition in Ctrl and S2HeKO but not in S3KO hepatocytes. Interestingly, S2HeKO cells spontaneously acquired mesenchymal features characteristic of epithelial-to-mesenchymal transition (EMT). Collectively, these results demonstrate that Smad2 suppresses hepatocyte growth and dedifferentiation independent of TGF-beta signaling. Smad2 is not required for TGF-beta-stimulated apoptosis, EMT, and growth inhibition in hepatocytes.

Topics: Animals; Apoptosis; Carbon Tetrachloride Poisoning; Cell Differentiation; Cell Movement; Cell Proliferation; Cells, Cultured; Chemical and Drug Induced Liver Injury; Cyclin D1; Hepatocytes; Liver; Mesoderm; Mice; Mice, Knockout; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Liver fibrosis results from chronic damage to the liver by chronic hepatitis, alcohol, and toxic agents. A characteristic of liver fibrosis is an accumulation of extracellular matrix (ECM) protein, which distorts the hepatic architecture by forming a fibrous scar, and the subsequent development of regenerating nodules defines cirrhosis. Transforming growth factor (TGF)-beta1, one of the most powerful profibrogenic mediators, plays a major role in the development of liver cirrhosis and regulates ECM gene expression and matrix degradation. This study elucidates the changes of TGF-beta1-mediated signals during liver fibrogenesis by using RNA interference. In this experiment, the TGF-beta1 siRNAs reduced the expression of TGF-beta1 in the livers of CCl(4) injection compared with those of control group, and the expression of type I collagen and alpha-smooth muscle actin was decreased. In conclusion, this study demonstrates that TGF-beta1 siRNAs inhibit TGF-beta1 expression in the murine model of liver cirrhosis and might be a good therapeutic strategy to prevent liver cirrhosis in human.

Topics: Actins; Animals; Carbon Tetrachloride Poisoning; Collagen Type I; Extracellular Matrix; Liver; Liver Cirrhosis, Experimental; Mice; Mice, Inbred C57BL; RNA Interference; RNA, Small Interfering; Transforming Growth Factor beta; Transforming Growth Factor beta1

The aim of this study was to determine whether transforming growth factor-beta1 (TGF-beta1) induces the synthesis, release and gene expression of urokinase-type plasminogen activator (uPA) in hepatic stellate cells. In addition to stimulating collagen production, TGF-beta1 induced the morphological and phenotypical changes characteristic of hepatic stellate cell activation. However, these changes accentuated in cells previously activated with acetaldehyde. TGF-beta1 increased to 2-fold uPA activity in lysates from quiescent cells, and to 3.5-fold in activated cells, and induced uPA gene expression to the same extent in both activated and non-activated cells. TGF-beta1 had a modest stimulatory action on the release of uPA into the conditioned medium, but reduced acetaldehyde-induced release, as demonstrated by Western blot analysis. In accord, whereas TGF-beta1 produces no effect on uPA activity in the conditioned media from quiescent cells, it significantly reduces the stimulatory action of acetaldehyde. These results show that the activity and gene expression of uPA are regulated by both acetaldehyde and TGF-beta1 and that the proteolytic activity in the extracellular space is reduced by the influence of TGF-beta1. Further studies on the molecular mechanisms responsible for the regulation of the plasminogen system by TGF-beta1 and other molecules in the presence of acetaldehyde will contribute to a better understanding of the processes involved in fibrogenesis.

Topics: Acetaldehyde; Animals; Blotting, Western; Carbon Tetrachloride Poisoning; Collagen; Culture Media, Conditioned; Hepatocytes; Liver; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA; Transforming Growth Factor beta; Transforming Growth Factor beta1; Urokinase-Type Plasminogen Activator

To observe the effect of taurine on hepatic stellate cell's apoptosis induced by carbon tetrachloride (CCl4) in rats and to study its protective mechanisms.. CCl4-induced rat hepatic fibrosis was treated by taurine. Serum alanine aminotransferase (ALT), plasma protein, hyaluronic acid (HA), procollagen III (PC III), hepatic microsomal drug-metabolizing enzyme and anti-transforming growth factor beta1 (TGF-beta1) were determined. In addition, hepatic stellate cell's apoptosis and the pathological changes of liver tissue were observed under light microscope.. The activity of serum ALT and the levels of serum HA, PC III were markedly reduced by taurine treatment. The hepatic cytochrome P450 (Cyt. P450) and cytochrome b5 (Cytb5) contents were increased by the same treatment. In addition, taurine could significantly inhibit the expression of TGF-beta1, promote the hepatic stellate cell's apoptosis, and relieve hepatic fibrosis.. Taurine fulfills a role in promoting hepatic stellate cell's apoptosis in the case of hepatic fibrosis, it mitigates the liver injury, decreases the expression of TGF-beta1, and relieves hepatic fibrosis.

Topics: Animals; Apoptosis; Carbon Tetrachloride Poisoning; Hepatocytes; Liver; Liver Cirrhosis; Male; Random Allocation; Rats; Rats, Wistar; Taurine; Transforming Growth Factor beta; Transforming Growth Factor beta1

To investigate the effects of adeno-associated virus (AAV) mediated expression of human interferon-gamma for gene therapy in experimental hepatic fibrosis in vitro and in vivo.. We constructed the recombinant AAV encoding human INF-gamma (rAAV- INF-gamma) and took the primary rat hepatic stellate cells and carbon tetrachloride induced rats as the experimental hepatic fibrosis model in vitro and in vivo. Immunocytochemistry analysis was used to reveal the expression of alpha-SMA, the marker protein expressed in hepatic stellate cells. The mRNA expression of TGF-beta, TIMP-1, and MMP-13 were analyzed by RT-PCR method. In vivo study, the hydroxyproline content in liver and serum AST, ALT were also detected.. In vitro study, AAV vector could mediated efficient expression of human INF-gamma, which inhibit the activation of hepatic stellate cells, decrease the expression of alpha-SMA and mRNA of TIMP-1, TGF-beta, with the MMP-13 unchanged. In vivo study, the histological examination revealed that rAAV- INF-gamma could inhibit the progression of the hepatic fibrosis. In the rAAV-INF-gamma induced group, the hydroxyproline content and serum AST, ALT level were decreased to 177+/-28 microg/g wet liver, 668.5+/-140.0, 458.4+/-123.5 U/L, compare with the fibrosis control group 236+/-31 microg/g wet liver, 1 019.1+/-276.3, 770.5+/-154.3 U/L, respectively (P<0.01). mRNA expression of TIMP-1 in the rAAV-INF-gamma induced rat liver was decreased while no significant change was observed in TGF-beta and MMP-13.. All these results indicated that rAAV-INF-gamma has potential effects for gene therapy of hepatic fibrosis, which could inhibit the progression of hepatic fibrosis.

Topics: Animals; Carbon Tetrachloride Poisoning; Cells, Cultured; Collagenases; Dependovirus; Disease Models, Animal; Disease Progression; Humans; Interferon-gamma; Liver; Liver Cirrhosis, Experimental; Liver Function Tests; Matrix Metalloproteinase 13; Rats; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Virion

To study the effect of IL-10 on the expression of growth factors--transforming growth factor-beta1 (TGF-beta1), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF) of hepatic stellate cells (HSCs) of hepatic fibrosis rat and the anti-fibrogenic role of exogenous IL-10.. Hepatic fibrosis was induced by CCl(4) administration intra-peritoneally. Sixty clean male Sprague-Dawley (SD) rats were randomly divided into three groups: normal control group (GN, 8 rats), hepatic fibrosis model group (GC, 28 rats) and IL-10 treated group (GI, 24 rats). At the beginning of the 7th and 11th wk, rats in each group were routinely perfused with pronase E and type IV collagenase through a portal vein catheter and the suspension obtained from the liver was spun by centrifugation with 11% Nycodenz density gradient to isolate HSCs. Histological examination was used to determine the degree of hepatic fibrosis. RT-PCR was employed to analyze mRNA expression from freshly isolated cells. Immunocytochemistry was performed to detect protein expression in primary cultured HSCs.. Rat hepatic fibrosis was developed with the increase of injection frequency of CCl(4), and HSCs were successfully isolated. At the 7th and 11th wk, TGF-beta1, EGF, and HGF mRNA in GC increased obviously compared with GN (P = 0.001/0.042, 0.001/0.001, 0.001/0.001) and GI (P = 0.001/0.007, 0.002/0.001, 0.001/0.001). For TGF-beta1, no difference was observed between GI and GN. For EGF, mRNA level in GI increased compared with GN during the 7th wk (P = 0.005) and 11th wk (P = 0.049). For HGF, mRNA level in GI decreased compared with GN at the 7th wk (P = 0.001) and 11th wk (P = 0.021). Between these two time points, TGF-beta1 expression at the 7th wk was higher than that of the 11th wk (P = 0.049), but for EGF, the former was lower than the latter (P = 0.022). As for PDGF mRNA, there was no significant difference between these groups, but difference seemed to exist in protein levels. Results by immunocytochemistry of TGF-beta1 and EGF were paralleled with the above findings.. The expression of TGF-beta1, EGF and HGF increased in HSC of hepatic fibrosis rat and decreased after treatment with IL-10. IL-10 plays an anti-fibrogenic role by suppressing growth factors expression.

Topics: Animals; Base Sequence; Carbon Tetrachloride Poisoning; DNA Primers; Epidermal Growth Factor; Gene Expression Regulation; Hepatocyte Growth Factor; Interleukin-10; Liver Diseases; Male; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

Angiotensin II has pro-fibrotic function in the liver. Blockade of the renin-angiotensin-aldosterone-system (RAAS) attenuates hepatic fibrosis. The aim of the present study was to determine the mechanism of angiotensin-converting enzyme inhibitor (ACEI) on the progression of rat hepatic fibrosis.. Forty male Wistar rats were divided into three groups. Model group (Mo): The rats were injected subcutaneously with 40% of CCl(4) 0.25 mL/100 g. Perindopril group (Pe): The rats were injected subcutaneously with 40% of CCl(4). Perindopril, equivalent to 2 mg/(kg.d), was administrated. Control group (Nc): the rats were treated with olive oil only. After 4 and 6 wk, the rats were killed. The liver sections were stained with Masson. The protein expressions of AT1R, TGF-beta1 and PDGF-BB were examined by Western blot. Nuclear factor kappaB (NF-kappaB) DNA binding activity was examined by EMSA (Electrophoretic gel mobility shift assay). Matrix metalloproteinase-2,9 (MMP-2,9) activity was assessed by zymography. Serum laminin (LN) and hyaluronic acid (HA) were measured using radioimmunoassays.. Using Western blot, we clearly provided direct evidence for the expression of AT1R in liver. The expression was up-regulated when fibrogenesis occurred. Perindopril treatment significantly reduced mean fibrosis score, protein levels of AT1R, TGF-beta1 and PDGF-BB, serum levels of HA and LN, and the activity of MMP-2,9. NF-kappaB DNA binding activity markedly increased in model group, perindopril treatment considerably reduced NF-kappaB DNA binding activity.. Perindopril attenuates CCl(4)-induced hepatic fibrogenesis of rat by inhibiting TGF-beta1, PDGF-BB, NF-kappaB and MMP-2,9.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Becaplermin; Carbon Tetrachloride Poisoning; Male; Matrix Metalloproteinase Inhibitors; NF-kappa B; Perindopril; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Rats; Rats, Wistar; Transforming Growth Factor beta; Transforming Growth Factor beta1

To evaluate the effects of Ginkgo biloba extract (EGB) on CCl(4)-induced liver fibrosis and to investigate the underlying mechanisms.. Rats were divided into the following groups: normal control group, CCl(4) model group, low dose EGB group, moderate dose EGB group and high dose EGB group. The rat liver fibrosis model was induced by intraperitoneal injection of CCl(4) twice a week for 8 weeks. The model rats of the three EGB treated groups were given 0.25 g/kg, 0.5 g/kg, 1.0 g/kg of EGB by stomach tubes every day. At the end of the eighth week, the blood and liver specimens were obtained. The expressions of nuclear factor kappaB (NF-kappaB) P65, and alpha-smooth muscle actin (alpha-SMA) were detected by immunohistochemistry. Radioimmunoassay was exploited to evaluate serum hyaluronic acid (HA) and laminin (LN) levels. Electrophoretic mobility shift assay (EMSA) was used to confirm the nuclear translocation activity of NF-kappaB in liver tissues. The mRNA expression of transforming growth factor-beta1 (TGFbeta1) and collagen I was determined by RT-PCR. Malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in liver tissues and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the sera were also examined.. CCl(4) administration induced liver fibrosis, which was inhibited by EGB in a dose-dependent manner. The histopathologic scores of liver fibrosis, the levels of serum ALT, AST, HA and LN were significantly lower in the rats treated with EGB compared with those not treated (P <0.01 or P <0.05). SOD and GSH-Px activities were notably elevated and MDA content was significantly lower in the rats treated with EGB (P <0.01 or P <0.05), indicating reduced oxidative stress. Immunohistochemical staining demonstrated inhibition of hepatic stellate cell (HSC) activation (in terms of alpha-SMA expression) and NF-kappaB P65 expression in the livers of the EGB-treated rats. As determined by EMSA and RT-PCR, activation of NF-kappaB, the mRNA expression of TGFbeta1 and collagen I were significantly higher in model group rats, but obviously lower in EGB treated rats.. EGB is able to ameliorate liver injury and prevent rats from CCl(4)-induced liver fibrosis by suppressing oxidative stress. This process may be related to inhibiting the expression of TGFbeta1 and the induction of NF-kappaB on HSC activation.

Topics: Animals; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Drugs, Chinese Herbal; Ginkgo biloba; Liver Cirrhosis, Experimental; Male; NF-kappa B; Phytotherapy; Plant Leaves; Rats; Rats, Wistar; Transforming Growth Factor beta

The cirrhotic liver manifests dysregulated hepatocyte growth by poor regenerative capacity, formation of regenerative nodules, and malignant transformation to hepatocellular carcinoma. The purpose of this study was to determine if dysregulated hepatocyte growth occurs through deficient apoptosis.. Hepatocytes were isolated from normal and CCl(4)-treated mice and treated with TGFbeta, TNFalpha, and UV-C, known apoptotic agents.. Cirrhotic hepatocytes were less sensitive to TGFbeta- (45+/-5 vs. 15+/-3%; P<0.003), TNFalpha- (59+/-21 vs. 21+/-8%; P=0.02), and UV-C-induced (31+/-4 vs. 17+/-4%; P<0.03) apoptosis compared to normal hepatocytes. In normal hepatocytes, TGFbeta-induced apoptosis occurred through a ROS-, MPT-, and caspase-dependent pathway. Cirrhotic hepatocytes lacked caspase activation, had decreased procaspase-8 expression, failed to undergo the MPT, and had increased basal ROS activity compared to normal hepatocytes. After treatment with trolox, an antioxidant that reduced basal ROS activity, cirrhotic hepatocytes underwent apoptosis in response to TGFbeta treatment.. These findings suggest that increased ROS activity in cirrhotic hepatocytes plays a critical role in mediating cirrhotic hepatocyte resistance to apoptosis. Cirrhotic hepatocyte resistance to TGFbeta-induced apoptosis is ROS-dependent and is a mechanism of dysregulated growth in the chronically inflamed liver.

Topics: Animals; Apoptosis; Carbon Tetrachloride Poisoning; Caspases; Cell Culture Techniques; Hepatocytes; Immunohistochemistry; Intracellular Membranes; Liver Cirrhosis, Experimental; Male; Mice; Mice, Inbred BALB C; Mitochondria, Liver; Permeability; Reactive Oxygen Species; Transforming Growth Factor beta

To study the effect of leflunomide on CCl4-induced hepatic fibrosis in rats.. Hepatic fibrosis was induced by subcutaneous injection with 50% CCl4 in Sprague-Dawley rats. The amount of CCl4 administered was 1 mg/kg. The alanine aminotransferase (ALT), aspartate aminotransferase (AST), nitric oxide (NO) levels in plasma and hydroxyproline (Hyp) contents in liver tissue were assayed by spectrophotometry. The hyaluronic acid (HA) and procollagen III (PC III) were assessed by radioimmunoassay. The transforming growth factor-beta1 (TGF-beta1) in serum was determined by ELISA. The nuclear factor-kappa B (NF-kappaB) in liver tissue was examined by immunohistochemistry. Liver samples collected after 12 weeks of CCl4 treatment were stained with hematoxylin and eosin.. Leflunomide (1, 3, and 9 mg/kg) significantly decreased indices of liver and spleen, the serum transaminase (AST, ALT) activities, HA and PC III levels, and Hyp contents in liver tissue in rats of hepatic fibrosis. Histopathological examination showed leflunomide had inhibitory effect on fibrogenesis and formation of pseudolobulus. Furthermore, leflunomide significantly inhibited NF-kappaB expression in liver tissue, and reduced elevated serum TGF-kappa1 and NO levels in rats of hepatic fibrosis.. Leflunomide showed inhibitory action on hepatic fibrosis induced by CCl4 in rats.

Topics: Adjuvants, Immunologic; Alanine Transaminase; Animals; Aspartate Aminotransferases; Carbon Tetrachloride Poisoning; Collagen Type III; Hydroxyproline; Isoxazoles; Leflunomide; Liver; Liver Cirrhosis, Experimental; Male; NF-kappa B; Nitric Oxide; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Transforming Growth Factor beta1

To investigate the effects of the proprietary Chinese Medicine "anti-hepatic-fibrosis 268" on hepatic fibrosis and the related mechanisms.. The model of CCl4-induced hepatic damage was established in SD rats. 54 male rats were divided into four groups, namely high dose and low dose "anti-hepatic-fibrosis 268" groups, colchicine control group, and model control group. Using Masson stain and light microscope, the authors examined the rats' hepatic tissues and counted the hepatic fiber components, then examined and counted TGF-beta1, alpha-SMA, FN, Type I, III collagen by means of immunohistochemical technique. The groups were compared and the internal relationships of the data were analyzed.. The levels of FN, LN, Type I and III collagen, TGF-beta1, and alpha-SMA of the CCl4 damaged rats increased (P<0.01). After 3 weeks of high dose "anti-hepatic-fibrosis 268" treatment, the levels of TGF-beta1, alpha-SMA, FN, LN, Type I and III collagen decreased (P<0.01) and the degree hepatic fibrosis took a favorable turn significantly (P<0.05) as compared with the model control. In the rats of the low dose group, the levels of TGF-beta1, alpha-SMA, FN, Type III collagen significantly decreased (P<0.05), the levels of LN, Type I collagen were not different from the model control; The hepatic fibrosis improved to a certain extent (P<0.05).. The mechanism of reversing hepatic fibrosis by "anti-hepatic-fibrosis 268" in this experiment is that the medicine regulates TGF-beta1 and further affects alpha-SMA, thus resulting in the decline of FN, Type I, III collagen levels in liver extracellular matrix.

Topics: Actins; Animals; Astragalus Plant; Astragalus propinquus; Carbon Tetrachloride Poisoning; Collagen Type I; Collagen Type III; Drugs, Chinese Herbal; Fibronectins; Liver; Liver Cirrhosis, Experimental; Male; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Transforming Growth Factor beta1

To investigate the therapeutic effects of perindopril, an angiotensin-converting enzyme inhibitor, and valsartan, an angiotensin II receptor blocker on TGFbeta1 and TGFbeta receptor II mRNA, Smad3 and Smad7 on rat liver fibrosis.. 60 Wistar rats were randomly divided into four groups (each group, n=15). Group 1 rats were not treated and served as healthy controls. The rats of groups 2,3,and 4 were injected with CCl(4) which induced liver fibrosis. After four weeks, group 3 rats started a treatment of perindopril, and group 4 rats with valsartan. All rats were sacrificed at the eighth week and their blood and livers were collected for analysis. The effects of perindopril and valsartan were evaluated by the levels of transforming growth factor-beta1 (TGFb1), and TGF receptor (TGFb1RII) mRNA in liver tissues by RT-PCR, the expressions and sites of TGFb1, Smad3 and Smad7 in liver tissue by immunohistochemical staining. The liver histopathology was also examined with HE staining, and the hydroxyproline in the liver and serum hyaluronic acid (HA) were examined using biochemsitry and RIA.. Compared with the control group, the levels of TGFb1, TGFb1RII mRNA and the expression Smad3 were significantly decreased in the two treated groups, and the expression of Smad7 was also remarkably increased in the livers of rats treated with perindopril or valsartan. The histological changes of fibrosis, the hydroxyproline in the livers and HA were also improved in the treated rats.. Perindopril and valsartan have a protective effect on liver injury and can inhibit hepatic fibrosis induced by CCl(4) in rats. Their mechanisms may be associated with their effects of down-regulating TGFb1, TGFb1RII mRNA and smad3, and up-regulating Smad7 which then resulted in suppressing the activation of hepatic stellate cells.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Female; Liver Cirrhosis, Experimental; Male; Perindopril; Random Allocation; Rats; Rats, Wistar; Receptors, Transforming Growth Factor beta; RNA, Messenger; Smad3 Protein; Smad7 Protein; Tetrazoles; Transforming Growth Factor beta; Transforming Growth Factor beta1; Valine; Valsartan

To explore the effect of Dahuang Zhechong Pill (DHZCP) on transforming growth factor-beta 1 (TGF-beta 1) in rats hepatic stellate cells.. Liver fibrosis model rats were induced by CCl4 compound factor, and treated with DHZCP in three different dosages (ordinary, double and triple) separately. TGF-beta 1 and alpha-smooth muscle actin (alpha-SMA) synthesis expression, and grades of fibrosis were observed.. TGF-beta 1 and alpha-SMA expression in the group treated with ordinary dose of DHZCP was insignificantly different from those in the control group (P > 0.05), but the expression attenuated significantly after treatment with double or triple dose of DHZCP in the portal area and discontinuous fibrous septum (P < 0.05, P < 0.01).. DHZCP of larger dosage could inhibit the hepatic stellate cells proliferation and secretion of TGF-beta 1, and reduce the genesis of collagen, so as to weaken the auto-secretion amplifying response of the cells, which might be one of the chief mechanisms of DHZCP in antagonizing liver fibrosis.

Topics: Actins; Animals; Carbon Tetrachloride Poisoning; Drugs, Chinese Herbal; Extracellular Matrix; Female; Hepatocytes; Liver Cirrhosis; Male; Rats; Transforming Growth Factor beta; Transforming Growth Factor beta1

To investigate the effect of rhein on liver fibrosis induced by the exposure of carbon tetrachloride (CCl4)/ethanol in rats.. Male Wistar rats were divided into four study groups (n=10 each group): healthy controls, CCl4/ethanol-injured rats left untreated, and CCl4/ethanol-injured rats treated with rhein of low-dose (25 mg/kg) and high-dose (100 mg/kg). Rhein was given once a day since rat received CCl4/ethanol injury. After administration of rhein for 6 weeks rats were killed. The following parameters were determined: the activity of alanine aminotransferase (ALT), hyalauronic acid (HA) and procollagen type III (PC-III) concentrations in serum, liver malondialdehyde (MDA) level, the degree of liver fibrosis, and the expression of alpha-smooth muscle actin (alpha-SMA) and transforming growth factor-beta1 (TGF-beta1) in liver tissue.. The treatment of rhein markedly reduced the ALT activity, HA and PC-III concentrations, and liver MDA level in CCl4/ethanol-injured rats (P<0.01). It also improved significantly histological changes of fibrosis and decreased the expression of alpha-SMA and TGF-beta1 in liver of these rats (P<0.05 or P<0.01).. Rhein has protective effect on liver injury and can inhibit liver fibrosis induced by CCl4/ethanol in rats. The mechanisms possibly contribute to its action of antioxidant and anti-inflammatory activity, also associated with its effect of inhibiting TGF-beta1 and suppressing the activation of hepatic stellate cells.

Topics: Animals; Anthraquinones; Antioxidants; Carbon Tetrachloride Poisoning; Liver Cirrhosis, Experimental; Male; Mixed Function Oxygenases; Procollagen; Rats; Rats, Wistar; Transforming Growth Factor beta

Since there is still debate about the ability of the aged liver to regenerate, we compared some aspects of this response in young, adult and old rodents. 2, 6, 12 and 19-month-old rats were intraperitoneally injected with CCl(4) (3mg/kg) or left untreated (CT) and killed either 2h (group A) or 24h (group B) after intoxication. Liver injury was checked histologically and by assaying transaminases. mRNA levels of albumin (Alb), c-fos, c-myc, hepatocyte growth factor (HGF), transforming growth factor (TGF)-alpha and TGF-beta1 were also analyzed. Heat shock protein (HSP)70 gene expression was evaluated, and liver GSH content. Transaminases and histology show more damage in aged rats. Alb mRNA was reduced starting at 12 months in group A and at all ages in group B; c-fos and c-myc mRNAs reached the highest levels in 6-month-old rats and the lowest in those aged 12 and 19 months of group A. In group B, c-fos was detectable only in 6-month animals, but c-myc at all ages. HGF, TGF-alpha and TGF-beta1 mRNAs were up-regulated in treated rats, but to a lesser extent in the aged. HSP70 mRNA, absent in CT, was significantly increased at the age of 6 months, undetectable in the oldest rats in group A; in group B it was only visible in 6-month animals. GSH content was reduced with aging. In conclusion, during aging the liver regenerative machinery is preserved but its activation is reduced and delayed.

Topics: Aging; Alanine Transaminase; Albumins; Animals; Aspartate Aminotransferases; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Cell Cycle; Gene Expression; Glutathione; Hepatocyte Growth Factor; HSP70 Heat-Shock Proteins; Liver; Male; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-myc; Rats; Rats, Sprague-Dawley; Transforming Growth Factor alpha; Transforming Growth Factor beta; Transforming Growth Factor beta1

Acute liver injury induced by CCl4 injection (0.5 ml/kg b.w.) was compared between Mini and Wistar rats. Mini rats (Jcl:Wistar-TgN (ARGHGEN)1Nts strain) are Wistar-derived transgenic animals in which the expression of growth hormone (GH) gene is suppressed by the presence of an antisense transgene. The hepatic lesion appeared earlier and its recovery was delayed in Mini rats compared to in Wistar rats. The degree of the liver injury was more severe in Mini rats than in Wistar rats, and this corresponded well with the changes in serum AST level. Moreover, in accordance with the localization of CYP2E1-positive hepatocytes in the early stage after CCl4 treatment, the initial lesion characterized by ballooning of hepatocytes developed in the centrilobular zone in Wistar rats while it appeared in the middle zone in Mini rats. The changes in the percentage of PCNA-positive cells and the levels of HGF and TGF-beta1 mRNAs were clearly different between the two strains. These results indicate that the response of the liver to CCl4 is different between GH-suppressed Mini rats and Wistar rats.

Topics: Alkaline Phosphatase; Animals; Animals, Genetically Modified; Apoptosis; Aspartate Aminotransferases; Carbon Tetrachloride; Carbon Tetrachloride Poisoning; Cell Count; Chemical and Drug Induced Liver Injury; Cytochrome P-450 CYP2E1; Hepatocyte Growth Factor; Hepatocytes; Immunoenzyme Techniques; In Situ Nick-End Labeling; Liver; Proliferating Cell Nuclear Antigen; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Species Specificity; Transforming Growth Factor beta

Lines of evidence suggested a possible link between leptin and hepatic fibrosis; however, whether leptin modulates the fibrogenesis in the liver remains unclear. The purpose of this study, therefore, was to evaluate the effect of leptin on inflammatory and profibrogenic responses in the liver caused by hepatotoxic chemicals. Male C57Bl/6 mice were given carbon tetrachloride (CCl(4)) (0.1 microL/g body weight [BW], intraperitoneally [IP]) and/or recombinant murine leptin (1 microg/g BW, IP) simultaneously, and sacrificed up to 72 hours later. Further, some mice were given thioacetamide (TAA; 200 microg/g BW, IP) and leptin 3 times per week for 4 weeks to evaluate the effect of leptin on chronic fibrogenic responses. A simultaneous injection of leptin enhanced acute CCl(4)-induced necroinflammatory and subsequent fibrotic changes in the hepatic lobules. The steady-state messenger RNA (mRNA) levels of alpha1(I) procollagen and heat shock protein 47 (HSP47) in the liver were potentiated when leptin was injected together with CCl(4). Expression of alpha smooth muscle actin (alpha-SMA) in the liver after CCl(4) treatment was also augmented markedly in combination with leptin. Further, leptin increased transforming growth factor beta1 (TGF-beta1) mRNA in the liver 24 hours after acute CCl(4) about 4-fold higher than CCl(4) alone. Moreover, leptin enhanced hepatic fibrosis and induction of alpha1(I) procollagen mRNA caused by chronic TAA administration. Collectively, these findings indicated that leptin augments both inflammatory and profibrogenic responses in the liver caused by hepatotoxic chemicals. It is postulated that the increase in systemic leptin levels enhances up-regulation of TGF-beta1, leading to activation of stellate cells, thereby augmenting the fibrogenic response in the liver.

Topics: Actins; Animals; Carbon Tetrachloride Poisoning; Chemical and Drug Induced Liver Injury; Endotoxins; Heat-Shock Proteins; HSP47 Heat-Shock Proteins; Leptin; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Muscle, Smooth; Portal System; Procollagen; RNA, Messenger; Thioacetamide; Transaminases; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

To study the antifibrotic effects of matrine in vitro a nd in vivo.. Rat hepatic stellate cell HSC-T6 and mouse fibroblast cell NIH3T3 proliferation stimulated with serum and platelet-derived growth factor (PDGF) was measured b y crystal violet staining assay. Collagen synthesis stimulated with serum and transforming growth factor beta1 (TGF-beta1) was determined by [3H]proline incorporation. Liver fibrosis was induced by carbon tetrachloride (CCl4) in rats an d evaluated with plasma hyaluranic acid level and hepatic hydroxyproline content.. Matrine (1-2 mmol/L) markedly reduced serum-driven proliferation and collagen synthesis of HSC-T6 cells as well as NIH3T3 cells. PDGF-driven proliferative activity and TGF-beta1-driven collagen synthesis in HSC-T6 cel ls were attenuated by matrine (0.25-2 mmol/L) in a concentration-dependent manner. In vivo matrine (50 mg/kg and 100 mg/kg) significantly decreased serum hyaluranic acid levels and hepatic hydroxyproline contents in rats treated with CCl4.. Inhibition of PDGF and TGF-beta1 actions on hepatic stellate cell by matrine might provide a possible mechanism of its antifibrotic activities.

Topics: 3T3 Cells; Alkaloids; Animals; Carbon Tetrachloride Poisoning; Cell Division; Cell Line; Collagen; Hepatocytes; Hyaluronic Acid; Hydroxyproline; Liver Cirrhosis; Male; Matrines; Mice; Platelet-Derived Growth Factor; Quinolizines; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor beta (TGF-beta) as well as tumor necrosis factor alpha (TNF-alpha) gene expression are up-regulated in chronically inflamed liver. These cytokines were investigated for their influence on apoptosis and proliferation of activated hepatic stellate cells (HSCs). Spontaneous apoptosis in activated HSC was significantly down-regulated by 53% +/- 8% (P <.01) under the influence of TGF-beta and by 28% +/- 2% (P <.05) under the influence of TNF-alpha. TGF-beta and TNF-alpha significantly reduced expression of CD95L in activated HSCs, whereas CD95 expression remained unchanged. Furthermore, HSC apoptosis induced by CD95-agonistic antibodies was reduced from 96% +/- 2% to 51 +/- 7% (P <.01) by TGF-beta, and from 96% +/- 2% to 58 +/- 2% (P <.01) by TNF-alpha, suggesting that intracellular antiapoptotic mechanisms may also be activated by both cytokines. During activation, HSC cultures showed a reduced portion of cells in the G0/G1 phase and a strong increment of G2-phase cells. This increment was significantly inhibited (G1 arrest) by administration of TGF-beta and/or TNF-alpha to activated cells. In liver sections of chronically damaged rat liver (CCl4 model), using desmin and CD95L as markers for activated HSC, most of these cells did not show apoptotic signs (TUNEL-negative). Taken together, these findings indicate that TGF-beta and/or TNF-alpha both inhibit proliferation and also apoptosis in activated HSC in vitro. Both processes seem to be linked to each other, and their inhibition could represent the mechanism responsible for prolonged survival of activated HSC in chronic liver damage in vivo.

Topics: Animals; Apoptosis; Carbon Tetrachloride Poisoning; Cell Cycle; Cell Division; Cells, Cultured; Fas Ligand Protein; fas Receptor; Gene Expression Regulation; Kinetics; Liver; Membrane Glycoproteins; Rats; Rats, Wistar; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Transforming growth factor-beta (TGF-beta) is a family of multifunctional proteins that regulate hepatocyte proliferation, and biosynthesis of the extracellular matrix. In this study we examined whether modulation of TGF-beta receptor expression contributes to the liver diseases.. The mRNA expression of TGF-beta1, TGF-beta type I receptor (TGFbetaRI), TGF-beta type II receptor (TGFbetaRII) and TGF-beta type III receptor (TGFbetaRIII) in rat livers injured by CCl4 administration was studied by Northern blotting. The mRNA expression patterns were confirmed by in situ hybridization.. The peak of TGF-beta1 mRNA expression was observed 48 h after acute intoxication with CCl4 in nonparenchymal cells. However, the levels of TGFbetaRI and TGFbetaRII mRNA expression decreased from 24 h to 48 h and from 12 h to 48 h, respectively, and returned to the normal level by 72 h. TGFbetaRII mRNA expression was depressed more and for longer than that of TGFbetaRI mRNA. Analysis in separated hepatocytes and nonparenchymal cells from the injured livers indicated that the mRNA changes occurred in hepatocytes. Nonparenchymal cells expressed TGFbetaRI and TGFbetaRII mRNAs at constant levels during liver regeneration. TGFbetaRIII mRNA, which also decreased after 12 h, was not apparent in hepatocytes but only in nonparenchymal cells.. These observations suggest that: (i) whenever TGF-beta1 is increased in CCl4-treated livers, it may induce liver fibrogenesis via nonparenchymal cells; (ii) the mitoinhibitory effect of TGF-beta1 on hepatocytes is transiently relieved by down-regulation of TGF-beta receptors for 72 h post-damage; and (iii) the resistance to TGF-beta growth inhibition between 24 to 48 h may be predominantly due to down-regulation of the expression of TGFbetaRII.

Topics: Animals; Carbon Tetrachloride Poisoning; Down-Regulation; In Situ Hybridization; Liver; Liver Regeneration; Male; Rats; Rats, Wistar; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Hepatic lipocytes are perisinusoidal cells that have been thought to be analogous to tissue pericytes, a cell type with purported vasoregulatory properties. However, we and others have recently demonstrated that lipocytes acquire markers of smooth muscle cells or myofibroblasts only after liver injury, via a process termed "activation." In this study, we document lipocyte contractility on collagen lattices and examine the importance of activation in this process. In culture, lipocytes became contractile only after spreading and activating, coincident with expression of smooth muscle alpha actin, a marker of activation (1990. Virchows Arch. B Cell Pathol. 59:349). After 5 d in culture, lipocytes induced rapid and sustained contraction of collagen lattices (to 43.7 +/- 2.3% of their original size 24 h after detachment). There was no contraction of lattices containing hepatocytes. Scanning electron microscopy demonstrated intimate associations of lipocyte cell membranes and collagen fibrils. Reduction in cell volume during contraction was also prominent. Lattice contraction by lipocytes was proportional to cell number. Serum was a potent stimulator of lipocyte contraction, as were endothelin types 1, 2, and 3; the effect of serum and endothelin 1 were additive. Neither thrombin, angiotensin-II, serotonin, nor the cytokines PDGF and TGF beta induced contraction. Cytochalasin B treatment resulted in concentration-dependent inhibition of contraction. As a test of the in vivo relevance of the culture findings, lipocytes were isolated from fibrotic animals and examined immediately after adherence. Whereas lipocytes from normal liver were initially compact, smooth muscle alpha actin negative and noncontractile, cells from animals with hepatic injury due to CCl4 displayed an activated appearance, expressed smooth muscle alpha actin, and were contractile immediately after adherence. Additionally, IFN-gamma, an agent which blocks lipocyte activation (1992. Hepatology. 16:776), inhibited lipocyte contraction. The data document that normal (i.e., quiescent) lipocytes are not contractile, but that activation is associated with the development of contractility. These findings suggest that a role for lipocytes in organ contraction or vasoregulation may be confined to injured, not normal liver.

Topics: Adipocytes; Angiotensin II; Animals; Carbon Tetrachloride Poisoning; Cells, Cultured; Collagen; Cytochalasin B; Endothelins; Liver; Liver Cirrhosis, Experimental; Male; Microscopy, Electron, Scanning; Platelet-Derived Growth Factor; Rats; Rats, Sprague-Dawley; Serotonin; Thrombin; Transforming Growth Factor beta