transforming-growth-factor-beta and Chemical-and-Drug-Induced-Liver-Injury

Unbalanced Th1/Th2 T-cell responses in the liver are a characteristic of hepatic inflammation and subsequent liver fibrosis. The recently discovered Th17 cells, a subtype of CD4(+) T-helper cells mainly producing IL-17 and IL-22, have initially been linked to host defense against infections and to autoimmunity. Their preferred differentiation upon TGFβ and IL-6, two cytokines abundantly present in injured liver, makes a contribution of Th17 cells to hepatic inflammation very likely. Indeed, initial studies in humans revealed activated Th17 cells and Th17-related cytokines in various liver diseases. However, functional experiments in mouse models are not fully conclusive at present, and the pathogenic contribution of Th17 cells to liver inflammation might vary upon the disease etiology, for example, between infectious and autoimmune disorders. Understanding the chemokines and chemokine receptors promoting hepatic Th17 cell recruitment (possibly CCR6 or CCR4) might reveal new therapeutic targets interfering with Th17 migration or differentiation in liver disease.

Topics: Animals; Autoimmune Diseases; Autoimmunity; Cell Differentiation; Cell Movement; Chemical and Drug Induced Liver Injury; Concanavalin A; Disease Models, Animal; Humans; Infections; Inflammation; Interleukin-17; Interleukin-22; Interleukins; Liver; Liver Diseases; Mice; Receptors, Chemokine; T-Lymphocytes, Regulatory; Th17 Cells; Transforming Growth Factor beta

Topics: Animals; Autophagy; Autophagy-Related Protein 7; Cell Proliferation; Chemical and Drug Induced Liver Injury; Choline Deficiency; Disease Models, Animal; Ethionine; Extracellular Matrix; Gene Expression Regulation; Liver; Liver Cirrhosis; Mice, Knockout; Microscopy, Electron, Transmission; Pyridines; Stem Cells; Transcription Factors; Transforming Growth Factor beta

Topics: Acetaminophen; Activins; Animals; Autophagy; Bone Morphogenetic Proteins; Chemical and Drug Induced Liver Injury; Liver; Mice; Signal Transduction; Transforming Growth Factor beta

Leflunomide-induced liver injury has been an important problem since its approval. Although, severe cases of leflunomide-induced liver injury leading to hospitalization are rare, the risk is higher with concurrent liver disease or use of other hepatotoxic drugs. The current study was conducted to investigate the potential protective effects of carvedilol and crocin alone and in combination against leflunomide-induced hepatic injury and to clarify the possible mechanism(s) through which carvedilol and crocin may elicit their effects. Fifty male albino mice were allocated into five groups: normal control group, leflunomide group, carvedilol group, crocin group, and combination group. These groups were given vehicle, leflunomide, leflunomide plus carvedilol, leflunomide plus crocin, and leflunomide plus combination of carvedilol and crocin, respectively. The study was conducted for 8 weeks, and different parameters were assessed. The results demonstrated that leflunomide significantly increased the serum levels of AST, ALT, ALP, hepatic MDA, nitrite, mTOR gene, PI3K gene, TGF-β, and the pathological changes alongside with the significant decrease of serum albumin, total protein, hepatic catalase, and GSH. While the coadministration of carvedilol, crocin and their combination with leflunomide significantly decreased the serum levels of AST, ALT, ALP, hepatic MDA, mTOR gene, PI3K gene, TGF-β, and the pathological changes alongside with the significant elevation of serum albumin, total protein, hepatic catalase, and GSH. This study is suggesting several solutions for Leflunomide-induced hepatotoxicity demonstrated by the protective effect of the antihypertensive drug carvedilol, the natural product crocin, and their combination which was demonstrated to be superior to each drug alone.

Topics: Animals; Carvedilol; Catalase; Chemical and Drug Induced Liver Injury; Chemical and Drug Induced Liver Injury, Chronic; Leflunomide; Liver; Male; Mice; Oxidative Stress; Phosphatidylinositol 3-Kinases; Serum Albumin; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

Silver nanoparticles (AgNPs) can accumulate in various organs after oral exposure. The main objective of the current study is to evaluate the renal toxicity induced by AgNPs after repeated oral exposure and to determine the relevant molecular mechanisms.. In this study, 40 male Wistar rats were treated with solutions containing 30, 125, 300, and 700 mg/kg of AgNPs. After 28 days of exposure, histopathological changes were assessed using hematoxylin-eosin (H&E), Masson's trichrome, and periodic acid-Schiff (PAS) staining. Apoptosis was quantified by TUNEL and immunohistochemistry of caspase-3, and the level of expression of the mRNAs of growth factors was determined using RT-PCR.. Histopathologic examination revealed degenerative changes in the glomeruli, loss of tubular architecture, loss of brush border, and interrupted tubular basal laminae. These changes were more noticeable in groups treated with 30 and 125 mg/kg. The collagen intensity increased in the group treated with 30 mg/kg in both the cortex and the medulla. Apoptosis was much more evident in middle-dose groups (i.e., 125 and 300 mg/kg). The results of RT-PCR indicated that Bcl-2 and Bax mRNAs upregulated in the treated groups (p < 0.05). Moreover, the data related to EGF, TNF-α, and TGF-β1 revealed that AgNPs induced significant changes in gene expression in the groups treated with 30 and 700 mg/kg compared to the control group.. Our observations showed that AgNPs played a critical role in in vivo renal toxicity.

Topics: Animals; Apoptosis; Blood Urea Nitrogen; Body Weight; Caspase 3; Chemical and Drug Induced Liver Injury; Creatinine; Epidermal Growth Factor; Extracellular Matrix Proteins; Gene Expression; Immunohistochemistry; In Situ Nick-End Labeling; Kidney; Male; Metal Nanoparticles; Organ Size; Rats, Wistar; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Neutrophils were classically described as powerful effectors of acute inflammation, and their main purpose was assumed to be restricted to pathogen killing through production of oxidants. As consequence, neutrophils also may lead to significant collateral damage to the healthy tissues, and after performing these tasks, these leukocytes are supposed to die within tissues. However, there is a growing body of evidence showing that neutrophils also play a pivotal role in the resolution phases of inflammation, because they can modulate tissue environment due to secretion of different kind of cytokines. Drug-induced liver injury (DILI) is a worldwide concern being one of the most prevalent causes of liver transplantation, and is well established that there is an intense neutrophil recruitment into necrotic liver during DILI. However, information if such abundant granulocyte infiltration is also linked to the tissue repairing phase of hepatic injury is still largely elusive. Here, we investigated the dynamics of neutrophil trafficking within blood, bone marrow, and liver during hepatic inflammation, and how changes in their gene expression profile could drive the resolution events during acetaminophen (APAP)-induced liver injury. We found that neutrophils remained viable during longer periods following liver damage, because they avidly patrolled necrotic areas and up-regulated pro-resolutive genes, including Tgfb, Il1r2, and Fpr2. Adoptive transference of "resolutive neutrophils" harvested from livers at 72 h after injury to mice at the initial phases of injury (6 h after APAP) significantly rescued organ injury. Thus, we provide novel insights on the role of neutrophils not only in the injury amplification, but also in the resolution phases of inflammation.

Topics: Acetaminophen; Animals; Cell Survival; Chemical and Drug Induced Liver Injury; Female; Liver; Mice; Neutrophil Infiltration; Neutrophils; Receptors, Formyl Peptide; Receptors, Interleukin-1 Type II; Transforming Growth Factor beta; Up-Regulation

Liver disease is common and often life-threatening. Sinomenine (SIN) is an active ingredient extracted from . In vivo experiments, mice were randomly divided into six groups (n=10): control group, model group, SIN (25 mg/kg) group, SIN (50 mg/kg) group, SIN (100 mg/kg) group and SIN (100 mg/kg) + SRI-011381 group. Alanine transaminases (ALT), aspartate transaminases (AST) and alkaline phosphatase (ALP) were detected. The pathological lesion was measured by HE staining. Apoptosis was measured by TUNEL staining. In vitro experiments, BRL-3A cells were treated with APAP (7.5 mM) and then subjected to various doses of SIN (10, 50 and 100 μg/mL) at 37°C for 24 h. Inflammatory factors and oxidative stress index were measured by ELISA. The expression of proteins was detected by Western blot.. The results showed that compared with the control group, the levels of ALT, AST and ALP in the serum of APAP-induced mice were significantly increased, followed by liver histological damage and hepatocyte apoptosis. Besides, APAP reduced the activity of SOD and GSH-Px, while increasing the content of MDA and LDH. Notably, APAP also promoted the expression of NLRP3, ASC, caspase-1 and IL-1β. Interestingly, SIN treatment dose-dependently reduced APAP-induced liver injury and oxidative stress, inhibited the activation of NLRP3 inflammasomes, and reduced the levels of inflammatory cytokines. In vitro studies have shown that SIN treatment significantly reduced the viability of BRL-3A cells and oxidative stress and inflammation. In addition, the Western blotting analysis showed that SIN inhibited the activation of TGF-β/Smad pathway in a dose-dependent manner in vitro and in vivo. These effects were significantly reversed by TGF-β/Smad activator SRI-011381 or TGF-β overexpression.. The study indicates that SIN attenuates APAP-induced acute liver injury by decreasing oxidative stress and inflammatory response via TGF-β/Smad pathway in vitro and in vivo.

Topics: Acetaminophen; Animals; Apoptosis; Cells, Cultured; Chemical and Drug Induced Liver Injury; Dose-Response Relationship, Drug; Inflammation; Mice; Mice, Inbred C57BL; Morphinans; Oxidative Stress; Smad Proteins; Structure-Activity Relationship; Transforming Growth Factor beta

Liver fibrosis is a disease caused by long-term damage that is related to a number of factors. The current research on the treatment of liver fibrosis mainly focuses on the activation of hepatic stellate cell, in addition to protecting liver cells. byakangelicin has certain anti-inflammatory ability, but its effect on liver fibrosis is unclear. This study aims to explore whether byakangelicin plays a role in the development of liver fibrosis and to explore its mechanism. We determined that byakangelicin has a certain ability to resist fibrosis and reduce liver cell damage in a model of carbon tetrachloride-induced liver fibrosis in mice. Thereafter, we performed further verification in vitro. The signalling pathways of two important pro-fibrotic cytokines, transforming growth factor-β and platelet-derived growth factor, were studied. Results showed that byakangelicin can inhibit related pathways. According to the hepatoprotective effect of byakangelicin observed in animal experiments, we studied the effect of byakangelicin on 4-HNE-induced hepatocyte (HepG2) apoptosis and explored its related pathways. The results showed that byakangelicin could attenuate 4-HNE-induced hepatocyte apoptosis via inhibiting ASK-1/JNK signalling. In conclusion, byakangelicin could improve carbon tetrachloride-induced liver fibrosis and liver injury by inhibiting hepatic stellate cell proliferation and activation and suppressing hepatocyte apoptosis.

Topics: Animals; Apoptosis; Biopsy; Carbon Tetrachloride; Cells, Cultured; Chemical and Drug Induced Liver Injury; Cytokines; Disease Models, Animal; Furocoumarins; Hepatic Stellate Cells; Hepatocytes; Liver Cirrhosis; Male; Mice; Protective Agents; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

Hepatic fibrosis, characterized by an excessive extracellular matrix (ECM) accumulation, leading to scar-tissue formation is a growing health problem worldwide. Hepatocellular damage due to liver injury triggers inflammation and transdifferentiation of quiescent hepatic stellate cells (HSCs) into proliferative, contractile, and ECM-producing myofibroblasts. Involvement of the Janus kinase (JAK)-2 pathway in the pathogenesis of fibrosis has been reported earlier. However, in this study, we have investigated the effect of selective JAK2 antagonist TG101348 in fibroblasts and inflammatory macrophages and

Topics: 3T3 Cells; Animals; Carbon Tetrachloride; Cell Line, Tumor; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Hep G2 Cells; Humans; Liver; Liver Cirrhosis; Macrophages; Male; Mice; Mice, Inbred C57BL; Myofibroblasts; Nitrogen Oxides; Pyrrolidines; Signal Transduction; Sulfonamides; Transforming Growth Factor beta

Acute liver failure is a serious consequence of acetaminophen (APAP)-induced hepatotoxic liver injury with high rates of morbidity and mortality. Transforming growth factor beta 1 (TGFβ1) is elevated during liver injury and influences hepatocyte senescence during APAP-induced hepatotoxicity. This study investigated TGFβ1 signaling in the context of inflammation, necrotic cell death, and oxidative stress during APAP-induced liver injury. Male C57Bl/6 mice were injected with 600 mg/kg APAP to generate liver injury in the presence or absence of the TGFβ receptor 1 inhibitor, GW788388, 1 h prior to APAP administration. Acetaminophen-induced liver injury was characterized using histological and biochemical measures. Transforming growth factor beta 1 expression and signal transduction were assessed using immunohistochemistry, Western blotting and ELISA assays. Hepatic necrosis, liver injury, cell proliferation, hepatic inflammation, and oxidative stress were assessed in all mice. Acetaminophen administration significantly induced necrosis and elevated serum transaminases compared with control mice. Transforming growth factor beta 1 staining was observed in and around areas of necrosis with phosphorylation of SMAD3 observed in hepatocytes neighboring necrotic areas in APAP-treated mice. Pretreatment with GW788388 prior to APAP administration in mice reduced hepatocyte cell death and stimulated regeneration. Phosphorylation of SMAD3 was reduced in APAP mice pretreated with GW788388 and this correlated with reduced hepatic cytokine production and oxidative stress. These results support that TGFβ1 signaling plays a significant role in APAP-induced liver injury by influencing necrotic cell death, inflammation, oxidative stress, and hepatocyte regeneration. In conclusion, targeting TGFβ1 or downstream signaling may be a possible therapeutic target for the management of APAP-induced liver injury.

Topics: Acetaminophen; Animals; Antioxidants; Apoptosis; Benzamides; Cell Death; Chemical and Drug Induced Liver Injury; Glutathione; Hepatocytes; Inflammation; JNK Mitogen-Activated Protein Kinases; Liver; Liver Failure, Acute; Male; Mice; Mice, Inbred C57BL; Necrosis; Oxidative Stress; Phosphorylation; Protective Agents; Pyrazoles; Regeneration; Signal Transduction; Transforming Growth Factor beta

Progression of fibrosis and the development of cirrhosis are responsible for the liver related morbidity and mortality associated with chronic liver diseases. There is currently a great unmet need for effective anti-fibrotic strategies. Stem cells play a central role in wound healing responses to restore liver homeostasis following injury. Here we tested the hypothesis that extracellular vesicles (EVs) isolated from induced pluripotent stem cells (iPSC) modulate hepatic stellate cell (HSCs) activation and may have anti-fibrotic effects. Human iPSCs were generated by reprogramming primary skin fibroblasts. EVs were isolated by differential centrifugation, quantified by flow cytometry (FACS) and characterized by dynamic light scattering (DLS) and electron microscopy (TEM). Primary human HSCs were activated with TGFβ (10 ng/mL) and exposed to iPSC-EVs. Efficacy of iPSC-EVs was tested on HSC in vitro and in two murine models of liver injury (CCl4 and bile duct ligation). Characterization of iPSC-derived EVs by flow cytometry identified a large population of EVs released by iPSC, primarily with a diameter of 300 nm and that could be visualized by TEM as round, cup-shaped objects. Fluorescent tracing assays detected iPSC-EVs in HSC cytosol after a short incubation and EV uptake by HSCs resulted in both decrease of pro-fibrogenic markers αSMA, CollagenIα1, Fibronectin and TIMP-1 and HSC pro-fibrogenic responses such as chemotaxis and proliferation. Genomics analyses of iPSC-EV miRNA cargo revealed 22 highly expressed miRNAs, among which miR-92a-3p resulted the most abundant. Transcriptome analysis identified 60 genes down-modulated and 235 up-regulated in TGF-β-primed HSC in presence or absence of iPSC-EVs. Intravenous injection of iPSC-EVs in CCl4 and bile duct ligation-induced liver fibrosis resulted in anti-fibrotic effects at protein and gene levels. Results of this study identify iPSC-EVs as a novel anti-fibrotic approach that may reduce or reverse liver fibrosis in patients with chronic liver disease.

Topics: Animals; Apoptosis; Cell Movement; Cell Proliferation; Chemical and Drug Induced Liver Injury; Chemotaxis; Disease Models, Animal; Extracellular Vesicles; Hepatic Stellate Cells; Humans; Induced Pluripotent Stem Cells; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Transcriptome; Transforming Growth Factor beta

Activation of TGFB (transforming growth factor β) promotes liver fibrosis by activating hepatic stellate cells (HSCs), but the mechanisms of TGFB activation are not clear. We investigated the role of ECM1 (extracellular matrix protein 1), which interacts with extracellular and structural proteins, in TGFB activation in mouse livers.. We performed studies with C57BL/6J mice (controls), ECM1-knockout (ECM1-KO) mice, and mice with hepatocyte-specific knockout of EMC1 (ECM1. ECM1-KO mice spontaneously developed liver fibrosis and died by 2 months of age without significant hepatocyte damage or inflammation. In liver tissues of mice, we found that ECM1 stabilized extracellular matrix-deposited TGFB in its inactive form by interacting with αv integrins to prevent activation of HSCs. In liver tissues from patients and in mice with CCl. ECM1, produced by hepatocytes, inhibits activation of TGFB and its activation of HSCs to prevent fibrogenesis in mouse liver. Strategies to increase levels of ECM1 in liver might be developed for treatment of fibrosis.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Sub-Family B Member 4; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Extracellular Matrix Proteins; Hepatic Stellate Cells; Hepatitis, Alcoholic; Hepatitis, Viral, Human; Humans; Liver; Liver Cirrhosis, Alcoholic; Liver Cirrhosis, Experimental; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Signal Transduction; Transforming Growth Factor beta

One of the therapeutic options for the treatment of fulminant hepatitis is repopulation of intrahepatic regulatory cells because their pool is significantly reduced during acute liver failure. Although it is known that mesenchymal stem cells (MSCs), which have beneficent effects in the therapy of fulminant hepatitis, may promote expansion of regulatory T cells (Tregs) and regulatory B cells (Bregs), the role of these regulatory cells in MSC-mediated attenuation of acute liver injury is unknown. Herewith, we described the molecular mechanisms involved in the crosstalk between MSCs and liver regulatory cells and analyzed the potential of MSC-based therapy for the expansion of intrahepatic regulatory cells in mouse model of acute liver failure. MSC-dependent attenuation of α-galactosylceramide (α-GalCer)-induced acute liver injury in mice was accompanied with an increased presence of interleukin (IL) 10-producing CD4

Topics: Adoptive Transfer; Animals; Cell Proliferation; Cells, Cultured; Chemical and Drug Induced Liver Injury; Coculture Techniques; Disease Models, Animal; Galactosylceramides; Indoleamine-Pyrrole 2,3,-Dioxygenase; Interleukin-10; Liver; Lymphocyte Activation; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice, Inbred C57BL; Natural Killer T-Cells; Paracrine Communication; Signal Transduction; T-Lymphocytes, Regulatory; Time Factors; Transforming Growth Factor beta

Acute liver injury (ALI) is highly lethal acute liver failure caused by different etiologies. Transforming growth factor β (TGF-β) is a multifunctional cytokine and a well-recognized inducer of apoptotic and necrotic cell death in hepatocytes. Latent TGF-β is activated partly through proteolytic cleavage by a serine protease plasma kallikrein (PLK) between the R58 and L59 residues of its propeptide region. Recently, we developed a specific monoclonal antibody to detect the N-terminal side LAP degradation products ending at residue R58 (R58 LAP-DPs) that reflect PLK-dependent TGF-β activation. This study aimed to explore the potential roles of PLK-dependent TGF-β activation in the pathogenesis of ALI. We established a mouse ALI model via the injection of anti-Fas antibodies (Jo2) and observed increases in the TGF-β1 mRNA level, Smad3 phosphorylation, TUNEL-positive apoptotic hepatocytes and R58-positive cells in the liver tissues of Jo2-treated mice. The R58 LAP-DPs were observed in/around F4/80-positive macrophages, while macrophage depletion with clodronate liposomes partly alleviated the Jo2-induced liver injury. Blocking PLK-dependent TGF-β activation using either the serine proteinase inhibitor FOY305 or the selective PLK inhibitor PKSI-527 or blocking the TGF-β receptor-mediated signaling pathway using SB431542 significantly prevented Jo2-induced hepatic apoptosis and mortality. Furthermore, similar phenomena were observed in the mouse model of ALI with the administration of acetaminophen (APAP). In summary, R58 LAP-DPs reflecting PLK-dependent TGF-β activation may serve as a biomarker for ALI, and targeting PLK-dependent TGF-β activation has potential as a therapeutic strategy for ALI.

Topics: Acetaminophen; Acute Lung Injury; Animals; Antibodies, Monoclonal; Benzamides; Biomarkers; Chemical and Drug Induced Liver Injury; Dioxoles; Disease Models, Animal; fas Receptor; Latent TGF-beta Binding Proteins; Macrophages; Male; Mice, Inbred C57BL; Plasma Kallikrein; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Preclinical Research Baogan Yihao (BGYH) is a traditional Chinese herbal medicine for the treatment of chronic liver diseases. In this study, the effects of BGYH on dimethylnitrosamine (DMN)-induced liver fibrosis were investigated using a rat model. BGYH alleviate liver damage, as indicated by decreased levels of AST, ALT, γ-GT, and AKP. BGYH also prevented collagen deposition and reduced pathological tissue injury in liver tissue. In fibrosis, high levels of α-SMA and TGF-β in liver tissue were markedly attenuated by BGYH. The inhibitory effect of BGYH on HSC-T6 proliferation demonstrated that BGYH exhibited significant hepatoprotective and antifibrogenic effects on DMN-induced liver injury. These findings suggest that BGYH may have therapeutic potential in the prevention and therapy of liver fibrosis. Drug Dev Res 78 : 155-163, 2017. © 2017 Wiley Periodicals, Inc.

Topics: Actins; Animals; Cell Line; Chemical and Drug Induced Liver Injury; Dimethylnitrosamine; Disease Models, Animal; Drugs, Chinese Herbal; Female; Humans; Male; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

Gut-derived microbial antigens trigger the innate immune system during acute liver injury. During recovery, regulatory immunity plays a role in suppressing inflammation; however, the precise mechanism underlying this process remains obscure. Here, we find that recruitment of immune-regulatory classical dendritic cells (cDCs) is crucial for liver tolerance in concanavalin A-induced acute liver injury. Acute liver injury resulted in enrichment of commensal Lactobacillus in the gut. Notably, Lactobacillus activated IL-22 production by gut innate lymphoid cells and raised systemic IL-22 levels. Gut-derived IL-22 enhanced mucosal barrier function and promoted the recruitment of regulatory cDCs to the liver. These cDCs produced IL-10 and TGF-β through TLR9 activation, preventing further liver inflammation. Collectively, our results indicate that beneficial gut microbes influence tolerogenic immune responses in the liver. Therefore, modulation of the gut microbiota might be a potential option to regulate liver tolerance.

Topics: Alanine Transaminase; Animals; Chemical and Drug Induced Liver Injury; Dendritic Cells; Gastrointestinal Microbiome; Histocompatibility Antigens Class II; Immune Tolerance; Immunity, Innate; Interferon-gamma; Interleukin-10; Interleukin-22; Interleukins; Intestinal Mucosa; Intestines; Lactobacillus; Liver; Male; Mice; Mice, Inbred C57BL; T-Lymphocytes, Regulatory; Toll-Like Receptor 9; 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

CD248 (endosialin) is a stromal cell marker expressed on fibroblasts and pericytes. During liver injury, myofibroblasts are the main source of fibrotic matrix.. To determine the role of CD248 in the development of liver fibrosis in the rodent and human setting.. CD248 expression was studied by immunostaining and quantitative PCR in both normal and diseased human and murine liver tissue and isolated hepatic stellate cells (HSCs). Hepatic fibrosis was induced in CD248(-/-) and wild-type controls with carbon tetrachloride (CCl4) treatment.. Expression of CD248 was seen in normal liver of humans and mice but was significantly increased in liver injury using both immunostaining and gene expression assays. CD248 was co-expressed with a range of fibroblast/HSC markers including desmin, vimentin and α-smooth muscle actin (α-SMA) in murine and human liver sections. CD248 expression was restricted to isolated primary murine and human HSC. Collagen deposition and α-SMA expression, but not inflammation and neoangiogenesis, was reduced in CD248(-/-) mice compared with wild-type mice after CCl4 treatment. Isolated HSC from wild-type and CD248(-/-) mice expressed platelet-derived growth factor receptor α (PDGFR-α) and PDGFR-β at similar levels. As expected, PDGF-BB stimulation induced proliferation of wild-type HSC, whereas CD248(-/-) HSC did not demonstrate a proliferative response to PDGF-BB. Abrogated PDGF signalling in CD248(-/-) HSC was confirmed by significantly reduced c-fos expression in CD248(-/-) HSC compared with wild-type HSC.. Our data show that deletion of CD248 reduces susceptibility to liver fibrosis via an effect on PDGF signalling, making it an attractive clinical target for the treatment of liver injury.

Topics: Actins; Angiogenesis Inducing Agents; Animals; Antigens, CD; Antigens, Neoplasm; Becaplermin; Carbon Tetrachloride; Cell Proliferation; Cells, Cultured; Chemical and Drug Induced Liver Injury; Chronic Disease; Collagen; Desmin; Fibrosis; Gene Expression; Hepatic Stellate Cells; Humans; Inflammation; Liver; Liver Cirrhosis; Mice; Mice, Inbred C57BL; Mice, Knockout; Neovascularization, Pathologic; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-sis; Receptor, Platelet-Derived Growth Factor alpha; Receptor, Platelet-Derived Growth Factor beta; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Vimentin

The ubiquitously expressed β2-spectrin (β2SP, SPTBN1) is the most common non-erythrocytic member of the β-spectrin gene family. Loss of β2-spectrin leads to defects in liver development, and its haploinsufficiency spontaneously leads to chronic liver disease and the eventual development of hepatocellular cancer. However, the specific role of β2-spectrin in liver homeostasis remains to be elucidated. Here, we reported that β2-spectrin was cleaved by caspase-3/7 upon treatment with acetaminophen which is the main cause of acute liver injury. Blockage of β2-spectrin cleavage robustly attenuated β2-spectrin-specific functions, including regulation of the cell cycle, apoptosis, and transcription. Cleaved fragments of β2-spectrin were physiologically active, and the N- and C-terminal fragments retained discrete interaction partners and activity in transcriptional regulation and apoptosis, respectively. Cleavage of β2-spectrin facilitated the redistribution of the resulting fragments under conditions of liver damage induced by acetaminophen. In contrast, downregulation of β2-spectrin led to resistance to acetaminophen-induced cytotoxicity, and its insufficiency in the liver promoted suppression of acetaminophen-induced liver damage and enhancement of liver regeneration.. β2-Spectrin, a TGF-β mediator and signaling molecule, is cleaved and activated by caspase-3/7, consequently enhancing apoptosis and transcriptional control to determine cell fate upon liver damage. These findings have extended our knowledge on the spectrum of β2-spectrin functions from a scaffolding protein to a target and transmitter of TGF-β in liver damage.

Topics: Acetaminophen; Animals; Caspase 3; Caspase 7; Cell Line; Chemical and Drug Induced Liver Injury; Chlorocebus aethiops; COS Cells; Down-Regulation; HEK293 Cells; HeLa Cells; Hep G2 Cells; Humans; Liver; Male; Mice; Mice, Inbred C57BL; Recombinant Proteins; Signal Transduction; Spectrin; Transforming Growth Factor beta

Thymoquinone (TQ) is a biologically active compound isolated from the seeds of Nigella sativa L. (Ranuculaceae). This study investigated the hepato-protective effect of TQ on liver injury through AMP-activated protein kinase (AMPK) signaling in hepatic stellate cells (HSCs). In vitro, TGF-β time-dependently attenuated liver kinase B-1 (LKB1) and AMPK phosphorylation, which were blocked by pretreatment with TQ and AICAR (an activator of AMPK). TQ significantly inhibited collagen-Ι, α-SMA, TIMP-1 and enhanced MMP-13 expression, contributing to prevent TGF-β-induced human HSCs activation. Moreover, TQ induced peroxisome proliferator activated receptor-γ (PPAR-γ) expression, which was inhibited by genetic deletion of AMPK. In vivo, C57BL/6 mice were fed with ethanol diet for 10 days, then administering a single dose of ethanol (5g/kg body weight) via gavage. TQ (20 or 40mg/kg) were given by gavage every day. TQ attenuated the increases in serum aminotransferase and hepatic triglyceride in mice fed with ethanol, while significantly activated LKB1 and AMPK phosphorylation. In addition, TQ enhanced the sirtuin 1 (SIRT1) expression. In conclusion, we demonstrate that AMPK pathway is a key therapeutic target for controlling liver injury and TQ confers hepato-protection against TGF-β-induced the activation of HSCs and ethanol-induced liver injury.

Topics: Adenylate Kinase; Alcoholism; AMP-Activated Protein Kinase Kinases; Animals; Benzoquinones; Binge Drinking; Chemical and Drug Induced Liver Injury; Hepatic Stellate Cells; Humans; Liver Function Tests; Male; Mice; Mice, Inbred C57BL; PPAR gamma; Protein Serine-Threonine Kinases; Sirtuin 1; Transforming Growth Factor beta; Up-Regulation

This study was designed to investigate the protective effects of bosentan an orally active non-peptide mixed ETA/ETB receptor antagonist, on liver injury in streptozotocin-induced diabetic rats.. 24 Albino-Wistar rats were randomly divided into 4 groups: healthy (Group 1), diabetic (Group 2) (60 mg/kg of streptozotocin i.p.), diabetic treated with bosentan 50 mg/kg (Group 3) and diabetic treated with bosentan 100 mg/kg (Group 4). The treatment of bosentan was initiated after streptozocin injection and continued for 60 days.. Liver from diabetic rats showed significant increase in malondialdehyde (MDA) level and significant decrease in glutathione (GSH), and superoxide dismutase (SOD) activity. Endothelin (ET-1), tumor necrosis factor (TNF-α) and transforming growth factor beta (TGF-β) gene expression significantly increased in the diabetic groups in the rat liver tissue. Bosentan treatment showed a significant up-regulatory effect on ET-1, TNF-α and TGF-β mRNA expression. Results from histopathological evaluation of the liver were in accordance with our biochemical and molecular results.. These data provide clear evidence that bosentan treatment is associated with promising hepatoprotective effect against diabetes-induced liver damage via reduction of cell inflammation and oxidative damage. These data suggest that ET receptors may be an important actor in diabetes-related liver damage, and blockage of these receptors may become a target for preventing diabetic complications in the future.

Topics: Animals; Bosentan; Chemical and Drug Induced Liver Injury; Diabetes Mellitus, Experimental; Endothelin Receptor Antagonists; Endothelin-1; Gene Expression Regulation; Glutathione; Liver; Male; Malondialdehyde; Rats; Streptozocin; Sulfonamides; Superoxide Dismutase; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Periostin actively contributes to tissue injury, fibrosis, atherosclerosis, and inflammatory diseases; however, its role in hepatic fibrosis is unclear. Herein, we revealed that periostin expression was significantly up-regulated in carbon tetrachloride- and bile duct ligation-induced mice with acute and chronic liver fibrosis. Deficiency in periostin abrogated the development of liver fibrosis in mice. Carbon tetrachloride treatment significantly increased α-smooth muscle actin, fibronectin, and collagen I levels in wild-type mice, which were unaffected in periostin-knockout mice. Periostin-deficient mice showed a significantly reduced area of collagen deposition and decreased levels of serum alanine aminotransferase and aspartate aminotransferase compared with wild-type mice after 2 weeks of carbon tetrachloride administration. Chemokine ligand 2, IL-6, IL-1β, tumor necrosis factor-α, and tissue inhibitor of metalloproteinases 1 mRNA levels were significantly lower in periostin-deficient mice than in wild-type mice after carbon tetrachloride treatment. Periostin colocalized with hepatic stellate cell-derived collagen I and α-smooth muscle actin in mouse acute and chronic fibrotic liver tissues. Transforming growth factor (TGF)-β1 markedly induced periostin expression in primary mouse hepatic stellate cells. Periostin-deficient mice showed significantly lower levels of TGF-β1 and TGF-β2 compared with wild-type mice after carbon tetrachloride treatment. High levels of periostin in patients with acute or chronic hepatitis correlated with TGF-β1 and TGF-β2 expression in serum from patients with hepatitis. Data indicate that periostin is a novel mediator of hepatic fibrosis development.

Topics: Animals; Cell Adhesion Molecules; Chemical and Drug Induced Liver Injury; Collagen; Hepatic Stellate Cells; Hepatitis; Humans; Inflammation; Liver; Liver Cirrhosis; Mice; Mice, Knockout; Transforming Growth Factor beta

The present study investigated the in vitro and in vivo effects of individual and combined doses of idebenone, carnosine and vitamin E on ameliorating some of the biochemical indices of nano-sized titanium dioxide (n-TiO2) in mice liver.. The in vitro cytotoxic effect of nano-sized anatase TiO2 (21 nm) on hepatic cell lines (HepG 2) was investigated. Additionally, n-TiO2 was orally administered (150 mg/kg/day) for 2 weeks, followed by a daily intragastric gavage of the aforementioned antioxidants for 1 month.. n-TiO2 induced significant cytotoxicity in hepatic cell lines and elevated the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), hepatic total antioxidant capacity (TAC) and nitrite/nitrate (NOx) levels. Meanwhile, glutathione-S-transferase (GST) activity was significantly reduced. Moreover, RT-PCR and western blot analysis showed that n-TiO2 significantly altered the mRNA and protein expressions of transforming growth factor-beta (TGF-β1) and Smad-2, as well as vascular endothelium growth factor (VEGF). Histopathological examination of hepatic tissue reinforced these results.. Idebenone, carnosine and vitamin E ameliorated the deviated parameters with the combination regimen demonstrating the most pronounced effect. Oxidative stress, liver fibrosis and angiogenesis may be implicated in n-TiO2-induced liver toxicity.

Topics: Alanine Transaminase; Angiogenesis Inhibitors; Animals; Antioxidants; Aspartate Aminotransferases; Carnosine; Cell Line, Tumor; Chemical and Drug Induced Liver Injury; Glutathione Transferase; Hep G2 Cells; Humans; Liver Cirrhosis; Male; Mice; Nitrates; Nitrites; Smad2 Protein; Titanium; Transforming Growth Factor beta; Ubiquinone; Vascular Endothelial Growth Factor A; Vitamin E

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

Fibrosis refers to the accumulation of excess extracellular matrix (ECM) components and represents a key feature of many chronic inflammatory diseases. Unfortunately, no currently available treatments specifically target this important pathogenic mechanism. MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally repress target gene expression and the development of miRNA-based therapeutics is being actively pursued for a diverse array of diseases. Because a single miRNA can target multiple genes, often within the same pathway, variations in the level of individual miRNAs can potently influence disease phenotypes. Members of the miR-29 family, which include miR-29a, miR-29b and miR-29c, are strong inhibitors of ECM synthesis and fibrosis-associated decreases in miR-29 have been reported in multiple organs. We observed downregulation of miR-29a/b/c in fibrotic livers of carbon tetrachloride (CCl4) treated mice as well as in isolated human hepatocytes exposed to the pro-fibrotic cytokine TGF-β. Importantly, we demonstrate that a single systemic injection of a miR-29a expressing adeno-associated virus (AAV) can prevent and even reverse histologic and biochemical evidence of fibrosis despite continued exposure to CCl4. The observed therapeutic benefits were associated with AAV transduction of hepatocytes but not hepatic stellate cells, which are the main ECM producing cells in fibroproliferative liver diseases. Our data therefore demonstrate that delivery of miR-29 to the hepatic parenchyma using a clinically relevant gene delivery platform protects injured livers against fibrosis and, given the consistent fibrosis-associated downregulation of miR-29, suggests AAV-miR-29 based therapies may be effective in treating a variety of fibroproliferative disorders.

Topics: Animals; Carbon Tetrachloride; Cell Line; Chemical and Drug Induced Liver Injury; Dependovirus; Extracellular Matrix; Gene Expression Regulation; Genetic Vectors; Hepatic Stellate Cells; Hepatocytes; Humans; Liver Cirrhosis; Mice; MicroRNAs; Transforming Growth Factor beta

Integrins are transmembrane heterodimeric receptors that contribute to diverse biological functions and play critical roles in many human diseases. Studies using integrin subunit knockout mice and inhibitory antibodies have identified important roles for nearly every integrin heterodimer and led to the development of a number of potentially useful therapeutics. One notable exception is the αvβ1 integrin. αv and β1 subunits are individually present in numerous dimer pairs, making it challenging to infer specific roles for αvβ1 by genetic inactivation of individual subunits, and αvβ1 complex-specific blocking antibodies do not yet exist. We therefore developed a potent and highly specific small-molecule inhibitor of αvβ1 to probe the function of this understudied integrin. We found that αvβ1, which is highly expressed on activated fibroblasts, directly binds to the latency-associated peptide of transforming growth factor-β1 (TGFβ1) and mediates TGFβ1 activation. Therapeutic delivery of this αvβ1 inhibitor attenuated bleomycin-induced pulmonary fibrosis and carbon tetrachloride-induced liver fibrosis, suggesting that drugs based on this lead compound could be broadly useful for treatment of diseases characterized by excessive tissue fibrosis.

Topics: Animals; Bleomycin; Cells, Cultured; Chemical and Drug Induced Liver Injury; Drug Design; Fibroblasts; Hydrocarbons, Brominated; Liver; Liver Cirrhosis, Experimental; Lung; Mice; Mice, Inbred C57BL; Molecular Targeted Therapy; Pulmonary Fibrosis; Receptors, Vitronectin; Signal Transduction; Transforming Growth Factor beta

A low dose of theophylline enhances histone deacetylase activity leading to inhibition of proinflammatory transcription, and inhibits lung fibroblast proliferation. The present work investigated the effect of lowdose theophylline on biochemical and histological pictures of liver tissues in rats with immunological hepatic injury induced by concanavalin A (Con A).. Ratswere assigned to control vehicle,model (Con A) and theophylline groups. Half of the animals in each group were sacrificed at the end of the 4th week and the other half were sacrificed at the end of the 8th week.. There was a time-dependent increase in the liver injury parameters by the end of the 4th and 8th weeks in the Con A treated group. Theophylline (20 mg/kg/day), produced a significant decrease in serum liver enzymes (ALT, AST), serum interferon gamma (IFN-γ) levels and the hepatic transforming growth factor-β (TGF-β) level. A significant decrease in liver tissue hydroxyproline content together with reduction in portal hypertension at the end of the 8th week was detected compared to the Con A group. Theophylline treated rats exhibited a significant decrease in hepatic vacuolation, apoptosis, leucocyte infiltration, and accumulation of collagen fibers in comparison to the Con A group. In addition, significant decreases in the area percentage of fibrosis and the area percentage of caspase +ve cells were reported compared to the Con A group.. Theophylline effectively reduced the inflammation of liver tissues and alleviated the liver damage by decreasing IFN-γ and TGF-β in liver tissues of rats with immunological hepatic injury.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Caspase 3; Chemical and Drug Induced Liver Injury; Drug Evaluation, Preclinical; Hydroxyproline; Interferon-alpha; Liver; Liver Cirrhosis; Male; Portal Pressure; Rats, Wistar; Theophylline; Transforming Growth Factor beta

At present, cirrhosis is an incurable liver disease. Transforming growth factor β (TGF‑β) is important in myofibroblast induction during the cirrhosis initiation process. The current approach in the development of hepatoprotective drugs depends on TGF‑β inhibition. San Huang Shel Shin Tang (SHSST) is a traditional herbal decoction able to exert a protective effect on the liver, however, similar to silymarin, it is limited by its hydrophobicity. In the present study, SHSST was modified with β‑cyclodextrin to form a hydrophilic complex, which improved its bioavailability. In the carbon tetrachloride‑induced acute injury animal model, the effects of pretreatment with silymarin, baicalein, SHSST and the SHSST‑β‑CD‑complex (SHSSTc) at a low and high dose were assessed. The biopsy results revealed marked liver protection following treatment with silymarin, baicalein and SHSST and these effects were improved further following pretreatment with SHSSTc. Protein analysis demonstrated that the hepatoprotective effects of silymarin occurred through inhibition of the TGF‑β/Smad‑3/connective tissue growth factor (CTGF) signaling pathway. SHSSTc exerted the same protective mechanism, however, SHSSTc suppressed CTGF level to a greater extent compared with the groups treated with SHSST or silymarin. Only pretreatment with SHSST and SHSSTc exhibited partial enhancement in the expression of proteins involved in the regulation of liver regeneration, including extracellular‑signal‑regulated kinase 5, phospho‑nuclear factor of activated T cells 3 and phospho‑GATA4.

Topics: Animals; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Connective Tissue Growth Factor; Cyclodextrins; Dose-Response Relationship, Drug; Flavanones; Interleukin-6; Liver; Protective Agents; Rats; Rats, Sprague-Dawley; Signal Transduction; Silymarin; Smad3 Protein; Transforming Growth Factor beta

Pyrrole-imidazole (PI) polyamide is a novel gene regulating agent that competitively inhibits transcription factor binding to the promoter of the specific target gene. Liver fibrosis is an integral stage in the development of chronic liver disease, and transforming growth factor β (TGFβ) is known to play a central role in the progression of this entity. The aim of this study was to evaluate the effect of PI polyamide targeting TGFβ1 on rat liver fibrosis. PI polyamide was designed to inhibit activator protein 1 (AP-1) transcription factor binding to the TGFβ1 gene promoter. The effect of PI polyamide on hepatic stellate cells was evaluated by real time polymerase chain reaction (PCR) in RI-T cells. To determine the effect of PI polyamide in vivo, PI polyamide was intravenously administered at a dose of 3 mg/kg/week in dimethylnitrosamine (DMN)-induced rat model of liver fibrosis. Treatment of RI-T cells with 1.0 µM PI polyamide targeting TGFβ1 significantly inhibited TGFβ1 mRNA expression. Azan staining showed that DMN treatment significantly increased areas of fibrous materials compared with controls. PI polyamide targeting TGFβ1 significantly decreased the fibrous area compared with DMN group. mRNA expression levels of α-smooth muscle actin and matrix metalloproteinase-2 were significantly increased in DMN-treated group compared with control. Treatment with TGFβ1 PI polyamide significantly decreased mRNA expression of these genes compared with DMN group. The novel gene regulator PI polyamide targeting TGFβ1 may be a feasible therapeutic agent for the treatment of chronic liver disease.

Topics: Actins; Animals; Cell Line; Chemical and Drug Induced Liver Injury; Dimethylnitrosamine; End Stage Liver Disease; Fibrosis; Gene Silencing; Hepatic Stellate Cells; Imidazoles; Liver; Liver Cirrhosis; Liver Cirrhosis, Experimental; Male; Matrix Metalloproteinase 2; Pyrroles; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor beta

Glycoprotein nonmetastatic melanoma B (Gpnmb), a transmembrane glycoprotein that is expressed in macrophages, negatively regulates inflammation. We have reported that Gpnmb is strongly expressed in the livers of rats fed a choline-deficient, L-amino acid-defined (CDAA) diet. However, the role of macrophage-expressed Gpnmb in liver injury is still unknown. This study aimed to clarify the characteristics of infiltrating macrophages that express Gpnmb, and the involvement of Gpnmb in the repair process in response to liver injury.. C57BL/6J, DBA/2J [DBA] and DBA/2J-Gpnmb+ [DBA-g+] mice were treated with a single intraperitoneal injection of carbon tetrachloride (CCl4) at a dose of 1.0 mL/kg body weight. Mice were sacrificed at predetermined time points, followed by measurement of serum alanine aminotransferase (ALT) levels and histological examination. Expression of Gpnmb, pro-/anti-inflammatory cytokines, and profibrotic/antifibrotic factors were examined by quantitative RT-PCR and/or Western blotting. Immunohistochemistry, fluorescent immunostaining and flow cytometry were used to determine the expression of Gpnmb, CD68, CD11b and α-SMA, phagocytic activity, and the presence of apoptotic bodies. We used quantitative RT-PCR and ELISA to examine TGF-β and MMP-13 expression and the concentrations and supernatants of isolated infiltrating hepatic macrophages transfected with siGpnmb.. In C57BL/6J mice, serum ALT levels increased at two days after CCl4 injection and decreased at four days. Gpnmb expression in the liver was stimulated four days after CCl4 injection. Histological examination and flow cytometry showed that Gpnmb-positive cells were almost positive for CD68-positive macrophages, contained engulfed apoptotic bodies and exhibited enhanced phagocytic activity. Isolated infiltrating hepatic macrophages transfected with siGpnmb showed high MMP-13 secretion. There was no significant difference in the magnitude of CCl4-induced liver injury between DBA-g+ and DBA mice. However, hepatic MMP-13 expression, as well as α-SMA expression and collagen production, increased significantly in DBA-g+ compared with DBA mice.. Gpnmb-positive macrophages infiltrate the liver during the recovery phase of CCl4-induced acute liver injury and contribute to the balance between fibrosis and fibrolysis in the repair process following acute liver injury.

Topics: Acute Disease; Alanine Transaminase; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Collagen; Eye Proteins; Liver; Liver Cirrhosis; Macrophages; Matrix Metalloproteinase 13; Matrix Metalloproteinase 9; Membrane Glycoproteins; Mice, Inbred C57BL; Mice, Inbred DBA; Phagocytosis; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Wound Healing

Hepatic stellate cells (HSCs) known as "master producers" and macrophages as "master regulators", are the key cell types that strongly contribute to the progression of liver fibrosis. Since Notch signaling regulates multiple cellular processes, we aimed to study the role of Notch signaling in HSCs differentiation and macrophages polarization and to evaluate its implication in liver fibrogenesis. Notch pathway components were found to be significantly upregulated in TGFβ-activated HSCs, inflammatory M1 macrophages, and in mouse and human fibrotic livers. Interestingly, inhibition of Notch using a selective γ-secretase inhibitor, Avagacestat, significantly inhibited TGFβ-induced HSC activation and contractility, and suppressed M1 macrophages. Additionally, Avagacestat inhibited M1 driven-fibroblasts activation and fibroblasts-driven M1 polarization (nitric oxide release) in fibroblasts and macrophages co-culture, and conditioned medium studies. In vivo, post-disease treatment with Avagacestat significantly attenuated fibrogenesis in CCl4-induced liver fibrosis mouse model. These effects were attributed to the reduction in HSCs activation, and inhibition of inflammatory M1 macrophages and upregulation of suppressive M2 macrophages. These findings suggest that Notch signaling plays a crucial role in HSC activation and M1/M2 polarization of macrophages in liver fibrosis. These results provide new insights for the development of novel therapies against liver fibrosis through modulation of Notch signaling.

Topics: 3T3 Cells; Animals; Cell Communication; Cell Differentiation; Cell Line; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Fibroblasts; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Macrophage Activation; Macrophages; Mice; Oxadiazoles; Receptors, Notch; Signal Transduction; Sulfonamides; Transforming Growth Factor beta

In human chronic liver disease, there is association between ductular reaction (DR) and fibrosis; yet, the mechanism triggering its onset and its role in scar formation remains unknown. Since we previously showed that osteopontin (OPN) is highly induced during drug-induced liver fibrosis, we hypothesised that OPN could drive oval cells (OC) expansion and DR and signal to hepatic stellate cells (HSC) to promote scarring.. In vivo studies demonstrated increased OPN expression in biliary epithelial cells (BEC) and in OC in thioacetamide (TAA)-treated mice. OPN ablation protected mice from TAA and bile duct ligation-induced liver injury, DR and scarring. This was associated with greater hepatocyte proliferation, lower OC expansion and DR along with less fibrosis, suggesting that OPN could activate the OC compartment to differentiate into BEC, which could then signal to HSC to enhance scarring. Since TAA-treated wild-type mice and cirrhotic patients showed TGF-β(+) BEC, which were lacking in TAA-treated Opn(-/-) mice and in healthy human explants, this suggested that OPN could regulate TGF-β, a profibrogenic factor. In vitro experiments confirmed that recombinant OPN (rOPN) decreases hepatocyte proliferation and increases OC and BEC proliferation. To evaluate how BEC regulate collagen-I production in HSC, co-cultures were established. Co-cultured BEC upregulated OPN and TGF-β expression and enhanced collagen-I synthesis by HSC. Lastly, recombinant TGF-β (rTGFβ) and rOPN promoted BEC proliferation and neutralisation of OPN and TGF-β reduced collagen-I expression in co-cultured HSC.. OPN emerges as a key matricellular protein driving DR and contributing to scarring and liver fibrosis via TGF-β.

Topics: Animals; Cell Proliferation; Chemical and Drug Induced Liver Injury; Coculture Techniques; Hepatic Duct, Common; Hepatocytes; Immunohistochemistry; Ki-67 Antigen; Liver Cirrhosis; Mice, Inbred C57BL; Mice, Inbred Strains; Osteopontin; Oxidative Stress; Transforming Growth Factor beta

n-3 polyunsaturated fatty acids (PUFA) ameliorate fatty liver in experimental models, but their effects on inflammation and fibrosis during steatohepatitis are either controversial or lacking. We compared the effects of supplementation with olive oil (OO) alone or OO and n-3 PUFA on the development and progression of experimental steatohepatitis.. Balb/C mice (≥5 mice/group) were fed a methionine- and choline-deficient (MCD) diet or a control diet for 4 or 8 weeks. At the same time, mice were supplemented with n-3 PUFA (eicosapentaenoic and docosahexahenoic acid, 25 mg together with 75 mg OO), or OO alone (100 mg), two times a week by intragastric gavage.. After 8 weeks, mice on MCD/n-3 had higher ALT levels compared to MCD/OO and more severe scores of inflammation, including a significant increase in the number of lipogranulomas (26.4 ± 8.4 vs. 5.1 ± 5 per field, P < 0.001). Intrahepatic expression of TNF-α and CCL2 was higher in MCD/n-3 mice at both time points. In addition, increased expression of the profibrogenic genes TIMP-1 and TGF-β, and more severe histological scores of fibrosis were evident in MCD/n-3 mice. After 8 week of MCD diet, portal pressure was higher in mice receiving n-3 than in those on OO alone (5.1 ± 1.4 vs. 7.0 ± 0.9 mmHg, P < 0.05). Analysis of hepatic fatty acid profile showed that supplementation resulted in effective incorporation of n-3 PUFA.. In a murine model of steatohepatitis, supplementation with n-3 PUFA and OO is associated with more severe necro-inflammation and fibrosis than in mice treated with OO only.

Topics: Animals; Biomarkers; Chemical and Drug Induced Liver Injury; Choline Deficiency; Dietary Supplements; Disease Models, Animal; Fatty Acids, Omega-6; Inflammation Mediators; Liver; Liver Cirrhosis; Male; Methionine; Mice, Inbred BALB C; Necrosis; Non-alcoholic Fatty Liver Disease; Olive Oil; Plant Oils; Time Factors; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta

Artemisia capillaris, also called "InJin" in Korean, has been widely used to treat various hepatic disorders in traditional Oriental medicine.. The purpose of this study is to evaluate the hepatoprotective effect of Artemisia capillaris (aqueous extract, WAC) on alcoholic liver injury.. Liver injury was induced by oral administration of 30% alcohol (10 mL/kg, twice per day) plus pyrazole (PRZ, 30 mg/kg) with/without WAC (50, 100mg/kg, orally once per day) or silymarin (50mg/kg) for 10 days. The hepatoprotective effects were assessed by observing histopathological changes, hepatic transaminase enzymes, hepatic oxidation and antioxidant parameters, inflammatory cytokines, and alcohol metabolic enzymes in serum and hepatic gene expression level, respectively.. Alcohol-PRZ treatment drastically increased the serum levels of aspartate transaminase (AST), alanine transaminase (ALT), and malondialdehyde (MDA) levels in serum and liver tissues while these changes were significantly ameliorated by WAC administration (p<0.05 or 0.01). The prominent microvesicular steatosis and mild necrosis in hepatic histopathology were induced by alcohol-PRZ treatment, but notably attenuated by WAC administration. Moreover, the alcohol-PRZ treatment-induced depletions of the antioxidant components including glutathione content, total antioxidant capacity (TAC), activities of glutathione peroxidase (GSH-Px), reductase (GSH-Rd), catalase, and superoxide dismutase (SOD) were significantly ameliorated by WAC administration (p<0.05, except GSH-Rd). These results were in accordance with the modulation of NF-E2-related factor (Nrf2) and heme oxygenase-1 (HO-1) gene expression. Alcohol-PRZ treatment increased the levels of tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) in hepatic tissues. However they were significantly normalized by WAC administration (p<0.05 or 0.01). In addition, WAC administration significantly attenuated the alterations of aldehyde dehydrogenase (ALDH) level in serum and hepatic gene expressions of ALDH and alcohol dehydrogenase (ADH).. These results support the relevance in clinical use of Artemisia capillaris for alcohol-associated hepatic disorders. The underlying mechanisms may involve both enhancement of antioxidant activities and modulation of proinflammatory cytokines.

Topics: Alanine Transaminase; Alkaline Phosphatase; Animals; Artemisia; Aspartate Aminotransferases; Blood Glucose; Catalase; Chemical and Drug Induced Liver Injury; Ethanol; Glutathione; Glutathione Peroxidase; Glutathione Reductase; L-Lactate Dehydrogenase; Male; Phytotherapy; Plant Extracts; Protective Agents; Pyrazoles; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Serum Albumin; Superoxide Dismutase; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The liver plays an essential role in the body by regulating several important metabolic functions. Liver injury is associated with the distortion of these functions causing many health problems. Pharmaceutical drugs treat liver disorders but cause further damage to it. Hence, herbal drugs are used worldwide and are becoming increasingly popular.. The hepatoprotective activity of Phyllanthus niruri (PN) was evaluated against liver cirrhosis induced by thioacetamide (TAA) in male Sprague Dawley rats. Rats received intraperitoneal injections of thioacetamide (TAA, 200 mg/kg, b.w. three times weekly) for eight weeks. Daily treatments with plant extract (200 mg/kg) were administered orally for eight weeks. At the end of the study, hepatic damage was evaluated by monitoring transforming growth factor (TGFβ), collagen α1 (Collα1), matrix metalloproteinase-2 (MMP2) and tissue inhibitor of matrix metalloproteinase-1 (TIMP1) gene expression by real-time PCR. Moreover, different chromatographic techniques including column chromatography, thin layer chromatography, and Ultra Performance Liquid Chromatography (UPLC) with Liquid Chromatography/Mass Spectrometry (LC/MS) were used to isolate the active constituents of the plant.. The results revealed that treatment with PN significantly reduced the effect of thioacetamide toxicity and exhibited effective hepatoprotective activity. The mechanism of the hepatoprotective effect of PN is proposed to be by normalizing ROSs. Additionally, PN treatment regulated the expression of TGFβ, Collα1, MMP2, and TIMP1 genes. In the active fraction of P. niruri, the isolated chemical constituents were 4-O-caffeoylquinic acid and quercetin 3-O-rhamnoside.. The results of the present study indicate that PN ethanol extracts possess hepatoprotective activity that is most likely because of the isolated chemical constituents.

Topics: Animals; Antioxidants; Chemical and Drug Induced Liver Injury; Gene Expression Profiling; Liver; Liver Cirrhosis; Male; Matrix Metalloproteinase 2; Phyllanthus; Phytotherapy; Plant Extracts; Quercetin; Quinic Acid; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1; 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

Chemotherapy-associated liver injury (CALI) has been linked to increased morbidity and poorer disease-specific outcomes in patients undergoing resection of colorectal liver metastases (CRLM). The aim of this study was to assess the contribution of tumour-related factors to the development of FOLFOX-induced liver injury.. We assessed the effect of FOLFOX treatment on the murine liver either in the presence or absence of CRLM to evaluate the contribution of both chemotherapy and tumour death to the development of CALI.. In the presence of liver metastases, there was increased hepatic expression of plasminogen activator inhibitor-1 (146-fold; P<0.01) and vWF (2.4-fold; P<0.01) transcript as compared with sham-operated controls. In addition, we detected large clusters of megakaryocytes in the spleen of FOLFOX-treated tumour-bearing animals. The livers of FOLFOX-treated animals also showed changes in matrix remodelling genes such as TGFβ (P<0.01), MMP2 (P<0.001), TIMP1 (P<0.001) and Pro-Collagen I (P<0.05) which was exacerbated in the presence of tumour. These genes have previously been demonstrated to have a key role in FOLFOX-induced liver injury.. It appears that the toxicity of FOLFOX chemotherapy is enhanced by tumour-related factors.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Chemical and Drug Induced Liver Injury; Collagen Type I; Colorectal Neoplasms; Fluorouracil; Hepatic Veno-Occlusive Disease; Inflammation; Leucovorin; Liver; Liver Neoplasms; Matrix Metalloproteinase 2; Megakaryocytes; Mice; Mice, Inbred C57BL; Organoplatinum Compounds; Plasminogen Activator Inhibitor 1; Spleen; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta

During the acute liver injury, immune responses are provoked into eliciting inflammation in the acute phase. In the healing phase, the inflammation is terminated for wound healing and restoration of immune homeostasis. In this study, we sought to address how regulatory T cells (Tregs) are involved in the progression of liver injury and repair. In the acute phase, intrahepatic Tregs (CD4(+)FoxP3(+)Helios(+)) diminished promptly through apoptosis, which was followed by inflammation and tissue injury. In the healing phase, a new subset of Tregs (CD4(+)Foxp3(+)Helios(-)) was generated in correlation with the matrix metalloproteinase (MMP) cascade and transforming growth factor-beta (TGF-β) activation that were manifested mainly by hepatic stellate cells. Moreover, the induction of induced Tregs and wound healing were both impaired in mice lacking TGF-β signaling or MMPs. The depletion of induced Tregs also impeded wound healing for tissue repair. Together, this study demonstrates the mechanism that the loss of nTregs through apoptosis in the acute phase may facilitate inflammation, while regenerated Tregs through MMP9/13-dependent activation of TGF-β in the healing phase are critical to terminate inflammation and allow for wound healing.

Topics: Animals; Blotting, Western; Cells, Cultured; Chemical and Drug Induced Liver Injury; Flow Cytometry; Homeostasis; Liver; Matrix Metalloproteinase 13; Matrix Metalloproteinase 9; Mice; Mice, Transgenic; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

A recent report showed that the unfolded protein response (UPR) signaling was activated in the pathogenesis of carbon tetrachloride (CCl(4))-induced hepatic fibrosis. Phenylbutyric acid (PBA) is a well-known chemical chaperone that inhibits endoplasmic reticulum (ER) stress and unfolded protein response (UPR) signaling. In the present study, we investigated the effects of PBA on CCl(4)-induced hepatic fibrosis in mice. All mice were intraperitoneally (i.p.) injected with CCl(4) (0.15 ml/kg BW, twice per week) for 8 weeks. In CCl(4)+PBA group, mice were i.p. injected with PBA (150 mg/kg, twice per day) from the beginning of CCl(4) injection to the end. As expected, PBA significantly attenuated CCl(4)-induced hepatic ER stress and UPR activation. Although PBA alleviated, only to a less extent, hepatic necrosis, it obviously inhibited CCl(4)-induced tumor necrosis factor alpha (TNF-α) and transforming growth factor beta (TGF-β). Moreover, PBA inhibited CCl(4)-induced hepatic nuclear factor kappa B (NF-κB) p65 translocation and extracellular signal-regulated kinase (ERK) and c-Jun N-terminal Kinase (JNK) phosphorylation. Interestingly, CCl(4)-induced α-smooth muscle actin (α-SMA), a marker for the initiation phase of HSC activation, was significantly attenuated in mice pretreated with PBA. Correspondingly, CCl(4)-induced hepatic collagen (Col)1α1 and Col1α2, markers for the perpetuation phase of HSC activation, were inhibited in PBA-treated mice. Importantly, CCl(4)-induced hepatic fibrosis, as determined using Sirius red staining, was obviously attenuated by PBA. In conclusion, PBA prevents CCl(4)-induced hepatic fibrosis through inhibiting hepatic inflammatory response and HSC activation.

Topics: Animals; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Endoplasmic Reticulum Stress; Hepatic Stellate Cells; Inflammation; Injections, Intraperitoneal; Liver Cirrhosis, Experimental; Male; Mice; Phenylbutyrates; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Unfolded Protein Response

In many organs, myofibroblasts play a major role in the scarring process in response to injury. In liver fibrogenesis, hepatic stellate cells (HSCs) are thought to transdifferentiate into myofibroblasts, but the origins of both HSCs and myofibroblasts remain elusive. In the developing liver, lung, and intestine, mesothelial cells (MCs) differentiate into specific mesenchymal cell types; however, the contribution of this differentiation to organ injury is unknown. In the present study, using mouse models, conditional cell lineage analysis has demonstrated that MCs expressing Wilms tumor 1 give rise to HSCs and myofibroblasts during liver fibrogenesis. Primary MCs, isolated from adult mouse liver using antibodies against glycoprotein M6a, undergo myofibroblastic transdifferentiation. Antagonism of TGF-β signaling suppresses transition of MCs to mesenchymal cells both in vitro and in vivo. These results indicate that MCs undergo mesothelial-mesenchymal transition and participate in liver injury via differentiation to HSCs and myofibroblasts.

Topics: Animals; Biliary Tract; Carbon Tetrachloride; Cell Lineage; Cell Transdifferentiation; Cells, Cultured; Chemical and Drug Induced Liver Injury; Epithelium; Gene Expression; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Liver Regeneration; Membrane Glycoproteins; Mesoderm; Mice; Mice, Transgenic; Myofibroblasts; Nerve Tissue Proteins; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; WT1 Proteins

Small fish models have been used for decades in carcinogenicity testing. Demonstration of common morphological changes associated with specific mechanisms is a clear avenue by which data can be compared across divergent phyletic levels. Dimethylnitrosamine, used in rats to model human alcoholic cirrhosis and hepatic neoplasia, is also a potent hepatotoxin and carcinogen in fish. We recently reported some striking differences in the mutagenicity of DMN in lambda cII transgenic medaka fish vs. Big Blue(®) rats, but the pre-neoplastic and neoplastic commonalities between the two models are largely unknown. Here, we focus on these commonalities, with special emphasis on the TGF-β pathway and its corresponding role in DMN-induced hepatic neoplasia. Similar to mammals, hepatocellular necrosis, regeneration, and dysplasia; hepatic stellate cell and "spindle cell" proliferation; hepatocellular and biliary carcinomas; and TGF-β1 expression by dysplastic hepatocytes all occurred in DMN-exposed medaka. Positive TGF-β1 staining increased with increasing DMN exposure in bile preductular epithelial cells, intermediate cells, immature hepatocytes and fewer mature hepatocytes. Muscle specific actin identified hepatic stellate cells in DMN-exposed fish. Additional mechanistic comparisons between animal models at different phyletic levels will continue to facilitate the interspecies extrapolations that are so critical to toxicological risk assessments.

Topics: Animals; Animals, Genetically Modified; Biliary Tract Neoplasms; Biomarkers; Carcinogenicity Tests; Carcinogens; Carcinoma, Hepatocellular; Cell Proliferation; Chemical and Drug Induced Liver Injury; Dimethylnitrosamine; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Fish Diseases; Liver; Liver Neoplasms; Male; Mutagenicity Tests; Oryzias; Rats; Signal Transduction; Species Specificity; Transforming Growth Factor beta

The aim of this study was to evaluate the possible protective effects of quercetin (QE) on liver injury caused by chronic toluene exposure in rats. The rats were randomly allotted into one of three experimental groups: control, toluene treated and toluene treated with QE; each group contained 10 animals. The control group received 1 ml physiologic serum, and toluene treatment was performed by inhalation of 3000 ppm toluene, for 8 h/day and 6 days/week for 12 weeks. The rats in the QE-treated groups were given QE (15 mg/kg, intraperitoneal) once a day for 12 weeks starting just after toluene exposure. Tissue samples were obtained for histopathological investigation. To date, no histopathological changes in the liver of rats after chronic toluene exposure by QE treatment have been reported. Light microscopic evaluation of liver tissue samples of toluene-exposed rats revealed enlarged sinusoids filled with blood. In addition, some of the hepatocytes showed loss of cytoplasm, and some had a hyperchromatic nucleus. QE treatment attenuated alterations in liver histology. The alpha smooth muscle actin, transforming growth factor beta-positive cells and the activity of terminal deoxynucleotidyl transferase dUTP nick end labeling in the toluene-treated group were observed to be reduced with QE treatment. The data indicate that QE attenuates toluene-induced liver injury, but further investigation is still absolutely necessary.

Topics: Actins; Animals; Chemical and Drug Induced Liver Injury; Immunohistochemistry; In Situ Nick-End Labeling; Liver; Liver Cirrhosis; Male; Protective Agents; Quercetin; Rats; Rats, Wistar; Statistics, Nonparametric; Toluene; Toxicity Tests, Chronic; Transforming Growth Factor beta

(Z)2-(5-(4-methoxybenzylidene)-2, 4-dioxothiazolidin-3-yl) acetic acid (MDA) is an aldose reductase (AR) inhibitor. Recent studies suggest that AR contributes to the pathogenesis of inflammation by affecting the nuclear factor κB (NF-κB)-dependent expression of cytokines and chemokines and therefore could be a novel therapeutic target for inflammatory pathology. The current study evaluated the in vivo role of MDA in protecting the liver against injury and fibrogenesis caused by CCl(4) in rats, and the underlying mechanisms.. A single injection of CCl(4) induced acute hepatitis, and repeated injections were used to induce hepatic fibrosis in rats. Therapeutic efficacy was assessed by comparison of the severity of hepatic injury and fibrosis in MDA-treated rats versus untreated controls.. MDA significantly protected the liver from injury by reducing the activity of serum alanine aminotransferase, and improving the histological architecture of the liver. MDA modulated NF-κB-dependent activation of inflammatory cytokines by reducing hepatic mRNA levels of tumor necrosis factor-α, interleukin-1β, inducible nitric oxide (NO) synthase and transforming growth factor-β. In addition, MDA attenuated oxidative stress by increasing the content of hepatic glutathione. These favorable changes were associated with suppressed hepatic NF-κB activation by MDA. MDA treatment improved liver fibrosis in rats that received repeated CCl(4) injections. In vitro, MDA attenuated phosphorylation of IκB and activation of NF-κB, and thus prevented biosynthesis of NO in lipopolysaccharide-activated RAW264.7 cells.. The present study suggests that AR is a novel therapeutic anti-inflammatory target for the treatment of hepatitis and liver fibrosis.

Topics: Alanine Transaminase; Aldehyde Reductase; Animals; Carbon Tetrachloride; Cells, Cultured; Chemical and Drug Induced Liver Injury; Enzyme Inhibitors; Glutathione; I-kappa B Proteins; Interleukin-1beta; Liver; Liver Cirrhosis, Experimental; Macrophages; Male; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidative Stress; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Thiazolidinediones; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Activated hepatic stellate cells (HSCs) play a key role in hepatic fibrogenesis, and inhibition of HSC activation may prevent liver fibrosis. Acetaldehyde, the most deleterious metabolite of alcohol, triggers HSC activation in alcoholic liver injury. In the present study, we investigated the protective effect of sodium ferulate (SF), a sodium salt of ferulic acid that is rich in fruits and vegetables, on acetaldehyde-stimulated HSC activation using precision-cut liver slices (PCLSs). Rat PCLSs were co-incubated with 350 μM acetaldehyde and different concentrations of SF. Hepatotoxicity was assessed by measuring enzyme leakage and malondialdehyde content in tissue. α-Smooth muscle actin, transforming growth factor-β(1), and hydroxyproline were determined to assess the activation of HSCs. In addition, matrix metalloproteinase (MMP)-1 and the tissue inhibitor of metalloproteinase (TIMP-1) were determined to evaluate collagen degradation. SF prominently prevented the enzyme leakage in acetaldehyde-treated slices and also inhibited HSC activation and collagen production stimulated by acetaldehyde. In addition, SF increased MMP-1 expression and decreased TIMP-1 expression. These results showed that SF protected PCLSs from acetaldehyde-stimulated HSC activation and liver injury, which may be associated with the attenuation of oxidative injury and acceleration of collagen degradation.

Topics: Acetaldehyde; Actins; Animals; Antioxidants; Chemical and Drug Induced Liver Injury; Collagen; Coumaric Acids; Ethanol; Hepatic Stellate Cells; Hydroxyproline; Liver; Male; Malondialdehyde; Matrix Metalloproteinase 1; Oxidative Stress; Phytotherapy; Plant Extracts; Rats; Rats, Wistar; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta

Chinese prescription Fufang-Liu-Yue-Qing (FLYQ) has long been employed clinically to treat chronic hepatitis B, and we have reported its beneficial effects on liver fibrosis in vitro. The present study was investigated to verify protective effects of FLYQ on liver fibrosis in a rat model and to investigate the underlying mechanisms which have not been explored yet.. Liver fibrosis was established by intragastric administration of 2 ml/kg CCl(4) twice a week for 12 weeks. During the experiment, the model group received CCl(4) only, and the normal control group received an equal volume of saline. Treatment groups received not only CCl(4) for 12 weeks, but also the corresponding drugs, colchicine (1.00 mg/kg/day) or FLYQ (300, 150, 75 mg/kg/day) from 5 to 12 weeks.. Analysis experiments showed that FLYQ could significantly alleviate liver injury, as indicated by decreasing levels of ALT, AST, ALP, GGT, IL-6 and TNF-α. Moreover, FLYQ could effectively inhibit collagen deposition and reduce the pathological tissue damage. Research on mechanism showed that FLYQ was able to markedly reduce lipid peroxidation, recruit the anti-oxidative defense system, promote ECM degradation by modulating the levels of TIMP-1 and MMP-2, and induce HSC apoptosis by down-regulating bcl-2 mRNA, as well as inhibit the expressions of α-SMA and TGF-β(1) proteins.. Our results show that FLYQ is effective in attenuating hepatic injury and fibrosis in the CCl(4)-induced rat model, which should be developed as a new drug for treatment of liver fibrosis and even cirrhosis.

Topics: Actins; Animals; Antioxidants; Apoptosis; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Colchicine; Collagen; Down-Regulation; Drugs, Chinese Herbal; Interleukin-6; Lipid Peroxidation; Liver; Liver Cirrhosis, Experimental; Magnoliopsida; Male; Matrix Metalloproteinase 2; Phytotherapy; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-1; Transaminases; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Hepatic stellate cells (HSCs) undergo activation during the development of liver fibrosis. Transcription factor myocyte enhancer factor (MEF2) 2C plays a key role in this process. In the present study, we investigated the effect of hydroxysafflor yellow A (HSYA) on hepatic fibrosis and further investigated potential mechanisms in vivo. Sprague-Dawley rats were administered with CCl(4) together with or without HYSA for 12 weeks. The effect of HYSA on hepatic fibrosis was evaluated using hematoxylin-eosin and Van Gieson staining. Messenger RNA expression was quantified by real-time polymerase chain reaction, and protein was quantified by Western blot or immunohistochemistry. Our results revealed that CCl(4) treatment induced micronodular hepatic fibrosis with a pronounced deposition of collagen fibers. Treatment with HYSA resulted in a significant decrease in fibrosis, protein expression of α-SMA, and MEF-2C gene expression. This was accompanied by a decreased expression of Tβ-RI, Tβ-RII, MEKK3, MEK5, and phosphorylation of ERk5. HYSA alone had no effect on the measured parameters. Our findings demonstrate that HSYA protected, at least in part, the rat liver from CCl(4)-caused fibrogenesis through inhibition of hepatic stellate cell (HSC) activation, attenuation of transforming growth factor beta (TGF-β) signaling. HSYA may become a novel and promising agent for the inhibition of hepatic fibrosis.

Topics: Actins; Animals; Carbon Tetrachloride; Carthamus; Chalcone; Chemical and Drug Induced Liver Injury; Collagen; Gene Expression; Hepatic Stellate Cells; Liver; Liver Cirrhosis, Experimental; Male; MEF2 Transcription Factors; Mitogen-Activated Protein Kinase 7; Myogenic Regulatory Factors; Phosphorylation; Phytotherapy; Plant Extracts; Quinones; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta

Chronic arsenic exposure induces oxidative damage to liver leading to liver fibrosis. We aimed to define the effect of grape seed extract (GSE), an antioxidant dietary supplement, on arsenic-induced liver injury. First, Male Sprague-Dawley rats were exposed to a low level of arsenic in drinking water (30ppm) with or without GSE (100mg/kg, every other day by oral gavage) for 12months and the effect of GSE on arsenic-induced hepatotoxicity was examined. The results from this study revealed that GSE co-treatment significantly attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines and fibrogenic genes. Moreover, GSE reduced arsenic-stimulated Smad2/3 phosphorylation and protein levels of NADPH oxidase subunits (Nox2, Nox4 and p47phox). Next, we explored the molecular mechanisms underlying GSE inhibition of arsenic toxicity using cultured rat hepatic stellate cells (HSCs). From the in vitro study, we found that GSE dose-dependently reduced arsenic-stimulated ROS production and NADPH oxidase activities. Both NADPH oxidases flavoprotein inhibitor DPI and Nox4 siRNA blocked arsenic-induced ROS production, whereas Nox4 overexpression suppressed the inhibitory effects of GSE on arsenic-induced ROS production and NADPH oxidase activities, as well as expression of TGF-β1, type I procollagen (Coll-I) and α-smooth muscle actin (α-SMA) mRNA. We also observed that GSE dose-dependently inhibited TGF-β1-induced transactivation of the TGF-β-induced smad response element p3TP-Lux, and that forced expression of Smad3 attenuated the inhibitory effects of GSE on TGF-β1-induced mRNA expression of Coll-I and α-SMA. Collectively, GSE could be a potential dietary therapeutic agent for arsenic-induced liver injury through suppression of NADPH oxidase and TGF-β/Smad activation.

Topics: Animals; Arsenic; Cells, Cultured; Chemical and Drug Induced Liver Injury; Grape Seed Extract; Humans; Liver Cirrhosis; Male; NADPH Oxidases; Rats; Rats, Sprague-Dawley; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Adverse alcohol effects in the liver involve oxidative metabolism, fat deposition and release of fibrogenic mediators, including TGF-beta. The work presents an assessment of liver damaging cross-talk between ethanol and TGF-beta in hepatocytes.. To investigate TGF-beta effects on hepatocytes, microarray analyses were performed and validated by qRT-PCR, Western blot analysis and immunohistochemistry. The cellular state was determined by assessing lactate dehydrogenase, cellular glutathione, reactive oxygen species, lipid peroxidation and neutral lipid deposition. RNA interference was used for gene silencing in vitro.. TGF-beta is induced in mouse livers after chronic ethanol insult, enhances ethanol induced oxidative stress and toxicity towards cultured hepatocytes plus induces lipid-, oxidative stress metabolism- and fibrogenesis-gene expression signatures. Interestingly, TGF-beta down-regulates alcohol metabolizing enzyme Adh1 mRNA in cultured hepatocytes and liver tissue from TGF-beta transgenic mice via the ALK5/Smad2/3 signalling branch, with Smad7 as a potent negative regulator. ADH1 deficiency is a determining factor for the increased lipid accumulation and Cyp2E1 dependent toxicity in liver cells upon alcohol challenge. Further, ADH1 expression was decreased during liver damage in an intragastric ethanol infusion mouse model.. In the presence of ethanol, TGF-beta displays pro-steatotic action in hepatocytes via decreasing ADH1 expression. Low ADH1 levels are correlated with enhanced hepatocyte damage upon chronic alcohol consumption by favoring secondary metabolic pathways.

Topics: Alcohol Dehydrogenase; Animals; Cells, Cultured; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Down-Regulation; Ethanol; Hepatocytes; Lipid Metabolism; Lipid Peroxidation; Male; Mice; Mice, Inbred C57BL; Oxidative Stress; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) plays a pivotal role in liver fibrosis, because it activates hepatic stellate cells, stimulating extracellular matrix deposition. Cyclooxygenase-2 (COX-2) has been associated with TGF-beta because its inhibition decreases TGF-beta expression and collagen production in some cultured cell types.. The aim of this work was to evaluate the ability of celecoxib (a selective COX-2 inhibitor) to prevent and to reverse the liver fibrosis induced by CCl(4).. We established experimental groups of rats including vehicle and drug controls, damage induced by chronic CCl(4) administration and CCl(4) plus pharmacological treatment in both prevention and reversion models. We determined: alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transpeptidase, COX and metalloproteinase-2 and -9 activities, lipid peroxidation, glutathione levels, glycogen and collagen content and TGF-beta expression.. Celecoxib prevented and aided to the recovery of livers with necrotic and cholestatic damage. Celecoxib exhibited anti-oxidant properties by restoring the redox equilibrium (lipid peroxidation and glutathione levels). Glycogen was decreased by CCl(4), while celecoxib partially prevented and reversed this effect. Celecoxib inhibited COX-2 activity, decreased TGF-beta expression, induced metalloproteinase-2 activity and, consequently, prevented and reversed collagen accumulation.. Our findings indicate that celecoxib exerts strong antifibrogenic and fibrolytic effects in the CCl(4) model of cirrhosis.

Topics: Alanine Transaminase; Animals; Anti-Inflammatory Agents; Antioxidants; Aspartate Aminotransferases; Carbon Tetrachloride; Celecoxib; Chemical and Drug Induced Liver Injury; Collagen; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Disease Models, Animal; gamma-Glutamyltransferase; Glutathione; Glycogen; Liver; Liver Cirrhosis; Male; Malondialdehyde; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Pyrazoles; Rats; Rats, Wistar; Sulfonamides; Time Factors; Transforming Growth Factor beta

Translational studies have explored the therapeutic effects of stem cells, raising hopes for the treatment of numerous diseases. Here, we evaluated the therapeutic effect of chorionic plate-derived mesenchymal stem cells (CP-MSCs) isolated from human placenta and transplanted into rats with carbon tetrachloride (CCl(4))-injured livers. CP-MSCs were analyzed for hepatocyte-specific gene expression, indocyanine green (ICG) uptake, glycogen storage, and urea production following hepatogenic differentiation. PKH26-labeled CP-MSCs were directly transplanted into the livers of rats that had been exposed to CCl(4) (1.6 g/kg, twice per week for 9 weeks). Blood and liver tissue were analyzed at 1, 2, and 3 weeks post-transplantation. The expression of type I collagen (Col I) and matrix metalloproteinases (MMPs) was analyzed in rat T-HSC/Cl-6 hepatic stellate cells co-cultured with CP-MSCs following exposure to TGF-β. The expression levels of α-smooth muscle actin (α-SMA) and Col I were lower in transplanted (TP) rats than in non-transplanted (Non-TP) animals (P < 0.05), whereas the expression levels of albumin and MMP-9 were increased. TP rats exhibited significantly higher uptake/excretion of ICG than non-TP rats (P < 0.005). In addition, collagen synthesis in T-HSC/Cl-6 cells exposed to TGF-β was decreased by co-culture with CP-MSCs, which triggered the activation of MMP-2 and MMP-9. These results contribute to our understanding of the potential pathophysiological roles of CP-MSCs, including anti-fibrotic effects in liver disease, and provide a foundation for the development of new cell therapy-based strategies for the treatment of difficult-to-treat liver diseases.

Topics: Animals; Blotting, Western; Carbon Tetrachloride; Cell Differentiation; Cell Line; Cells, Cultured; Chemical and Drug Induced Liver Injury; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Humans; Liver Diseases; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Placenta; Pregnancy; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta

Phyllanthus rheedii Wight. (Euphorbiaceae) has been used by Muthuvan tribes of Kerala for curing liver diseases. The present study was conducted to assess the hepatoprotective activity of this plant. The ethanol extract of Phyllanthus rheedii was pharmacologically analysed for its preventive effect in d-galactosamine (d-GalN) induced liver damage in rats.. The levels of hepatotoxicity in various groups were quantified by different parameters of liver damage viz. serum levels of ALT, AST, LDH, GGT, ALP and total bilirubin. The effect of extract on the expression levels of pro-inflammatory cytokines like TNF-alpha, and TGF-beta were analysed by RT-PCR. Histological changes in the liver were evaluated by hematoxylin and eosin staining of paraffin processed liver sections. The antioxidant and choleretic activity of the extract were also analysed.. Comparison of serum values of control and extract treated groups have revealed that the d-GalN induced alterations in the serum and liver markers were normalized in extract treatment groups showing hepatoprotective activity of the extract. The extract also prevented the toxin induced histological changes in liver. The mRNA levels of TGF-beta and TNF-alpha in the liver were up regulated by the hepatotoxin. The extract treatment has normalized this change, giving light to the probable mechanism of action of the extract. It has showed marked antioxidant and choleretic activity. Preliminary phytochemical screening has revealed the presence of tannins, flavanoids and phenolics as major components.. This study concluded the ethanol extract of P. rheedii could be a promissory candidate for drug development and validated the tribal claim.

Topics: Animals; Chemical and Drug Induced Liver Injury; Ethanol; Female; Galactosamine; Gene Expression Regulation; Liver; Liver Diseases; Male; Phyllanthus; Phytotherapy; Plant Extracts; Rats; Rats, Wistar; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

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

The mechanisms of transmigration of inflammatory cells through the sinusoids are still poorly understood. This study aims to identify in vitro conditions (cytokine treatment) which may allow a better understanding of the changes in PECAM (platelet endothelial cell adhesion molecule)-1-gene-expression observed in vivo.. In this study we show by immunohistochemistry, that there is an accumulation of ICAM-1 (intercellular cell adhesion molecule-1) and ED1 positive cells in necrotic areas of livers of CCl4-treated rats, whereas there are few PECAM-1 positive cells observable. After the administration of CCl4, we could detect an early rise of levels of IFN-gamma followed by an enhanced TGF-beta protein level. As shown by Northern blot analysis and surface protein expression analysed by flow cytometry, IFN-gamma-treatment decreased PECAM-1-gene-expression in isolated SECs (sinusoidal endothelial cells) and mononuclear phagocytes (MNPs) in parallel with an increase in ICAM-1-gene-expression in a dose and time dependent manner. In contrast, TGF-beta-treatment increased PECAM-1-expression. Additional administration of IFN-gamma to CCl4-treated rats and observations in IFN-gamma-/- mice confirmed the effect of IFN-gamma on PECAM-1 and ICAM-1-expression observed in vitro and increased the number of ED1-expressing cells 12 h after administration of the toxin.. The early decrease of PECAM-1-expression and the parallel increase of ICAM-1-expression following CCl4-treatment is induced by elevated levels of IFN-gamma in livers and may facilitate adhesion and transmigration of inflammatory cells. The up-regulation of PECAM-1-expression in SECs and MNPs after TGF-beta-treatment suggests the involvement of PECAM-1 during the recovery after liver damage.

Topics: Animals; Carbon Tetrachloride; Cells, Cultured; Chemical and Drug Induced Liver Injury; Cytokines; Down-Regulation; Endothelial Cells; Gene Expression Regulation; Immunologic Factors; Interferon-alpha; Interferon-gamma; Liver; Male; Mononuclear Phagocyte System; Rats; Rats, Wistar; Transforming Growth Factor beta

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

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) accumulates and remains stable in the fatty tissues and liver of rodents for a long time. Considering the pronounced difference between species, long-term, low dose hepatic effects of TCDD were investigated after subcutaneous administration of TCDD into rhesus monkeys during pregnancy. Macroscopic and histopathological examination of the liver carried out 4 y after TCDD administration demonstrated intrahepatic focal fatty changes, infarction, hemorrhage, microthrombi-formation, sinusoidal ectasia, small hepatocyte hyperplasia, and increased number of alpha-smooth muscle actin (alpha-SMA)-positive cells. An electron microscopic study disclosed sinusoidal endothelial cell degeneration and injury in the liver of TCDD-treated monkeys. Western blot analysis showed downregulation of aryl hydrocarbon receptor (AhR) protein expression and decreased level of vascular endothelial (VE) cadherin but increased expression levels of CYP1A1 and transforming growth factor beta (TGF-beta) protein in the liver tissues. These changes observed in TCDD-exposed monkeys indicated sinusoidal endothelial cell injury and impairment in intrasinusoidal microcirculation. Infarction, focal fatty change, and microthrombi-formation are considered to be closely associated with intrahepatic circulatory impairment. Increased number of alpha-SMA-positive cells and decreased level of VE cadherin expression in the liver tissues might also be associated with sinusoidal endothelial cell injury. In addition, downregulation of AhR expression and increased CYP1A1 protein levels in the liver were consistent with persistent effects of TCDD. Although it has been reported that TCDD induced endothelial cell injury, this is the first report to describe vascular disorders and protein expression in the liver after injection with TCDD in a primate model.

Topics: Animals; Antigens, CD; Blotting, Northern; Cadherins; Chemical and Drug Induced Liver Injury; Cytochrome P-450 CYP1A1; Endothelial Cells; Fatty Liver; Female; Hemorrhage; Infarction; Injections, Subcutaneous; Liver Diseases; Macaca mulatta; Microscopy, Electron; Muscle, Smooth; Polychlorinated Dibenzodioxins; Pregnancy; Receptors, Aryl Hydrocarbon; Thrombosis; Time Factors; Transforming Growth Factor beta

Sustained hepatic inflammation induced by various causes can lead to liver fibrosis. Transcription factor NF-kappaB is important in regulating inflammatory responses, especially in macrophages. We presently investigated whether an NF-kappaB decoy, a synthetic oligodeoxynucleotide (ODN) imitating the NF-kappaB binding site, inhibited the inflammatory response after CCl(4) intoxication to prevent CCl(4)-induced hepatic injury and fibrosis. The NF-kappaB decoy was introduced into livers by injecting the spleens of mice, using a hemagglutinating virus of Japan (HVJ)-liposome method. ODN was transferred mainly to macrophages in normal or fibrotic livers. Increases in serum transaminases and production of inflammatory cytokines after a single challenge with CCl(4) were inhibited by the NF-kappaB decoy, which suppressed nuclear translocation of NF-kappaB in liver macrophages. Liver fibrosis induced by CCl(4) administration for 8 wk was suppressed by the NF-kappaB decoy, accompanied by diminished mRNA expression for transforming growth factor (TGF)-beta, procollagen type 1 alpha(1), and alpha-smooth muscle actin (SMA). In vitro, isolated liver macrophages showed increased DNA binding activity of NF-kappaB and inflammatory cytokine production after hydrogen peroxide treatment; both increases were inhibited significantly by the NF-kappaB decoy. In contrast, NF-kappaB decoy transferred to isolated hepatic stellate cells (HSC) had no effect on their morphological activation or alpha-SMA expression, although the decoy accelerated tumor necrosis factor (TNF)-alpha-induced apoptosis in activated HSC. The effect of NF-kappaB decoy suppressing fibrosis probably results mainly from anti-inflammatory effects on liver macrophages, with a possible minor contribution from its direct proapoptotic effect on activated HSC.

Topics: Actins; Active Transport, Cell Nucleus; Animals; Anti-Inflammatory Agents; Apoptosis; Carbon Tetrachloride; Cell Nucleus; Cells, Cultured; Chemical and Drug Induced Liver Injury; Collagen Type I; Cytokines; Disease Models, Animal; Gene Transfer Techniques; Genetic Therapy; Hydrogen Peroxide; Kupffer Cells; Liposomes; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Oligonucleotides; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sendai virus; Time Factors; Transfection; Transforming Growth Factor beta

Oxidative stress plays an important role in acetaminophen (APAP)-induced hepatotoxicity. In addition to inducing direct cellular damage, oxidants can activate transcription factors including NF-kappaB, which regulate the production of inflammatory mediators implicated in hepatotoxicity. Here, we investigated the role of APAP-induced oxidative stress and NF-kappaB in inflammatory mediator production. Treatment of mice with APAP (300 mg/kg, i.p.) resulted in centrilobular hepatic necrosis and increased serum aminotransferase levels. This was correlated with depletion of hepatic glutathione and CuZn superoxide dismutase (SOD). APAP administration also increased expression of the proinflammatory mediators, interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNFalpha), macrophage chemotactic protein-1 (MCP-1), and KC/gro, and the anti-inflammatory cytokine, interleukin-10 (IL-10). Pretreatment of mice with the antioxidant, N-acetylcysteine (NAC) prevented APAP-induced depletion of glutathione and CuZnSOD, as well as hepatotoxicity. NAC also abrogated APAP-induced increases in TNFalpha, KC/gro, and IL-10, but augmented expression of the anti-inflammatory cytokines interleukin-4 (IL-4) and transforming growth factor-beta (TGFbeta). No effects were observed on IL-1beta or MCP-1 expression. To determine if NF-kappaB plays a role in regulating mediator production, we used transgenic mice with a targeted disruption of the gene for NF-kappaB p50. As observed with NAC pretreatment, the loss of NF-kappaB p50 was associated with decreased ability of APAP to upregulate TNFalpha, KC/gro, and IL-10 expression and increased expression of IL-4 and TGFbeta. However, in contrast to NAC pretreatment, the loss of p50 had no effect on APAP-induced hepatotoxicity. These data demonstrate that APAP-induced cytokine expression in the liver is influenced by oxidative stress and that this is dependent, in part, on NF-kappaB. However, NF-kappaB p50-dependent responses do not appear to play a major role in the pathogenesis of toxicity in this model.

Topics: Acetaminophen; Acetylcysteine; Alanine Transaminase; Analgesics, Non-Narcotic; Animals; Blotting, Western; Chemical and Drug Induced Liver Injury; Chemokine CCL2; Chemokines; Gene Expression; Glutathione; Humans; Inflammation Mediators; Injections, Intraperitoneal; Interleukin-1; Interleukin-10; Interleukin-4; Liver; Macrophage Inflammatory Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Knockout; Necrosis; NF-kappa B p50 Subunit; Oxidative Stress; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Superoxide Dismutase; Time Factors; Transcription Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

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

Silymarin is a potent antioxidant, antiinflammatory and anti-fibrogenic agent in the liver, which is mediated by alteration of hepatic Kupffer cell function, lipid peroxidation, and collagen production. Especially, in hepatic fibrogenesis, mast cells are expressed in chronic inflammatory conditions, and promote fibroblast growth and stimulate production of the extracellular matrix by hepatic stellate cells.. We examined the inhibitory mechanism of silymarin on CCl(4)-induced hepatic cirrhosis in rats. At 4, 8, and 12 wk, liver tissues were examined histopathologically for fibrotic changes produced by silymarin treatment.. In the silymarin with CCl(4)-treated group, increase of hepatic stellate cells and TGF-beta1 production were lower than in the CCl(4)-treated group at early stages. Additionally, at the late fibrogenic stage, expressions of TGF-beta1 were weaker and especially not expressed in hepatocytes located in peripheral areas. Moreover, the number of mast cell in portal areas gradually increased and was dependent on the fibrogenic stage, but those of CCl(1)+silymarin-treated group decreased significantly.. Anti-fibrotic and antiinflammatory effects of silymarin were associated with activation of hepatic stellate cells through the expression of TGF-beta1 and stabilization of mast cells. These results suggest that silymarin prevent hepatic fibrosis through suppression of inflammation and hypoxia in the hepatic fibrogenesis.

Topics: Alanine Transaminase; Animals; Antioxidants; Aspartate Aminotransferases; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Collagen; Immunohistochemistry; Liver; Liver Cirrhosis; Male; Mast Cells; Rats; Rats, Wistar; Silymarin; Transforming Growth Factor beta; Transforming Growth Factor beta1

To determine the effects of dominant-negative TGF-beta receptor expression during liver regeneration in rats with dimethylnitrosamine (DMN)-induced liver injury.. Rats were first treated with DMN for 3 weeks, and then intravenously injected once with AdTbeta-TR, AdLacZ, or saline. Serial changes in hepatocyte proliferation and apoptosis were evaluated by immunohistochemistry using anti-Ki67 antibody, and TUNEL staining, respectively. The mRNA expression of regeneration factors (HGF, TGF-alpha, EGF, and IGF-I) and IL-6 were evaluated by real-time PCR and northern blotting.. Anti-TGF-beta molecular intervention up-regulated hepatocyte proliferation and inhibited apoptosis. In the AdTbeta-TR-treated rats, EGF and IGF-I mRNA expression levels were significantly increased at day 1 and remained high for 3 days after gene transfer; TGF-alpha mRNA expression levels were significantly increased at 2 to 5 days after gene transfer; HGF mRNA expression levels were significantly up-regulated at day 2 only after gene transfer; while IL-6 mRNA expression level tended to increase at day 1, but decreased thereafter.. In rats with DMN-induced liver injury, anti-TGF-beta molecular intervention therapy stimulates proliferation and reduces apoptosis of hepatocytes, and also up-regulates the transcription of various growth factors.

Topics: Animals; Apoptosis; Blotting, Northern; Cell Division; Chemical and Drug Induced Liver Injury; Chronic Disease; Dimethylnitrosamine; Gene Transfer Techniques; Growth Substances; Immunohistochemistry; In Situ Nick-End Labeling; Interleukin-6; Liver Diseases; Liver Regeneration; Male; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Up-Regulation

Macrophages may play a role in fibrogenesis. The kinetics and distribution of different macrophage populations were investigated immunohistochemically in hepatic lesions following acute hepatocyte injury induced in F344 rats by a single injection of thioacetamide (TAA) (300 mg/kg body weight, intraperitoneally). Hepatocyte degeneration or necrosis induced by TAA occurred mainly in the perivenular areas of hepatic lobules as early as post-injection (PI) days 1 and 3; fibrotic lesion development began in the damaged areas on day 1, and peaked on day 5; thereafter (PI days 7 and 10), the fibrotic areas decreased and were replaced by regenerated hepatocytes on PI days 15 and 20, indicating a remodelling process. In this rat model, the number of macrophages reacting with ED1 antibody (specific for exudate macrophages), ED2 (recognizing cell membrane antigens of resident macrophages, including Kupffer cells) and OX6 (recognizing MHC class II antigens expressed in antigen-presenting macrophages and dendritic cells) began to increase on PI day 1, peaking on PI day 3. The numbers gradually decreased on PI days 5 and 7; however, the statistically significant increase was maintained in respect of ED1-positive cells up to PI day 20, whereas no significant increase in ED2- and OX6-positive cells remained from PI day 10 onwards. Interestingly, of the ED1-, ED2- and OX6-positive cells, the OX6-positive cells were the least numerous. ED1- and OX6-positive cells appeared exclusively in the injured perivenular areas, whereas ED2-positive cells were present mainly in the mid-zonal areas and in smaller numbers in the perivenular areas. These findings indicated differences in kinetics and distribution between macrophage populations appearing in hepatic fibrosis. In addition, RT-PCR revealed that mRNA expression of osteopontin, a factor for induction and maintenance of macrophages in inflammation, was markedly increased on PI days 5, 7 and 10, suggesting a role in the pathogenesis of hepatic fibrosis.

Topics: Animals; Antibodies, Monoclonal; Biomarkers; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Hepatocytes; Immunohistochemistry; Injections, Intraperitoneal; Liver Cirrhosis; Liver Regeneration; Macrophages; Male; Osteopontin; Rats; Rats, Inbred F344; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sialoglycoproteins; Thioacetamide; Transforming Growth Factor beta; Transforming Growth Factor beta1

Genomic ablation of hepatocyte-specific fibroblast growth factor receptor (FGFR)4 in mice revealed a role of FGF signaling in cholesterol and bile acid metabolism and hepatolobular restoration in response to injury without effect on liver development or hepatocyte proliferation. Although the potential role of all 23 FGF polypeptides in the liver is still unclear, the most widely studied prototypes, FGF1 and FGF2, are present and have been implicated in liver cell growth and function in vitro. To determine whether FGF1 and FGF2 play a role in response to injury and fibrosis, we examined the impact of both acute and chronic exposure to carbon tetrachloride (CCl(4)) in the livers of FGF1- and FGF2-deficient mice. After acute CCl(4) exposure, FGF1(-/-)FGF2(-/-) mice exhibited an accelerated release of serum alanine aminotransferase similar to FGFR4 deficiency, but no effect on overall hepatolobular restoration or bile acid metabolism. FGF1(-/-)FGF2(-/-) mice exhibited a normal increase in alpha-smooth muscle actin and desmin associated with activation and migration of hepatic stellate cells to damage, but a reduced level of hepatic stellate cell-derived matrix collagen alpha1(I) synthesis. Liver fibrosis resulting from chronic CCl(4) exposure was markedly decreased in the livers of FGF1/FGF2-deficient mice. These results suggest an agonist role for FGF1 and FGF2 in specifically insult-induced liver matrix deposition and hepatic fibrogenesis and a potential target for the prevention of hepatic fibrosis.

Topics: Actins; Animals; Bile Acids and Salts; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Collagen Type I; Drug Administration Schedule; Extracellular Matrix; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Hepatectomy; Liver; Liver Cirrhosis; Liver Diseases; Liver Regeneration; Mice; Mice, Knockout; Muscle, Smooth; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

During chronic liver injury, transforming growth factor beta (TGF-beta) plays a prominent role in stimulating liver fibrogenesis by myofibroblast-like cells derived from hepatic stellate cells (HSCs). On the other hand, Smad 7 was recently shown to antagonize the TGF-beta-induced activation of signal-transducing Smads (2 and 3). In this study, we investigated the regulatory mechanisms of the TGF-beta signals in rat HSCs during acute liver injury and myofibroblasts (MFBs) during chronic liver injury, focusing on the roles of Smad 2 and antagonistic Smad 7. In acute liver injury, HSC-derived TGF-beta increased plasminogen activator inhibitor type 1 (PAI-1) and alpha2(I) procollagen (COL1A2) transcripts. Smad 2 in HSCs during liver injury and primary cultured HSCs were activated by an autocrine mechanism, because high levels of Smad 2 phosphorylation and induction of PAI-1 transcript by TGF-beta were observed in HSCs. Thereafter, Smad 7 induced by TGF-beta negatively regulated the Smad 2 action. These results indicated that endogenous TGFbeta-mediated Smad 7 in HSCs terminated the fibrotic signals mediated by signal-transducing Smads, and might be involved in the transient response to autocrine TGF-beta signal after acute liver injury. By contrast, Smad 7 was not induced by the autocrine TGF-beta signal, and constitutive Smad 2 activation was observed in MFBs throughout chronic liver injury, although Smad 7 could inhibit the TGF-beta signal requiring Smad 2 phosphorylation by activated TGF-beta receptor in cultured MFBs. This constitutive phosphorylation of Smad 2 by endogenous TGF-beta under a low level of Smad 7 could be involved in the progression of liver fibrosis.

Topics: Acute Disease; Animals; Carbon Tetrachloride; Cell Division; Cell Line; Cells, Cultured; Chemical and Drug Induced Liver Injury; Chronic Disease; Collagen; Collagen Type I; DNA-Binding Proteins; Fibroblasts; Gene Expression; Hepatocytes; Liver; Liver Diseases; Male; Phosphorylation; Plasminogen Activator Inhibitor 1; Promoter Regions, Genetic; Rats; Rats, Wistar; RNA, Messenger; Signal Transduction; Smad2 Protein; Smad7 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta

The acute-phase response (APR) represents a systemic reaction of the organism to multiple nonspecific inflammatory stimuli. In general, it is protective for the host, and hepatocytes are the main cells responding with alterations in the expression of a set of liver-specific proteins named the acute-phase proteins. We have previously shown that although a turpentine-induced APR is not fibrogenic per se, it enhances collagen deposition in rats treated with CCl(4) and up-regulates expression of hepatic alpha1(I) collagen and tissue inhibitor of metalloproteinases 1 (TIMP-1) messenger RNAs (mRNAs). In this report we extended our studies and showed that turpentine induced, in a time-dependent manner, expression of alpha1(I) and alpha1(IV) collagens, TIMP-1, and matrix-metalloproteinase 2 (MMP-2) mRNAs. We further showed that expression of these mRNAs occurs in hepatic stellate cells, but not in hepatocytes obtained 6 hours after the induction of an APR episode. These changes were accompanied by increased blood levels of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) without noticeable immediate changes in the expression of their respective mRNAs in the liver. In contrast to CCl(4)-induced liver damage, turpentine alone, whether administered as a single dose or as a weekly dose for 3 weeks did not up-regulate expression of transforming growth factor beta1 (TGF-beta1) mRNA and did not result in excess collagen deposition. Overall, these findings suggest that collagen deposition in the livers of rats with repeated APR episodes may be enhanced only when given together with a fibrogenic stimulus that activates hepatic stellate cells (HSCs) and/or up-regulates TGF-beta1 mRNA expression.

Topics: Acute-Phase Reaction; Animals; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Collagen; Interleukin-6; Irritants; Liver; Liver Diseases; Male; Matrix Metalloproteinase 2; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; RNA, Messenger; Time Factors; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Turpentine

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

Transforming growth factor beta (TGF-beta) is the most potent profibrogenic mediator in liver fibrosis. Although Smad proteins have been identified as intracellular mediators in the TGF-beta signaling pathway, the function of individual Smad proteins remains poorly understood. The aim of this study was to explore the contribution of Smad3 in mediating TGF-beta responses in a model of acute liver injury in vivo and in culture-activated hepatic stellate cells (HSCs). Wild-type, Smad3 heterozygous or Smad3 homozygous knockout mice were treated with a single intragastric administration of CCl(4). After 72 hours, the induction of hepatic collagen alpha1(I) and alpha2(I) messenger RNA (mRNA) levels in Smad3 knockout mice was only 42% and 64%, respectively, of the levels induced in wild-type mice. However, smooth muscle alpha-actin (alpha-SMA) was expressed at a slightly higher level in livers from knockout mice compared with wild-type mice. In culture-activated HSCs from Smad3 knockout mice, collagen alpha1(I) mRNA was 73% of wild-type HSCs, but alpha-SMA expression was the same. HSCs from knockout mice showed a higher proliferation rate than wild-type HSCs. Smad3-deficient HSCs did not form TGF-beta1-induced Smad-containing DNA-binding complexes. In conclusion, (1) maximal expression of collagen type I in activated HSCs requires Smad3 in vivo and in culture; (2) Smad3 is not necessary for HSC activation as assessed by alpha-SMA expression; (3) Smad3 is necessary for inhibition of proliferation of HSCs, which might be TGF-beta-dependent; and (4) Smad3 is required for TGF-beta1-mediated Smad-containing DNA-binding complex formation in cultured HSCs.

Topics: Actins; Animals; Carbon Tetrachloride; Cattle; Cell Division; Cells, Cultured; Chemical and Drug Induced Liver Injury; Collagen; DNA; DNA-Binding Proteins; Enzyme Activation; Fetal Blood; Hepatocytes; In Situ Hybridization; Liver Diseases; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; Muscle, Smooth; Platelet-Derived Growth Factor; RNA, Messenger; Smad3 Protein; Trans-Activators; 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

Many acute and chronic liver diseases are often associated with atypical ductular proliferation (ADP). These ADPs have gained increasing interest since a number of recent observations suggest that ADPs may represent progenies of the putative liver stem cell compartment. In this study, we show that feeding mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) results in persistent proliferation of primitive ductules with poorly defined lumens. Similar to oval cell proliferation in other rodent models as well as in various human liver diseases, DDC-induced ADP originated from the portal tract, spread into the hepatic lobule, and was associated closely with appearance of hepatocytes harboring an antigen (A6), which normally is expressed in biliary epithelium. Furthermore, DDC treatment severely inhibited the regenerative capacity of mice after partial hepatectomy. The development of ADP was selectively blocked in DDC-fed TGF-beta1 transgenic mice producing active TGF-beta1 in the liver and no accumulation of new hepatocytes expressing the A6 antigen was observed. Moreover, the transforming growth factor beta1 (TGF-beta1) transgenic mice did not survive beyond 3 weeks from starting the DDC-containing diet. The results suggest that persistent activation of the hepatic stem cell compartment is essential for liver regeneration in the DDC model and that active TGF-beta1 may negatively control activation of stem cells in the liver. These data further emphasize the relevance of the DDC model as an experimental tool for studying chronic liver diseases.

Topics: Animals; Bile Ducts, Intrahepatic; Cell Division; Chemical and Drug Induced Liver Injury; Chronic Disease; Common Bile Duct; Dicarbethoxydihydrocollidine; Disease Models, Animal; Epithelial Cells; Hepatectomy; Ligation; Liver; Liver Diseases, Alcoholic; Liver Regeneration; Mice; Mice, Transgenic; Transforming Growth Factor beta

The effect of human placental extract (HPE) on liver regeneration in rats was investigated. After intravenous administration of HPE to a-naphthylisothiocyanate (ANIT)-intoxicated rats, the labeling index in hepatocytes was significantly increased to a level 16.5 times higher than that of the control. A 1/500 dilution of HPE directly stimulated DNA synthesis of the hepatocytes in primary culture. HPE heated at 121 degrees C did not stimulate the labeling index in vivo or hepatocyte DNA synthesis in primary culture, suggesting that HPE contains heat-unstable but potent mitogens for hepatocytes. HPE contains hepatocyte growth factor (HGF), but the mitogenic effect of HPE cannot be explained by the effect exerted by HGF alone, since both the labeling index in vivo and hepatocellular DNA synthesis in vitro stimulated by HPE were much higher than those stimulated by HGF alone when the applied doses of HGF were set to be almost the same level between each case. When HPE was fractionated on a heparin-sepharose column, the mitogenic effect of HPE was found to be located mainly in the heparin-bound fraction. Hepatocyte DNA synthesis induced by this fraction was enhanced cooperatively by the heparin-unbound fraction, suggesting that there are some modulators in the heparin-unbound fraction which enhance the proliferative activity of the heparin-bound fraction by a synergetic mechanism. Both HPE and heated HPE completely recovered the biochemical marker activity for liver function (glutamic-pyruvic transaminase, GPT; alkaline phosphatase, ALP; lactate dehydrogenase, LAP; gamma-glutamyltransferase, gamma-GTP activities and the bilirubin concentration) almost to the control level in the serum of ANIT-intoxicated rats, indicating that HPE also contains a heat-stable fraction which repairs liver function.

Topics: Animals; Bilirubin; Biomarkers; Cells, Cultured; Chemical and Drug Induced Liver Injury; DNA; Heparin; Hepatocyte Growth Factor; Liver Function Tests; Liver Regeneration; Male; Mitogens; Placenta; Rats; Rats, Wistar; Stimulation, Chemical; Tissue Extracts; Transforming Growth Factor beta

The role of the anti-inflammatory cytokine interleukin-10 (IL-10) was investigated in the mouse model of liver injury induced by carbon tetrachloride (CCl4). To address the role of endogenous IL-10 production, acute hepatitis was induced by CCl4 in C57Bl/6 IL-10 gene knock out (KO) and wild-type (WT) mice. After CCl4 challenge, serum and liver levels of tumor necrosis factor-alpha (TNF-) and serum levels of transforming growth factor-beta 1 (TGF-beta1) increased and were significantly higher in IL-10 KO mice, whereas IL-6 serum levels were only slightly increased compared with WT mice. At histological examination, the livers disclosed a significantly more prominent neutrophilic infiltration in IL-10 KO mice 12 and 24 hours after CCl4 injection. In contrast, hepatocyte necrosis, evaluated by histological examination and serum alanine aminotransferase levels, was only marginally affected. The proliferative response of hepatocytes, assessed by the proliferating cell nuclear-antigen labeling index, was significantly increased in IL-10 KO mice, compared with WT mice 48 hours after CCl4 injection. Finally, repeated CCl4 injections led to more liver fibrosis in IL-10 KO mice after 7 weeks. In conclusion, endogenous IL-10 marginally affects the hepatocyte necrosis although it controls the acute inflammatory burst induced by CCl4. During liver repair, it limits the proliferative response of hepatocytes and the development of fibrosis.

Topics: Animals; Carbon Tetrachloride; Cell Division; Cell Movement; Chemical and Drug Induced Liver Injury; Injections; Interleukin-10; Interleukin-6; Liver; Liver Cirrhosis, Experimental; Mice; Mice, Inbred C57BL; Mice, Knockout; Necrosis; Neutrophils; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Hepatic silicosis, cirrhosis, liver cell adenoma, and carcinomas developed in nude mice (NCr-Nu) given quartz by the subcutaneous and intraperitoneal routes. Syrian golden hamsters (15:16 EHS:cr) given quartz by both routes developed extensive fibrosis and cirrhosis and had higher morbidity and mortality rates after 3 months. Crystalline silica (quartz) induces fibrosis, adenomas, and carcinomas in the lungs of Fisher 344 rats, but certain strains of mice and hamsters are resistant to quartz-induced pulmonary carcinogenesis. Pulmonary fibrosis, however, is minimal in mice and absent in hamsters who received quartz intratracheally. To determine whether species differences are due to organ-specific rather than species-specific factors, susceptibility of the liver to quartz toxicity was investigated in nude mice and hamsters. The present study shows that the differential manifestations of quartz toxicity by these rodent species are dependent on factors that are organ-specific rather than host-specific. At 3 months, hepatocytes in mice were immunostained with intracellular transforming growth factor (TGF) beta 1 (LC 1-30) but not with TGF-beta 1 latency-associated peptide (LAP) protein (266-278); at 12 months, hepatocytes were immunostained with TGF-beta 1 LAP (266-278) but not with TGF-beta 1 (LC1-30). The hepatocytes of hamsters at 3 months showed immunoreactivities to TGF-beta 1 LAP (266-278) and TGF-beta 1 (LC1-30); immunostaining to TGF-beta 1 (LC1-30) was detected in nonparenchymal cells. Extracellular TGF-beta 1 (CC1-30) was detected in the silicotic granulomas and fibrous tissue in livers of both species. Quartz-induced liver carcinoma did not express TGF-beta 1 LAP (266-278) and LC (1-30) proteins, but these were detected in the cells of the adenoma in the same liver. Control animals showed no hepatic lesions nor immunoreactivity to TGF-beta 1. The spatial and temporal patterns of expression of TGF-beta 1, TGF-beta 2, TGF-beta receptor type II messenger RNAs (mRNAs), and TGF-beta 1 proteins in the different hepatic lesions suggests that TGF-beta isoforms may play a role in the pathogenesis of quartz-induced fibrosis, cirrhosis, liver cell adenoma, and carcinoma.

Topics: Adenoma, Liver Cell; Animals; Blotting, Northern; Chemical and Drug Induced Liver Injury; Cricetinae; Female; Immunohistochemistry; Liver; Liver Cirrhosis, Experimental; Liver Diseases; Liver Neoplasms, Experimental; Male; Mesocricetus; Mice; Mice, Nude; Microscopy, Electron; Microscopy, Immunoelectron; Quartz; Receptors, Transforming Growth Factor beta; RNA, Messenger; Silicosis; Transforming Growth Factor beta

Specific binding of 125I-labeled human recombinant HGF to the primary cultured rat hepatocytes or liver plasma membranes was observed to be temperature- and time-dependent. Scatchard analysis indicated the presence of a single class of high affinity receptors with a dissociation constant (Kd) of 24-32 pM, a value in good accord with half maximum dose for HGF activity and a receptor density of about 500-600 sites/cell. Affinity cross-linking of the receptor with 125I-HGF revealed the HGF receptor in rat liver membranes to be a polypeptide of Mr approximately 220,000. After partial hepatectomy, specific binding of 125I-HGF to the membranes of residual livers decreased by 60-70% between 3 and 6 h, and was scanty at 12 h after hepatectomy. After one week, the binding was recovered to the 1.7 fold level in the untreated rat liver. This rapid down-regulation of HGF receptors was also observed in plasma membranes of rat livers in the presence of hepatitis induced by CCl4. We propose that HGF which can be immediately supplied to the liver after hepatic injury will function as a trigger for regeneration of this organ.

Topics: Animals; Carbon Tetrachloride; Cell Membrane; Cells, Cultured; Chemical and Drug Induced Liver Injury; Cross-Linking Reagents; DNA; Down-Regulation; Epidermal Growth Factor; Growth Substances; Hepatectomy; Hepatocyte Growth Factor; Humans; Liver; Liver Regeneration; Male; Molecular Weight; Rats; Rats, Inbred Strains; Recombinant Proteins; Transforming Growth Factor alpha; Transforming Growth Factor beta