thioacetamide and Liver-Cirrhosis

Thioacetamide (TAA) undergoes bioactivation in the liver by the CYP450 2E1 enzyme, resulting in the formation of TAA-S-oxide and TAA-S-dioxide. TAA-S-dioxide induces oxidative stress via lipid peroxidation of the hepatocellular membrane. A single TAA dose (50-300 mg/kg) administration initiates hepatocellular necrosis around the pericentral region after its covalent binding to macromolecules in the liver. Intermittent TAA administration (150-300 mg/kg, weekly thrice, for 11-16 weeks) activates transforming growth factor (TGF)-β/smad3 downstream signaling in injured hepatocytes, causing hepatic stellate cells (HSCs) to acquire myofibroblast like phenotype. The activated HSCs synthesize a variety of extracellular matrix, leading to liver fibrosis, cirrhosis, and portal hypertension. The TAA induced liver injury varies depending on the animal model, dosage, frequency, and routes of administration. However, TAA induces hepatotoxicity in a reproducible manner, and it is an ideal model to evaluate the antioxidant, cytoprotective, and antifibrotic compounds in experimental animals.

Topics: Animals; Hepatic Stellate Cells; Hepatocytes; Liver; Liver Cirrhosis; Thioacetamide; Transforming Growth Factor beta

The liver, a component of the gastrointestinal tract, is one of the most important organs in the human body. The liver performs over 500 functions to promote physiological homeostasis. In addition, the liver acts as a screen, by metabolizing substances carried by blood coming from the digestive tract before they enter the systemic circulation. This vital function exposes the hepatic tissue to hepatotoxic agents, which can lead to liver damage if the organ's repair and regenerative capacity is insufficient. Several conditions such as persistent exposure to hepatitis C and B viruses, alcohol, and drugs can provoke this disbalance, eventually leading to liver cirrhosis, which is an irreversible and life-threatening condition. This paradigm of irreversibility began to be reconsidered when several studies showed that hepatic fibrosis is potentially reversible after cessation of exposure to the hepatotoxic agent or eradication of the primary disease. In the context of basic research in liver fibrosis and cirrhosis, it is essential to keep in mind that the capacity of the organ to recover spontaneously might be a significant limitation to long-term studies that use experimental models of liver cirrhosis. Here, we review animal models where liver cirrhosis is experimentally induced. We focus on a surgery-based model, i.e., bile duct ligation (BDL), and hepatotoxic drugs such as carbon tetrachloride (CCl

Topics: Animals; Carbon Tetrachloride; Disease Models, Animal; Liver; Liver Cirrhosis; Liver Cirrhosis, Experimental; Models, Theoretical; Rodentia; Thioacetamide

Since 1900 bc, several therapeutic activities have been attributed to the rhizomes of the plant Curcuma longa for a variety of diseases, including liver disorders. Curcumin, the main active compound obtained from this plant, was first isolated two centuries ago and its structure as diferuloylmethane was determined in 1910. Curcumin has shown anti-inflammatory, anti-oxidant, antifungal, antibacterial and anticancer activities. The pharmacological properties of curcumin were reviewed recently and focused mainly on its anticancer properties. However, its beneficial activity on liver diseases (known centuries ago, and demonstrated recently utilizing animal models) has not being reviewed in depth until now. The curcumin ability to inhibit several factors like nuclear factor-kappaB, which modulates several pro-inflammatory and profibrotic cytokines as well as its anti-oxidant properties, provide a rational molecular basis to use it in hepatic disorders. Curcumin attenuates liver injury induced by ethanol, thioacetamide, iron overdose, cholestasis and acute, subchronic and chronic carbon tetrachloride (CCl(4)) intoxication; moreover, it reverses CCl(4) cirrhosis to some extent. Unfortunately, the number of studies of curcumin on liver diseases is still very low and investigations in this area must be encouraged because hepatic disorders constitute one of the main causes of worldwide mortality.

Topics: Animals; Carbon Tetrachloride; Cholestasis; Curcuma; Curcumin; Humans; Iron; Liver; Liver Cirrhosis; Liver Cirrhosis, Biliary; Liver Diseases; Thioacetamide

The present study determined the regenerative effect of bone marrow-derived stem cells (BMSCs) on thioacetamide (TA)-induced liver fibrosis in rats. A total of 30 male Wistar rats were randomly divided into sham control and treatment groups. The rats of the sham control group were subdivided into three groups and sampled on the 14th, 18th, and 20th weeks after fibrosis induction. The rats of the treatment group were subdivided into two groups and sampled on the 4th and 6th weeks after BMSCs treatment. Fibrosis was induced by the intraperitoneal administration of 200 mg/kg of TA twice a week for a period of 14 weeks. All the animals underwent liver function tests and histopathologic evaluation 4 and 6 weeks after BMSCs transplantation. The BMSCs were characterized using osteogenic induction and reverse transcription-polymerase chain reaction. The BMSCs were plastic adherent, spindle-shaped, and positive for osteogenic differentiation. They expressed CD73 and were negative for CD45. The infiltration of inflammatory cells and deposition of collagen fibers were noticed after TA administration. A significant decline in inflammatory cells and a healing process were detected 4 weeks after cell transplantation. The amelioration in hepatic tissue was significant 6 weeks after cell therapy. Following the injection of BMSCs, a nonsignificant decrease was visible in aspartate transaminase level; however, this decline was significant for alanine aminotransferase level. The alkaline phosphatase and albumin levels showed an increasing trend after cell administration. The transplantation of BMSCs resulted in a significant regenerative effect after hepatic injuries. Therefore, it was shown that BMSCs transplantation can open a new window and be a therapy of choice in the amelioration of liver fibrosis.

Topics: Animals; Bone Marrow; Liver Cirrhosis; Male; Mesenchymal Stem Cell Transplantation; Rats; Rats, Wistar; Regeneration; Thioacetamide

Chronic intraperitoneal injection of thioacetamide (TAA) in rats has been used as an animal model of human cirrhosis to study the effects of the disease on drug metabolism. However, TAA inhibits P450 enzymes directly and independently of cirrhosis. We investigated the effects of chronic cirrhosis in rats, induced by 10 weeks of intraperitoneal TAA, on the P450 enzymes after a 10-day washout period to eliminate TAA. Liver histology and serum biomarkers of hepatic function confirmed cirrhosis in all animals. Microsomal total P450 content, P450 reductase activity and ethoxycoumarin O-deethylase activity, a general marker of P450 activity, were significantly reduced by 30%-50% in cirrhotic animals. Additionally, the protein content and Michaelis-Menten kinetics of the activities of CYP2D, CYP2E1 and CYP3A were investigated. Whereas cirrhosis reduced the microsomal protein contents of CYP2D and CYP3A by 70% and 30%, respectively, the protein contents of CYP2E1 were not affected. However, the activities of all the tested isoenzymes were substantially lower in the cirrhotic livers. It is concluded that the TAA model of cirrhosis that incorporates a 10-day washout period after intraperitoneal injection of the chemical to rats produces isoenzyme-selective reductions in the P450 proteins or activities, which are independent of the direct inhibitory effects of TAA.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Cytochrome P-450 CYP2E1; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Fibrosis; Humans; Injections, Intraperitoneal; Liver; Liver Cirrhosis; Microsomes, Liver; Rats; Thioacetamide

Deep-frying is a common cooking practice worldwide, and after repeated heating's, the oil undergoes various chemical reactions, including hydrolysis, polymerization, lipid oxidation, and the Maillard reaction. Studies have pointed out that oxidized dietary frying oil may cause teratogenesis in mice and increase cancer and cardiovascular risks. The liver is the main organ involved in dietary nutrient catabolism, detoxification, bile production, and lipid metabolism. Nevertheless, the effects of oxidized frying oil exposure on the activation of hepatic stellate cells (HSCs) and liver fibrosis are still unclear. In this study, we showed that exposure to oxidized frying oil enhanced the sensitivity of HSCs to transforming growth factor (TGF)-β1-induced α-smooth muscle actin (α-SMA), collagen 1a2, collagen 1a1, metalloproteinase-2, and phosphorylated smad2/3 activation. In both carbon tetrachloride (CCl

Topics: Animals; Carbon Tetrachloride; Dietary Fats, Unsaturated; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Matrix Metalloproteinase 2; Mice; Thioacetamide; Transforming Growth Factor beta1

Liver fibrosis is a worldwide public health problem due to its life-threatening complications, including portal hypertension, liver failure, cirrhosis, and hepatocellular carcinoma (HCC). Liver fibrosis is the net result of a complex excessive accumulation of extracellular matrix (ECM). Activation of hepatic stellate cells (HSCs) are the cause of deposition of ECM and are commonly recognized as a key step in liver fibrosis. The aim of this study was to investigate the effect of foreskin-derived mesenchymal stem cells treated with boron compounds on liver fibrosis. Rats were injected intraperitoneally with thioacetamide (TAA) at a dose of 150 mg/kg except sham and control groups' rats. Thioacetamide (TAA), foreskin-derived mesenchymal stem cells (TAA + FSDMSC), FSDMSC treated with boric acid (TAA + FSDMSC + BA), FSDMSC treated with sodium pentaborate pentahydrate (TAA + FSDMSC + NaB), control and sham groups were studied. Boron compound treated foreskin-derived mesenchymal stem cells were injected into the tail vein, and evaluations were conducted after 4 weeks and liver tissues were obtained for structural, immunohistochemical, and western blot studies and blood samples were taken for biochemical analysis. FSDMSC (BA) alleviates TAA-induced rats liver fibrosis, and BA showed a positive effect on foreskin-derived mesenchymal stem cells viability. After using BA-treated mesenchymal stem cells, we observed that there was regression in the fibrotic areas at TAA-induced liver fibrosis. The result demonstrates that the contribution of TAA + FSDMSC and TAA + FSDMSC (NaB) at the level of structure is not effective in regression of fibrosis in TAA-generated liver fibrosis. We concluded that FSDMSC treated with BA may be a factor in the regression of fibrosis.

Topics: Animals; Carcinoma, Hepatocellular; Fibrosis; Foreskin; Liver; Liver Cirrhosis; Liver Neoplasms; Male; Mesenchymal Stem Cells; Rats; Thioacetamide

Toxic chemicals such as carbon tetrachloride and thioacetamide (TAA) are reported to induce hepato-nephrotoxicity. The potential protective outcome of the antidiabetic and pleiotropic drug metformin against TAA-induced chronic kidney disease in association with the modulation of AMP-activated protein kinase (AMPK), oxidative stress, inflammation, dyslipidemia, and systemic hypertension has not been investigated before. Therefore, 200 mg/kg TAA was injected (via the intraperitoneal route) in a model group of rats twice a week starting at week 3 for 8 weeks. The control rats were injected with the vehicle for the same period. The metformin-treated group received 200 mg/kg metformin daily for 10 weeks, beginning week 1, and received TAA injections with dosage and timing similar to those of the model group. All rats were culled at week 10. It was observed that TAA induced substantial renal injury, as demonstrated by significant kidney tissue damage and fibrosis, as well as augmented blood and kidney tissue levels of urea, creatinine, inflammation, oxidative stress, dyslipidemia, tissue inhibitor of metalloproteinases-1 (TIMP-1), and hypertension. TAA nephrotoxicity substantially inhibited the renal expression of phosphorylated AMPK. All these markers were significantly protected by metformin administration. In addition, a link between kidney fibrosis and these parameters was observed. Thus, metformin provides profound protection against TAA-induced kidney damage and fibrosis associated with the augmentation of the tissue protective enzyme AMPK and inhibition of oxidative stress, inflammation, the profibrogenic gene TIMP-1, dyslipidemia, and hypertension for a period of 10 weeks in rats.

Topics: AMP-Activated Protein Kinases; Animals; Down-Regulation; Dyslipidemias; Fibrosis; Hypertension; Inflammation; Liver; Liver Cirrhosis; Metformin; Oxidative Stress; Rats; Renal Insufficiency, Chronic; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1; Up-Regulation

In chronic liver diseases, liver fibrosis occurs due to excessive extracellular matrix (ECM) protein accumulation. Approximately 2 million deaths occur yearly due to liver disease, while cirrhosis is the 11th most common cause of death. Therefore, newer compounds or biomolecules must be synthesized to treat chronic liver diseases. In this aspect, the present study focuses on the assessment of the anti-inflammatory and antioxidant impact of Bacterial Protease (BP) produced by a new mutant strain of bacteria (Bacillus cereus S6-3/UM90) and 4,4'-(2,5-dimethoxy-1,4-phenylene) bis (1-(3-ethoxy phenyl)-1H-1,2,3-triazole) (DPET) in the treatment of early stage of liver fibrosis induced by thioacetamide (TAA). Sixty male rats were divided into six groups, ten rats each as follows: (1) Control group, (2) BP group, (3) TAA group, (4) TAA-Silymarin (S) group, (5) TAA-BP group, and (6) TAA-DPET group. Liver fibrosis significantly elevated liver function ALT, AST, and ALP, as well as anti-inflammatory interleukin 6 (IL-6) and VEGF. The oxidative stress parameters (MDA, SOD, and NO) were significantly increased with a marked reduction in GSH. Expression of MAPK and MCP-1 was unregulated in the TAA group, with downregulation of Nrf

Topics: Animals; Anti-Inflammatory Agents; Endopeptidases; Fibrosis; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Male; Oxidative Stress; Peptide Hydrolases; Rats; Thioacetamide; Vascular Endothelial Growth Factor A

Ginseng, when used as a food and nutritional supplement, has the ability to regulate human immunity. Here, the potential anti-hepatic fibrosis effect of ginsenoside Rd (Rd), one of the protopanaxadiol types of ginsenoside, was investigated. We established a hepatic fibrosis model using intraperitoneal injection of thioacetamide (TAA) for five weeks in mice. In addition, an

Topics: Animals; ERRalpha Estrogen-Related Receptor; Ginsenosides; Hepatic Stellate Cells; Humans; Inflammasomes; Inflammation; Liver; Liver Cirrhosis; Mice; NLR Family, Pyrin Domain-Containing 3 Protein; Thioacetamide; Transforming Growth Factor beta

Various therapeutic approaches are conducted for regression of liver fibrosis and prevent possible further carcinogenic transformation. This study was aimed to assess the prospective therapeutic potential of bromelain against thioacetamide (TAA)-induced liver fibrosis using in-vitro and in vivo approaches. In vitro study, HSC-T6 cell line was used to evaluate the effect of bromelain on HSC-T6 cell viability and apoptosis. In vivo, Rats were treated by TAA for 6 weeks for induction of hepatic fibrosis followed by post treatment by different doses of bromelain and silymarin for further 4 weeks to assess the regression of hepatic fibrosis. The in-vitro findings indicated that bromelain hindered the proliferation of HSCs in concentration dependent manner compared with the untreated cells. The in vivo study revealed that treatment of TAA fibrotic rats with different doses of bromelain and silymarin induced a significant restoration in liver function biomarkers, attenuation of oxidative stress, upregulation of total antioxidant capacity and thereby decline of fibrotic biomarkers and improving histopathological and immunohistochemical changes. In conclusion, This study indicates that bromelain can regress TAA induced hepatic fibrosis in rats via inhibiting HSCs activation, α-SMA expression and the ECM deposition in hepatic tissue in addition to its antioxidants pathway, these findings prove the promising therapeutic potential of bromelain as a novel therapeutic approach for chronic hepatic fibrotic diseases.

Topics: Animals; Antioxidants; Biomarkers; Bromelains; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Rats; Silymarin; Thioacetamide

Lycopene is a fat-soluble carotenoid pigment that gives tomatoes their red color and capacity for scavenging free radicals. The current study was designed to evaluate the effect of lycopenesupplementation on blood glucose, lipid profile and electrolyte homeostasis in thioacetamide induced liver cirrhosis. Experimental period was consisted of 12 weeks, divided into two phases (each of six weeks). For this purpose 24 male albino wistar rats were randomly distributed into four groups (n=6). Group I served as control, Group II received thioacetamide (200mg/kg b.w, i.p, twice a week) in the first phase and then saline in the second phase. Group III received thioacetamidein the first phase and lycopene in the second phase. Group IV received saline in the first phase and lycopene in the second phase. Thioacetamide toxicity was evidenced by decrease in body weight, plasma glucose and HDL level, plasma and intra-erythrocyte sodium and potassium and increase in liver weight, plasma total cholesterol, triglyceride and LDL level. While lycopene administration resulted in increased body weight, HDL level, plasma and intra-erythrocyte sodium and potassium and decreased liver weight, plasma cholesterol, triglyceride, LDL and plasma glucose level. Thus, confirms the protective role of lycopene in thioacetamide induced liver cirrhosis.

Topics: Animals; Blood Glucose; Cholesterol; Dietary Supplements; Electrolytes; Homeostasis; Liver Cirrhosis; Lycopene; Male; Potassium; Rats; Rats, Wistar; Saline Solution; Thioacetamide; Triglycerides; Weight Gain

Topics: Animals; Erythropoietin; Liver Cirrhosis; Male; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Signal Transduction; Thioacetamide; Toll-Like Receptor 4

Drug-induced liver fibrosis models are used in normal and immunosuppressed small animals for transplantation and regenerative medicine to improve liver fibrosis. Although large animal models are needed for pre-clinical studies, they are yet to be established owing to drug sensitivity in animal species and difficulty in setting doses. In this study, we evaluated liver fibrosis by administering thioacetamide (TA) to normal microminipig and thymectomized microminipig; 3 times for 1 week (total duration: 8 weeks). The pigs treated with TA showed elevated blood cytokine levels and a continuous liver injury at 8 weeks. RNA-seq of the liver showed increased expression of fibrosis-related genes after TA treatment. Histopathological examination showed degenerative necrosis of hepatocytes around the central vein, and revealed fibrogenesis and hepatocyte proliferation. TA treatment caused CD3-positive T cells and macrophages scattered within the hepatic lobule to congregate near the center of the lobule and increased αSMA-positive cells. Thymectomized pigs showed liver fibrosis similar to that of normal pigs, although the clinical signs tended to be milder. This model is similar to pathogenesis of liver fibrosis reported in other animal models. Therefore, it is expected to contribute to research as a drug discovery and pre-clinical transplantation models.

Topics: Animals; Cell Proliferation; Cytokines; Liver Cirrhosis; Swine; Thioacetamide

Hepatic encephalopathy (HE) is one of the most debilitating cerebral complications of liver cirrhosis. The one-year survival of patients with liver cirrhosis and severe encephalopathy is less than 50%. Recent studies have indicated that neuroinflammation is a new player in the pathogenesis of HE, which seems to be involved in the development of cognitive impairment. In this study, we demonstrated neurobehavioral and neuropathological consequences of liver cirrhosis and tested the therapeutic potential of the tumor necrosis factor-α (TNF-α) inhibitor, etanercept. Sixty male adult Wistar albino rats (120-190 g) were allocated into four groups, where groups I and IV served as controls. Thioacetamide (TAA; 300 mg/kg) was intraperitoneally injected twice a week for five months to induce liver cirrhosis in group II (n = 20). Both TAA and etanercept (2 mg/kg) were administered to group III (n = 20). At the end of the experiment, spatial learning was assessed using Morris water maze. TNF-α was detected in both serum and hippocampus. The excised brains were also immunohistochemically stained with glial fibrillary acidic protein (GFAP) to estimate both the number and integrity of hippocampal astrocytes. Ultrastructural changes in the hippocampus were characterized by transmission electron microscopy. The results showed that blocking TNF-α by etanercept was accompanied by a lower TNF-α expression and a higher number of GFAP-positive astrocytes in the hippocampus. Etanercept intervention alleviated the neuronal and glial degenerative changes and impeded the deterioration of spatial learning ability. In conclusion, TNF-α is strongly involved in the development of liver cirrhosis and the associated encephalopathy. TNF-α blockers may be a promising approach for management of hepatic cirrhosis and its cerebral complications.

Topics: Animals; Brain Diseases; Disease Models, Animal; Etanercept; Hepatic Encephalopathy; Hippocampus; Humans; Liver Cirrhosis; Male; Rats; Rats, Wistar; Spatial Learning; Thioacetamide; Tumor Necrosis Factor-alpha

The protective effect of biochanin A (BCA) on the histopathology, immunohistochemistry, and biochemistry of thioacetamide (TAA)-induced liver cirrhosis in vivo was investigated. There was a significant reduction in liver weight and hepatocyte propagation, with much lower cell injury in rat groups treated with BCA (25 mg/kg and 50 mg/kg) following a TAA induction. These groups had significantly lower levels of proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA). The liver homogenates showed increased antioxidant enzyme activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), as well as decreased malondialdehyde (MDA) levels. The serum biomarkers associated with liver function, namely alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma glutamyl transaminase (GGT), returned to normal levels, comparable to those observed in both the normal control group and the reference control group. Taken together, the normal microanatomy of hepatocytes, the inhibition of PCNA and α-SMA, improved antioxidant enzymes (SOD, CAT, and GPx), and condensed MDA with repairs of liver biomarkers validated BCA's hepatoprotective effect.

Topics: Alanine Transaminase; Animals; Antioxidants; Aspartate Aminotransferases; Chemical and Drug Induced Liver Injury; Liver; Liver Cirrhosis; Oxidative Stress; Proliferating Cell Nuclear Antigen; Rats; Rats, Wistar; Superoxide Dismutase; Thioacetamide

Hepatic stellate cells activation (HSCs) plays a crucial role in the pathogenesis of liver fibrosis. Specific microRNAs have been suggested to affect the activation of HSCs via various signalling pathways including TGF-β/smads and Wnt/β-catenin pathways. Dasatinib is a multitarget inhibitor of many tyrosine kinases has recently studied for its anti-fibrotic effects in a variety of fibrous diseases. This study investigated the role of modulation of miRNA-378 and miRNA-17 in the pathogenesis of liver fibrosis through altering Wnt/β-catenin and TGF-β/smads pathways and evaluated the beneficial effect of the tyrosine kinase inhibitor, dasatinib, in thioacetamide-induced liver fibrosis model in mice. Treatment with dasatinib down-regulated miRNA-17 expression, leading to the restoration of WiF-1 and smad-7 which cause the inhibition of both Wnt/β-catenin and TGF-β/smads signalling. In addition, it upregulated miRNA-378 leading to the decrease of Wnt-10 which contributes to the suppression of activated HSCs.

Topics: Animals; beta Catenin; Dasatinib; Dose-Response Relationship, Drug; Liver Cirrhosis; Male; Mice; MicroRNAs; Molecular Structure; Smad7 Protein; Structure-Activity Relationship; Thioacetamide; Transforming Growth Factor beta; Wnt Signaling Pathway

Cirrhosis refers to irreversible liver damage where healthy tissue is replaced by scar tissue, resulting in impaired liver function. There is no cure and current treatments only prevent further liver damage; thus, novel therapeutic options are urgently needed. Here, we report a new approach that enables the formation of self-assembled 3D spheroids of adipose-derived stem cells (ADSCs) and murine hepatocytes (AML12) via reconstituted collagen fibers. Compared with the spheroids formed in the commercially available EZSHERE dish, the collagen fiber-based ADSC/hepatocyte spheroids offer a notable benefit in structure formation and paracrine factor secretion. To test the regenerative capability of the collagen fiber-based 3D ADSC/hepatocyte spheroids, a rat model of thioacetamide (TAA)-induced liver cirrhosis was employed. The transplantation of the collagen fiber-based 3D ADSC/hepatocyte spheroids show an improvement in liver function and ameliorates pathological liver cirrhosis in TAA-treated rats. In summary, our data show collagen fiber-based self-assembled 3D ADSC/hepatocyte spheroids to possess the excellent regenerative capacity in response to TAA-induced liver injury, promising an alternative therapeutic strategy for liver cirrhosis.

Topics: Animals; Collagen; Disease Models, Animal; Hepatocytes; Humans; Liver Cirrhosis; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Rats, Sprague-Dawley; Spheroids, Cellular; Thioacetamide

Egyptian Purslane (Portulaca oleracea) is rich in omega-3 fatty acids and a wide range of vitamins and phyto-constituents that were absorbed slowly due to their high molecular weights. Therefore, this study was designed to accelerate the absorption of these phyto-constituents and hence increase their bioavailability by incorporating silver (Ag-NPs) and zinc oxide nanoparticles (ZnO-NPs) due to their impressive properties.. The major phyto-constituents and different biological activities were quantified in aqueous extract before and after incorporating metal nanoparticles (M-NPs). The efficiency of ZnO-P. nano-extract was studied on cell cycle and apoptosis of human hepatocellular carcinoma (HEPG-2) cells. Then, both Ag- and ZnO-P. nano-extracts were studied against hepatic fibrosis induced by thioacetamide (TAA) in rats through undergoing different hematological and biochemical measurements in addition to the histopathological examination in hepatic tissues compared to the extract itself.. The ZnO-P. nano-extract showed significantly (P<0.05) higher antioxidant and scavenging activity due to the existence of higher total polyphenolic content. Also, it exhibited a significantly (P<0.05) higher inhibitory effect on acetyl cholinesterase (AChE) activity and higher cytotoxic activity against HEPG-2 cells. Therefore, ZnO-P. nano-extract was studied against the cell cycle and apoptosis of HEPG-2 cells compared to the extract itself. It was found that ZnO-P. nano-extract was safer than Ag-P. nano-extract. Both Ag- and ZnO- P. nano-extracts were studied against the hepatic fibrosis induced by thioacetamide (TAA) compared to the native extract. It was noticed that ZnO-P. nano-extract exhibited an ameliorative effect against hepatic fibrosis by decreasing levels of inflammatory and fibrotic markers significantly (P<0.05) more than Ag-P. nano-extract. Furthermore, it improved the antioxidant status of the hepatic tissue in addition to restoring the histopathological architecture of liver tissue.. ZnO-P. nano-extract showed higher in vitro and in vivo biological activities than Ag-P. nano-extract and native P. extract itself.

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Hep G2 Cells; Humans; Liver Cirrhosis; Metal Nanoparticles; Plant Extracts; Portulaca; Rats; Silver; Thioacetamide; Zinc Oxide

Liver cirrhosis is the result of a vicious cycle of both chronic oxidative stress and inflammation. NADPH oxidase-4 (NOX4) and its companion, NOD-like receptor protein 3 (NLRP3) inflammasome, are emerging as therapeutic targets of liver fibrosis.. Baicalin (BA), a natural flavone, has been investigated for its therapeutic potential against cirrhosis induced by thioacetamide (TAA) (200 mg/kg, twice/week) for 12 weeks in Sprague-Dawley rats. Two doses of BA were administered (25 and 75 mg/kg/day, orally, a week after TAA was stopped and continued for 4 weeks).. BA was able to reduce fibrosis visualized by Masson trichrome and immunohistochemical staining of the hepatic α-smooth muscle actin (α-SMA) and transforming growth factor-β1. Moreover, BA was able to ameliorate inflammation by reducing hepatic NLRP3 inflammasome subunits, NLRP3 and caspase-1, both parts of the complex responsible for the activation of different interleukins (IL), measured as IL-1β. In addition, BA was able to reduce hepatic nuclear factor kappa B (NF-κB)-driven inflammation through IL-6. BA targeted inflammation through its anti-oxidant ability evidenced by the enhancement of the hepatic superoxide dismutase (SOD) and reduced glutathione (GSH) activity and level, respectively, and the reduction of both hepatic malondialdehyde (MDA) and nitric oxide (NOx) contents. Treatment with BA significantly decreased TAA-induced elevation in hepatic NOX4, a key enzyme for reactive oxygen species (ROS) generation, as well as, inducible nitric oxide synthase (iNOS).. therefore, the study could conclude, the anti-fibrotic effect of BA through TGF- β1/NOX4/NF-κB/NLRP3 pathway, exerting both anti-inflammatory and anti-oxidant effects.

Topics: Animals; Antioxidants; Fibrosis; Flavonoids; Inflammasomes; Inflammation; Liver; Liver Cirrhosis; Male; NADPH Oxidase 4; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Thioacetamide

The number of patients with liver diseases has increased significantly with the progress of global industrialization. Hepatic fibrosis, one of the most common liver diseases diagnosed in many developed countries, occurs in response to chronic liver injury and is primarily driven by the development of inflammation. Earlier immunological studies have been focused on the importance of the innate immune response in the pathophysiology of steatohepatitis and fibrosis, but recently, it has also been reported that adaptive immunity, particularly B cells, plays an essential role in hepatic inflammation and fibrosis. However, despite recent data showing the importance of adaptive immunity, relatively little is known about the role of B cells in the pathogenesis of steatohepatitis fibrosis. In this study, a single-cell-based, high-dimensional mass cytometric investigation of the peripheral blood mononuclear cells collected from mice belonging to three groups [normal chow (NC), thioacetamide (TAA), and 11beta-HSD inhibitor drug] was conducted to further understand the pathogenesis of liver fibrosis through reliable noninvasive biomarkers. Firstly, major immune cell types and their population changes were qualitatively analyzed using UMAP dimensionality reduction and two-dimensional visualization technique combined with a conventional manual gating strategy. The population of B cells displayed a twofold increase in the TAA group compared to that in the NC group, which was recovered slightly after treatment with the 11beta-HSD inhibitor drug. In contrast, the populations of NK cells, effector CD4

Topics: Animals; CD8-Positive T-Lymphocytes; Humans; Inflammation; Leukocytes, Mononuclear; Liver Cirrhosis; Mice; Thioacetamide

Liver fibrosis is a chronic wound-healing response to liver injury of various origins and represents a major health problem.. The current study endeavored to investigate the repressing effect of fisetin on hepatic fibrosis induced by thioacetamide (TAA) in rats.. Rats were injected with TAA (200 mg/kg) intraperitoneally twice per week for 6 weeks to induce liver fibrosis. Fisetin (50 and 100 mg/kg/day) or silymarin (50 mg/kg/day) were given orally on a daily basis along with TAA. Liver function parameters, oxidative stress, inflammatory and fibrogenic biomarkers as well as wnt3a, β-catenin, glycogen synthase kinase 3 (GSK-3β) and cyclin D1 were estimated. Histoapthological and immunohistochemical examinations were performed.. Fisetin restored normal liver functions, increased reduced glutathione (GSH) level and decreased malondialdehyde (MDA), as well as inflammatory biomarkers including; tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6). Additionally, it lessened transforming growth factor β1 (TGF-β1), collagen I and tissue inhibitor of metalloproteinase-1 (TIMP-1) levels as well as elevated matrix metalloproteinase-9 (MMP-9) hepatic content. Furthermore, fisetin significantly suppressed wnt3a gene expression associated with decreased β-catenin and increased GSK-3β levels. Moreover, fisetin decreased the progress of histologic hepatic fibroplasia and diminished hepatic expression of α-SMA and cyclin D1.

Topics: Animals; beta Catenin; Biomarkers; Cyclin D1; Flavonols; Glycogen Synthase Kinase 3 beta; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Matrix Metalloproteinase 9; Rats; Silymarin; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1; Wnt Signaling Pathway

Liver fibrosis is a common chronic hepatic disease. This study was done to examine the effect of pyridoxamine against thioacetamide-induced hepatic fibrosis. Animals were divided into four groups (1) control group; (2) Thioacetamide group (200 mg/kg, i.p.) twice a week for eight weeks; (3) Pyridoxamine-treated group treated with pyridoxamine (100 mg/kg/day, i.p.) for eight weeks; (4) Thioacetamide and pyridoxamine group, in which pyridoxamine was given (100 mg/kg/day, i.p.) during thioacetamide injections. Thioacetamide treatment resulted in hepatic dysfunction manifested by increased serum levels of bilirubin, gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Oxidative stress was noted by increased hepatic lipid peroxidation and decreased glutathione (GSH). Increased concentrations of total nitrite/nitrate, advanced glycation end products (AGEs), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), matrix metalloproteinases (MMP-2&9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were noticed in hepatic tissues. Immunostaining sections also revealed overexpression of MMP-2, MMP-9 and collagen IV. Liver fibrosis was confirmed by severe histopathological changes. Pyridoxamine improved the assessed parameters. Moreover, histopathological and immunohistological studies supported the ability of pyridoxamine to reduce liver fibrosis. The findings of the present study provide evidence that pyridoxamine is a novel target for the treatment of liver fibrosis.

Topics: Animals; Glycation End Products, Advanced; Liver; Liver Cirrhosis; Matrix Metalloproteinase 2; Matrix Metalloproteinases; Oxidative Stress; Pyridoxamine; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1

Ecballium elaterium (EE), widely used plant in Mediterranean medicine, showed anticancer activity. This study aimed to investigate EE effects on liver fibrosis in an animal model of thioacetamide (TAA). Intraperitoneal administration of TAA was performed twice weekly for four weeks in C57BL6J mice. Livers were extracted and serum were evaluated for inflammatory markers (H&E staining, ALT, AST, ALP), pro-inflammatory cytokines, fibrosis (Sirius red staining, Masson's trichrome, α-smooth muscle actin and collagen III), and metabolic (cholesterol, triglyceride, C-peptide, and fasting-blood-sugar) profiles. Glutathione, glutathione peroxidase, and catalase liver antioxidant markers were assessed. Tissue-resident NK cells from mice livers were functionally assessed for activating receptors and cytotoxicity. Compared to vehicle-treated mice, the TAA-induced liver injury showed attenuation in the histopathology outcome following EE treatment. In addition, EE-treated mice resulted in decreased serum levels of ALT, AST, and ALP, associated with a decrease in IL-20, TGF-β, IL-17, IL-22 and MCP-1 concentrations. Moreover, EE-treated mice exhibited improved lipid profile of cholesterol, triglycerides, C-peptide, and FBS. EE treatment maintained GSH, GPX, and CAT liver antioxidant activity and led to elevated counts of tissue-resident NK (trNK) cells in the TAA-mice. Consequently, trNK demonstrated an increase in CD107a and IFN-γ with improved potentials to kill activated hepatic-stellate cells in an in vitro assay. EE exhibited antifibrotic and antioxidative effects, increased the number of trNK cells, and improved metabolic outcomes. This plant extract could be a targeted therapy for patients with advanced liver injury.

Topics: Animals; Antioxidants; C-Peptide; Disease Models, Animal; Glutathione; Humans; Killer Cells, Natural; Liver; Liver Cirrhosis; Mice; Oxidative Stress; Thioacetamide

The present study aimed to investigate the hepatoprotective effect of sesamol (SML), a nutritional phenolic compound obtained from sesame seeds, in liver fibrosis induced by thioacetamide (TAA) in rats and to explore the underlying mechanisms. Thirty-two male Sprague-Dawley rats were equally divided into four groups: control, TAA, TAA + SML 50 mg/kg, and TAA + SML 100 mg/kg groups. Liver functions and hepatic contents of glutathione (GSH) and malondialdehyde (MDA) were measured colorimetrically. Gene expressions of lysophosphatidic acid receptor (LPAR)-1 and -3, connective tissue growth factor (CTGF), transforming growth factor (TGF)-β1, small mothers against decapentaplegic (Smad)-3 and -7, α-smooth muscle actin (α-SMA), and cytokeratin 19 (CK19) were analyzed by qRT-PCR. Moreover, phosphorylated Smad3 (pSmad3) was quantified by ELISA. Additionally, TGF-β1, α-SMA, CK19, and vascular endothelial growth factor (VEGF) protein concentrations were semi-quantitatively analyzed by immunostaining of liver sections. SML treatment markedly improved liver index and liver functions. Moreover, SML protected against liver fibrosis in a dose-dependent manner as indicated by down-regulation of LPAR1, LPAR3, CTGF, TGF-β1/Smad3, and α-SMA expressions and a decrease in pSmad3 level, as well as an up-regulation of Smad7 expression. In addition, SML suppressed ductular reaction hinted by the decrease in CK19 expression. These results reveal the anti-fibrotic effect of SML against liver fibrosis that might be attributed to down-regulation of LPAR1/3 expressions, inhibition of TGF-β1/Smad3 pathway, and ductular reaction.

Topics: Animals; Benzodioxoles; Liver; Liver Cirrhosis; Male; Phenols; Rats; Rats, Sprague-Dawley; Receptors, Lysophosphatidic Acid; Signal Transduction; Thioacetamide; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

The Hedgehog (Hh) pathway has emerged as a potential target for effectual hepatic repair based on convincing clinical and preclinical evidence that proves its significance in regulating hepatic damage. The purpose of this study is to probe the effect of quercetin on liver fibrosis through the modulation of the Hh pathway. Healthy male Wistar rats were divided into four groups (n = 10). The control group was treated with saline, rats in the remaining three groups received twice a week intoxication with intraperitoneal injections of thioacetamide (200 mg/kg) for the induction of hepatic fibrosis for 6 weeks. After 28 days of quercetin and silymarin treatment, histological changes, serum biochemical index, antioxidant enzyme activity, key mediators of Hh pathway and inflammation were analyzed. Serological analysis showed statistically improved cholesterol, H.D.L-Cholesterol, and L.D.L-Cholesterol in the treatment groups. Superoxide dismutase and glutathione levels were found to be increased after the treatment with quercetin and silymarin. mRNA expression of important mediators of the Hh signaling, and inflammation including Shh, Ihh, Ptch-1, Smo, Hhip, Gli-3, TNF-α, NFκ-β, and Socs-3 were significantly downregulated after the use of quercetin and silymarin. Quercetin also minimized the thioacetamide-induced histopathological changes, as confirmed by a lower degree of hepatic lobule degeneration, the intralobular occurrence of inflammatory cells, and a lower degree of hepatocytic necrosis. Sudan Black B staining showed remarked lipids improvements in the treatment groups. Taken together, these findings demonstrate that quercetin could ameliorate hepatic fibrosis by antagonizing the hedgehog pathway and also suggest the hedgehog pathway as a potential therapeutic target for the treatment of liver fibrosis.

Topics: Animals; Antioxidants; Hedgehog Proteins; Inflammation; Liver; Liver Cirrhosis; Male; Oxidative Stress; Quercetin; Rats; Rats, Wistar; Silymarin; Thioacetamide

This study aimed at investigating the chemical composition and the hepatoprotective activities of Plumbago indica L. and P. auriculata Lam. LC-MS/MS analyses for the hydroalcoholic extracts of the aerial parts of the two Plumbago species allowed the tentative identification of thirty and twenty-five compounds from P. indica and P. auriculata, respectively. The biochemical and histopathological alterations associated with thioacetamide (TAA)-induced liver fibrosis in rats were evaluated in vivo where rats received the two extracts at three different dose levels (100, 200 and 400 mg/kg p.o, daily) for 15 consecutive days with induction of hepatotoxicity by TAA (200 mg/kg/day, i.p.) at 14th and 15th days. Results of the present study showed a significant restoration in liver function biomarkers viz. alanine transaminase (ALT), aspartate transaminase (AST), gamma glutamyl transferase and total bilirubin. The liver homogenates exhibited increased levels of antioxidant biomarkers: reduced glutathione (GSH) and catalase (CAT), accompanied with decline in malondialdehyde (MDA). Furthermore, treated groups exhibited a significant suppression in liver inflammatory cytokines: tumor necrosis factor-α (TNF-α) and interlukin-6 (IL-6), and fibrotic biomarker: alpha smooth muscle relaxant. Histopathological examination of the liver showed normality of hepatocytes. Noteworthy, P. indica extract showed better hepatoprotective activity than P. auriculata, particularly at 200 mg/kg. To sum up, all these results indicated the hepatoprotective properties of both extracts, as well as their antifibrotic effect was evidenced by reduction in hepatic collagen deposition. However, additional experiments are required to isolate their individual secondary metabolites, assess the toxicity of the extracts and explore the involved mechanism of action.

Topics: Animals; Antioxidants; Biomarkers; Chemical and Drug Induced Liver Injury; Chromatography, Liquid; Liver; Liver Cirrhosis; Oxidative Stress; Phytochemicals; Plant Extracts; Plumbaginaceae; Rats; Rats, Wistar; Tandem Mass Spectrometry; Thioacetamide

Chronic liver injury can lead to hepatic failure and the only available method of treatment would be liver transplantation. The link between inflammation (TNF-α), nuclear factor-kappa B (NF-kB), nitrosative stress (iNOS) and hypoxia-inducible factor-1α (HIF-1α) in thioacetamide (TAA) induced liver fibrosis, and hypertension with and without the incorporation of the anti-inflammatory and antioxidant resveratrol (RES) has not been investigated before. Consequently, we injected rats with either 200 mg/kg TAA for 8 weeks starting at week 2 (model group) or pretreated them before TAA injections with RES (20 mg/kg) for 2 weeks and continued them on RES and TAA until being culled at week 10 (protective group). In the model group, we documented the induction of hepatic fibrosis and upregulation of tumor necrosis factor-α (TNF-α), NF-kB, inducible nitric oxide synthase (iNOS), HIF-1α and the profibrotic biomarkers alpha-smooth muscle actin (α-SMA) and matrix metalloproteinase-9 (MMP-9) that was significantly (p ≤ 0.0014) ameliorated by RES. RES also significantly (p ≤ 0.0232) reduced triglycerides (TG), cholesterol (CHOL), very low-density lipoprotein (vLDL-C), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure, and heart rate (HR) induction by TAA. Also, a significant (p < 0.0001) positive correlation between TNF-α/NF-kB/iNOS/HIF-1α axis-mediated fibrosis and hypertension and liver injury biomarkers was observed. These findings suggest that in the hepatotoxic compound, TAA is associated with TNF-α/NF-kB/iNOS/HIF-1α-mediated fibrosis and hypertension, whilst being inhibited by RES.

Topics: Animals; Biomarkers; Chemical and Drug Induced Liver Injury, Chronic; Hypertension; Hypoxia-Inducible Factor 1, alpha Subunit; Liver; Liver Cirrhosis; NF-kappa B; Nitric Oxide Synthase Type II; Rats; Resveratrol; Thioacetamide; Tumor Necrosis Factor-alpha

Efficient anti-fibrotic therapies are required for the treatment of liver cirrhosis. Hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) and cyclooxygenase-2 (COX-2) inhibitors have been reported to have anti-fibrotic effects. Here, we investigated whether combined treatment with a statin and a COX-2 inhibitor has synergistic anti-fibrotic effects.. The effects of treatment strategies incorporating both simvastatin and a COX-2 inhibitor, NS-398, were investigated using an immortalized human hepatic stellate cell line (LX-2) and a hepatic fibrosis mouse model developed using thioacetamide (TAA) in drinking water. Cellular proliferation was investigated via 5-bromo-2-deoxyuridine uptake. Pro- and anti-apoptotic factors were investigated through Western blotting and real-time polymerase chain reaction analysis.. The evaluation of the anti-proliferative effects on LX-2 cells showed that the observed effects were more pronounced with combination therapy than with single-drug therapy. Moreover, hepatic fibrosis and collagen deposition decreased significantly in TAA-treated mice in response to the combined treatment strategy. The mechanisms underlying the anti-fibrotic effects of the combination therapy were investigated. The effects of the combination therapy were correlated with increased expression levels of extracellular signal-regulated kinase 1/2 signaling molecules, upregulation of the Bax/Bcl-2 signaling pathway, inhibition of the transforming growth factor-β signaling pathway, and inhibition of tissue inhibitor of matrix metalloproteinases 1 and 2.. The combination of simvastatin and NS-398 resulted in a synergistic anti-fibrotic effect through multiple pathways. These findings offer a theoretical insight into the possible clinical application of this strategy for the treatment of advanced liver diseases with hepatic fibrosis.

Topics: Animals; Cyclooxygenase 2 Inhibitors; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Mice; Nitrobenzenes; Simvastatin; Sulfonamides; Thioacetamide

Minimal hepatic encephalopathy (MHE) is a common neuropsychiatric complication in patients with cirrhosis. Alterations in monoamine neurotransmitters have been associated with the pathogenesis of MHE. We investigated the levels of hippocampal noradrenergic neurotransmitter in a rat model of thioacetamide-induced chronic liver failure-related MHE, and their role in cognitive impairment.. 18 male Sprague-Dawley (SD) rats were equally divided in MHE and control groups. A rat model of MHE was established by intraperitoneal injection of thioacetamide (TAA) for 12 weeks. Cognitive function was assessed using the Morris water maze (MWM) test and locomotor activity and exploratory behavior assessed with open field test. The concentration of hippocampal noradrenaline (NE) was detected by ELISA, and the magnetic susceptibility value in the hippocampus was detected by quantitative susceptibility mapping. Hippocampal iron content was quantified by Prussian blue staining.. MHE rats performed significantly poorer than their control counterparts in the MWM test, as seen by decreased number of platform crossings and time in the target quadrant, and increased path length to reach the target zone (P < 0.05 for all parameters). In the open field test, the MHE group exhibited lower locomotor activity and exploratory behavior than the control group (P < 0.05 for all parameters). We detected pronounced iron staining in the hippocampus of MHE rats, whereas no iron-stained particles were found in control rats. We observed an imbalance of inflammatory (increased pro- and decreased anti-) cytokines in the hippocampus of MHE rats. Further analysis of the data showed that the level of hippocampal noradrenaline in MHE rats was significantly lower than that of control rats (P < 0.05). We observed a correlation between the level of inflammatory cytokine and noradrenaline land susceptibility value in the rat hippocampus of the MHE group.. Our results suggest that MHE associated with TAA-induced chronic liver failure is associated with alterations in noradrenergic neurotransmission. We propose that iron imbalance in the brain might lead to reduction in the levels of noradrenaline, and cognitive impairment.

Topics: Animals; Brain; Cognitive Dysfunction; Cytokines; End Stage Liver Disease; Hepatic Encephalopathy; Liver Cirrhosis; Male; Norepinephrine; Rats; Rats, Sprague-Dawley; Thioacetamide

Upon chronic damage to the liver, multiple cytokines stimulate hepatic stellate cells (HSCs), causing the alterations of gene expression profiles and thus leading to HSC activation, a key step in liver fibrogenesis. Activated HSCs are the dominant contributors to liver fibrosis. Bromodomain containing protein 4 (BrD4), an important epigenetic reader, was demonstrated to concentrate on hundreds of enhancers associated with genes involved in multiple profibrotic pathways, thereby directing HSC activation and the fibrotic responses. The present studies were designed to examine the effect of transforming growth factor beta-1 (TGFβ1), the most potent pro-fibrotic cytokine, on BrD4 expression in HSCs and, if so, elucidated the underlying mechanisms in vitro and in vivo. The experiments employed the heterogeneous TGFβ1 knockout (TGFβ1

Topics: Animals; Cell Cycle Proteins; Chemical and Drug Induced Liver Injury, Chronic; Early Growth Response Protein 1; Epigenesis, Genetic; Fibrosis; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Mice; Nuclear Proteins; Smad3 Protein; Thioacetamide; Transcription Factors; Transforming Growth Factor beta1

The detailed understanding of fibrogenesis has been hampered by a lack of important functional quiescence characteristics and an in vitro model to recapitulate hepatic stellate cell (HSC) activation. In our study, we establish robust endoderm- and mesoderm-sourced quiescent-like induced HSCs (iHSCs) derived from human pluripotent stem cells. Notably, iHSCs present features of mature HSCs, including accumulation of vitamin A in the lipid droplets and maintained quiescent features. In addition, iHSCs display a fibrogenic response and secrete collagen I in response to hepatoxicity caused by thioacetamide, acetaminophen, and hepatitis B and C virus infection. Antiviral therapy attenuated virally induced iHSC activation. Interestingly, endoderm- and mesoderm-derived iHSCs showed similar iHSC phenotypes. Therefore, we provide a novel and robust method to efficiently generate functional iHSCs from hESC and iPSC differentiation, which could be used as a model for hepatocyte toxicity prediction, anti-liver-fibrosis drug screening, and viral hepatitis-induced liver fibrosis.

Topics: Hepatic Stellate Cells; Hepatocytes; Humans; Liver Cirrhosis; Pluripotent Stem Cells; Thioacetamide

Edaravone (EDA), a strong novel free radical scavenger, have been demonstrated to exert neurovascular protective effects clinically. Furthermore, EDA can suppress the lung injury, pulmonary fibrosis and skin fibrosis, while the precise effects and mechanisms of EDA on liver injury and fibrosis remain unclear. The effects of EDA on the Thioacetamide (TAA)-induced liver fibrosis were evaluated by sirius red staining, α-SMA immunohistochemistry. The percentages of immune cell subsets were analyzed by flow cytometry. Immunofluorescence assay was performed to identify the fibrotic properties of hepatic stellate cells (HSCs). Western blot and qPCR were used to detect the levels of liver fibrosis-related molecules and IL-1β. EDA displayed a hepatic protective role in TAA-induced chronic liver fibrosis via inhibiting monocyte/macrophages recruitment and IL-1β production of macrophages. Mechanically, EDA inhibited of NF-κB signal pathway and reactive oxygen species (ROS) production in macrophages. Moreover, EDA treatment indirectly suppressed the activation of HSCs by decreasing the IL-1β secretion of macrophages. Together, EDA protects against TAA-induced liver fibrosis via decreasing the IL-1β production of macrophages, thereby providing a feasible solution for clinical treatment of liver fibrosis.

Topics: Edaravone; Fibrosis; Hepatic Stellate Cells; Humans; Liver; Liver Cirrhosis; Macrophages; Thioacetamide

Hepatic fibrosis is a progressive consequence of injury to the liver cells. Liver fibrosis causes hepatic dysfunction and also plays a key role in the pathogenesis of other chronic ailments. Dehydrozingerone (DHZ) is a half-structural analogue of curcumin and is known to have several therapeutic benefits. However, the impact of DHZ on liver fibrosis was not investigated. The current investigation attempted to determine the anti-fibrotic effect of DHZ against thioacetamide-induced liver fibrosis in rats and TGF-β-induced differentiation in human HSC-LX2 cells and to uncover the possible mechanisms. In in-vivo, DHZ significantly reduced the TAA-induced liver index and ameliorated the liver functional parameters. TAA elevated the fibrotic marker's expression in TAA control, on the other hand, DHZ treatment significantly mitigated the same in mRNA and protein levels. Additionally, these findings were supported by histological investigations and immunohistochemistry studies of the fibrotic marker's expressions. DHZ treatment effectively reduced oxidative stress by increasing catalase activity and decreased the expression of inflammatory markers (myeloperoxidase and neutrophil-elastase) in liver tissues. Additionally, collagen staining and histological findings confirmed that DHZ administration significantly reduced TAA induced pathological deformities and elevated collagen levels. In-vitro results showed that TGF-β-induced differentiation was suppressed by DHZ treatment in a dose-dependent manner. Mechanistic approaches in HSC-LX2 and liver tissues revealed that DHZ treatment mitigated fibrosis by modulating the MAPK-pathway. Overall, these results show that DHZ exhibited anti-fibrotic action by reducing fibrotic markers and their activities through regulation of the MAPK-pathway, suggesting that DHZ may be a promising therapeutic molecule for liver fibrosis.

Topics: Animals; Biomarkers; Collagen; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Rats; Thioacetamide; Transforming Growth Factor beta

Hepatic fibrosis is caused by chronic injury due to toxic, infectious, or metabolic causes, and it may progress to cirrhosis and hepatocellular carcinoma. There is currently no antifibrotic therapy authorized for human use; however, there are promising studies using cell therapies. There are also no animal models that exactly reproduce human liver fibrosis that can be used to better understand the mechanisms of its regression and identify new targets for treatment and therapeutic approaches. On the other hand, mesenchymal stem cells (MSC) have experimentally demonstrated fibrosis regression effects, but it is necessary to have an animal model of advanced liver fibrosis to evaluate the effect of these cells. The aim of this work was to establish a protocol for the induction of advanced liver fibrosis in rats using thioacetamide (TAA), which will allow us to perform trials using MSC as a possible therapy for fibrosis regression. For this purpose, we selected 24 female rats and grouped them into three experimental groups: the control group (G-I) without treatment and groups II (G-II) and III (G-III) that received TAA by intraperitoneal injection for 24 weeks. Then, 1 × 106/kg adipose mesenchymal stem cells (ASCs) were infused intravenously. Groups G-I and G-II were sacrificed 7 days after the last dose of ASC, and G-III was sacrificed 8 weeks after the last ASC infusion, all with xylazine/ketamine (40 mg/kg). The protocol used in this work established a model of advanced hepatic fibrosis as corroborated by METAVIR tests of the histological lesions; by the high levels of the markers

Topics: Animals; Disease Models, Animal; Female; Humans; Liver Cirrhosis; Mesenchymal Stem Cells; Rats; Thioacetamide

Liver fibrosis is a chronic progressive disease, its resolution still unclear, and the current study explored the role of melatonin in modulation of interleukin-6 (IL-6), interleukin-4 (IL-4), transforming growth factor beta1 (TGF-β1) and urokinase plasminogen activator receptor-associated protein/Endo180 (uPARAP/Endo180) pathway in thioacetamide (TAA)-induced hepatotoxicity. Thirty two adult Sprague-Dawley rats were divided into four groups: vehicle control group, TAA-induced liver fibrosis group that was left untreated, melatonin administration before and along with TAA and melatonin along with TAA group. TTA-induced massive liver necrosis, fibrosis around portal tract and increases serum levels of liver enzymes and total bilirubin when compared with control vehicle group. While both melatonin pretreatment and treatment retained liver parenchyma and liver enzymes quite similar to control group and reduced TAA-induced liver injury. Notably, melatonin pretreatment and treatment increased collagen degradation in TAA liver injury by19, 31.7-fold respectively evidence by collagen percentage area. Melatonin also decreased the amount of thiobarbituric acid reactive compounds and retained the reduced glutathione and superoxide dismutase to basal level quite similar to control group. Additionally, melatonin significantly (P value ≤0.05) decreased the levels of TGF-β1, epidermal growth factor (EGF), hydroxyproline, tissues IL-6, caspase-3, and receptor interacting serine/threonine kinase1 (RIPK1), fibrillin-1, and - smooth muscle actin in the liver tissues while significantly (P value ≤0.05) increasing the levels of IL-4 and uPARAP/Endo180. Due to its anti-inflammatory, anti-apoptotic, and antioxidant capabilities as well as its ability to decrease hepatic stellate cell activation and fibrogenesis, these data imply that melatonin has a powerful anti-fibrotic effect.

Topics: Animals; Apoptosis; Collagen; Interleukin-4; Interleukin-6; Liver; Liver Cirrhosis; Male; Melatonin; Oxidative Stress; Rats; Rats, Sprague-Dawley; Receptors, Urokinase Plasminogen Activator; Thioacetamide; Transforming Growth Factor beta1

Collagen production by activated hepatic stellate cells (HSCs) to encapsulate injury is part of the natural wound-healing response in injured liver. However, persistent activation of HSCs can lead to pathological fibrogenesis. Such persistent HSC activation could be mediated by norepinephrine (NE), a reaction product of dopamine beta-hydroxylase (DBH).. To investigate the potential paracrine role of NE in hepatotoxin thioacetamide (TAA)-induced liver fibrosis.. In TAA-treated mice, fibrotic liver tissue showed significant increases in the mRNA expression of DBH up to 14-fold and collagen up to 7-fold. Immunohistochemical staining showed increased DBH protein expression in fibrotic liver tissue. Parenchymal hepatocyte cell line HepG2 expressed DBH and secreted NE, and the conditioned medium of HepG2 cells promoted collagenesis in nonparenchymal HSC cell line LX-2. TAA treatment increased DBH expression by 170% in HepG2 cells, as well as increased NE by 120% in the conditioned medium of HepG2 cells. The conditioned medium of TAA-treated HepG2 cells was used to culture LX-2 cells, and was found to increase collagen expression by 80% in LX-2 cells. Collagen expression was reduced by pre-treating HepG2 cells with siRNA targeting DBH or by adding NE antagonists to the conditioned medium.. Finally, TAA-induced oxidative stress in HepG2 cells was associated with induction of DBH expression. Collectively, our results suggest a potential role for DBH/NE-mediated crosstalk between hepatocytes and HSCs in fibrogenesis.. From a therapeutic standpoint, antagonism of DBH/NE induction in hepatocytes might be a useful strategy to suppress pathological fibrogenesis.

Topics: Animals; Culture Media, Conditioned; Hepatic Stellate Cells; Hepatocytes; Liver Cirrhosis; Mice; Norepinephrine; Thioacetamide

Liver fibrosis, which still does not have a standard treatment due to its complex pathogenesis, is an important cause of mortality and morbidity. In this study, it was aimed to examine the possible protective and antifibrotic effects of tyrosol on the liver through histopathologic, immunohistochemical, biochemical, and molecular methods in rats with chronic liver damage induced by thioacetamide (TAA). The study was carried out in four groups with eight rats in each group. Created groups are, respectively, control, TAA, tyrosol and TAA +tyrosol. Chronic liver damage was induced in the TAA and TAA +tyrosol groups by the addition of TAA (200 mg/L) to drinking water. Tyrosol (20 mg/kg/b.w./daily) was administered by oral gavage to tyrosol and TAA +tyrosol groups for 10 weeks. The results of this study demonstrate that the consumption of tyrosol alleviated the histopathologic changes such as inflammation, degeneration, and especially fibrosis induced by TAA in the liver. In addition, administration of tyrosol significantly attenuated alpha-smooth muscle actin (α-SMA) expression and apoptosis expression. Biochemically, it was determined that tyrosol increased glutathione (GSH) level, glutathione peroxidase (GSH.Px), and catalase (CAT) activities and showed antioxidant efficacy by reducing malondialdehyde (MDA) level. Moreover, it reduced inflammation and fibrosis by decreasing gene expression levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and transforming growth factor-beta (TGF-β1). Western blot analysis also revealed similar results in TGF-β1 expression. As a result, tyrosol suppressed fibrogenesis thanks to its antioxidant, anti-inflammatory, and anti-apoptotic effects and showed an antifibrotic effect in the liver. PRACTICAL APPLICATIONS: It is stated that tyrosol, a natural phenolic antioxidant found in olive oil, has neuroprotective, cardioprotective, anti-inflammatory, and anticancer properties. In this study, tyrosol suppressed fibrogenesis thanks to its antioxidant, anti-inflammatory, and anti-apoptotic effects and showed an antifibrotic effect in the liver. Olive oil has an important place in the Mediterranean diet, which reduces the incidence of chronic diseases. It is thought that the anti-fibrotic effect of tyrosol plays a role in this feature. As a result, it is thought that tyrosol can be used to prevent or slow down chronic liver diseases.

Topics: Animals; Liver Cirrhosis; Oxidative Stress; Phenylethyl Alcohol; Rats; Thioacetamide

This study has been designed to investigate the role of vanillin either as prophylaxis or treatment in liver regeneration augmentation and liver fibrosis regression in thioacetamide (TAA) induced liver damage.. Animals were injected with TAA to induce liver injury (200mg/kg twice weekly) for 8 weeks. In vanillin prophylaxis group; rats were administered vanillin (100 mg/Kg; IP, daily) from day 1 of TAA injection for 8 weeks. In vanillin treatment group; rats were confronted with the same dose of TAA injection for 8 weeks then treated with vanillin (100 mg/Kg, IP, daily) for 4 weeks. ALT, AST activities, serum albumin, hepatic GSH, MDA, HGF, VEGF, IL-6 and TNF-α levels were measured and also, MMP-2, TIMP-1 and cyclin D gene expression were determined. Liver sections were stained with H&E and Sirius red and immunostained for Ki-67 and α-SMA for histological and immunohistological changes analysis.. Vanillin improved liver function and histology. Also, showed a remarkable increase in hepatic HGF and VEGF level, and up-regulation of cyclin D1 expression accompanied by a significant up-regulation of MMP-2 and down- regulation of TIMP-1. All these effects were accompanied by TNF-α, IL-6 and oxidative stress significant attenuation.. In conclusion, vanillin enhanced liver regeneration in TAA induced liver damage model; targeting growth factors (HGF, VEGF) and cellular proliferation marker cyclin D1. As well as stimulating fibrosis regression by inhibition of ECM accumulation and enhancing its degradation.

Topics: Animals; Benzaldehydes; Cell Proliferation; Cyclin D1; Intercellular Signaling Peptides and Proteins; Liver; Liver Cirrhosis; Liver Regeneration; Male; Oxidative Stress; Rats; Rats, Wistar; Thioacetamide

The monosaccharide mannose has gained recent interest for its beneficial effect against certain inflammatory disorders. Nevertheless, the influence of mannose on experimentally-induced liver fibrosis and the ensued inflammation is still not fully clear to date.. The current study investigated the outcomes of treating rats with mannose (0.2 ml of 20% w/v, oral gavage) 30 min before the twice weekly intoxication with thioacetamide (TAA) (200 mg/kg, intraperitoneal) for a total period of 8 weeks.. The data indicated that mannose markedly dampened TAA-induced liver fibrosis, as indicated by lowering the fibrotic bridges shown by Masson's trichrome staining. This effect was consistent with reducing TAA-induced hepatocellular injury, as evidenced biochemically (serum ALT and AST activities) and pathologically (necroinflammation score). These hepatoprotective effects mediated by mannose were attributed to i) reversing TAA-induced rise in malondialdehyde (MDA) and decrease in reduced glutathione (GSH) expressions in the liver, ii) limiting TAA-induced release of the proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), iii) impairing TAA-induced activation of hepatic stellate cells by downregulating α-smooth muscle actin expression (α-SMA), and more importantly, iv) dampening TAA-induced fibrogenesis driven by transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF).. Mannose may be a valuable candidate for preventing oxidative stress, inflammation and fibrogenesis in the liver.

Topics: Animals; Cytokines; Hepatic Stellate Cells; Inflammation; Liver; Liver Cirrhosis; Male; Malondialdehyde; Mannose; Oxidative Stress; Rats; Rats, Sprague-Dawley; Signal Transduction; Thioacetamide

Small- and intermediate-conductance Ca

Topics: Actins; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Line; Collagen; Diet, High-Fat; Fatty Liver; Hepatic Stellate Cells; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Modafinil; Non-alcoholic Fatty Liver Disease; Small-Conductance Calcium-Activated Potassium Channels; Stereoisomerism; Thioacetamide

Antifibrotic agent for the treatment of liver fibrosis has not been developed so far. Long term treatment of chronic hepatitis B patients with antiviral drugs tenofovir disoproxil fumarate (TDF) and entecavir (ETV) results in the regression of liver fibrosis, but the underlying mechanism has not been clarified. Therefore, we aimed to investigate the direct impact of TDF and ETV on liver fibrosis.. Activated hepatic stellate cell (HSC) cell lines were used to evaluate the effects of TDF and ETV. After treatment with each antiviral agent, cell viability, morphology, apoptotic features, autophagy and antifibrosis signalling pathways were examined. Then, collagen deposition, fibrosis markers and activated HSCs were measured in liver tissues of the liver fibrosis model mice.. After TDF treatment, the viabilities of LX2 and HSC-T6 cells were decreased, and the cells exhibited apoptotic features, but ETV did not induce these effects. Cleavage of PARP and Caspase-3 and the inhibition of the antiapoptotic gene Bcl-xl indicated activated HSC apoptosis following TDF treatment. TDF simultaneously increased autophagy, which also regulated apoptosis through crosstalk. TDF inactivated the PI3K/Akt/mTOR signalling pathway, which was associated with the activation of both apoptosis and autophagy. In the liver fibrosis mouse model, the fibrotic area and activated HSC markers were decreased by TDF but not ETV treatment. Additionally, apoptotic cells were concentrated in the periportal fibrotic area after TDF treatment, which indicated the specific antifibrotic effect of TDF.. TDF directly ameliorates liver fibrosis by downregulating the PI3K/Akt/mTOR signalling pathway, which results in the apoptosis of activated HSCs. The antifibrotic effects of TDF indicate that it may be a therapeutic agent for the treatment of liver fibrosis.

Topics: Animals; Antiviral Agents; Apoptosis; Gene Expression Regulation; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Tenofovir; Thioacetamide; TOR Serine-Threonine Kinases

The objective of the current study is to investigate the effect of rice bran oil (RBO) on hepatic fibrosis as a characteristic response to persistent liver injuries. Rats were randomly allocated into five groups: the negative control group, thioacetamide (TAA) group (thioacetamide 100 mg/kg thrice weekly for two successive weeks, ip), RBO 0.2 and 0.4 groups (RBO 0.2mL and 0.4 mL/rat/day, po) and standard group (silymarin 100 mg/kg/day, po) for two weeks after TAA injection. Blood and liver tissue samples were collected for biochemical, molecular, and histological analyses. Liver functions, oxidative stress, inflammation, liver fibrosis markers were assessed. The obtained results showed that RBO reduced TAA-induced liver fibrosis and suppressed the extracellular matrix formation. Compared to the positive control group, RBO dramatically reduced total bilirubin, AST, and ALT blood levels. Furthermore, RBO reduced MDA and increased GSH contents in the liver. Simultaneously RBO downregulated the NF-κβ signaling pathway, which in turn inhibited the expression of some inflammatory mediators, including Cox-2, IL-1β, and TNF-α. RBO attenuated liver fibrosis by suppressing the biological effects of TGF-β1, α-SMA, collagen I, hydroxyproline, CTGF, and focal adhesion kinase (FAK). RBO reduced liver fibrosis by inhibiting hepatic stellate cell activation and modulating the interplay among the TGF-β1 and FAK signal transduction. The greater dosage of 0.4 mL/kg has a more substantial impact. Hence, this investigation presents RBO as a promising antifibrotic agent in the TAA model through inhibition of TGF-β1 /FAK/α-SMA.

Topics: Actins; Albumins; Animals; Becaplermin; Biomarkers; Collagen Type I; Connective Tissue Growth Factor; Cyclooxygenase 2; Cytokines; Focal Adhesion Protein-Tyrosine Kinases; Gas Chromatography-Mass Spectrometry; Globulins; Glutathione; Hydroxyproline; Inflammation Mediators; Liver; Liver Cirrhosis; Male; Malondialdehyde; NF-kappa B; Oxidative Stress; Proto-Oncogene Proteins c-akt; Rats, Wistar; Rice Bran Oil; Signal Transduction; Thioacetamide; Transaminases; Transforming Growth Factor beta1

One of the main factors which played a key role in the prevention of liver disorders such as hepatic inflammation, fibrosis, and carcinogenesis would be the vitamin D axis. Therefore, the current research was designed to evaluate the role of Vitamin D (Vit D)   as a protective agent against liver damage caused by Thioacetamide (TAA). In the current study, 18 male Wistar rats were randomly allocated into three equal groups (n=6): in group 1(G1) the animals were considered as the control group and did not receive any supplement in drinking water; in group 2 (G2) TAA was administrated to the drinking water at a dose of 300 mg/L; in group 3 (G3) TAA was administrated to the drinking water at a dose of 300 mg/L plus vitamin D at a dose of 0.5 mg/100g body (intraperitoneal) for 8 weeks. At the end of the experiment, the animals were sacrificed and the liver was dissected and removed for histopathology. Histopathological evaluations were used to evaluate the possible adverse effects of TAA on the liver. Several hepatic damages were observed in the G2 group such as lobular disorder, some degrees of degeneration in hepatocytes and enlargement of the hepatic capillaries, and focal necrotic areas. Hepatic fibrosis was observed around portal areas and central veins. Bridging fibrous septa were formed between portal veins. The recorded data in this study showed that Vit D has some beneficial effects in protecting the liver from fibrosis and toxic damages. The recorded data showed that liver damages in the G3 group were partially prevented or cured. In conclusion, it is evident that the Vit D played a pivotal role as an antioxidant and anti-fibrotic agent, therefore it would be the best supplement for liver protection against damages due to toxin entrance into the animal's body.

Topics: Animals; Liver Cirrhosis; Male; Protective Agents; Rats; Rats, Wistar; Thioacetamide; Vitamin D

In liver cirrhosis, intestinal mucus barrier is rarely studied.. This study aimed to investigate whether mucus barrier in ileum is altered in cirrhotic rats and its underlying mechanisms.. Thioacetamide was injected to induce liver cirrhosis in rats. Serum from portal vein blood, and ileum and liver tissues were obtained for further analysis. Goblet cell-like Ls174T cells were cultured for in vitro experiments.. The ileal mucus was thin, loose, and porous with small bubbles in cirrhotic rats. mRNA expressions of Muc2 and TFF3 were also down-regulated in cirrhotic rats. Bacteria located near to crypts and LPS were increased in the serum from portal vein in cirrhotic rats. Smaller theca area and few goblet cells were found in cirrhotic rats compared with control. Increased proliferation of ileal epithelia was observed in cirrhotic rats. Notch1, Dll1, and Hes1 expressions were enhanced, and KLF4 expression was suppressed in ileum of cirrhotic rats. In Ls174T cells, EDTA and NICD plasmid induced NICD and Hes1 expression and suppressed KLF4 concomitantly, and mucus expression almost vanished in these cells. NICD plasmid induced more proliferation in Ls174T cells. Oppositely, after DBZ treatment, NICD and Hes1 were inhibited along with augmentation of KLF4 and increased mucous expression in Ls174T cells, while proliferation of the cells was suppressed.. In cirrhotic rats, mucus barrier was impaired. This might be attributed to increased proliferation and decreased differentiation of epithelia, which might be mediated by Notch1-Hes1-KLF4 signaling.

Topics: Animals; Cell Line, Tumor; Homeostasis; Humans; Ileum; Intestinal Mucosa; Kruppel-Like Factor 4; Liver Cirrhosis; Male; Rats; Rats, Sprague-Dawley; Receptor, Notch1; Thioacetamide

Cirrhotic cardiomyopathy is a condition where liver cirrhosis is associated with cardiac dysfunction. Triggers and blockers of cirrhotic cardiomyopathy are poorly understood, which might compromise the prognosis of chronic liver disease patients. We tested whether exercise training would reduce liver damage induced by thioacetamide and prevent liver cirrhosis-associated cardiomyopathy. Wistar rats were divided into three groups: control, thioacetamide (TAA), or TAA plus exercise. Thioacetamide increased liver weight and serum alanine aminotransferase and aspartate aminotransferase levels. Also, TAA treatment was involved with hepatic nodule formation, fibrotic septa, inflammatory infiltration, and hepatocyte necrosis. The exercise group presented with a reduction in liver injury status. We found that liver injury was associated with disordered cardiac hypertrophy as well as diastolic and systolic dysfunction. Exercise training attenuated cirrhosis-associated cardiac remodeling and diastolic dysfunction and prevented systolic impairment. These results provided insights that exercise training can mitigate cirrhotic cardiomyopathy phenotype. Graphical Abstract Exercise training attenuated liver injury as well as cirrhosis-associated cardiac remodeling and diastolic dysfunction and prevented systolic impairment.

Topics: Animals; Atrial Function, Left; Biomarkers; Cardiomyopathies; Disease Models, Animal; Exercise Therapy; Exercise Tolerance; Humans; Liver; Liver Cirrhosis; Male; Myocardium; Physical Conditioning, Human; Rats, Wistar; Thioacetamide; Ventricular Function, Left

Topics: Animals; Lactobacillales; Liver Cirrhosis; Male; Rats; Rats, Wistar; Thioacetamide

Activation of hepatic stellate cells is a central event in hepatic fibrogenesis that offers multiple potential sites for therapeutic interventions. Peroxisome proliferator-activated receptors are implicated in liver fibrosis. We aimed to evaluate the effect of bezafibrate and pioglitazone on a thioacetamide (TAA) rat model of liver fibrosis and to clarify the possible underlying mechanisms. Rats received intraperitoneal injections of TAA for 6 weeks. Daily oral treatments with bezafibrate or pioglitazone were started with the first day of TAA intoxication. Serum liver function tests, hepatic malondialdehyde (MDA), total nitrite and nitrate (NOx), superoxide dismutase, and hepatic histopathology were assessed to evaluate hepatic damage. Alpha smooth muscle actin (α-SMA) and tissue inhibitor metalloproteinase-1 (TIMP-1) and caspase-3 were also assessed. The TAA group experienced significant deterioration of liver functions, increased oxidative stress, and increased liver tissue NOx. Administration of bezafibrate or pioglitazone resulted in significant improvement of all liver functions and reduced oxidative stress in hepatic tissues. Only administration of bezafibrate significantly reduced NOx levels. Liver tissues from the TAA-treated group showed disrupted normal architecture. Administration of bezafibrate or pioglitazone attenuated this picture. Stronger α-SMA expression was detected in the TAA group. Treatment with bezafibrate or pioglitazone decreased the α-SMA expression.

Topics: Actins; Animals; Bezafibrate; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Liver Function Tests; Male; Malondialdehyde; Nitrites; Pioglitazone; Rats; Thioacetamide

Topics: Animals; Glutathione; Interleukin-6; Liver; Liver Cirrhosis; Male; Malondialdehyde; Nitric Oxide; Oxidative Stress; Protective Agents; Rats, Sprague-Dawley; Superoxide Dismutase; Thioacetamide; Tumor Necrosis Factor-alpha; Vinca Alkaloids

Liver fibrosis is a reversible wound-healing response to acute or chronic liver injury that can progress to cirrhosis and liver cancer. Finding new strategies for prevention and management of liver fibrosis is urgently needed. It is known that hepatic stellate cell (HSC) is the primary source of extracellular matrix that drives liver fibrosis progression. Herein, we carried out a comprehensive secretome profiling to identify NMN-induced changes in secretory proteins and found that NMN suppressed the secretion of profibrotic protein and oxidoreductase in activated HSC (LX-2) cells, while real-time quantitative PCR analysis revealed that NMN downregulated profibrotic gene expression, resulting in HSC inactivation. Next, we demonstrated that nicotinamide mononucleotide (NMN) reduced the accumulation of liver extracellular matrix in thioacetamide (TAA) and carbon tetrachloride (CCl

Topics: Animals; Dinoprostone; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Mice; Nicotinamide Mononucleotide; Thioacetamide

Decreased cardiac contractility has been observed in cirrhosis, but the mechanisms that initiate and maintain cardiac dysfunction are not entirely understood.. We test the hypothesis that cirrhotic cardiomyopathy is related to deterioration of myocardial contractility due to alterations in calcium-handling proteins expression. In addition, we evaluated whether cardiac pro-inflammatory cytokine levels are associated with this process.. Cirrhosis was induced by thioacetamide (TAA, 100 mg/kg/i.p., twice weekly for eight weeks). The myocardial performance was evaluated in isolated left ventricle papillary muscles under basal conditions and after inotropic challenge. The cardiac calcium handling protein expression was detected by Western blotting. Cardiac TNF-α and IL-6 levels were measured by ELISA.. Thioacetamide induced liver cirrhosis, which was associated with cirrhotic cardiomyopathy characterized by in vivo left ventricular diastolic and systolic dysfunction as well as cardiac hypertrophy. In vitro baseline myocardial contractility was lower in cirrhosis. Also, myocardial responsiveness to post-rest contraction stimulus was declined. Protein expression for RYR2, SERCA2, NCX, pPBL Ser. Our study demonstrates that cirrhotic cardiomyopathy is associated with decreased cardiac contractility with alteration of phospholamban phosphorylation in association with higher cardiac pro-inflammatory IL-6 levels. These findings provided molecular and functional insights about the effects of liver cirrhosis on cardiac function.

Topics: Animals; Calcium-Binding Proteins; Cardiomyopathies; Interleukin-6; Liver Cirrhosis; Male; Myocardial Contraction; Myocardium; Phosphorylation; Random Allocation; Rats; Rats, Wistar; Thioacetamide

Pathological bacterial translocation from the disrupted intestinal barrier leads to substantial complications and mortality in liver cirrhosis. Vitamin D is reported as beneficial to gut barriers in some animal models. However, its effect on cirrhotic bacterial translocation is unknown. The authors aim to investigate the effects of calcitriol on bacterial translocation in cirrhotic rats.. Cirrhotic rats are administrated with a 2-week course of active vitamin D. Calcitriol treatment attenuates intestinal permeability, reduces bacterial translocation, and enriches potentially beneficial gut microbiota in cirrhotic rats that may enable it as a potential therapeutic agent to prevent cirrhotic complications.

Topics: Animals; Bacteria; Calcitriol; Cholecalciferol; Colon; Cytochrome P-450 CYP3A; Cytokines; Feces; Gastrointestinal Microbiome; Gene Expression Regulation; Hydroxyproline; Intestines; Liver Cirrhosis; Male; Rats, Sprague-Dawley; Thioacetamide; Tight Junction Proteins

Liver fibrogenesis is a complex scar-forming process in the liver. We suggested that the liver first responded to chronic injuries with gradual changes, then reached the critical state and ultimately resulted in cirrhosis rapidly. This study aimed to identify the tipping point and key molecules driving liver fibrosis progression. Mice model of liver fibrosis was induced by thioacetamide (TAA), and liver tissues were collected at different time-points post-TAA administration. By dynamic network biomarker (DNB) analysis on the time series of liver transcriptomes, the week 9 post-TAA treatment (pathologically relevant to bridging fibrosis) was identified as the tipping point just before the significant fibrosis transition, with 153 DNB genes as key driving factors. The DNB genes were functionally enriched in fibrosis-associated pathways, in particular, in the top-ranked DNB genes, Tgfb3 negatively regulated Mmp13 in the interaction path and they formed a bistable switching system from a dynamical perspective. In the in vitro study, Tgfb3 promoted fibrogenic genes and down-regulate Mmp13 gene transcription in an immortalized mouse HSC line JS1 and a human HSC line LX-2. The presence of a tipping point during liver fibrogenesis driven by DNB genes marks not only the initiation of significant fibrogenesis but also the repression of the scar resolution.

Topics: Animals; Biomarkers; Disease Models, Animal; Liver; Liver Cirrhosis; Male; Matrix Metalloproteinase 13; Mice; Mice, Inbred C57BL; Thioacetamide; Transforming Growth Factor beta3

Hepatocellular carcinoma (HCC) arises in a cirrhotic, pro-angiogenic microenvironment. Inhibiting angiogenesis is a key mode of action of multikinase inhibitors and current non-cirrhotic models are unable to predict treatment response. We present a novel mouse cirrhotic model of xenotransplant that predicts the natural biology of HCC and allows personalized therapy.. Cirrhosis was induced in NOD Scid gamma mice with 4 months of thioacetamide administration. Patient derived xenografts (PDXs) were created by transplant of human HCC subcutaneously into non-cirrhotic mice and intra-hepatically into both cirrhotic and non-cirrhotic mice. The applicability of cirrhotic PDXs for drug testing was tested with 16 days of either sorafenib or lenvatinib. Treatment response was evaluated by MRI.. 8 out of 19 (42%) human HCC engrafted in the cirrhotic model compared with only 3 out of 19 (16%) that engrafted in the subcutaneous non-cirrhotic model. Tumor vasculature was preserved in the cirrhotic model but was diminished in the non-cirrhotic models. Metastasis developed in 3 cirrhotic PDX lines and was associated with early HCC recurrence in all 3 corresponding patients (100%), compared with only 5 out of 16 (31%) of the other PDX lines, P = .027. The cirrhotic model was able to predict response and non-response to lenvatinib and sorafenib respectively in the corresponding patients. Response to lenvatinib in the cirrhotic PDX was associated with reduction in CD34, VEGFR2 and CLEC4G immunofluorescence area and intensity (all P ≤ .03).. A clinically relevant cirrhotic PDX model preserves tumor angiogenesis and allows prediction of response to multikinase inhibitors for personalized therapy.

Topics: Adult; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; Disease Models, Animal; Female; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Mice; Mice, Inbred NOD; Mice, SCID; Middle Aged; Neovascularization, Pathologic; Phenylurea Compounds; Precision Medicine; Prognosis; Protein Kinase Inhibitors; Quinolines; Sorafenib; Thioacetamide; Tumor Cells, Cultured; Tumor Microenvironment; Xenograft Model Antitumor Assays

Liver cirrhosis is the main chronic liver disease and is considered a catabolic disease. Cirrhotic patients have a low energy intake and high energy expenditure at rest, leading to metabolic disorders. Malnutrition is associated with complications of cirrhosis and has been shown that a nutritional intervention with increase of energy intake improves the survival of cirrhotic patients. Therefore, our aim was to evaluate the effect of a high sucrose diet in the liver of animals with cirrhosis induced by thioacetamide and investigate the mechanism involved.. Male Wistar rats were divided into three groups: Control; Thioacetamide; and Thioacetamide + high sucrose diet. The thioacetamide was administrated (100 mg kg. The administration of thioacetamide was associated with fibrosis and inflammatory infiltrate in the liver and increased levels of transaminases enzymes. The high sucrose diet promoted a reduction of theses parameters in cirrhotic rats. The malnutrition observed in cirrhotic rats was attenuated by the high sucrose diet shown by the improvements in weight loss, subcutaneous fat, and caloric intake. The high sucrose diet also attenuated the oxidative stress present in the liver of animals with thioacetamide-induced cirrhosis.. The high sucrose diet had anti-inflammatory and anti-oxidant effects in the liver of animals with thioacetamide-induced cirrhosis. In addition, the high sucrose diet also improved malnutrition and catabolism present in cirrhosis. Thus, a high sucrose diet may be a therapeutic option for cirrhotic patients in a catabolic state.

Topics: Animals; Chemical and Drug Induced Liver Injury; Diet; Dietary Sucrose; Inflammation; Liver; Liver Cirrhosis; Liver Cirrhosis, Experimental; Male; Oxidative Stress; Rats; Rats, Wistar; Sucrose; Thioacetamide

Exposure to environmental chemicals, particularly those with persistent and bioaccumulative properties have been linked to liver diseases. Induction of fibrotic pathways is considered as a pre-requirement of chemical induced liver fibrosis. Here, we applied 3D in vitro human liver microtissues (MTs) composed of HepaRG, THP-1 and hTERT-HSC that express relevant hepatic pathways (bile acid, sterol, and xenobiotic metabolism) and can recapitulate key events of liver fibrosis (e.g. extracellular matrix-deposition). The liver MTs were exposed to a known profibrotic chemical, thioacetamide (TAA) and three representative environmental chemicals (TCDD, benzo [a] pyrene (BaP) and PCB126). Both TAA and BaP triggered fibrotic pathway related events such as hepatocellular damage (cytotoxicity and decreased albumin release), hepatic stellate cell activation (transcriptional upregulation of α-SMA and Col1α1) and extracellular matrix remodelling. TCDD or PCB126 at measured concentrations did not elicit these responses in the 3D liver MTs system, though they caused cytotoxicity in HepaRG monoculture at high concentrations. Reduced human transcriptome (RHT) analysis captured molecular responses involved in liver fibrosis when MTs were treated with TAA and BaP. The results suggest that 3D, multicellular, human liver microtissues represent an alternative, human-relevant, in vitro liver model for assessing fibrotic pathways induced by environmental chemicals.

Topics: Benzo(a)pyrene; Extracellular Matrix; Humans; Liver; Liver Cirrhosis; Thioacetamide

Topics: Animals; Cannabinoids; Dose-Response Relationship, Drug; Inflammation; Liver Cirrhosis; Male; MicroRNAs; Rats; Rats, Wistar; Signal Transduction; Structure-Activity Relationship; Thioacetamide; Toll-Like Receptor 4; Transcription Factor RelA

Chronic liver injury is characterised by continuous or repeated epithelial cell loss and inflammation. Hepatic wound healing involves matrix deposition through activated hepatic stellate cells (HSCs) and the expansion of closely associated Ductular Reactions and liver progenitor cells (LPCs), which are thought to give rise to new epithelial cells. In this study, we used the murine thioacetamide (TAA) model to reliably mimic these injury and regeneration dynamics and assess the impact of a recovery phase on subsequent liver injury and fibrosis. Age-matched naïve or 6-week TAA-treated/4-week recovered mice (C57BL/6 J, n = 5-9) were administered TAA for six weeks (C57BL/6 J, n = 5-9). Sera and liver tissues were harvested at key time points to assess liver injury biochemically, by real-time PCR for fibrotic mediators, Sirius Red staining and hydroxyproline assessment for collagen deposition as well as immunofluorescence for inflammatory, HSC and LPC markers. In addition, primary HSCs and the HSC cell line LX-2 were co-cultured with the well-characterised LPC line BMOL and analysed for potential changes in expression of fibrogenic mediators. Our data demonstrate that recovery from a previous TAA insult, with LPCs still present on day 0 of the second treatment, led to a reduced TAA-induced disease progression with less severe fibrosis than in naïve TAA-treated animals. Importantly, primary activated HSCs significantly reduced pro-fibrogenic gene expression when co-cultured with LPCs. Taken together, previous TAA injury established a fibro-protective molecular and cellular microenvironment. Our proof-of principle HSC/LPC co-culture data demonstrate that LPCs communicate with HSCs to regulate fibrogenesis, highlighting a key role for LPCs as regulatory cells during chronic liver disease.

Topics: Animals; Cells, Cultured; Chemical and Drug Induced Liver Injury; Coculture Techniques; Disease Models, Animal; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Liver Regeneration; Male; Mice; Mice, Inbred C57BL; Stem Cells; Thioacetamide

Stem cell treatments using scaffolds for liver disease have been well studied. However, macro-encapsulation of mesenchymal stem cells (MSCs) to minimize or inhibit stem cell homing has not been evaluated. Here, we conducted a proof-of-concept study using MSCs macro-encapsulated in poly lactic-co-glycolic acid in liver disease models.. Poly lactic-co-glycolic acid semipermeable membranes (surface pore size up to 40 μm) were used as the macro-encapsulation system. Macro-encapsulated pouches were loaded with MSCs and sealed. Each pouch was implanted in the subcutaneous region of the dorsum or interlobular space of the liver. Acute liver injury was induced using thioacetamide intraperitoneal injection thrice a week. For the chronic liver fibrosis model, thioacetamide dose was gradually increased, starting from 100 to 400 mg/kg over 16 weeks (thrice a week).. In the acute liver injury model, the treated groups showed decreased liver inflammation and necrosis compared with the control. Hepatic fibrosis decreased in the treated group in the chronic liver fibrosis model compared with that in the control group. Encapsulated MSCs exhibited changed cell morphology and characteristics after implantation, showing increased periodic acid-Schiff staining and CYP2E1 expression. Migration and homing of MSCs into the liver was not observed. Under hypoxic conditions, macro-encapsulated MSCs secreted more growth hormones, including vascular endothelial growth factor, platelet-derived growth factor, angiopoietin-2, and placental growth factor, than monolayered MSCs in vitro.. Macro-encapsulated MSCs attenuate hepatic inflammation and fibrosis by upregulating hypoxia-induced growth hormone secretion in liver disease models.

Topics: Animals; Disease Models, Animal; Female; Fibrosis; Inflammation; Liver; Liver Cirrhosis; Liver Diseases; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Placenta Growth Factor; Thioacetamide; Vascular Endothelial Growth Factor A

Splenomegaly is usually taken as a consequence of liver cirrhosis. However, as a risk factor for cirrhosis, the impacts of spleen-liver axis on the development of cirrhosis are largely unknown. This study focused on the impacts of splenomegaly on the development of cirrhosis and assessment of the effects of celecoxib, a selective COX-2 inhibitor, on the splenomegaly and cirrhotic liver.. Liver cirrhosis was induced by thioacetamide (TAA). Sixty rats were randomly divided into control, TAA-16w, TAA + celecoxib groups and normal, TAA + sham, TAA + splenectomy groups. Hepatic stellate cells (HSCs) or hepatocytes were co-cultured with splenocytes from those groups.. Splenocytes of cirrhotic rats stimulated the HSCs activation and induced hepatocyte apoptosis via enhancing oxidative stress. The hepatic levels of NOX-4 and the in situ O. Splenomegaly contributed to the development of liver cirrhosis through enhancing oxidative stress in liver. Celecoxib could effectively ameliorate liver cirrhosis via reducing inflammatory cytokines and immune cells derived from spleen and suppressing oxidative stress.

Topics: Animals; Apoptosis; Celecoxib; China; Cyclooxygenase 2 Inhibitors; Disease Models, Animal; Hepatic Stellate Cells; Hepatocytes; Inflammation; Liver; Liver Cirrhosis; Liver Cirrhosis, Experimental; Male; Oxidative Stress; Rats; Rats, Sprague-Dawley; Signal Transduction; Spleen; Splenomegaly; Thioacetamide

Liver fibrosis is a continuous wound healing response caused by chronic liver injury, and the activation of hepatic stellate cells (HSCs) is considered as the main event for it. Core fucosylation catalyzed by FUT8 refers to adding the fucosyl moiety to the innermost GlcNAc residue of N-linked oligosaccharides and is involved in many biological processes such as cell differentiation, migration, and signaling transduction. Aberrant core fucosylation is associated with a variety of diseases including cardiovascular disease, tumors and neuroinflammation, but much less is understood in liver fibrosis. Herein, we reported FUT8 mRNA level was increased in patients with liver fibrosis from GEO database and positively correlated with fibrosis progression. FUT8 expression and the core fucosylation were also elevated in TAA-induced mouse liver fibrosis model, and were mainly distributed in the fibrous septum of mouse liver. TGF-β1, as the most pro-fibrogenic cytokine, could promote the expression of FUT8 and total core fucosylation levels in HSCs in vitro. However, up-regulation of FUT8 in turn inhibited TGF-β1-induced trans-differentiation, migration and pro-fibrogenic signaling pathways in HSCs. In conclusion, our results suggest that the up-regulation of FUT8 inhibits TGF-β1-induced HSC activation in a negative feedback loop, and provide potential new therapeutic strategy for liver fibrosis by targeting FUT8.

Topics: Animals; Cell Line; Cell Movement; Cell Transdifferentiation; Disease Models, Animal; Fucosyltransferases; Gene Expression; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Male; Mice, Inbred C57BL; Rats; Signal Transduction; Thioacetamide; Transforming Growth Factor beta1; Up-Regulation

Intrahepatic cholangiocarcinoma (iCCA) is closely correlated with hepatic progenitor cell (HPC) expansion and liver fibrosis. Brahma-related gene 1 (Brg1), an enzymatic subunit of the switch/sucrose nonfermentable complex that is critical in stem cell maintenance and tumor promotion, is prominently up-regulated in both HPCs and iCCA; however, its role in this correlation remains undefined.. A retrospective cohort study indicated that high Brg1 expression suggests poor prognosis in patients with iCCA. In chronically injured livers induced by a 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet or bile duct ligation surgery, HPCs were dramatically activated, as indicated by their enhanced expression of Brg1 and a subset of stem cell markers; however, Brg1 ablation in HPCs strongly suppressed HPC expansion and liver fibrosis. Furthermore, in a chemically induced iCCA model, inhibition of Brg1 by a specific inhibitor or inducible gene ablation markedly improved histology and suppressed iCCA growth. Mechanistically, in addition to transcriptionally promoting both Wnt receptor genes and target genes, Brg1 was found to bind to the β-catenin/transcription factor 4 transcription complex, suggesting a possible approach for regulation of Wnt/β-catenin signaling.. We have demonstrated the function of Brg1 in promoting HPC expansion, liver cirrhosis, and, ultimately, iCCA development in chronically injured livers, which is largely dependent on Wnt/β-catenin signaling. Our data suggest that therapies targeting Brg1-expressing HPCs are promising for the treatment of liver cirrhosis and iCCA.

Topics: Adult; Aged; Animals; Azabicyclo Compounds; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cholangiocarcinoma; Disease-Free Survival; DNA Helicases; Female; Gene Expression Regulation, Neoplastic; Humans; Liver; Liver Cirrhosis; Male; Mice; Mice, Transgenic; Middle Aged; Neoplasm Recurrence, Local; Neoplasms, Experimental; Nuclear Proteins; Prognosis; Pyridines; Retrospective Studies; Stem Cells; Thioacetamide; Transcription Factors; Up-Regulation; Wnt Signaling Pathway

Liver fibrosis is a public health burden that is highly associated with morbidity and mortality. Therefore, this study aims to explore the anti-fibrotic effects of low dose of paclitaxel (PTX) against thioacetamide (TAA)-induced liver fibrosis in rats and the possible mechanisms involved. TAA was administered at a dose of 200 mg/kg twice weekly for 6 weeks in rats to induce liver fibrosis similar to that in humans. Liver dysfunction was shown by increased alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and γ-glutamyl transferase, along with histopathological changes. Liver fibrosis was confirmed by Masson's Trichome staining, increased collagen content, and elevated α-smooth muscle actin (α-SMA) protein expression. In addition, TAA induced liver apoptosis as indicated by the increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in liver tissues. This study demonstrated that the administration of PTX (0.3 mg/kg/i.p.) three times a week for 6 weeks significantly alleviated functional and biochemical changes induced by TAA in addition to improving the liver architecture. PTX attenuated liver fibrosis as reflected by the decreased collagen content and α-SMA protein expression. Additionally, PTX attenuated liver apoptosis as indicated by the decreased TUNEL-positive cells. Moreover, PTX prevented TAA-induced elevation of transforming growth factor-β1 (TGF-β1), platelet-derived growth factor-BB (PDGF-BB), and tissue inhibitor of metalloproteinase 1 (TIMP-1) levels in liver tissues. These findings suggest that the low dose of PTX prevented TAA-induced liver fibrosis in rats, possibly by inhibiting the expression of TGF-β1 and PDGF-BB and subsequently suppressing the apoptosis and the expression of TIMP-1.

Topics: Animals; Disease Models, Animal; Liver Cirrhosis; Male; Paclitaxel; Rats; Rats, Sprague-Dawley; Thioacetamide

Persistent chronic liver diseases increase the scar formation and extracellular matrix accumulation that further progress to liver fibrosis and cirrhosis. Nevertheless, there is no antifibrotic therapy to date. The ketogenic diet is composed of high fat, moderate to low-protein, and very low carbohydrate content. It is mainly used in epilepsy and Alzheimer's disease. However, the effects of the ketogenic diet on liver fibrosis remains unknown. Through ketogenic diet consumption, β-hydroxybutyrate (bHB) and acetoacetate (AcAc) are two ketone bodies that are mainly produced in the liver. It is reported that bHB and AcAc treatment decreases cancer cell proliferation and promotes apoptosis. However, the influence of bHB and AcAc in hepatic stellate cell (HSC) activation and liver fibrosis are still unclear. Therefore, this study aimed to investigate the effect of the ketogenic diet and ketone bodies in affecting liver fibrosis progression. Our study revealed that feeding a high-fat ketogenic diet increased cholesterol accumulation in the liver, which further enhanced the carbon tetrachloride (CCl

Topics: 3-Hydroxybutyric Acid; Acetoacetates; Actins; Animals; Becaplermin; Carbon Tetrachloride; Catalase; Cell Proliferation; Cholesterol; Collagen Type I; Cytochrome P-450 CYP1A2; Desmin; Diet, Ketogenic; Disease Progression; Gene Expression Regulation; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Primary Cell Culture; Severity of Illness Index; Superoxide Dismutase; Superoxide Dismutase-1; Thioacetamide; Transforming Growth Factor beta

Chronic liver diseases are associated with excessive deposition of extracellular matrix proteins. This so-called fibrosis can progress to cirrhosis and impair vital functions of the liver. We examined whether the receptor tyrosine kinase (RTK) class III inhibitor Crenolanib affects the behavior of hepatic stellate cells (HSC) involved in fibrogenesis. Rats were treated with thioacetamide (TAA) for 18 weeks to trigger fibrosis. After TAA treatment, the animals received Crenolanib for two weeks, which significantly improved recovery from liver fibrosis. Because Crenolanib predominantly inhibits the RTK platelet-derived growth factor receptor-β, impaired HSC proliferation might be responsible for this beneficial effect. Interestingly, blocking of RTK signaling by Crenolanib not only hindered HSC proliferation but also triggered their specification into hepatic endoderm. Endodermal specification was mediated by p38 mitogen-activated kinase (p38 MAPK) and c-Jun-activated kinase (JNK) signaling; however, this process remained incomplete, and the HSC accumulated lipids. JNK activation was induced by stress response-associated inositol-requiring enzyme-1α (IRE1α) in response to Crenolanib treatment, whereas β-catenin-dependent WNT signaling was able to counteract this process. In conclusion, the Crenolanib-mediated inhibition of RTK impeded HSC proliferation and triggered stress responses, initiating developmental processes in HSC that might have contributed to improved recovery from liver fibrosis in TAA-treated rats.

Topics: Animals; Becaplermin; Benzimidazoles; Biomarkers; Cell Differentiation; Cell Proliferation; Endoderm; Hepatic Stellate Cells; JNK Mitogen-Activated Protein Kinases; Liver Cirrhosis; Male; Models, Biological; p38 Mitogen-Activated Protein Kinases; Piperidines; Rats, Wistar; Thioacetamide; Wnt Signaling Pathway

25-Hydroxylprotopanaxadiol-3β, 12β, 20-triol (25-OH-PPD or AD-2) belongs to dammarane ginsenoside, and is commonly obtained from the acidic hydrolysate of total ginsensides of Panax ginseng. This study investigated the potential mechanism of AD-2 toward improving thioacetamide (TAA)-induced hepatic fibrosis in mice. Mice were divided into seven groups: control group, TAA model group, TAA + AD-2 (5, 10, and 20 mg/kg) groups, TAA + silymarin (100 mg/kg) group, and TAA + Fu Fang Biejia (FFBj; 300 mg/kg) group. All mice were treated to intraperitoneal TAA injection to establish a hepatic fibrosis model, and drugs were administered orally. The mechanism and related pathways underlying the AD-2-mediated action against hepatic fibrosis were explored by Western blotting and immunohistochemical staining. After AD-2 treatment, the expression levels of Lipin-1, SREBP1, and F4/80 significantly decreased, meanwhile the protein expressions levels of IL1β, IL1R1, IL18, Bax, Bid, Bcl-2, and cFlips also decreased. Furthermore, AD-2 inhibited RAF and MEK pathways. The results demonstrate that AD-2 can alleviate hepatic fibrosis. The mechanism is likely related to the regulation of lipid accumulation, inflammatory response, apoptosis pathway, and Raf-MEK signaling pathways, which provide a basis for clinical research for the treatment of hepatic fibrosis. PRACTICAL APPLICATION: Ginsenoside is one of the main active ingredients of ginseng, and can alleviate the symptoms of various diseases, for example, hepatic fibrosis. This paper mainly used Western blotting to explore its possible mechanism of action. The goal was to provide a reference for the development of traditional Chinese medicines for hepatic fibrosis.

Topics: Animals; Chemical and Drug Induced Liver Injury, Chronic; Ginsenosides; Inflammation; Liver Cirrhosis; Male; Mice; Mitogen-Activated Protein Kinase Kinases; Panax; raf Kinases; Signal Transduction; Thioacetamide

Liver fibrosis is the result of long-term liver injury and has a high incidence worldwide. Protocatechuic acid (PCA) is ubiquitous in vegetables, nuts, brown rice and herbal medicines, which is reported to possess anti-asthmatic, anti-cancer, and anti-oxidation properties. Our research aimed to investigate the effect of PCA on liver fibrosis.

Topics: Animals; Cell Line; Hepatic Stellate Cells; Hydroxybenzoates; Inflammation; Kidney; Liver; Liver Cirrhosis; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Models, Animal; Rats; Signal Transduction; Spleen; Thioacetamide; Transaminases; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Liver fibrosis is defined as excessive accumulation of extracellular matrix, and results from maladaptive wound healing processes that occur in response to chronic liver injury and inflammation. The main etiologies of liver fibrosis include nonalcoholic fatty liver disease (NAFLD), chronic viral hepatitis, as well as alcoholic and cholestatic liver disease. In patients, liver fibrosis typically develops over several decades and can progress to cirrhosis, and liver failure due to replacement of functional liver tissue with scar tissue. Additionally, advanced fibrosis and cirrhosis are associated with an increased risk for the development of hepatocellular carcinoma. On a cellular level, hepatic fibrosis is mediated by activated hepatic stellate cells, the primary fibrogenic cell type of the liver. Murine models are employed to recapitulate, understand, and therapeutically target mechanisms of fibrosis and hepatic stellate cell activation. Here, we summarize different mouse models of liver fibrosis focusing on the most commonly used models of toxic, biliary, and metabolically induced liver fibrosis, triggered by treatment with carbon tetrachloride (CCl

Topics: Animals; Carbon Tetrachloride; Diet, High-Fat; Disease Models, Animal; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Mice; Mice, Inbred BALB C; Pyridines; Thioacetamide

Induction of deactivation and apoptosis of hepatic stellate cells (HSCs) are principal therapeutic strategies for liver fibrosis. Krüppel-like factor 14 (KLF14) regulates various biological processes, however, roles, mechanisms and implications of KLF14 in liver fibrosis are unknown.. KLF14 expression was detected in human, rat and mouse fibrotic models, and its effects on HSCs were assessed. Chromatin immunoprecipitation assays were utilized to investigate the binding of KLF14 to peroxisome proliferator-activated receptor γ (PPARγ) promoter, and the binding of enhancer of zeste homolog 2 (EZH2) to KLF14 promoter. In vivo, KLF14-overexpressing adenovirus was injected via tail vein to thioacetamide (TAA)-treated rats to investigate the role of KLF14 in liver fibrosis progression. EZH2 inhibitor EPZ-6438 was utilized to treat TAA-induced rat liver fibrosis.. KLF14 expression was remarkably decreased in human, rat and mouse fibrotic liver tissues. Overexpression of KLF14 increased LD accumulation, inhibited HSCs activation, proliferation, migration and induced G2/M arrest and apoptosis. Mechanistically, KLF14 transactivated PPARγ promoter activity. Inhibition of PPARγ blocked the suppressive role of KLF14 overexpression in HSCs. Downregulation of KLF14 in activated HSCs was mediated by EZH2-regulated histone H3 lysine 27 trimethylation. Adenovirus-mediated KLF14 overexpression ameliorated TAA-induced rat liver fibrosis in PPARγ-dependent manner. Furthermore, EPZ-6438 dramatically alleviated TAA-induced rat liver fibrosis. Importantly, KLF14 expression was decreased in human with liver fibrosis, which was significantly correlated with EZH2 upregulation and PPARγ downregulation.. KLF14 exerts a critical anti-fibrotic role in liver fibrosis, and targeting the EZH2/KLF14/PPARγ axis might be a novel therapeutic strategy for liver fibrosis.

Topics: Animals; Apoptosis; Benzamides; Biphenyl Compounds; Cell Movement; Cell Proliferation; Disease Models, Animal; Down-Regulation; Enhancer of Zeste Homolog 2 Protein; G2 Phase Cell Cycle Checkpoints; Hepatic Stellate Cells; Humans; Kruppel-Like Transcription Factors; Liver Cirrhosis; Mice; Morpholines; PPAR gamma; Promoter Regions, Genetic; Pyridones; Rats; RNA Interference; RNA, Small Interfering; Thioacetamide

Liver fibrosis results from many chronic injuries and may often progress to cirrhosis and hepatocellular carcinoma (HCC). In fact, up to 90% of HCC arise in a cirrhotic liver. Conversely, stress is implicated in liver damage, worsening disease outcome. Hence, stress could play a role in disrupting liver homeostasis, a concept that has not been fully explored. Here, in a murine model of TAA-induced liver fibrosis we identified nerve growth factor (NGF) to be a crucial regulator of the stress-induced fibrogenesis signaling pathway as it activates its receptor p75 neurotrophin receptor (p75NTR), increasing liver damage. Additionally, blocking the NGF decreased liver fibrosis whereas treatment with recombinant NGF accelerated the fibrotic process to a similar extent than stress challenge. We further show that the fibrogenesis induced by stress is characterized by specific changes in the hepatoglycocode (increased β1,6GlcNAc-branched complex N-glycans and decreased core 1 O-glycans expression) which are also observed in patients with advanced fibrosis compared to patients with a low level of fibrosis. Our study facilitates an understanding of stress-induced liver injury and identify NGF signaling pathway in early stages of the disease, which contributes to the established fibrogenesis.

Topics: Animals; Gene Expression Regulation; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Nerve Growth Factor; Polysaccharides; Receptors, Nerve Growth Factor; Stress, Physiological; Thioacetamide

Liver fibrosis is a significant health problem that can cause serious illness and death. Unfortunately, a standard treatment for liver fibrosis has not been approved yet due to its complicated pathogenesis. The current study aimed at assessing the anti-fibrotic effect of taurine against thioacetamide induced liver fibrosis in rats through the modulation of toll like receptor 4/nuclear factor kappa B signaling pathway. Both concomitant and late taurine treatment (100 mg/kg, IP, daily) significantly reduced the rise in serum ALT and AST activities and significantly reversed the decrease in serum albumin and total protein. These results were confirmed by histopathological examinations and immunehistochemical inspection of α-SMA, caspase-3 and NF-κB. The antioxidant potential of taurine was verified by a marked increase of GSH content and a reduction of MDA level in liver tissue. The anti-fibrotic effects of taurine were evaluated by investigating the expression of TLR4, NF-κB. The protein levels of IL-6, LPS, MyD88, MD2, CD14, TGF-β1 and TNF-α were determined. Docking studies were carried out to understand how taurine interacts inside TLR4-MD2 complex and it showed good binding with the hydrophobic binding site of MD2. We concluded that the anti-fibrotic effect of taurine was attributable to the modulation of the TLR4/NF-κB signaling.

Topics: Actins; Animals; Antioxidants; Caspase 3; Gene Expression Regulation; Humans; Liver Cirrhosis; Lymphocyte Antigen 96; Molecular Docking Simulation; NF-kappa B; Protein Binding; Rats; Serum Albumin; Signal Transduction; Taurine; Thioacetamide; Toll-Like Receptor 4

Schisandra chinensis, a traditional Chinese medicine for liver protection, can significantly improve liver fibrosis. However, it is still unclear which active components in Schisandra chinensis play an anti-fibrosis role.. The purpose of present study was to observe the anti-fibrosis effect of schisantherin A (SCA) on liver fibrosis and explore its underlying mechanism.. The liver fibrosis model of mice was constructed by the progressive intraperitoneal injection of thioacetamide (TAA), and SCA (1, 2, and 4 mg/kg) was administered by gavage for 5 weeks. The biochemical indicators and inflammatory cytokines were measured, changes in the pathology of the mice liver were observed by hematoxylin & eosin (H&E) and Masson stainings for studying the anti-fibrosis effect of SCA. A hepatic stellate cell (HSCs) activation model induced by transforming growth factor-β1 (TGF-β1) was established, and the effect of SCA on the HSCs proliferation was observed by MTT assay. The expressions of target proteins related to transforming growth factor-β-activated kinase 1 (TAK1)/mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways were evaluated by western blotting, immunohistochemistry or immunofluorescence analysis, to explore the potential mechanism of SCA.. SCA could significantly ameliorate the pathological changes of liver tissue induced by TAA, and reduce the serum transaminase level, the hydroxyproline level and the expression of α-smooth muscle actin (α-SMA) and collagen 1A1 (COL1A1) proteins in the liver tissue. SCA could significantly lower the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in the serum and liver tissue, and down-regulate the expression of target proteins related to TAK1/MAPK and NF-κB pathways in the liver tissue. The in vitro studies demonstrated that SCA significantly inhibited the proliferation and activation of HCS-T6 cells induced by TGF-β1, decreased TNF-α and IL-6 levels, and inhibited the TAK1 activation induced by TGF-β1 and then the expression of MAPK and NF-κB signaling pathway-related proteins.. Together, SCA can ameliorate the liver fibrosis induced by TAA and the HSC-T6 cell activation induced by TGF-β1 in mice, and its mechanism may be to inhibit the HSCs activation and inflammatory response by inhibiting TGF-β1 mediated TAK1/MAPK and signal pathways.

Topics: Animals; Cell Line; Cyclooctanes; Cytokines; Dioxoles; Hepatic Stellate Cells; Lignans; Liver Cirrhosis; Male; MAP Kinase Kinase Kinases; Mice, Inbred ICR; Mitogen-Activated Protein Kinases; NF-kappa B; Rats; Signal Transduction; Thioacetamide; Transforming Growth Factor beta1

Fibrosis is a common outcome of nearly all chronic diseases of liver that results in changes of its functions which requires medical attention. The current research aims to investigate the potential anti-fibrotic efficacy of Carvacrol against thioacetamide (TAA)-induced liver fibrosis in male rats using Ursodeoxycholic acid (UDCA) as a reference anti-fibrotic product. Carvacrol (25 and 50 mg/kg) markedly declined TAA-increased serum liver enzymes; alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) as well as total bilirubin (TB) and direct bilirubin (DB) levels as well as increased levels of total protein (TP) and albumin. Carvacrol significantly reduced glutathione depletion (GSH), Nitric oxide (NO

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Cymenes; Disease Progression; Liver; Liver Cirrhosis; Male; Oxidative Stress; Phosphoric Diester Hydrolases; Rats, Wistar; Thioacetamide; Thioredoxins

Demand for a cure of liver fibrosis is rising with its increasing morbidity and mortality. Therefore, it is an urgent issue to investigate its therapeutic candidates. Liver fibrosis progresses following 'multi-hit' processes involving hepatic stellate cells, macrophages, and hepatocytes. The NOD-like receptor protein 3 (NLRP3) inflammasome is emerging as a therapeutic target in liver fibrosis. Previous studies showed that the anti-rheumatic agent auranofin inhibits the NLRP3 inflammasome; thus, this study evaluates the antifibrotic effect of auranofin in vivo and explores the underlying molecular mechanism. The antifibrotic effect of auranofin is assessed in thioacetamide- and carbon tetrachloride-induced liver fibrosis models. Moreover, hepatic stellate cell (HSC), bone marrow-derived macrophage (BMDM), kupffer cell, and hepatocyte are used to examine the underlying mechanism of auranofin. Auranofin potently inhibits activation of the NLRP3 inflammasome in BMDM and kupffer cell. It also reduces the migration of HSC. The underlying molecular mechanism was inhibition of cystine-glutamate antiporter, system Xc. Auranofin inhibits system Xc activity and instantly induced oxidative burst, which mediated inhibition of the NLRP3 inflammasome in macrophages and HSCs. Therefore, to the best of our knowledge, we propose the use of auranofin as an anti-liver fibrotic agent.

Topics: Amino Acid Transport System y+; Animals; Apoptosis; Auranofin; Carbon Tetrachloride; Cells, Cultured; Humans; Inflammasomes; Interleukin-1beta; Kupffer Cells; Liver; Liver Cirrhosis; Macrophages; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Pyroptosis; Thioacetamide

Macrophages contribute to liver fibrogenesis by the production of a large variety of cytokines. ATF6 is associated with the activation of macrophages. The present study aimed to investigate the role of ATF6 in the expression of macrophage-derived cytokines and liver fibrogenesis after acute liver injury. Following thioacetamide (TAA)-induced acute liver injury, the characteristics of the occurrence of liver fibrosis and the secretion of cytokines by macrophages were first described. Then, the role of various cytokines secreted by macrophages in activating hepatic stellate cells (HSCs) was tested in vitro. Finally, endoplasmic reticulum stress (ER-stress) signals in macrophages were detected following liver injury. siRNA was used to interfere with the expression of ATF6 in macrophages to verify the influence of ATF6 on cytokine expression and liver fibrogenesis after liver injury. A single intraperitoneal injection of TAA induced acute liver injury. The depletion of macrophages attenuated acute liver injury, while it inhibited liver fibrogenesis. During acute liver injury, macrophages secrete a variety of cytokines. Most of these cytokines promoted the activation of HSCs, but the effect of IL-1α was most significant. In the early stage of acute liver injury, ER-stress signals in macrophages were activated. Interference of ATF6 expression suppressed the secretion of cytokines by macrophages and attenuated liver fibrogenesis. Overall, in the early stage of acute liver injury, ATF6 signals promoted the expression of macrophage-derived cytokines to participate in liver fibrogenesis, and IL-1α exhibited the most significant role in promoting the activation of HSCs and liver fibrogenesis.

Topics: Activating Transcription Factor 6; Animals; Cells, Cultured; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Endoplasmic Reticulum Stress; Gene Expression Regulation; Humans; Interleukin-1alpha; Liver; Liver Cirrhosis; Macrophage Activation; Macrophages; Mice; Mice, Inbred C57BL; RNA, Small Interfering; Thioacetamide

Present study was designed to evaluate the effects of coffee on liver function tests and liver antioxidant enzymes in thioacetamide induced liver cirrhosis in rats. Experimental study period was consisted of eighteen weeks divided into two phases. Therefore 24 rats were distributed randomly into four groups (n=6). Group I served as control. In phase I, group II and III received thioacetamide (200mg/kg body weight intraperitoneally twice a week) and group IV received saline for 12 weeks. In phase II, group II received saline while group III and IV received an oral dose of coffee (0.4mg/Kg b.w) daily for 6 weeks. At the end of the study period rats were sacrificed and blood was collected to get serum and liver was homogenized for the determination of antioxidant enzymes. Marked increase in serum total and direct bilirubin, ALT, AST whereas reduced ALP was observed in test group. The reduced tissue SOD activity and increased tissue catalase and tissue MDA activity were also observed in test group. However, coffee consumption in group III in phase II significantly restored liver biomarkers and the tissue antioxidant enzymes SOD, catalase and MDA activities. In conclusion, thioacetamide induced liver cirrhosis can be prevented by coffee supplementation.

Topics: Alanine Transaminase; Alkaline Phosphatase; Animals; Aspartate Aminotransferases; Bilirubin; Catalase; Coffee; Dietary Supplements; Lipid Peroxidation; Liver Cirrhosis; Liver Cirrhosis, Experimental; Malondialdehyde; Rats; Superoxide Dismutase; Thioacetamide

Transforming growth factor-β (TGF-β) has been identified as an inducer of hepatocyte epithelial-mesenchymal transition (EMT), which triggers liver fibrosis. Death-associated protein 6 (Daxx) is known to be associated with the TGF-β-induced apoptotic pathway, but the function of Daxx in liver fibrosis remains unknown. This study aimed to elucidate the role of Daxx in liver fibrosis. We used liver fibrosis tissues from humans and mice to assess Daxx expression. EMT properties and TGF-β signaling pathway activation were investigated in the Daxx-overexpressing FL83B cell line. The therapeutic effect of Daxx was investigated in a mouse model of liver fibrosis by the hydrodynamic injection of plasmids. The expression of Daxx was markedly decreased in hepatocytes from fibrotic human and mouse livers, as well as in hepatocytes treated with TGF-β in vitro. The overexpression of Daxx inhibited the EMT process by interfering with the TGF-β-induced phosphorylation of Smad2. Coimmunoprecipitation analysis confirmed that Daxx reduced the transcriptional activity of Smad2 by binding to its MH1 domain and interfering with Smad2 acetylation. In addition, the therapeutic delivery of Daxx alleviated liver fibrosis in a thioacetamide-induced fibrosis mouse model. Overall, our results indicate that Daxx could be a potential therapeutic target to modulate fibrogenesis, as well as a useful biomarker for liver fibrosis.

Topics: Acetylation; Animals; Cell Nucleus; Co-Repressor Proteins; Disease Models, Animal; Epithelial-Mesenchymal Transition; HEK293 Cells; Hepatocytes; Humans; Liver; Liver Cirrhosis; Mice, Inbred C57BL; Molecular Chaperones; Phosphorylation; Protein Binding; Protein Domains; Signal Transduction; Smad2 Protein; Thioacetamide; Transcription, Genetic; Transforming Growth Factor beta

Morinda elliptica L. (Rubiaceae) is a phytomedicinal herb, used to treat gastrointestinal complications in Peninsular Malaysia. The study evaluates the in vivo hepatoprotective activity of ethanolic extract of M. elliptica stem in thioacetamide (TAA) induced liver fibrosis in male Sprague Drawly rats. Thirty adult rats were divided into five groups of six rats each. Rats of the normal control group received intraperitoneal injections (i. p.) of vehicle 10% Tween-20, 5 ml/kg, and hepatotoxic group 200 mg/kg TAA three times per week respectively. Three supplementary groups were treated with TAA plus daily oral silymarin (50 mg/kg) or M. elliptica (250 or 500 mg/kg). After 8 weeks of treatment, all rats were sacrificed. Liver fibrosis was assessed by gross macroscopic and microscopic tissue analysis, histopathological, and biochemical analysis. The livers of the TAA treated group showed uniform coarse granules, hepatocytic necrosis with lymphocytes infiltration. Contrary, the livers of M. elliptica treated groups (250 and 500 mg/kg) were much smoother and the cell damage was much lesser. The livers of M. elliptica treated groups rats showed elevated activity of SOD and CAT with a significant decrease in MDA level at p < .0001. The level of liver damage parameters, that is, ALP, ALT, and AST, bilirubin, total protein, and albumin were restored to the normal comparable to silymarin. M. elliptica stem extract significantly promoted normal rat liver architecture with significant perfections in biochemical parameters. The molecular contents of M. elliptica with hepatoprotective influence could be discovered, is the future prospective of this study.

Topics: Animals; Chemical and Drug Induced Liver Injury; Liver; Liver Cirrhosis; Morinda; Plant Extracts; Rats; Rats, Wistar; Thioacetamide

Autophagy and apoptosis are important players in the progression of hepatic fibrosis via activation of hepatic stellate cells (HSCs). Despite the recently depicted antifibrotic effects of alpha-lipoic acid (ALA), however, its modulatory effects on HSCs autophagy remain unverified. Our study aimed to elucidate the underlying antifibrotic mechanisms through which ALA mediates HSC autophagy and apoptosis. Liver fibrosis was induced via thioacetamide (TAA) intoxication in rats; TAA-intoxicated rats were treated with either silymarin or ALA. Effect of ALA on biochemical parameters and immunohistopathological examinations was measured and compared to silymarin. ALA restored normal hepatic architecture (S1 vs. S4), liver functions, hepatic glutathione, and transforming growth factor-β1 levels. ALA ameliorated hepatic levels of malondialdehyde, platelet-derived growth factor, tissue inhibitor metalloproteinases-1, hydroxyproline, and expression of alpha-smooth muscle actin. Moreover, ALA significantly reduced messenger RNA expression of LC3-II genes and triggered caspase-3 expression. Interestingly, ALA exhibited superior activities over silymarin regarding suppression of proliferation, activation and autophagy of HSCs, collagen deposition, and induction of HSCs apoptosis. In conclusion, treatment of TAA-intoxicated rats with ALA inhibited autophagy and induced apoptotic clearance of activated HSCs. Accordingly, this study provides mechanistic insights into the possible applicability of ALA in the treatment of hepatic fibrosis.

Topics: Animals; Apoptosis; Autophagy; Biomarkers; Disease Models, Animal; Liver Cirrhosis; Male; Oxidative Stress; Protective Agents; Rats; Rats, Sprague-Dawley; Silymarin; Thioacetamide; Thioctic Acid; Transforming Growth Factor beta1

Topics: Actins; Animals; Antioxidants; Benzoquinones; Biomarkers; Cell Line; Cell Survival; Collagen Type I; Gene Expression Regulation; Hepatic Stellate Cells; Inflammation; Liver Cirrhosis; Male; Mice; Mice, Inbred BALB C; Oxidative Stress; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1

Although transplantation is the only definitive treatment for liver cirrhosis, there remains a shortage of donors, necessitating that novel treatments be developed. We aimed to establish a liver fibrosis model in Macaca fascicularis that can help accelerate preclinical research. Liver fibrosis was induced by administering thioacetamide (TAA) and carbon tetrachloride (CCl

Topics: Animals; Disease Models, Animal; Disease Progression; Liver; Liver Cirrhosis; Liver Function Tests; Macaca fascicularis; Thioacetamide

The present study was designed to investigate the hepatoprotective potential of dimethyl fumarate (DMF) against thioacetamide (TAA)-induced liver damage. Wistar rats were treated with DMF (12.5, 25, and 50 mg/kg/day, orally) and TAA (200 mg/kg intraperitoneally, every third day) for 6 consecutive weeks. TAA exposure significantly reduced body weight, increased liver weight and index, and intervention with DMF did not ameliorate these parameters. DMF treatment significantly restored TAA-induced increase in the levels of aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase, total bilirubin, uric acid, malondialdehyde, reduced glutathione, and histopathological findings such as inflammatory cell infiltration, deposition of collagen, necrosis, and bridging fibrosis. DMF treatment significantly ameliorated TAA-induced hepatic stellate cell activation, increase in inflammatory cascade markers (NACHT, LRR, and PYD domains-containing protein 3; NLRP3, apoptosis-associated speck like protein containing a caspase recruitment domain; ASC, caspase-1, nuclear factor-kappa B; NF-κB, interleukin-6), fibrogenic makers (α-smooth muscle actin; ɑ-SMA, transforming growth factor; TGF-β1, fibronectin, collagen 1) and antioxidant markers (nuclear factor (erythroid-derived 2)-like factor 2; Nrf2, superoxide dismutase-1; SOD-1, catalase). The present findings concluded that DMF protects against TAA-induced hepatic damage mediated through the downregulation of inflammatory cascades and upregulation of antioxidant status.

Topics: Animals; Antioxidants; Apoptosis; Dimethyl Fumarate; Disease Models, Animal; DNA Damage; Hepatic Stellate Cells; Liver Cirrhosis; Male; NF-E2-Related Factor 2; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Rats; Rats, Wistar; Signal Transduction; Thioacetamide; Treatment Outcome

The SRC kinase family comprises non-receptor tyrosine kinases that are ubiquitously expressed in all cell types. Although Src is reportedly activated in pulmonary and renal fibrosis, little is known regarding its role in liver fibrosis. This study investigated whether the inhibition of Src protects against liver fibrosis. The expression of Src was upregulated in thioacetamide (TAA)-induced fibrotic mouse liver and cirrhosis of patients, and phospho-Src was upregulated during activation of hepatic stellate cells (HSC). In addition, Src inhibition reduced the expression of α-smooth muscle actin (αSMA) in primary HSCs and suppressed transforming growth factor β (TGF-β)-induced expression of connective tissue growth factor (CTGF) in hepatocytes. Src inhibitor Saracatinib also attenuated TAA-induced expression of type I collagen, αSMA, and CTGF in mouse liver tissues. The antifibrotic effect of Src inhibitors was associated with the downregulation of smad3, but not of signal transducer and activator of transcription 3 (STAT3). In addition, Src inhibition increased autophagy flux and protected against liver fibrosis. These results suggest that Src plays an important role in liver fibrosis and that Src inhibitors could be treat liver fibrosis.

Topics: Animals; Autophagy; Benzodioxoles; Cells, Cultured; Connective Tissue Growth Factor; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Male; Mice, Inbred C57BL; Phosphorylation; Quinazolines; src-Family Kinases; STAT3 Transcription Factor; Thioacetamide; Transforming Growth Factor beta; Up-Regulation

Orthotopic liver transplantation (OLT) is the only curative treatment for refractory chronic liver failure in liver cirrhosis. However, the supply of donated livers does not meet the demand for OLT due to donor organ shortage. Cell therapy using hepatocyte-like cells derived from human induced pluripotent stem cells (hiPSC-HLCs) is expected to mitigate the severity of liver failure, postpone OLT and ameliorate the insufficient liver supply. For the successful clinical translation of hiPSC-based cell therapy against liver cirrhosis, realistic animal models are required. In this study, we created a nonhuman primate (NHP) liver fibrosis model by repeated administrations of thioacetamide (TAA) and evaluated the short-term engraftment of hiPSC-HLCs in the fibrotic liver. The NHP liver fibrosis model reproduced well the pathophysiology of human liver cirrhosis including portal hypertension. Under immunosuppressive treatment, we transplanted ALBUMIN-GFP reporter hiPSC-HLC aggregates into the fibrotic livers of the NHP model via the portal vein. Fourteen days after the transplantation, GFP-expressing hiPSC-HLC clusters were detected in the portal areas of the fibrotic livers. These results will facilitate preclinical studies using the NHP liver fibrosis model and help establish iPSC-based cell therapies against liver cirrhosis.

Topics: Animals; Cell Line; Disease Models, Animal; Female; Hepatocytes; Humans; Induced Pluripotent Stem Cells; Liver; Liver Cirrhosis; Macaca fascicularis; Thioacetamide

Transforming growth factor β (TGFβ) is a crucial factor in fibrosis, and transcriptional intermediary factor 1γ (TIF1γ) is a negative regulator of the TGFβ pathway; however, its role in liver fibrosis is unknown. In this study, mesenchymal stem cells derived from human embryonic stem cells (hE-MSCs) that secrete hepatocyte growth factor (HGF) were used to observe the repair of thioacetamide (TAA)-induced liver fibrosis. Our results showed that TIF1γ was significantly decreased in LX2 cells when exposed to TGFβ1. Such decrease of TIF1γ was significantly prevented by co-culture with hE-MSCs. Interaction of TIF1γ with SMAD2/3 and binding to the promoter of the α-smooth muscle gene (αSMA) suppressed αSMA expression. Phosphorylation of cAMP response element-binding protein (CREB) and binding on the TIF1γ promoter region induced TIF1γ expression. Furthermore, hepatic stellate cell-specific TIF1γ-knockout mice showed aggravation of liver fibrosis. In conclusion, loss of TIF1γ aggravates fibrosis, suggesting that a strategy to maintain TIF1γ during liver injury would be a promising therapeutic approach to prevent or reverse liver fibrosis.

Topics: Actins; Animals; Cells, Cultured; Cyclic AMP Response Element-Binding Protein; Hepatic Stellate Cells; Hepatocyte Growth Factor; Humans; Liver Cirrhosis; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice, Knockout; Mice, Nude; Mice, Transgenic; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Reproducibility of Results; Smad Proteins; Thioacetamide; Transcription Factors; Up-Regulation

This study evaluates the possible protective effects of gallic acid (GaA) and ferulic acid (FeA) against an experimentally induced liver fibrosis by thioacetamide (TAA) in rats. Animals were divided into: Control group, GaA group (20 mg/kg/day, p.o), FeA (20 mg/kg/day, p.o), TAA group (receiving 250 mg/kg twice/week, I.P), TAA + GaA group, TAA + FeA group (received the same previous doses) and TAA+silymarin group (received silymarin at 100 mg/kg/day+TAA as mentioned above). After 6 consecutive weeks, animals were sacrificed and the assessment of liver functions, oxidative stress biomarkers and histopathological examination of the liver tissues were performed. In addition, the effect on TGF-β1/Smad3 signaling and the expression of miR-21, miR-30 and miR-200 were evaluated. The results showed that administration of GaA or FeA with TAA induced a significant reduction in serum ALT, AST and ALP activities and protected the integrity of liver tissues. Furthermore, they increased the activities of the hepatic antioxidant enzymes; superoxide dismutase and catalase while decreased malondialdehyde content to a normal level. The hepatic expression of TGF-β1, phosphorylated and total Smad3 proteins were significantly decreased. In addition, miR-21 expression was downregulated while miR-30 and miR-200 expressions were upregulated by administration of gallic acid or ferulic acid. In conclusion, gallic and ferulic acids exhibit hepatoprotective and antioxidant effects against TAA-induced liver fibrosis in rats. These effects are mediated through inhibition of TGF-β1/Smad3 signaling and differentially regulating the hepatic expression level of miR-21, miR-30 and miR-200.

Topics: Animals; Coumaric Acids; Down-Regulation; Gallic Acid; Gene Expression; Liver; Liver Cirrhosis; Male; MicroRNAs; Protective Agents; Rats, Wistar; Signal Transduction; Smad3 Protein; Thioacetamide; Transforming Growth Factor beta1; Up-Regulation

Due to their bacterial ancestry, many components of mitochondria share structural similarities with bacteria. Release of molecular danger signals from injured cell mitochondria (mitochondria-derived damage-associated molecular patterns, mito-DAMPs) triggers a potent inflammatory response, but their role in fibrosis is unknown. Using liver fibrosis resistant/susceptible mouse strain system, we demonstrate that mito-DAMPs released from injured hepatocyte mitochondria (with mtDNA as major active component) directly activate hepatic stellate cells, the fibrogenic cell in the liver, and drive liver scarring. The release of mito-DAMPs is controlled by efferocytosis of dying hepatocytes by phagocytic resident liver macrophages and infiltrating Gr-1(+) myeloid cells. Circulating mito-DAMPs are markedly increased in human patients with non-alcoholic steatohepatitis (NASH) and significant liver fibrosis. Our study identifies specific pathway driving liver fibrosis, with important diagnostic and therapeutic implications. Targeting mito-DAMP release from hepatocytes and/or modulating the phagocytic function of macrophages represents a promising antifibrotic strategy.

Topics: Adult; Aged; Aged, 80 and over; Alarmins; Animals; Apoptosis; Disease Models, Animal; Disease Progression; Female; Hepatic Stellate Cells; Hepatocytes; Humans; Liver; Liver Cirrhosis; Macrophages; Male; Mice; Middle Aged; Mitochondria; Non-alcoholic Fatty Liver Disease; Phagocytosis; Thioacetamide; Young Adult

HSCs (hepatic stellate cells) contribute to the excessive extracellular matrix (ECM) deposition, inflammatory pathways and crucial cell-cell interactions in hepatic disease leading to fibrosis. P2x7R is considered a potential orchestrater in liver fibrosis. For this reason, this work explored the role of P2x7R in liver fibrosis and the mechanism by which P2x7R in macrophages promotes fibrogenesis. In a model of liver fibrosis induced by administration of thioacetamide (TAA), inhibition of P2x7R with its selective inhibitor A438079 reversed TAA-induced liver damage and fibrosis. The mechanism was linked to inhibition of P2x7R-NLRP3 inflammasome activation and thereby infiltration of macrophages and neutrophils into the liver. This result indicated that the P2x7R-TLR4-NLRP3 axis is involved in the process of TGF-β-mediated ECM deposition in HSCs. Ectopic overexpression of P2x7R lowered the threshold of extracellular matrix (ECM) deposition and maintained HSCs in an activated state. The culture medium of THP-1 macrophages stimulated by LPS/ATP aggravated ECM deposition in HSCs by activating P2x7R. Additionally, IL-1β secreted by LPS / ATP activated macrophages amplified fibrosis. These data indicate that P2x7R plays a key regulative role in the activation and maintenance of HSCs promoted by macrophages. Thus, pharmacological inhibition of P2x7R could be a potential therapeutic mechanism to treat human liver fibrosis.

Topics: Animals; Cell Culture Techniques; Cell Line, Tumor; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Extracellular Matrix; Feedback, Physiological; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C57BL; Purinergic P2X Receptor Antagonists; Pyridines; Receptors, Purinergic P2X7; Tetrazoles; Thioacetamide

Endoplasmic reticulum (ER) stress is an important mechanism in the progression of chronic and acute liver diseases, especially in the progression and recovery of liver fibrosis. Excessive and long-term ER stress induces apoptosis. ER stress-induced apoptosis is considered to be an important pathway in the development of liver fibrosis. Cyclooxygenase-2 (COX-2) induction is also closely related to ER stress. In our previous studies, we showed that celecoxib, a COX-2 inhibitor, improves liver fibrosis and portal hypertension. However, the role and mechanism of celecoxib in alleviating liver fibrosis remain unclear.. To investigate whether celecoxib alleviates liver fibrosis by inhibiting hepatocyte apoptosis. Cirrhosis was induced by intraperitoneal injections of thioacetamide (TAA) for 16 wk (injection dose is 200 mg/kg per 3 d for the first 8 wk and 100 mg /kg per 3 d after 8 wk). Thirty-six male Sprague-Dawley rats were randomly divided into three groups, namely, control group, TAA group, and TAA + celecoxib group. In the last 8 wk, TAA-induced cirrhotic rats received celecoxib (20 mg/kg/day) or the vehicle by gastric gavage. After 16 wk, the rats were sacrificed, and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (ALB) were detected. The hepatic fibrosis areas were evaluated by Sirius red staining and the degree of fibrosis was assessed by measuring the level of hydroxyproline. ER stress levels were evaluated by detecting the marker proteins glucose-regulated protein 78 (GRP78), CCAAT/enhancer binding protein homologous protein (CHOP), PKR-like ER protein kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 alpha (IRE1α). Apoptosis levels were evaluated by detecting caspase-12 and caspase-3.. The serum ALT and AST levels in the liver were significantly reduced by celecoxib; however, the serum ALB had no significant changes. Celecoxib significantly reduced the degree of liver fibrosis and the levels of hydroxyproline (-38% and -25.7%, respectively,. Therapeutic administration of celecoxib effectively reduces hepatic apoptosis in TAA-induced cirrhotic rats. The mechanism of action may be attributed to the suppression of CHOP expression, which subsequently inhibits ER stress.

Topics: Animals; Apoptosis; Celecoxib; Endoplasmic Reticulum Stress; Endoribonucleases; Hepatocytes; Liver Cirrhosis; Male; Protein Serine-Threonine Kinases; Rats; Rats, Sprague-Dawley; Thioacetamide

Adult stem cells, such as bone marrow mesenchymal stem cells (BMSCs), are postdevelopmental cells found in many bone tissues. They are capable of multipotent differentiation and have low immune-rejection characteristics. Hepatocytes may become inflamed and produce a large number of free radicals when affected by drugs, poisoning, or a viral infection. The excessive accumulation of free radicals in the extracellular matrix (ECM) eventually leads to liver fibrosis. This study aims to investigate the restorative effects of mouse bone marrow mesenchymal stem cells (mBMSCs) on thioacetamide (TAA)-induced damage in hepatocytes. An in vitro transwell co-culture system of HepG2 cells were co-cultured with mBMSCs. The effects of damage done to TAA-treated HepG2 cells were reflected in the overall cell survival, the expression of antioxidants (SOD1, GPX1, and CAT), the ECM (COL1A1 and MMP9), antiapoptosis characteristics (BCL2), and inflammation (TNF) genes. The majority of the damage done to HepG2 by TAA was significantly reduced when cells were co-cultured with mBMSCs. The signal transducer and activator of transcription 3 (STAT3) and its phosphorylated STAT3 (p-STAT3), as related to cell growth and survival, were detected in this study. The results show that STAT3 was significantly decreased in the TAA-treated HepG2 cells, but the STAT3 and p-STAT3 of HepG2 cells were significantly activated when the TAA-treated HepG2 co-cultured with mBMSCs. Strong expression of interleukin (Il6) messenger RNA in co-cultured mBMSCs/HepG2 indicated mBMSCs secret the cytokines IL-6, which promotes cell survival through downstream STAT3 activation and aid in the recovery of HepG2 cells damaged by TAA.

Topics: Animals; Bone Marrow Cells; Cell Differentiation; Cell Proliferation; China; Coculture Techniques; Hep G2 Cells; Hepatocytes; Humans; Liver Cirrhosis; Mesenchymal Stem Cells; Mice; Mice, Mutant Strains; Phosphorylation; Signal Transduction; STAT3 Transcription Factor; Thioacetamide

The present research work was designed to evaluate the effects of curcumin supplementation on various biochemical parameters in rats with thioacetamide (TAA) induced liver cirrhosis. For this purpose 24 male Albino Wistar rats were randomly distributed into four groups (n=6).Group I served as control, Group II and Group III received thioacetamide 200mg/kg b.w, i.p, twice a week for 12 weeks in first phase. In second phase Group II received saline and Group III received curcumin 50mg/kg b.w/day, i.p for 12 weeks, in second phase, Group IV received curcumin 50mg/kg b.w/day, i.p, for 12 weeks, in first phase and saline in second phase. Evaluation of histopathological and biochemical parameters was carried out by liver histopathology and estimation of total and direct bilirubin, liver specific enzymes, antioxidant enzymes, MDA level, plasma and intraerythrocyte sodium and potassium respectively. Histopathology of liver showed highest degree of fibrosis and nodule formation, significant alteration in biochemical parameters indicated development of severe liver cirrhosis. Curcumin treatment showed reduced amount of fibrosis and significant reduction in level of liver biomarkers, reversal of antioxidant enzymes (SOD and GSH), MDA level, catalase activity and regain of electrolyte homeostasis. These findings confirm the protective role of curcumin in liver cirrhosis.

Topics: Animals; Antioxidants; Biomarkers; Curcumin; Homeostasis; Liver; Liver Cirrhosis; Male; Oxidative Stress; Protective Agents; Rats; Rats, Wistar; Thioacetamide

Hepatic fibrosis is a reversible would-healing response following chronic liver injury of different aetiologies and represents a major worldwide health problem. Up to date, there is no satisfactory drugs treated for liver fibrosis. The present study was to investigate hepatoprotection of yangonin against liver fibrosis induced by thioacetamide (TAA) in mice and further to clarify the involvement of farnesoid X receptor (FXR) in vivo and in vitro. Yangonin treatment remarkably ameliorated TAA-induced liver injury by reducing relative liver weight, as well as serum ALT and AST activities. Moreover, yangonin alleviated TAA-induced accumulation of bile acids through increasing the expression of bile acid efflux transporters such as Bsep and Mrp2, and reducing hepatic uptake transporter Ntcp expression, all of these are FXR-target genes. The liver sections stained by H&E indicated that the histopathological change induced by TAA was improved by yangonin. Masson and Sirius red staining indicated the obvious anti-fibrotic effect of yangonin. The mechanism of anti-fibrotic effect of yangonin was that yangonin reduced collagen content by regulating the genes involved in hepatic fibrosis including COL1-α1 and TIMP-1. Besides, yangonin inhibited hepatic stellate cell activation by reducing TGF-β1 and α-SMA expression. In addition, yangonin protected against TAA-induced hepatic inflammation via its inhibition of NF-κB and TNF-α. These hepatoprotective effects of yangonin were abrogated by guggulsterone which is a FXR antagonist. In vitro experiment further demonstrated dose-dependent activation of FXR by yangonin using dual-luciferase reporter assay. In summary, yangonin produces hepatoprotection against TAA-induced liver fibrosis via FXR activation.

Topics: Animals; Chemical and Drug Induced Liver Injury; Cytokines; Hep G2 Cells; Humans; Liver; Liver Cirrhosis; Male; Mice, Inbred C57BL; Pregnenediones; Protective Agents; Pyrones; Receptors, Cytoplasmic and Nuclear; Thioacetamide

Bone marrow-derived fibrocytes (FC) represent a unique cell type, sharing features of both mesenchymal and hematopoietic cells. FC were shown to specifically infiltrate the injured liver and participate in fibrogenesis. Moreover, FC exert a variety of paracrine functions, thus possibly influencing the disease progression. However, the overall contribution of FC to liver fibrosis remains unclear. We aimed to study the effect of a specific FC depletion, utilizing a herpes simplex virus thymidine kinase (HSV-TK)/Valganciclovir suicide gene strategy. Fibrosis was induced by oral thioacetamide (TAA) administration in C57BL/6J mice. Hepatic hydroxyproline content was assessed for the primary readout. The HSV-TK model enabled the specific depletion of fibrocytes. Hepatic hydroxyproline content was significantly reduced as a result of the fibrocyte ablation (-7.8%; 95% CI: 0.7-14.8%;

Topics: Animals; Female; Hematopoietic Stem Cells; Liver Cirrhosis; Male; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Myofibroblasts; Thioacetamide

To facilitate translational drug development for liver fibrosis, preclinical trials need to be run in parallel with clinical research. Liver function estimation by gadoxetate-enhanced dynamic contrast-enhanced MRI (DCE-MRI) is being established in clinical research, but still rarely used in preclinical trials. We aimed to evaluate feasibility of DCE-MRI indices as translatable biomarkers in a liver fibrosis animal model.. Liver fibrosis was induced in Sprague-Dawley rats by thioacetamide (200 mg, 150 mg, and saline for the high-dose, low-dose, and control groups, respectively). Subsequently, DCE-MRI was performed to measure: relative liver enhancement at 3-min (RLE-3), RLE-15, initial area-under-the-curve until 3-min (iAUC-3), iAUC-15, and maximum-enhancement (Emax). The correlation coefficients between these MRI indices and the histologic collagen area, indocyanine green retention at 15-min (ICG-R15), and shear wave elastography (SWE) were calculated. Diagnostic performance to diagnose liver fibrosis was also evaluated by receiver-operating-characteristic (ROC) analysis.. Animal model was successful in that the collagen area of the liver was the largest in the high-dose group, followed by the low-dose group and control group. The correlation between the DCE-MRI indices and collagen area was high for iAUC-15, Emax, iAUC-3, and RLE-3 but moderate for RLE-15 (r, - 0.81, - 0.81, - 0.78, - 0.80, and - 0.51, respectively). The DCE-MRI indices showed moderate correlation with ICG-R15: the highest for iAUC-15, followed by iAUC-3, RLE-3, Emax, and RLE-15 (r, - 0.65, - 0.63, - 0.62, - 0.58, and - 0.56, respectively). The correlation coefficients between DCE-MRI indices and SWE ranged from - 0.59 to - 0.28. The diagnostic accuracy of RLE-3, iAUC-3, iAUC-15, and Emax was 100% (AUROC 1.000), whereas those of RLE-15 and SWE were relatively low (AUROC 0.777, 0.848, respectively).. Among the gadoxetate-enhanced DCE-MRI indices, iAUC-15 and iAUC-3 might be bidirectional translatable biomarkers between preclinical and clinical research for evaluating histopathologic liver fibrosis and physiologic liver functions in a non-invasive manner.

Topics: Animals; Area Under Curve; Contrast Media; Disease Models, Animal; Drug Evaluation, Preclinical; Feasibility Studies; Gadolinium DTPA; Humans; Liver; Liver Cirrhosis; Liver Function Tests; Magnetic Resonance Imaging; Male; Rats; Rats, Sprague-Dawley; Thioacetamide

Clusterin is a glycoprotein that is expressed in most human tissues and found in body fluids. In our previous studies we demonstrated that clusterin has a protective effect against hepatic lipid accumulation and renal fibrosis; however, the role of clusterin in hepatic fibrosis is unknown. Here, we examined whether clusterin had protective effects against hepatic fibrosis using in vitro and in vivo models. Clusterin was upregulated in the livers of human cirrhotic patients and in thioacetamide (TAA)-induced and bile duct ligation mouse models of liver fibrosis. Loss and overexpression of clusterin promoted and attenuated hepatic fibrosis after TAA injection, respectively. In addition, we found that clusterin attenuates hepatic fibrosis by inhibiting the activation of hepatic stellate cells and Smad3 signaling pathways. Thus, clusterin plays an important role in hepatic fibrosis.

Topics: Animals; Cell Line; Clusterin; Disease Models, Animal; Down-Regulation; Gene Knockout Techniques; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Male; Mice; Signal Transduction; Smad3 Protein; Thioacetamide; Up-Regulation

Tumor necrosis factor-α (TNF-α)-driven inflammatory reaction plays a crucial role in the initiation of liver fibrosis. We herein attempted to design genetically engineered adipose-derived stem cells (ASCs) producing etanercept (a potent TNF-α inhibitor), and to determine the anti-fibrotic potential of the secretome released from the etanercept-synthesizing ASCs (etanercept-secretome). First, we generated the etanercept-synthesizing ASCs by transfecting the ASCs with mini-circle plasmids containing the gene insert encoding for etanercept. We subsequently collected the secretory material released from the etanercept-synthesizing ASCs and determined its anti-fibrotic effects both in vitro (in thioacetamide [TAA]-treated AML12 and LX2 cells) and in vivo (in TAA-treated mice) models of liver fibrosis. We observed that while etanercept-secretome increased the viability of the TAA-treated AML12 hepatocytes (

Topics: Adipose Tissue; Cell Line; Etanercept; Humans; Interleukin-6; Liver Cirrhosis; Stem Cells; Thioacetamide; Tumor Necrosis Factor-alpha

The associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) procedure promotes the proliferation of the future liver remnant, but evidence to support the feasibility of ALPPS in livers with fibrosis is needed. Therefore the aim of this study was to establish a fibrotic ALPPS model in the rat to compare the capacity of regeneration in the remnant liver with or without fibrosis.. In our study we first established a thioacetamide-induced fibrotic ALPPS model in rats. Then the ALPPS-induced regenerative capacities of normal and fibrotic liver were compared in this animal model. In addition, markers of regeneration, including the proliferative index and cyclin D1 and proliferating cell nuclear antigen levels, as well as various indicators of liver function were determined to evaluate the quality of the hepatic regeneration.. Compared with that of the sham group (opening of the peritoneal cavity with no further operative manipulation), the proliferation of the future liver remnant in fibrotic rat liver after the ALPPS procedure was increased on postoperative days 1, 2, and 5 (P < .039 each). In addition, the proliferative response was greater in the ALPPS group than in the ligation group subjected only to portal vein ligation of the left lateral, left middle, right, and caudate lobes (P = .099, P = .006, and P = .020 on postoperative days 1, 2, and 5, respectively). In contrast, the ALPPS-induced regenerative capacity in the fibrotic rat livers was attenuated compared with that in the normal liver on postoperative days 1, 2, and 5 (P < .031 for each) after stage I and on postoperative day 5 after stage II of the ALPPS procedure (P < .005). This attenuated the recovery of liver function, and the greater mortality rate indicated that functional proliferation was either delayed or not as extensive in the fibrotic rat livers.. Through establishing a rat model of thioacetamide-induced liver fibrosis, we found that ALPPS-derived liver regeneration was present and feasible in fibrotic livers, but this effect was attenuated compared with that in normal liver.

Topics: Animals; Biomarkers; Cyclin D1; Disease Models, Animal; Feasibility Studies; Hepatectomy; Ki-67 Antigen; Ligation; Liver; Liver Cirrhosis; Liver Regeneration; Portal Vein; Proliferating Cell Nuclear Antigen; Random Allocation; Rats, Sprague-Dawley; Thioacetamide

The potential inhibitory effect of the antidiabetic and anti-inflammatory drug, metformin on thioacetamide (TAA)-induced hepatotoxicity associated with the inhibition of mammalian target of rapamycin (mTOR)-hypoxia-inducible factor-1α (HIF-1α) axis has not been investigated before. Therefore, we tested whether metformin can protect against liver injuries including fibrosis induced by TAA possibly via the downregulation of mTOR-HIF-1α axis and profibrogenic and inflammatory biomarkers. Rats either injected with TAA (200 mg/kg; twice a week for 8 weeks) before being killed after 10 weeks (model group) or were pretreated with metformin (200 mg/kg) daily for 2 weeks before TAA injections and continued receiving both agents until the end of the experiment, at Week 10 (protective group). Using light and electron microscopy examinations, we observed in the model group substantial damage to the hepatocytes and liver tissue such as collagen deposition, infiltration of inflammatory cells, and degenerative cellular changes with ballooned mitochondria that were substantially ameliorated by metformin. Metformin also significantly ( p < 0.05) inhibited TAA-induced HIF-1α, mTOR, the profibrogenic biomarker α-smooth muscle actin, tissue inhibitor of metalloproteinases-1, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), alanine aminotransferase (ALT) and aspartate aminotransferase in harvested liver homogenates and blood samples. In addition, a significant ( p < 0.01) positive correlation between hypoxia scoring (HIF-1α) and the serum levels of TNF-α ( r = 0.797), IL-6 ( r = 0.859), and ALT ( r = 0.760) was observed. We conclude that metformin protects against TAA-induced hepatic injuries in rats, which is associated with the inhibition of mTOR-HIF-1α axis and profibrogenic and inflammatory biomarkers; thus, may offer therapeutic potential in humans.

Topics: Animals; Biomarkers; Chronic Disease; Hepatocytes; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; Liver; Liver Cirrhosis; Male; Metformin; Protective Agents; Rats; Signal Transduction; Thioacetamide; TOR Serine-Threonine Kinases

Liver cells isolated from pre-clinical models are essential tools for studying liver (patho)physiology, and also for screening new therapeutic options. We aimed at developing a new antibody-free isolation method able to obtain the four main hepatic cell types (hepatocytes, liver sinusoidal endothelial cells [LSEC], hepatic macrophages [HMΦ] and hepatic stellate cells [HSC]) from a single rat liver. Control and cirrhotic (CCl

Topics: Albumins; Animals; Capillaries; Carbon Tetrachloride; Cell Separation; Cell Survival; Centrifugation; Endothelial Cells; Hepatic Stellate Cells; Hepatocytes; Lipopolysaccharides; Liver; Liver Cirrhosis; Macrophages; Rats; Rats, Wistar; Thioacetamide; Urea

Accumulating evidence has revealed the pivotal role of epigenetic regulation in the pathogenesis of liver disease. However, the epigenetic mechanism that accounts for hepatic stellate cells (HSCs) activation in liver fibrosis remains largely unknown.. Primary HSCs were used to screen the differentially expressed histone H3 lysine methyltransferases and demethylases during HSC activation. Loss-of-function experiments were applied to determine the cellular functions of KDM4D in HSCs. Transcriptome analysis was applied to explore the downstream targets of KDM4D. Real-time qPCR, western blotting, immunohistochemical staining, and chromatin immunoprecipitation were performed to uncover the underlying mechanism concerning KDM4D during liver fibrogenesis.. KDM4D was identified as a remarkable up-regulated histone H3 demethylase during HSC activation. The overexpression profile of KDM4D was confirmed in three fibrosis animal models and human fibrotic liver tissues. In vitro Kdm4d knockdown impaired the collagen gel contraction and migration capacity of primary HSCs. In established CCl. KDM4D facilitates TLR4 transcription through demethylation of H3K9, thus activating TLR4/NF-κB signaling pathways in HSCs, contributing to HSC activation and collagen crosslinking, further, hepatic fibrosis progression. FUND: Shanghai New Hundred Talents Program, Shanghai Municipal Commission of Health and Family Planning, Key Developing Disciplines Program, Shanghai Key disciplines program of Health and Family Planning and Shanghai Sailing Program.

Topics: Animals; Carbon Tetrachloride; Cell Line; Epigenesis, Genetic; Gene Regulatory Networks; Hepatic Stellate Cells; Histones; Humans; Jumonji Domain-Containing Histone Demethylases; Liver Cirrhosis; Male; Mice; Rats; Signal Transduction; Thioacetamide; Toll-Like Receptor 4; Transcription, Genetic; Up-Regulation

Liver cirrhosis is associated with increased morbidity and mortality with important health and social consequences; however, an effective treatment has not been found yet. Previous reports have shown some beneficial effects of stevioside (SVT) in different diseases, but the ability of SVT to inhibit liver cirrhosis has not been reported. Therefore, we studied the potential of this diterpenoid to inhibit liver cirrhosis induced by thioacetamide, a model that shares many similarities with the human disease, and investigated the possible underlying molecular mechanism using in vivo and in vitro approaches. Cirrhosis was induced in male Wistar rats by chronic thioacetamide administration (200 mg/kg) intraperitoneally three times per week. Rats received saline or SVT (20 mg/kg) two times daily intraperitoneally. In addition, co-cultures were incubated with either lipopolysaccharide or ethanol. Liver fibrosis, hepatic stellate cells activation, metalloproteinases activity, canonical and non-canonical Smads pathway and expression of several profibrogenic genes were evaluated. Thioacetamide activated hepatic stellate cells and distorted the liver parenchyma with the presence of abundant thick bands of collagen. In addition, thioacetamide up-regulated the protein expression of α-smooth muscle actin, transforming growth factor-β1, metalloproteinases-9,-2 and -13 and overstimulate the canonical and non-canonical Smad pathways. SVT administration inhibited all of these changes. In vitro, SVT inhibited the up-regulation of several genes implicated in cirrhosis when cells were exposed to lipopolysaccharides or ethanol. We conclude that SVT inhibited liver damage by blocking hepatic stellate cells activation, down-regulating canonical and non-canonical profibrotic Smad pathways.

Topics: Actins; Animals; Cell Line; Collagen Type I; Collagenases; Deoxycytosine Nucleotides; Diterpenes, Kaurane; Down-Regulation; Fibrosis; Glucosides; Hepatic Stellate Cells; Humans; Liver; Liver Cirrhosis; Lymphokines; Male; MAP Kinase Signaling System; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-myc; Rats; Rats, Wistar; Signal Transduction; Smad Proteins; Thioacetamide; Transforming Growth Factor beta1; Up-Regulation

Our previous study has confirmed that maltol can attenuate alcohol-induced acute hepatic damage and prevent oxidative stress in mice. Therefore, maltol might have the capacity to improve thioacetamide (TAA)-induced liver fibrosis. The purpose of this work was to explore the antifibrotic efficacy and underlying mechanisms of maltol for TAA-treated mice. Progressive liver fibrosis was established with a dose-escalating protocol in which the mice received TAA intraperitoneal three times a week for a total duration of 9 weeks. The injection doses of TAA were 50 mg/kg for the first week, 100 mg/kg for the second and third weeks, and 150 mg/kg for the rest of the injections. Maltol with doses of 50 and 100 mg/kg was given by gavage after 4 weeks of intraperitoneal injection of TAA, respectively, once daily for 5 weeks. Results indicated that TAA intraperitoneal injection significantly increased serum activities of alanine aminotransferase (ALT) (52.93 ± 13.21 U/L vs 10.22 ± 3.36 U/L) and aspartate aminotransferase (AST) (67.58 ± 25.84 U/L vs 39.34 ± 3.89 U/L); these elevations were significantly diminished by pretreatment with maltol. Additionally, maltol ameliorated TAA-induced oxidative stress with attenuation in MDA ( p < 0.05 or p < 0.01) content; evident elevation in the GSH levels, GSH/GSSG ratio ( p < 0.05 or p < 0.01), and superoxide dismutase (SOD) ( p < 0.01); and restored liver histology accompanied by a decrease of α-smooth muscle actin (α-SMA) expression. Furthermore, maltol significantly suppressed the transforming growth factor-β1 (TGF-β1) expression and the PI3K/Akt pathway. This study suggested that maltol alleviated experimental liver fibrosis by suppressing the activation of HSCs and inducing apoptosis of activated HSCs through TGF-β1-mediated PI3K/Akt signaling pathway. These findings further clearly suggested that maltol is a potent therapeutic candidate for the alleviation of liver fibrosis.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Hepatic Stellate Cells; Humans; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred ICR; Oxidative Stress; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pyrones; Signal Transduction; Superoxide Dismutase; Thioacetamide; Transforming Growth Factor beta1

Rebaudioside A (Reb A) is a diterpenoid isolated from the leaves of Stevia rebaudiana (Bertoni) that has been shown to possess pharmacological activity, including anti-inflammatory and antioxidant properties. However, the ability of Reb A to prevent liver injury has not been evaluated. Therefore, we aimed to study the potential of Reb A (20 mg/kg; two times daily intraperitoneally) to prevent liver injury induced by thioacetamide (TAA) administration (200 mg/kg; three times per week intraperitoneally). In addition, cocultures were incubated with either lipopolysaccharide or ethanol. Antifibrotic, antioxidant and immunological responses were evaluated. Chronic TAA administration produced considerable liver damage and distorted the liver parenchyma with the presence of prominent thick bands of collagen. In addition, TAA upregulated the expression of α-smooth muscle actin, transforming growth factor-β1, metalloproteinases 9, 2 and 13, and nuclear factor kappaB and downregulated nuclear erythroid factor 2. Reb A administration prevented all of these changes. In cocultured cells, Reb A prevented the upregulation of genes implicated in fibrotic and inflammatory processes when cells were exposed to ethanol and lipopolysaccharide. Altogether, our results suggest that Reb A prevents liver damage by blocking oxidative processes via upregulation of nuclear erythroid factor 2, exerts immunomodulatory effects by downregulating the nuclear factor-κB system and acts as an antifibrotic agent by maintaining collagen content.

Topics: Animals; Antioxidants; Cells, Cultured; Collagen; Disease Models, Animal; Diterpenes, Kaurane; Gene Expression; Lipid Peroxidation; Liver; Liver Cirrhosis; Male; Oxidative Stress; Rats; Rats, Wistar; Stevia; Thioacetamide

This study was designed to evaluate hematological disorders and the orchestrating roles of hepcidin and IL-6 in rat models of thioacetamide (TAA) and carbon tetrachloride (CCl4) hepatotoxicity.. Rats were intraperitoneally injected with TAA (10 mg/100 g rat weight dissolved in isosaline) or CCl4 (100 μL/100 g rat weight diluted as 1:4 in corn oil) twice weekly for eight consecutive weeks to induce subchronic liver fibrosis. Blood and tissue samples were collected and analyzed.. CCl4 but not TAA significantly decreased the RBCs, Hb, PCV, and MCV values with minimal alterations in other erythrocytic indices. Both hepatotoxins showed leukocytosis, granulocytosis, and thrombocytopenia. By the end of the experiment, the erythropoietin level increased in the CCl4 model. The serum iron, UIBC, TIBC, transferrin saturation%, and serum transferrin concentration values significantly decreased, whereas that of ferritin increased in the CCl4 model. TAA increased the iron parameters toward iron overload. RT-PCR analysis revealed increased expression of hepatic hepcidin and IL-6 mRNAs in the CCl4 model and suppressed hepcidin expression without significant effect on IL-6 in the TAA model.. These data suggest differences driven by hepcidin and IL-6 expression between CCl4 and TAA liver fibrosis models and are of clinical importance for diagnosis and therapeutics of liver diseases.

Topics: Animals; Blood Chemical Analysis; Carbon Tetrachloride; Hepcidins; Injections, Intraperitoneal; Interleukin-6; Iron; Leukocytosis; Liver Cirrhosis; Male; Rats; Thioacetamide; Thrombocytopenia; Transferrin

In liver fibrosis, conversion of fibroblasts to profibrogenic myofibroblasts significantly drives the development of the disease. A crucial role of cyclic adenosine monophosphate (cAMP) in regulation of fibroblast function has been reported. Increase in cAMP levels has been found to decrease fibroblast proliferation, inhibit their conversion to myofibroblast, and stimulate their death. cAMP is generated by adenyl cyclase (AC), and degraded by cyclic nucleotide phosphodiesterase (PDE). In this study, the antifibrotic effect of a PDE inhibitor, cilostazol (Cilo), on a rat model of liver fibrosis induced by thioacetamide (TAA) was investigated. Four groups of rats were used; the first group received the vehicles and served as the normal control group, while liver fibrosis was induced in the other groups using (TAA, 200 mg/kg/biweekly for 8 successive weeks, ip). The last two groups were treated with Cilo (50 and 100 mg/kg/day, po, respectively). Induction of liver fibrosis in TAA-treated rats was observed as evidenced by the biochemical and histopathological findings. On the other hand, a potent antifibrotic effect was observed in the groups treated with Cilo, with preference to the higher dose. In these groups, a significant increase in the liver content of cAMP was demonstrated that was accompanied by reduction in the hepatic expression of key fibrogenic cytokines, growth factors, and inflammatory biomarkers, including interleukin-6, tumor necrosis factor-alpha, nuclear factor kappa B, and transforming growth factor-beta as compared to TAA group. Moreover, amelioration of TAA-induced oxidative stress and apoptosis in the liver has been observed. These findings reveal the antifibrotic effect of Cilo against TAA-induced liver fibrosis in rats, and suggest regulation of cAMP pathway, together with the modulation of oxidative stress, inflammation, and apoptosis as mechanistic cassette underlines this effect.

Topics: Animals; Apoptosis; Biomarkers; Cilostazol; Cyclic AMP; Inflammation; Liver; Liver Cirrhosis; Oxidative Stress; Rats; Thioacetamide; Up-Regulation

Ginseng is a type of medicinal and edible homologous plant that is very common in medicine, food and even cosmetics. Ginsenosides are the main active constituents of ginseng, which has many pharmacological activities. AD-2 is a type of ginsenoside extracted from ginseng and prepared in large quantities in our laboratory. However, the anti-fibrosis effects and mechanism of ginsenosides are rarely reported. In this study, the anti-fibrosis pharmacodynamics of AD-2 were evaluated. The results revealed that AD-2 could reduce the expression of collagen I, TIMP-1 and MMP-13, inhibit the deposition of extracellular matrix, and play an role in anti-hepatic fibrosis. The mechanism and related pathways of AD-2 against liver fibrosis have also been studied. Inflammatory factors (including TNF-α, IL-1β, caspase-1 and IL-6) associated with hepatic fibrosis, and the p-JNK and the p38-ERK pathways, have been shown to be associated with the anti-fibrotic effect of AD-2. In conclusion, our study reveals that AD-2, as a small-molecule, targeted drug for improving liver function, needs further study.

Topics: Animals; Chemical and Drug Induced Liver Injury, Chronic; Collagen Type I; Cytokines; Disease Models, Animal; Ginsenosides; Liver; Liver Cirrhosis; Male; MAP Kinase Signaling System; Mice; p38 Mitogen-Activated Protein Kinases; Panax; Plant Extracts; Plants, Medicinal; Signal Transduction; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1

Chronic liver diseases feature excessive collagen and matrix protein deposition or crosslinking that characterises fibrosis, leads to scar tissue, and disrupts liver functions. There is no effective treatment. This study investigated whether treatment with selective histone deacetylase (HDAC) inhibitors might specifically reduce type 2 inflammation in the injured liver, thereby attenuating fibrogenesis in mice.. Thioacetamide (TAA) was used to induce hepatic inflammation, fibrosis, and liver damage in female C57BL/6 mice, similar to the clinical features of chronic human liver disease. We used eight inhibitors of different human HDAC enzymes to probe histological (IHC and TUNEL), biochemical and immunological changes (flow cytometry, qPCR, Legendplex, and ELISA) in pathology, fibrosis, hepatic immune cell flux, and inflammatory cytokine expression.. Inhibitors of class I, but not class II, HDAC enzymes potently suppressed chronic hepatic inflammation and fibrosis in mice, attenuating accumulation and activation of IL-33-dependent, but not IL-25-dependent, group 2 innate lymphoid cells (ILC2) and inhibiting type 2 inflammation that drives hepatic stellate cells to secrete excessive collagen and matrix proteins.. The results show that potent and selective inhibitors of class I only HDAC enzymes profoundly inhibit hepatocyte death and type 2 inflammation to prevent TAA-induced liver fibrosis in mice. The specific HDAC enzymes identified here may be key promoters of inflammation in chronic liver fibrosis.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Female; Histone Deacetylase Inhibitors; Histone Deacetylases; Inflammation; Ligands; Liver Cirrhosis; Mice; Mice, Inbred C57BL; Thioacetamide

The renin-angiotensin system (RAS) has a substantial role in liver fibrosis, cirrhosis, and portal hypertension. Hence, targeting RAS through angiotensin-converting enzyme (ACE) inhibitors can mend hepatic fibrosis; the current study was designed to examine the potential fibrosis inhibition activity of perindopril using a rat model of liver fibrosis induced by thioacetamide (TAA). Four groups of rats were used throughout this study, Group I (control group); rats received the vehicle. TAA was used for inducing liver fibrosis in rats by intraperitoneal injection of 200-mg/kg body weight twice a week for 6 weeks. Group II served as (TAA group). Rats of Groups III and IV were given perindopril at doses of 2 and 8 mg/kg 2 weeks after TAA administration and continued concomitantly with TAA till the end of the experiment. Injection of TAA resulted in a significant increase in aminotransferases' activities and bilirubin with a significant decrease in serum albumin and total protein and a significant decrease in hepatic content of GSH and SOD. Additionally, TAA injection raised the hepatic content of TGF-β1, α-SMA, TNF-α, and level of MDA. Histological and immunohistochemical data presented marked fibrosis in liver sections of TAA-administrated rats with increased collagen deposition, elevated METAVIR scoring, and increased expression of α-SMA, caspase-3, and AT1R. Oral dosing of perindopril for 4 weeks concomitant with TAA could mend the altered parameters near to normal values and abolished the ongoing fibrosis extension. In conclusion, these results demonstrated that perindopril, as ACE inhibitor, could grant a superior remedial nominee in preventing liver fibrosis progression through targeting angiotensin II formation.

Topics: Angiotensin II; Animals; Liver Cirrhosis; Male; Perindopril; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Renin-Angiotensin System; Signal Transduction; Thioacetamide

Hepatic fibrosis is characterized by persistent deposition of extracellular matrix proteins and occurs in chronic liver diseases. The aim of the present study is to investigate whether estrogen deficiency (ED) potentiates hepatic fibrosis in a thioacetamide (TAA)-treated rat model. Fibrosis was induced via intraperitoneal injection (i.p.) of TAA (150 mg/kg/day) for four weeks in ovariectomized (OVX) female, sham-operated female, or male rats. In TAA-treated OVX rats, the activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and γ-glutamyl transferase (GGT) were significantly increased compared to those in TAA-treated sham-operated OVX rats or TAA-treated male rats. Furthermore, α-smooth muscle actin (α-SMA) expression was significantly increased compared to that in TAA-treated sham-operated rats. This was accompanied by the appearance of fibrosis biomarkers including vimentin, collagen-I, and hydroxyproline, in the liver of TAA-treated OVX rats. In addition, ED markedly reduced total glutathione (GSH) levels, as well as catalase (CAT) and superoxide dismutase (SOD) activity in TAA-treated OVX rats. In contrast, hepatic malondialdehyde (MDA) levels were elevated in TAA-treated OVX rats. Apoptosis significantly increased in TAA-treated OVX rats, as reflected by elevated p53, Bcl-2, and cleaved caspase 3 levels. Significant increases in interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) concentrations were exhibited in TAA-treated OVX rats, and this further aggravated fibrosis through the transforming growth factor-β (TGF-β)/Smad pathway. Our data suggest that ED potentiates TAA-induced oxidative damage in the liver, suggesting that ED may enhance the severity of hepatic fibrosis in menopausal women.

Topics: Animals; Apoptosis; Biomarkers; Body Weight; Disease Models, Animal; Estrogens; Hepatocytes; Liver Cirrhosis; Liver Function Tests; Male; Organ Size; Oxidation-Reduction; Oxidative Stress; Rats; Rats, Sprague-Dawley; Thioacetamide

Liver fibrosis is a challenging global health problem resulting from chronic liver injury with no treatment currently available. It has been shown that activators for different peroxisome proliferator-activated receptor (PPAR) isoforms (α, γ, and δ) can affect different pathways in liver fibrosis. To evaluate the effects of the dual PPAR-α/γ agonist saroglitazar (SGZ) against thioacetamide (TAA)-induced fibrosis in rats, SGZ was administered for 6 weeks together with TAA injection. Administration of SGZ ameliorated TAA-induced elevation in hepatic biomarkers. SGZ was able to inhibit periportal and intralobular fibrous connective tissue proliferation, to decrease hydroxyproline content, and to lower alpha smooth muscle actin (α-SMA) protein expression. To unearth the antifibrotic mechanism of SGZ, the role of several fibrotic markers was studied. SGZ possesses inhibitory effect on protein levels of leptin, transforming growth factor-beta 1 (TGF-β1) and platelet-derived growth factor-BB (PDGF-BB). Furthermore, SGZ rectified matrix degradation through decreasing tissue inhibitor of metalloproteinases-1 (TIMP-1). This study suggests that SGZ could have a possible antifibrotic effect via suppression of leptin that can repress TGF-β1 and PDFG-BB, with subsequent inhibition of TIMP-1.

Topics: Actins; Animals; Becaplermin; Leptin; Liver; Liver Cirrhosis; Male; Phenylpropionates; PPAR alpha; PPAR gamma; Pyrroles; Rats, Sprague-Dawley; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta1

Liver cirrhosis is a scene profitable to the advance of hepatocellular carcinoma (HCC). The current work was engrossed to weigh the potential role of Cichorium intybus linn against thioacetamide (TAA)-induced liver cirrhosis and their probable underlying biochemical and molecular mechanisms. farnesoid-X-receptor (FXR) expression, proliferating cell nuclear antigen (PCNA) immunoreactivity, and activated AMP protein kinase (pAMPK), sirtuin-1 (SIRT1), and interleukin-6 (IL6) levels were estimated in hepatic tissue by real-time PCR, immunohistochemistry, and immunoassay, respectively. C. intybus linn supplementation caused a significant improvement in serum liver enzymes, albumin, bilirubin levels, tissues redox status and hepatic histological features in addition to decreased IL6 level, hydroxylproline content, and PCNA immunoreactivity. On contrary, increased pAMPK/SIRT1 levels and upregulated FXR gene expression were observed. C. intybus linn could feasibly protect against TAA-induced hepatic damage, fibrosis, and cirrhosis by relieving oxidative stress and by interruption of the inflammatory pathway via AMPK/SIRT1/FXR signaling. PRACTICAL APPLICATIONS: No specific therapies are available until now to target the underlying mechanisms for protection against liver diseases. Herbal protection is widely available and cheap with no side effect. Cichorium intybus linn, a natural supplement, is proved in this current work to have the potential of being hepatoprotectant, antioxidant, and anti-inflammatory agents, thus reducing the risk of hepatic cirrhosis.

Topics: Adenylate Kinase; Animals; Body Weight; Cell Proliferation; Chemical and Drug Induced Liver Injury; Cichorium intybus; Dietary Supplements; Gene Expression Regulation; Hepatocytes; Inflammation; Liver; Liver Cirrhosis; Male; Organ Size; Oxidation-Reduction; Proliferating Cell Nuclear Antigen; Rats; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Signal Transduction; Sirtuin 1; Thioacetamide

The present research studied the influence of zinc oxide nanoparticles (ZnO-NPs; 5, 7.5, and 10 mg/kg, i.p.) on the liver and kidney injuries motivated by thioacetamide (TAA; 100 mg/kg, i.p.). Each treatment was carried out 3 times per week for 8 weeks. ZnO-NPs relieved the decrease of hepatic or renal reduced glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) induced by TAA. Moreover, ZnO-NPs lowered tissue malondialdehyde (MDA, an indicator for lipid peroxidation). TAA treatment led to a significant increase in plasma inflammatory markers (TNF-α, IL-6), liver enzymes (gamma-glutamyltransferase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and kidney function parameters (creatinine, urea, uric acid). However, these parameters were reduced after treatment with ZnO-NPs. In addition, the hepatic fibrosis markers, hydroxyproline level, and α-smooth muscle actin immunopositive stain were lowered by ZnO-NPs. The protective effect of ZnO-NPs in respect to biochemical changes was also confirmed by histopathological and immunohistochemistry studies in the liver and kidney sections. Our results suggested that ZnO-NPs may attenuate TAA toxicity via suppression of oxidative stress.

Topics: Actins; Animals; Biomarkers; Cytokines; Hydroxyproline; Inflammation; Kidney; Kidney Function Tests; Liver Cirrhosis; Liver Function Tests; Male; Nanoparticles; Oxidative Stress; Rats, Sprague-Dawley; Thioacetamide; Zinc Oxide

Topics: Alanine Transaminase; Alkaline Phosphatase; Angiotensin-Converting Enzyme 2; Animals; Aspartate Aminotransferases; Biomarkers; Collagen; Dose-Response Relationship, Drug; Gene Deletion; Liver; Liver Cirrhosis; Male; Mice, Inbred C57BL; Mice, Knockout; Peptidyl-Dipeptidase A; Thioacetamide

The potential antifibrotic effects of melatonin against induced hepatic fibrosis were explored.. Rats were allocated into four groups: placebo; thioacetamide (TAA) (200mg/kg bwt, i.p twice weekly for two months); melatonin (5mg/kgbwt, i.p daily for a week before TAA and continued for an additional two months); and melatonin plus TAA. Hepatic fibrotic changes were evaluated biochemically and histopathologically. Hepatic oxidative/antioxidative indices were assessed. The expression of hepatic proinflammatory cytokines (tumor necrosis factor-α, and interleukin-1β), fibrogenic-related genes (transforming growth factor-1β, collagen I, collagen, III, laminin, and autotaxin) and an antioxidant-related gene (thioredoxin-1) were detected by qRT-PCR.. In fibrotic rats, melatonin lowered serum aspartate aminotransferase, alanine aminotransferase, and autotaxin activities, bilirubin, hepatic hydroxyproline and plasma ammonia levels. Melatonin displayed hepatoprotective and antifibrotic potential as indicated by mild hydropic degeneration of some hepatocytes and mild fibroplasia. In addition, TAA induced the depletion of glutathione, glutathione s-transferase, glutathione peroxidase, superoxide dismutase, catalase, and paraoxonase-1 (PON-1), while inducing the accumulation of malondialdehyde, protein carbonyl (C=O) and nitric oxide (NO), and DNA fragmentation. These effects were restored by melatonin pretreatment. Furthermore, melatonin markedly attenuated the expression of proinflammatory cytokines and fibrogenic genes via the upregulation of thioredoxin-1 mRNA transcripts.. Melatonin exhibits potent anti-inflammatory, antioxidant and fibrosuppressive activities against TAA-induced hepatic fibrogenesis via the suppression of oxidative stress, DNA damage, proinflammatory cytokines and fibrogenic gene transcripts. In addition, we demonstrate that the antifibrotic activity of melatonin is mediated by the induction of thioredoxin-1 with attenuation of autotaxin expressions.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antioxidants; Cytokines; Hydroxyproline; Liver; Liver Cirrhosis; Liver Function Tests; Male; Melatonin; Oxidative Stress; Protective Agents; Rats; Rats, Wistar; Thioacetamide

Various somatic tissue-derived mesenchymal stromal cells (MSCs) have been considered as an attractive therapeutic tool for treatment of liver diseases in which the secretion of soluble factors or extracellular vesicles (EVs) is the most probable mechanism. The experimental application of human embryonic stem cell-derived MSC (ES-MSC) increased rapidly and showed promising results, in vitro and in vivo. However, possible therapeutic effects of human ES-MSC and their EVs on Thioacetamide (TAA)-induced chronic liver injury have not been evaluated yet. Our data indicated that human ES-MSC can significantly suppress the proliferation of peripheral blood mononuclear cells compared to bone marrow (BM)-MSC and adipose (AD)-MSC. Moreover, ES-MSC increased the secretion of anti-inflammatory cytokines (i.e., TGF-β and IL-10) and decreased IFN-γ, compared to other MSCs. ES-MSC EVs demonstrated immunomodulatory activities comparable to parental cells and ameliorated cirrhosis in TAA-induced chronic rat liver injury, that is, reduction in fibrosis and collagen density, necrosis, caspase density, portal vein diameter, and transaminitis. The gene expression analyses also showed upregulation in collagenases (MMP9 and MMP13), anti-apoptotic gene (BCL-2) and anti-inflammatory cytokines (TGF-β1 and IL-10) and down-regulation of major contributors to fibrosis (Col1α, αSMA, and TIMP1), pro-apoptotic gene (BAX) and pro-inflammatory cytokines (TNFα and IL-2) following treatment with ES-MSC and ES-MSC-EV. These results demonstrated that human ES-MSC and ES-MSC EV as an off-the-shelf product, that needs further assessment to be suggested as an allogeneic product for therapeutic applications for liver fibrosis.

Topics: Adipose Tissue; Animals; Apoptosis; Bone Marrow Cells; Cell Line; Cell Survival; Chronic Disease; Cytokines; Disease Models, Animal; Extracellular Vesicles; Hepatocytes; Human Embryonic Stem Cells; Humans; Immunomodulation; Liver; Liver Cirrhosis; Male; Mesenchymal Stem Cells; Rats, Wistar; Thioacetamide

The common marmoset (Callithrix jacchus) is a nonhuman primate that is used for preclinical research on stem cell transplantation therapies due to its similarity to human beings as well as its small size, enabling researchers to perform experiments without preparing a large number of cells. In this study, we developed a marmoset hepatic fibrosis model for regenerative medicine research. Six female marmosets aged 4-6 years were administered thioacetamide (TAA) at a dose of 2.5-40 mg/kg two or three times a week. Hepatic fibrosis was assessed by liver biopsy when blood chemistry indicated liver damage. Administration of TAA increased total bile acid, aspartate aminotransferase, and total bilirubin and decreased serum albumin levels. Following more than 11 weeks of continuous injection of TAA, histological analyses detected hepatic fibrosis in all animals. Type IV collagen 7S serum levels in animals with hepatic fibrosis were significantly higher than in normal animals as a possible marker of hepatic fibrosis in marmosets. Serial liver biopsies following the last administration of TAA revealed that induced fibrosis remained up to 11 weeks. The results suggest that continuous TAA administration induces persistent hepatic fibrosis in the common marmoset and this nonhuman primate hepatic fibrosis model have the possibility to evaluate the therapeutic effects of test samples to ameliorate hepatic fibrosis.

Topics: Animals; Biomarkers; Callithrix; Collagen Type IV; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Liver; Liver Cirrhosis; Regenerative Medicine; Thioacetamide

Lysophosphatidic acid is a lipid mediator that is supposed to be implicated in hepatic fibrosis. Silymarin and caffeine are natural compounds known for their anti-inflammatory and antioxidant effects. Our study aimed to explore the effect of silymarin, caffeine, and their combination on lysophosphatidic acid receptor 1 (LPAR1) pathway in thioacetamide (TAA)-induced hepatic fibrosis.. Hepatic fibrosis was induced in male Sprague-Dawley rats by intraperitoneal injection of 200 mg/kg of TAA twice a week for 8 weeks. Silymarin (50 mg/kg), caffeine (50 mg/kg), and their combination (50 mg/kg silymarin + 50 mg/kg caffeine) were orally given to rats every day for 8 weeks along with TAA injection. Liver functions were measured. Histopathological examination of liver tissues was performed using hematoxylin and eosin and Masson's trichrome staining. mRNA expressions of LPAR1, transforming growth factor beta 1 (TGF-β1), connective tissue growth factor (CTGF), and alpha smooth muscle actin (α-SMA) were measured using RT-PCR. LPAR1 tissue expression was scored using immunohistochemistry.. Silymarin, caffeine, and their combination significantly improved liver function. They caused significant decrease in fibrosis and necro-inflammatory scores. Combination of silymain and caffeine caused a significant decrease in the necro-inflammatory score than the single treatment with silymarin or caffeine. In addition, silymarin, caffeine, and their combination significantly decreased hepatic LPAR1, TGF-β1, CTGF, and α-SMA gene expressions and LPAR1 tissue expression.. Silymarin, caffeine, and their combination protect against liver fibrosis through down-regulation of LPAR1, TGF-β1, and CTGF.

Topics: Animals; Antioxidants; Caffeine; Down-Regulation; Drug Therapy, Combination; Gene Expression; Liver Cirrhosis; Male; Rats; Rats, Sprague-Dawley; Receptors, Lysophosphatidic Acid; Silymarin; Thioacetamide

Although a plethora of signaling pathways are known to drive the activation of hepatic stellate cells in liver fibrosis, the involvement of connexin-based communication in this process remains elusive. Connexin43 expression is enhanced in activated hepatic stellate cells and constitutes the molecular building stone of hemichannels and gap junctions. While gap junctions support intercellular communication, and hence the maintenance of liver homeostasis, hemichannels provide a circuit for extracellular communication and are typically opened by pathological stimuli, such as oxidative stress and inflammation. The present study was set up to investigate the effects of inhibition of connexin43-based hemichannels and gap junctions on liver fibrosis in mice. Liver fibrosis was induced by administration of thioacetamide to Balb/c mice for eight weeks. Thereafter, mice were treated for two weeks with TAT-Gap19, a specific connexin43 hemichannel inhibitor, or carbenoxolone, a general hemichannel and gap junction inhibitor. Subsequently, histopathological analysis was performed and markers of hepatic damage and functionality, oxidative stress, hepatic stellate cell activation and inflammation were evaluated. Connexin43 hemichannel specificity of TAT-Gap19 was confirmed in vitro by fluorescence recovery after photobleaching analysis and the measurement of extracellular release of adenosine-5'-triphosphate. Upon administration to animals, both TAT-Gap19 and carbenoxolone lowered the degree of liver fibrosis accompanied by superoxide dismutase overactivation and reduced production of inflammatory proteins, respectively. These results support a role of connexin-based signaling in the resolution of liver fibrosis, and simultaneously demonstrate the therapeutic potential of TAT-Gap19 and carbenoxolone in the treatment of this type of chronic liver disease.

Topics: Animals; Anti-Inflammatory Agents; Carbenoxolone; Cells, Cultured; Connexin 43; Hepatic Stellate Cells; Liver Cirrhosis; Male; Mice; Mice, Inbred BALB C; Peptide Fragments; Superoxide Dismutase; Thioacetamide

Hepatic fibrosis is a pathological process that eventually leads to the development of cirrhosis and liver cancer by various types of chronic liver disease. To date, there is no standard treatment for the progression of liver fibrosis. This study aims to investigate the hepatoprotection of auraptene (AUR), a simple coumarin contained in the peels of citrus fruits such as grapefruit, against thioacetamide (TAA)-induced hepatic fibrosis in mice. The involvement of farnesoid X receptor (FXR) in the anti-fibrotic effect of AUR was further elucidated using in vivo and in vitro experiments. AUR was found to remarkably protect against liver injury induced by TAA in mice and maintain the homeostasis of bile acids via the regulation of FXR-target genes including Bsep, Mrp2, Ntcp, Cyp7a1 and Cyp8b1. Masson and Sirius red staining indicated a reduction of the collagen content in the liver of AUR treated mice. Furthermore, AUR inhibited the activation of hepatic stellate cells (HSCs) by down-regulating the expression of TGF-β1 and α-SMA and expressed anti-inflammatory effects by reducing the expression of NF-κB, TNF-α and IL-1β. However, the changes in these genes and protein as well as ameliorative liver histology induced by AUR were abrogated by FXR antagonist guggulsterone in vivo and FXR siRNA in vitro. Overall, AUR protects against TAA-induced hepatic fibrosis due to the reduction of toxic bile acids and inhibition of hepatic stellate cell (HSC) activation and inflammation, which were all in association with FXR activation. AUR might be efficacious for the prevention and treatment of hepatic fibrosis in mice.

Topics: Animals; Bile Acids and Salts; Citrus; Coumarins; Humans; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Multidrug Resistance-Associated Proteins; Organic Anion Transporters, Sodium-Dependent; Protective Agents; Receptors, Cytoplasmic and Nuclear; Symporters; Thioacetamide; Transforming Growth Factor beta1

Liver fibrosis is a major health issue leading to high morbidity and mortality. The potential anti-fibrotic activity and the effect of mesalazine on osteopontin (OPN), an extra cellular matrix (ECM) component were evaluated in TAA-induced liver fibrosis in rats. For this purpose, forty-two adult male Wistar rats were divided into six groups. All animals, except the normal control, were intraperitoneally injected with TAA (200 mg/kg) twice per week for 6 weeks. In the hepato-protective study, animals were administered mesalazine (50 and 100 mg/kg, orally) for 4 weeks before induction of liver fibrosis then concomitantly with TAA injection. In the hepato-therapeutic study, animals were administered mesalazine for 6 weeks after TAA discontinuation with the same doses. In both studies, mesalazine administration improved liver biomarkers through decreasing serum levels of AST, ALT and total bilirubin when compared to fibrotic group with significant increase in total protein and albumin levels. Mesalazine significantly decreased hepatic MDA level and counteracted the depletion of hepatic GSH content and SOD activity. Additionally, it limits the elevation of OPN and TGF-β1 concentrations and suppressed TNF-α as well as α-SMA levels in hepatic tissue homogenate. Histopathologically, mesalazine as a treatment showed a good restoration of the hepatic parenchymal cells with an obvious decreased intensity and retraction of fibrous proliferation, while as a prophylaxis it didn't achieve enough protection against the harmful effect of TAA, although it decreased the intensity of portal to portal fibrosis and pseudolobulation. Furthermore, mesalazine could suppress the expression of both α-SMA and caspase-3 in immunohistochemical sections. In conclusion, mesalazine could have a potential new indication as anti-fibrotic agent through limiting the oxidative damage and altering TNF-ɑ pathway as an anti-inflammatory drug with down-regulating TGF-β1, OPN, α-SMA and caspase-3 signaling pathways.

Topics: Actins; Animals; Biomarkers; Caspase 3; Immunohistochemistry; Liver; Liver Cirrhosis; Liver Function Tests; Male; Mesalamine; Osteopontin; Oxidative Stress; Rats, Wistar; Thioacetamide; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Chronic liver injury can cause cirrhosis and impaired liver regeneration, impairing organ function. Adult livers can regenerate in response to parenchymal insults, and multiple cellular sources have been reported to contribute to this response. In this study, we modeled human chronic liver injuries, in which such responses are blunted, without genetic manipulations, and assessed potential contributions of non-parenchymal cells (NPCs) to hepatocyte regeneration. We show that NPC-derived hepatocytes replenish a large fraction of the liver parenchyma following severe injuries induced by long-term thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) treatment. Through lineage tracing of biliary epithelial cells (BECs), we show that BECs are a source of new hepatocytes and gain an Hnf4α

Topics: Animals; Chronic Disease; Epithelial Cells; Hepatocytes; Humans; Liver Cirrhosis; Mice; Mice, Inbred Strains; Thioacetamide

Biomarker is the change associated with the disease. Blood is relatively stable because of the homeostatic mechanisms of the body. However, urine accumulates changes of the body, which makes it a better early biomarker source. Liver fibrosis is a reversible pathological condition, whereas cirrhosis, the end-stage of liver fibrosis, is irreversible. Consequently, noninvasive early biomarkers for fibrosis are desperately needed. In this study, differential urinary proteins were identified in the thioacetamide liver fibrosis rat model using tandem mass tagging and two-dimensional liquid chromatography tandem mass spectrometry. A total of 766 urinary proteins were identified, 143 and 118 of which were significantly changed in the TAA 1-week and 3-week groups, respectively. Multiple reaction monitoring (MRM)-targeted proteomics was used to further validate the abundant differentially expressed proteins. A total of 40 urinary proteins were statistically significant, 15 of which had been previously reported as biomarkers of liver fibrosis, cirrhosis or other related diseases and 10 of which had been reported to be associated with the pathology and mechanism of liver fibrosis. These differential proteins were detected in urine before the alanine aminotransferase and aspartate transaminase changes in the serum and before fibrosis was observed upon hematoxylin and eosin (HE) and Masson's staining.

Topics: Animals; Biomarkers; Disease Models, Animal; Liver Cirrhosis; Male; Mass Spectrometry; Proteomics; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Thioacetamide

Kupffer cells (KCs) contribute to liver fibrosis resolution by production of a large spectrum of matrix metalloproteinases (MMPs). MMP9 is a major MMP expressed by KCs. However, its role in liver fibrosis resolution remains unclear. In this study, rodent liver fibrosis was induced by intraperitoneal thioacetamide (TAA) and the resolution process was initiated by TAA withdrawal. The role of KC-derived MMP9 in fibrolysis was investigated by adoptive transfer of KCs with or without MMP9 following their depletion. The levels of serum alanine aminotransferase (ALT) and hepatic cytokines were measured during fibrosis regression. The mRNA levels of MMPs and tissue inhibitor of metalloproteinases (TIMPs) were analyzed as well. It was found that removing KCs delayed fibrosis resolution. Adoptive transfer of KCs from WT animals promoted liver fibrosis resolution, compared with transfer of KCs from MMP9-/- mice. Depletion of KCs also resulted in prolonged liver wound healing, which was reversed partially by transferred KCs from either WT or MMP9-/- mice. Likewise, the absence of KCs led to reduction in MMPs mRNA levels and elevation in TIMPs mRNA levels. The expression patterns of MMPs or TIMPs were restored by adoptive transfer of the wild-type but not MMP9-/- KCs. In addition, liver fibrosis resolution was accelerated in MMP9-/- mice by adoptive transferred KCs from WT animals, compared to the KCs from MMP9-/- mice. Overall, KC-derived MMP9 plays a critical role in fibrosis resolution, which might serve as the foundation for developing anti-fibrosis therapy.

Topics: Animals; Blotting, Western; Enzyme-Linked Immunosorbent Assay; Injections, Intraperitoneal; Kupffer Cells; Liver Cirrhosis; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Knockout; Reverse Transcriptase Polymerase Chain Reaction; Thioacetamide; Tissue Inhibitor of Metalloproteinases

Mesenchymal stem cells (MSCs) have been investigated to treat liver diseases, but the efficiency of MSCs to treat chronic liver diseases is conflicting. FGF21 can reduce inflammation and fibrosis. We established FGF21-secreting adipose derived stem cells (FGF21_ADSCs) to enhance the effects of ADSCs and transplanted them into thioacetamide (TAA)-induced liver fibrosis mice via the tail vein. Transplantation of FGF21_ADSCs significantly improved liver fibrosis by decreasing serum hyaluronic acid and reducing the expression of fibrosis-related factors such as α-smooth muscle actin (α-SMA), collagen and tissue inhibitor of metalloproteinase-1 (TIMP-1) compared with the Empty_ADSCs by inhibition of p-JNK, NF-κB and p-Smad2/3 signalling. α-lactoalbumin (LA) and lactotransferrin (LTF), secretory factors produced from FGF21_ADSCs inhibited TGF-β1-induced expression of α-SMA and collagen in LX-2 cells. These results suggest that transplantation of FGF21_ADSCs inhibited liver fibrosis more effectively than Empty_ADSCs, possibly via secretion of α-LA and LTF.

Topics: Actins; Animals; Collagen; Fibroblast Growth Factors; Hepatic Stellate Cells; Humans; Lactalbumin; Lactoferrin; Liver; Liver Cirrhosis; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Signal Transduction; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta1

Somatostatin and its analogues, which function by binding to somatostatin receptors (SSTRs) 1-5, play a protective role in liver cirrhosis. Hepatic SSTR-2 expression is up-regulated in subjects with liver cirrhosis. However, little is known about the mechanisms underlying this process. In the present study, we observed the up-regulation of hepatic SSTR-2 expression in thioacetamide (TAA)-induced cirrhotic rats and further showed that cyclooxygenase-2 (COX-2) might play a role in this process via the protein kinase C (PKC)-cAMP response element binding protein (CREB) signaling pathway. In vivo, the up-regulated SSTR-2 in liver cirrhosis was inhibited by the addition of a selective COX-2 inhibitor, such as celecoxib. In vitro, the up-regulation of COX-2 by either transfection with COX-2 plasmids or treatment with TAA increased levels of SSTR-2 and phosphorylated CREB (p-CREB) in the human hepatocyte cell line L02. Furthermore, the increase in SSTR-2 expression was inhibited by the addition of celecoxib and a PKC inhibitor. Moreover, for comparable DNA methylation levels in the region upstream of the hepatic SSTR-2 gene in normal and cirrhotic livers, DNA methylation may not contribute to the up-regulation of SSTR-2 expression in cirrhotic livers. In conclusion, the up-regulation of hepatic SSTR-2 might be induced by COX-2 via the PKC-CREB signaling pathway but is probably not induced by DNA methylation.

Topics: Animals; Cell Line; Cyclic AMP Response Element-Binding Protein; Cyclooxygenase 2; DNA Methylation; Hepatocytes; Humans; Liver Cirrhosis; Male; Plasmids; Rats; Rats, Sprague-Dawley; Receptors, Somatostatin; Thioacetamide

The development of complex in vitro hepatic systems and artificial liver devices has been hampered by the lack of reliable sources for relevant cell types, such as hepatic stellate cells (HSCs). Here we report efficient differentiation of human pluripotent stem cells into HSC-like cells (iPSC-HSCs). iPSC-HSCs closely resemble primary human HSCs at the transcriptional, cellular, and functional levels and possess a gene expression profile intermediate between that of quiescent and activated HSCs. Functional analyses revealed that iPSC-HSCs accumulate retinyl esters in lipid droplets and are activated in response to mediators of wound healing, similar to their in vivo counterparts. When maintained as 3D spheroids with HepaRG hepatocytes, iPSC-HSCs exhibit a quiescent phenotype but mount a fibrogenic response and secrete pro-collagen in response to known stimuli and hepatocyte toxicity. Thus, this protocol provides a robust in vitro system for studying HSC development, modeling liver fibrosis, and drug toxicity screening.

Topics: Cell Differentiation; Cells, Cultured; Coculture Techniques; Female; Hepatic Stellate Cells; Humans; Infant, Newborn; Liver Cirrhosis; Male; Models, Biological; Pluripotent Stem Cells; Thioacetamide; Wound Healing

Lactoferrin (LF) was previously suggested to have a protective effect against liver fibrosis by preventing hepatic stellate cells (HSCs) activation. The effect of LF on heat shock protein 47 (HSP47) has not yet been studied so this study was designed to investigate LF effect on HSP47 as a potential target for management of liver fibrosis and comparing it with silymarin (SM) in a thioacetamide (TAA)-induced liver fibrosis model. Rats were divided into four groups; normal control, TAA (TAA-treated), LF (LF + TAA-treated), and SM (SM + TAA-treated). After 6 weeks, both LF and SM improved the grade of cirrhosis, reduced collagen fibers deposition, inactivated HSCs, significantly decreased elevated liver enzymes, HSP47, hydroxyproline content, transforming growth factor-beta 1, matrix metalloproteinase-2, 8-hydroxydeoxyguanosine, malondialdehyde, nitric oxide levels and the percentage of alpha smooth muscle actin positive HSCs compared with TAA group. Moreover, LF significantly increased the total antioxidant capacity compared with TAA group. It could be concluded that LF is a promising antifibrotic drug and could be considered as one of the HSP47 inhibitors but SM is still more potent. © 2018 IUBMB Life, 70(8):795-805, 2018.

Topics: Animals; Antioxidants; Gene Expression Regulation; Hepatic Stellate Cells; HSP47 Heat-Shock Proteins; Humans; Lactoferrin; Liver; Liver Cirrhosis; Matrix Metalloproteinase 2; Rats; Silymarin; Thioacetamide; Transforming Growth Factor beta1

Berberine (BBR) is an isoquinoline alkaloid extracted from the roots, rhizomes and stems of coptis. Liver fibrosis is a worldwide health problem with no established therapy until now. The aim of our study is to investigate the efficacy of BBR on hepatic fibrosis induced in rats and to uncover other mechanisms. Rats were injected with thioacetamide (TAA) (200 mg/kg, i.p) twice per week for 6 weeks to induce fibrosis. Treated groups were gavaged with BBR (50 mg/kg/day, p.o) simultaneously with TAA injection. Hepatic antioxidant enzymes (catalase, SOD, GPx) were assessed in hepatic homogenate. Their activities were attenuated by TAA injection and elevated by BBR administration. Additionally, serum IL-6 and mRNA levels of IL-1β, IL-6, IL-10 and IFN-γ were evaluated as inflammatory markers. Our results showed that BBR suppressed the inflammation induced by TAA injection. Tissue expression of α-SMA (marker of activated HSCs), TGF-β1 and fibronectin were measured by immunohistochemistry as well as mRNA expressions of TGF-β1 and fibronectin were quantified as fibrotic markers. The collagen deposition in hepatic tissues was assessed by Masson's trichome staining. BBR significantly alleviated TGF-β1 production, decreased collagen and fibronectin deposition and consequently attenuated hepatic fibrogenesis. Akt pathway controls cell survival, proliferation, migration and adhesion. The relative phosphorylation of Akt was determined in hepatic homogenates that was increased with TAA injection and decreased by BBR treatment. Inhibition of Akt pathway has been linked to the intrinsic pathway of apoptosis. Caspase-3, caspase-9, Bcl-2 and Bax were quantified as apoptotic markers using qPCR and also caspase-3 by immunohistochemistry. BBR-treated rats showed an increase in the expression of apoptotic markers. Moreover, BBR-treated rats showed restoration of normal liver lobular architecture as shown by H&E staining. In conclusion, BBR is a potential therapeutic candidate for liver fibrosis owing to its antioxidant and anti-inflammatory activities.

Topics: Actins; Animals; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Berberine; Collagen; Cytokines; Fibronectins; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Male; Oxidative Stress; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Thioacetamide; Transforming Growth Factor beta1

Chronic liver damage leads to the onset of fibrogenesis. Rodent models for liver fibrosis have been widely used, but are less suitable for screening purposes. Therefore the aim of our study was to design a novel model for liver fibrosis in zebrafish embryos, suitable for high throughput screening. Furthermore, we evaluated the efficacy of mesenchymal stromal cells (MSCs) to inhibit the fibrotic process and thereby the applicability of this model to evaluate therapeutic responses. Zebrafish embryos were exposed to TAA or CCL4 and mRNA levels of fibrosis-related genes (Collagen-1α1, Hand-2, and Acta-2) and tissue damage-related genes (TGF-β and SDF-1a, SDF-1b) were determined, while Sirius-red staining was used to estimate collagen deposition. Three days after start of TAA exposure, MSCs were injected after which the fibrotic response was determined. In contrast to CCL4, TAA resulted in an upregulation of the fibrosis-related genes, increased extracellular matrix deposition and decreased liver sizes suggesting the onset of fibrosis. The applicability of this model to evaluate therapeutic responses was shown by local treatment with MSCs which resulted in decreased expression of the fibrosis-related RNA markers. In conclusion, TAA induces liver fibrosis in zebrafish embryos, thereby providing a promising model for future mechanistic and therapeutic studies.

Topics: Animals; Biomarkers; Chemokine CCL4; Disease Models, Animal; Disease Progression; Embryo, Nonmammalian; Fibroblasts; Gene Expression; Liver Cirrhosis; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Thioacetamide; Zebrafish

Liver fibrosis is a health concern that leads to organ failure mediated via production of inflammatory cytokines and fibrotic biomarkers. This study aimed to explore the protective effect of tadalafil, a phosphodiesterase-5 inhibitor, against thioacetamide (TAA)-induced liver fibrosis. Fibrosis was induced by administration of TAA (200 mg/kg, i.p.) twice weekly for 6 weeks. Serum transaminases activities, liver inflammatory cytokines, fibrotic biomarkers, and liver histopathology were assessed. TAA induced marked histopathological changes in liver tissues coupled with elevations in serum transaminases activities. Furthermore, hepatic content of nitric oxide and tumor necrosis factor-alpha, interleukin-6, and interleukin-1 beta were elevated, together with a reduction of interleukin-10 in the liver. In addition, TAA increased hepatic contents of transforming growth factor-beta, hydroxyproline, alpha-smooth muscle actin, and gene expression of collagen-1. Pretreatment with tadalafil protected against TAA-induced liver fibrosis, in a dose-dependent manner, as proved by the alleviation of inflammatory and fibrotic biomarkers. The effects of tadalafil were comparable with that of silymarin, a natural antioxidant, and could be assigned to its anti-inflammatory and anti-fibrotic properties.

Topics: Actins; Animals; Anti-Inflammatory Agents; Biomarkers; Collagen Type I; Cytokines; Gene Expression; Hydroxyproline; Inflammation; Interleukin-10; Interleukin-1beta; Interleukin-6; Liver; Liver Cirrhosis; Male; Nitric Oxide; Rats; Rats, Wistar; Tadalafil; Thioacetamide; Transaminases; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Cytoglobin (CYGB), discovered in hepatic stellate cells (HSCs), is known to possess a radical scavenger function, but its pathophysiological roles remain unclear. Here, for the first time, we generated a new transgenic (TG) mouse line in which both Cygb and mCherry reporter gene expression were under the control of the native Cygb gene promoter. We demonstrated that the expression of Cygb-mCherry was related to endogenous Cygb in adult tissues by tracing mCherry fluorescence together with DNA, mRNA, and protein analyses. Administration of a single dose (50 mg/kg) of thioacetamide (TAA) in Cygb-TG mice resulted in lower levels of alanine transaminase and oxidative stress than those in WT mice. After 10 weeks of TAA administration, Cygb-TG livers exhibited reduced neutrophil accumulation, cytokine expression and fibrosis but high levels of quiescent HSCs. Primary HSCs isolated from Cygb-TG mice (HSC

Topics: Animals; Artificial Gene Fusion; Cytoglobin; Gene Expression; Genes, Reporter; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Luminescent Proteins; Mice; Mice, Transgenic; Promoter Regions, Genetic; Red Fluorescent Protein; Thioacetamide

Allium senescens Linn. (Liliaceae) (ASL) has been traditionally used in Korea and other Asian countries for improving digestive and liver functions.. The anti-hepatofibrosis effect of ASL ethanol extract in cellular and experimental fibrosis rat model was investigated.. In vitro cell viability, cell cycle and apoptosis in hepatic stellate cells (HSCs) were studied using MTT assay, flow cytometry and Annexin V-FITC/PI staining. Thioacetamide (TAA; 200 mg/kg, i.p.)-induced liver fibrosis model using Sprague Dawley rats (n = 10) was developed in vivo by injecting TAA twice per week for 13 weeks. ASL (25 and 100 mg/kg) and silymarin (50 mg/kg) were administered through oral gavage 2 times per week from 7th to 13th week. Specific fibrotic-related biomarkers such as aspartate transaminase (AST), alanine transaminase (ALT), glutathione and hydroxyproline levels in serum were analyzed by spectrophotometer using commercial kits. Morphological, histopathological and fibrotic-related gene expression such as TGF-β, Col1α1 and α-SMA in liver tissues was estimated by hematoxylin and eosin staining, Picrosirius red stain and quantitative real-time polymerase chain reaction, respectively.. ASL (0.1 mg/mL) and silymarin (0.05 mg/mL) treatment induced apoptosis (4.06% and 8.67%) in activated HSC-T6 cells, compared with control group (3.7%). The altered morphology in activated primary HSCs was also restored by ASL (0.1 mg/mL) treatment. Further, ASL (100 and 25 mg/kg) ameliorated the TAA-induced altered fibrotic-related biomarkers, histopathological changes and fibrotic-related gene expression significantly (p < 0.05 ∼ p < 0.001).. ASL can potentially be developed as a therapeutic agent in the treatment of hepatic fibrosis.

Topics: Allium; Animals; Cell Line; Cell Proliferation; Cell Survival; Cells, Cultured; Disease Models, Animal; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Male; Plant Extracts; Rats; Rats, Sprague-Dawley; Thioacetamide; Treatment Outcome

Multiple etiologies of liver injury are associated with fibrosis in which the key event is the activation of hepatic stellate cells (HSCs). Although microRNAs (miRNAs) are reportedly involved in fibrogenesis, the complete array of miRNA signatures associated with the disease has yet to be elucidated. Here, deep sequencing analysis revealed that compared to controls, 80 miRNAs were upregulated and 21 miRNAs were downregulated significantly in the thioacetamide (TAA)-induced mouse fibrotic liver. Interestingly, 58 of the upregulated miRNAs were localized to an oncogenic miRNA megacluster upregulated in liver cancer. Differential expression of some of the TAA-responsive miRNAs was confirmed, and their human orthologs were similarly deregulated in TGF-β1-activated HSCs. Moreover, a functional analysis of the experimentally validated high-confidence miRNA targets revealed significant enrichment for the GO terms and KEGG pathways involved in HSC activation and liver fibrogenesis. This is the first comprehensive report of miRNAs profiles during TAA-induced mouse liver fibrosis.

Topics: Actins; Animals; Cell Line, Transformed; Collagen Type I; Collagen Type I, alpha 1 Chain; Disease Models, Animal; Gene Expression Profiling; Gene Expression Regulation; Hepatic Stellate Cells; Humans; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Molecular Sequence Annotation; Signal Transduction; Thioacetamide; Transforming Growth Factor beta1

The present study aimed to compare the therapeutic efficiency of nano-encapsulated and nano-emulsion carvacrol administration on liver injury in thioacetamide (TAA) treated rats.. To fulfill our target, we used sixty male albino rats classified into six groups as follow: control, nano-encapsulated carvacrol, nano-emulsion carvacrol, thioacetamide, treated nano-encapsulated carvacrol and treated nano-emulsion carvacrol groups. Blood samples were collected from all groups and the separated serum was used for analysis of the following biochemical parameters; aspartate aminotransferase (AST), alanine aminotransferase (ALT), S100 B protein, alpha fetoprotein (AFP) and caspase-3. The levels of malondialdehyde (MDA), reduced glutathione (GSH), nitric oxide (NO), monocyte chemoattractant protein-1(MCP-1) and hydroxyproline content were all evaluated in liver tissue homogenate. Histopathological examinations for liver tissues were also performed.. Thioacetamide induced hepatic damage in rats as revealed by the significant increase in the levels of serum ALT, AST and produced oxidative stress as displayed by the significant elevation in the levels of hepatic MDA and NO concomitant with a significant decrease in GSH. In addition, thioacetamide significantly increased serum S100B protein, alpha fetoprotein and caspase-3 along with hepatic MCP-1 and hydroxyproline; these results were confirmed by the histopathological investigation. In contrast, nano-encapsulated and nano-emulsion carvacrol were able to ameliorate these negative changes in the thioacetamide injected rats. However, the effect of the nano-encapsulated form of carvacrol was more prominent than the nano-emulsion form.. Nano-encapsulated and nano-emulsion carvacrol can ameliorate thioacetamide induced liver injury. These results could be attributed to the potential anti-inflammatory, antioxidant, and anti-apoptotic activities of carvacrol in addition to the effectiveness of the encapsulation technique that can protect carvacrol structure and increase its efficiency and stability. Moreover, nano-encapsulation of carvacrol is more efficient than nano-emulsion.

Topics: Alanine Transaminase; Animals; Antioxidants; Aspartate Aminotransferases; Caspase 3; Chemokine CCL2; Cymenes; Disease Models, Animal; Emulsions; Glutathione; Liver; Liver Cirrhosis; Male; Malondialdehyde; Monoterpenes; Nanoparticles; Oxidative Stress; Protective Agents; Rats; Thioacetamide

Nicotine exerts a number of physiological effects. The purpose of this study was to determine the effects of nicotine on thioacetamide (TAA)-induced liver fibrosis in mice.. For in vivo experiments, hepatic fibrosis was induced by TAA (0.25 g/kg, i.p.) three times a week for 6 weeks. Mice of TAA treated groups were administered daily with distilled water and nicotine (50 or 100 μg/mL) via gastrogavage throughout the experimental period. For in vitro experiments, HepG2 (human liver cancer cell line) and LX-2 (human hepatic stellate cell line) were used to determine oxidative stress and fibrosis, respectively.. Compared to control groups, TAA treated groups had significantly differences in serum alanine transferase and aspartate aminotransferase levels and nicotine accentuated liver injury. Moreover, nicotine increased the mRNA levels of TAA-induced transforming growth factor-β (TGF-β) and collagen type I alpha 1 in the liver. Nicotine also increased TAA-induced oxidative stress. Histological examination confirmed that nicotine aggravated the degree of fibrosis caused by TAA treatment. Additionally, nicotine enhanced hepatic stellate cell activation via promoting the expression of α-smooth muscle actin.. Oral administration of nicotine significantly aggravated TAA-induced hepatic fibrosis in mice through enhancing TGF-β secretion and TAA-induced oxidative stress. The increase in TGF-β levels might be associated with the strengthening of oxidative processes, subsequently leading to increased hepatic stellate cell activation and extracellular matrix deposition. These results suggest that patients with liver disease should be advised to abandon smoking since nicotine may exacerbate hepatic fibrosis.

Topics: Animals; Biomarkers; Cell Line; Chemical and Drug Induced Liver Injury; Collagen Type I, alpha 1 Chain; Dose-Response Relationship, Drug; Drug Synergism; Fungicides, Industrial; Gene Expression Regulation; Hep G2 Cells; Hepatic Stellate Cells; Hepatocytes; Humans; Liver; Liver Cirrhosis; Male; Mice, Inbred C57BL; Nicotine; Oxidative Stress; Random Allocation; Thioacetamide

Ductular reaction is a standard component of fibrotic liver tissue but its function is largely unknown. It is supposed to interact with the matrix producing myofibroblasts and compensate the declining regenerative capacity of hepatocytes. The relationship between the extent of fibrosis-ductular reaction, proliferative activity of hepatocytes and ductular reaction were studied sequentially in experimental hepatic fibrosis models.. Liver fibrosis/cirrhosis was induced in wild type and TGFβ overproducing transgenic mice by carbon tetrachloride and thioacetamide administration. The effect of thioacetamide was modulated by treatment with imatinib and erlotinib. The extent of ductular reaction and fibrosis was measured by morphometry following cytokeratin 19 immunofluorescent labeling and Picro Sirius staining respectively. The proliferative activity of hepatocytes and ductular reaction was evaluated by BrdU incorporation. The temporal distribution of the parameters was followed and compared within and between different experimental groups.. There was a strong significant correlation between the extent of fibrosis and ductular reaction in each experimental group. Although imatinib and erlotinib temporarily decreased fibrosis this effect later disappeared. We could not observe negative correlation between the proliferation of hepatocytes and ductular reaction in any of the investigated models.. The stringent connection between ductular reaction and fibrosis, which cannot be influenced by any of our treatment regimens, suggests that there is a close mutual interaction between them instead of a unidirectional causal relationship. Our results confirm a close connection between DR and fibrogenesis. However, since the two parameters changed together we could not establish a causal relationship and were unable to reveal which was the primary event. The lack of inverse correlation between the proliferation of hepatocytes and ductular reaction questions that ductular reaction can compensate for the failing regenerative activity of hepatocytes. No evidences support the persistent antifibrotic property of imatinib or erlotinib.

Topics: Animals; Carbon Tetrachloride; Cell Proliferation; Disease Models, Animal; Erlotinib Hydrochloride; Fibrosis; Imatinib Mesylate; Keratin-19; Liver; Liver Cirrhosis; Liver Regeneration; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Confocal; Protein Kinase Inhibitors; Thioacetamide; Transforming Growth Factor beta

Liver fibrosis is a major health problem. The current study evaluated the potential of icariin (ICA) to guard against thioacetamide (TAA)-induced liver fibrosis in rats.. Four groups of male rats were treated as follows: group 1 was the control group, group 2 was given TAA (200mg/kg), group 3 was administered ICA (50mg/kg) and TAA (200mg/kg), and group 4 was given ICA (50mg/kg) alone. Animal treatment was continued for four weeks.. Co-administration of ICA guarded against TAA hepatotoxicity as indicated by significant inhibition in the rise of serum ALT and AST activities and albumin concentrations. This was accompanied by inhibition of reduced glutathione depletion, superoxide dismutase exhaustion, and lipid peroxide accumulation. In addition, ICA inhibited the pathological alterations in liver architecture induced by TAA. The antifibrotic activity of ICA was verified by reduced hepatic collagen deposition in liver sections stained with Masson's trichrome and hepatic Col-1α mRNA and hydroxyproline contents compared to the TAA-treated group. The antiangiogenic activity of ICA was evidenced by lowered levels of mRNA of Ang-1 and protein expression of VEGF, PDGF-β, and CTGF immunohistochemically. Further, the anti-autophagic property of ICA was evidenced by amelioration of the decrease in mTOR and p70S6 kinase expression and an increase in TLR4, NFκB, IL1-β, and COX-2 immunohistochemically. Moreover, ICA antagonized the increase in HMGB1, TGF-β, and Beclin-1 and the decrease in BAMBI hepatic mRNA levels.. ICA inhibits TAA-induced liver fibrosis in rats, possibly via inhibition of angiogenesis and autophagy.

Topics: Angiogenesis Inhibitors; Animals; Autophagy; Biomarkers; Flavonoids; Liver Cirrhosis; Neovascularization, Pathologic; Rats; Thioacetamide

Cuscuta chinensis Lam. (Convolvulaceae) has been used as a traditional herbal remedy for treating liver and kidney disorders.. Anti-fibrotic effects of C. chinensis extract (CCE) in cellular and experimental animal models were investigated.. HSC-T6 cell viability, cell cycle and apoptosis were analysed using MTT assay, flow cytometry and Annexin V-FITC/PI staining techniques. Thioacetamide (TAA)-induced fibrosis model was established using Sprague Dawley rats (n = 10). Control, TAA, CCE 10 (TAA with CCE 10 mg/kg), CCE 100 (TAA with CCE 100 mg/kg) and silymarin (TAA with silymarin 50 mg/kg). Fibrosis was induced by TAA (200 mg/kg, i.p.) twice per week for 13 weeks. CCE and silymarin were administered orally two times per week from the 7th to 13th week. Fibrotic related gene expression (α-SMA, Col1α1 and TGF-β1) was measured by RT-PCR. Serum biomarkers, glutathione (GSH) and hydroxyproline were estimated by spectrophotometer using commercial kits.. CCE (0.05 and 0.1 mg/mL) and silymarin (0.05 mg/mL) treatment significantly (p < 0.01 and p < 0.001) induced apoptosis (11.56%, 17.52% for CCE; 16.50% for silymarin, respectively) in activated HSC-T6 cells, compared with control group (7.26%). Further, rat primary HSCs showed changes in morphology with CCE 0.1 mg/mL treatment. In in vivo studies, CCE (10 and 100 mg/kg) treatment ameliorated the TAA-induced altered levels of serum biomarkers, fibrotic related gene expression, GSH, hydroxyproline significantly (p < 0.05-0.001) and rescued the histopathological changes.. CCE can be developed as a potential agent in the treatment of hepatofibrosis.

Topics: Animals; Cell Line, Transformed; Cell Survival; Cells, Cultured; Cuscuta; Dose-Response Relationship, Drug; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Male; Plant Extracts; Rats; Rats, Sprague-Dawley; Thioacetamide

Currently most liver fibrosis research is performed in vivo, since suitable alternative in vitro systems which are able to recapitulate the cellular events leading to liver fibrosis are lacking. Here we aimed at generating a system containing cells representing the three key players of liver fibrosis (hepatocyte, Kupffer cells and stellate cells) and assess their response to pro-fibrotic compounds such as TGF-β1, methotrexate (MTX) and thioacetamide (TAA).. Human cell lines representing hepatocytes (HepaRG), Kupffer cell (THP-1 macrophages) and stellate cells (hTERT-HSC) were co-cultured using the InSphero hanging drop technology to generate scaffold-free 3D microtissues, that were treated with pro-fibrotic compounds (TGF-β1, MTX, TAA) for up to 14 days. The response of the microtissues was evaluated by determining the expression of cytokines (TNF-α, TGF-β1 and IL6), the deposition and secretion of ECM proteins and induction of gene expression of fibrosis biomarkers (e.g. αSMA). Induction of Nrf2 and Keap1, as key player of defence mechanism, was also evaluated.. We could demonstrate that the multicellular 3D microtissue cultures could be maintained in a non-activated status, based on the low expression levels of activation markers. Macrophages were activated by stimulation with LPS and hTERT-HSC showed activation by TGF-β1. In addition, MTX and TAA elicited a fibrotic phenotype, as assessed by gene-expression and protein-deposition of ECM proteins such as collagens and fibronectin. An involvement of the antioxidant pathway upon stimulation with pro-fibrotic compounds was also observed.. Here, for the first time, we demonstrate the in vitro recapitulation of key molecular and cellular events leading to liver fibrosis: hepatocellular injury, antioxidant defence response, activation of Kupffer cells and activation of HSC leading to deposition of ECM.

Topics: Cell Line; Extracellular Matrix; Hepatic Stellate Cells; Humans; Lipopolysaccharides; Liver Cirrhosis; Macrophages; Methotrexate; NF-E2-Related Factor 2; Thioacetamide; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

To evaluate a calcium activated potassium channel (KCa3.1) inhibitor attenuates liver disease in models of non-alcoholic fatty liver disease (NAFLD).. We have performed a series of. Upregulation of KCa3.1 expression was recorded in TAA-induced and high fat diet-induced liver disease. Treatment with Senicapoc decreased palmitic acid-driven HepG2 cell death. (. These data suggest that Senicapoc interrupts more than one node in progressive fatty liver disease by its anti-steatotic and anti-fibrotic activities, serving as a double-edged therapeutic sword.

Topics: Acetamides; Animals; Apoptosis; Biomarkers, Tumor; Diet, High-Fat; Fibrosis; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Inflammation; Intermediate-Conductance Calcium-Activated Potassium Channels; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Non-alcoholic Fatty Liver Disease; Palmitic Acid; Rats; Rats, Wistar; Thioacetamide; Trityl Compounds; Up-Regulation

Naringin (NR) is a flavanone glycoside extracted from grapefruits and citrus fruits. The aim of this study is to investigate the antifibrotic efficacy of NR in thioacetamide (TAA)-induced hepatic fibrosis in rats through evaluating NR effect on the PI3K/Akt pathway.. Hepatic fibrosis was induced in rats by intraperitoneal injection of TAA (200mg/kg) twice per week for 6weeks. Simultaneously, NR (40mg/kg/day, p.o.) was given along with TAA injection. The ratio of P-Akt/Akt was assessed in hepatic homogenate as well as antioxidant enzymes (catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx)) and lipid peroxidation marker, malondialdehyde (MDA). Serum level of interleukin (IL)-6 were measured using ELISA. Hepatic tissues were examined histopathologically using hematoxylin and eosin (H&E) and Masson trichome staining. Tissue expression of alpha smooth muscle actin (α-SMA), transforming growth factor β1 (TGF-β1), caspase-3 and fibronectin were scored immunohistochemically. Finally, the mRNA level of cytokine genes (IL-1β, IL-6, IL-10, interferon gamma (IFN-γ)), caspase-3, TGF-β1 and fibronectin were quantified using qPCR.. NR significantly suppressed Akt phosphorylation associated with increased number of caspase-3 positive cells especially in the fibrotic areas. Liver tissues of treated rats showed restoration of normal liver histology and decrease in collagen and fibronectin deposition. Furthermore, NR treatment ameliorated oxidative stress and inflammatory cytokine production.. NR alleviated experimental liver fibrosis through inhibition of PI3K/Akt pathway beside its anti-inflammatory and antioxidant effects. Therefore, NR is a promising therapeutic candidate for hepatic fibrosis.

Topics: Animals; Antioxidants; Caspase 3; Cytokines; Fibronectins; Flavanones; Interleukin-6; Liver Cirrhosis; Male; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Thioacetamide; Transforming Growth Factor beta1

This study aimed the integrative characterization of morphological, biochemical and molecular features of chemically-induced cirrhosis-associated hepatocarcinogenesis. Thus, male Wistar rats were submitted to a diethylnitrosamine (DEN)/thioacetamide (TAA)-induced model. Liver tissue was processed for global gene expression, histopathological and collagen evaluations; as well as immunohistochemical and oxidative stress analysis. Gene Ontology and functional analysis showed the upregulation of extracellular matrix deposition genes, such as collagen type I alpha 1 and 2 (Col1α1 and Col1α2) and tissue inhibitor of metalloproteinase 1 and 2 genes (Timp1 and Timp2). In agreement these findings, animals presented extensive liver cirrhosis with increased collagen deposition (Sirius red). Besides, the animals developed many glutathione S-transferase pi (GST-P)-positive preneoplastic lesions showing high cell proliferation (Ki-67), in keeping with the Gstp1 and Gstp2 increased gene expression. DEN/TAA-treated rats also showed the upregulation of tumorigenesis-related annexin A2 gene (Anxa2) and few neoplastic lesions (hepatocellular adenomas, carcinomas, and cholangiocarcinoma). In contrast, gene expression and activity of antioxidant enzymes were decreased (glutathione peroxidase, total glutathione-S-transferase, and catalase). The model featured remarkable similarities to human hepatocarcinogenesis. Our findings could bring up new molecular insights into cirrhosis-associated hepatocarcinogenesis, and provide a suitable animal model for the establishment of further diagnostic, preventive and therapeutic approaches.

Topics: Alanine Transaminase; Animals; Annexin A2; Aspartate Aminotransferases; Carcinogenesis; Collagen; Collagen Type I, alpha 1 Chain; Diethylnitrosamine; Gene Expression Regulation, Neoplastic; Glutathione Peroxidase; Glutathione Transferase; Liver Cirrhosis; Liver Neoplasms, Experimental; Male; Matrix Metalloproteinases; Oxidative Stress; Rats; Rats, Wistar; Thioacetamide; Tissue Inhibitor of Metalloproteinases

Minimal hepatic encephalopathy is more common than the acute syndrome. Losartan, the first angiotensin-II receptor blocker (ARB), and candesartan, another widely-used ARB, have protected against developing fibrogenesis, but there is no clear data about their curative antifibrotic effects. The current study was designed to examine their effects in an already-established model of hepatic fibrosis and also their effects on the associated motor dysfunction. Low-grade chronic liver failure (CLF) was induced in 3-month old Sprague-Dawley male rats using thioacetamide (TAA, 50 mg·kg-1·day-1) intraperitoneally for 2 weeks. The TAA-CLF rats were randomly divided into five groups (n=8) treated orally for 14 days (mg·kg-1·day-1) as follows: TAA (distilled water), losartan (5 and 10 mg/kg), and candesartan (0.1 and 0.3 mg/kg). Rats were tested for rotarod and open-field tests. Serum and hepatic biochemical markers, and hepatic histopathological changes were evaluated by H&E and Masson's staining. The TAA-CLF rats showed significant increases of hepatic malondialdehyde, hepatic expression of tumor necrosis factor-α (TNF-α), and serum ammonia, alanine aminotransferase, γ-glutamyl transferase, TNF-α, and malondialdehyde levels as well as significant decreases of hepatic and serum glutathione levels. All treatments significantly reversed these changes. The histopathological changes were moderate in losartan-5 and candesartan-0.1 groups and mild in losartan-10 and candesartan-0.3 groups. Only candesartan significantly improved TAA-induced motor dysfunction. In conclusion, therapeutic antifibrotic effects of losartan and candesartan in thioacetamide-induced hepatic fibrosis in rats are possibly through angiotensin-II receptor blocking, antioxidant, and anti-inflammatory activities. Improved motor dysfunction by candesartan could be attributed to better brain penetration and slower "off-rate" from angiotensin-II receptors. Clinical trials are recommended.

Topics: Alanine Transaminase; Ammonia; Angiotensin II Type 1 Receptor Blockers; Animals; Benzimidazoles; Biphenyl Compounds; Disease Models, Animal; End Stage Liver Disease; Enzyme-Linked Immunosorbent Assay; gamma-Glutamyltransferase; Glutathione; Liver; Liver Cirrhosis; Locomotion; Losartan; Male; Malondialdehyde; Motor Disorders; Random Allocation; Rats, Sprague-Dawley; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Tetrazoles; Thioacetamide; Treatment Outcome; Tumor Necrosis Factor-alpha

This study was designed to evaluate the effects of sylimarin supplementation on different biochemical parameters in thioacetamide induced cirrhotic rats. For this purpose 24 male Albino wistar rats were divided into four groups (n=6). Group I, remained healthy control rats, Group II, received thioacetamide (at a dose of 200mg/kg b.w, i.p, for 12 weeks, twice a week) in first phase and saline in second phase, Group III, received thioacetamide (200mg/kg b.w, i.p for 12 weeks, twice a week) in first phase and silymarin (orally at a dosage of 200mg/kg b.w, twice a week, for 8 weeks) in second phase and Group IV, received silymarin (orally at a dosage of 200mg/kg b.w, twice a week, for 8 weeks) in first phase and saline in second phase. Biochemical analysis was evaluated by total and direct bilirubin (Retiman and Franhel, 1957, Sherlock, 1951), liver specific enzymes, antioxidant enzymes [SOD (Kono et al., 1978), Catalase (Sinha et al., 1979), Glutathione reductase (Calberg and Mannervik, 1985) and MDA (Okhawa et al., 1979)] and plasma and intraerythrocyte sodium and potassium (Tabssum et al., 1996). Marked increase in total and direct bilirubin and ALT activity was the indicative markers of liver cirrhosis while reduced antioxidant activity (SOD and GSH) and increased MDA and Catalase levels and disturbed electrolyte homeostasis were observed in cirrhotic group. Silymarin supplementation markedly reduced total bilirubin and ALT activity and restored the antioxidant enzymes (SOD and GSH), MDA and catalase activity and electrolyte homeostasis. These results indicate that silymarin successively attenuates the thioacetamide induced liver cirrhosis.

Topics: Animals; Antioxidants; Bilirubin; Liver Cirrhosis; Male; Potassium; Protective Agents; Rats; Silymarin; Sodium; Thioacetamide

PU box binding protein (PU.1) is a critical transcription factor involved in many pathological processes. However, its exact role in activation of hepatic stellate cells (HSCs) and liver fibrosis was rarely reported. Here, we found that, in HSCs of PU.1

Topics: Animals; Cell Proliferation; Drug Resistance; Gene Expression Regulation; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Proto-Oncogene Proteins; Sirtuin 1; Thioacetamide; Trans-Activators; Transcription, Genetic

Thioacetamide (TAA) administration is widely used for induction of liver cirrhosis in rats, where reactive oxygen radicals (ROS) and nitric oxide (NO) participate in development of liver damage. Cardiac dysfunction is an important complication of liver cirrhosis, but the role of ROS or NO in cardiac abnormalities during liver cirrhosis is not well understood. This was investigated in animals after TAA-induced liver cirrhosis and temporal changes in oxidative stress, NO and mitochondrial function in the heart evaluated. TAA induced elevation in cardiac levels of nitrate before development of frank liver cirrhosis, without gross histological alterations. This was accompanied by an early induction of P38 MAP kinase, which is influenced by ROS and plays an important signaling role for induction of iNOS. Increased nitrotyrosine, protein oxidation and lipid peroxidation in the heart and cardiac mitochondria, suggestive of oxidative stress, also preceded frank liver cirrhosis. However, compromised cardiac mitochondrial function with a decrease in respiratory control ratio and increased mitochondrial swelling was seen later, when cirrhosis was evident. In conclusion, TAA induces elevations in ROS and NO in the heart in parallel to early liver damage. This leads to later development of functional deficits in cardiac mitochondria after development of liver cirrhosis.

Topics: Animals; Chemical and Drug Induced Liver Injury; Female; Heart Diseases; Lipid Peroxidation; Liver; Liver Cirrhosis; Male; Mitochondria, Heart; Mitochondrial Swelling; Myocytes, Cardiac; Nitrates; Nitric Oxide; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Rats, Wistar; Reactive Oxygen Species; Signal Transduction; Thioacetamide; Time Factors; Tyrosine

The epithelial-mesenchymal transition (EMT) is involved in many different types of cellular behavior, including liver fibrosis. In this report, we studied a novel function of RAR-related orphan receptor gamma (ROR-γ) in hepatocyte EMT during liver fibrosis. To induce EMT in vitro, primary hepatocytes and FL83B cells were treated with TGF-β1. Expression of ROR-γ was analyzed by Western blot in the fibrotic mouse livers and human livers with cirrhosis. To verify the role of ROR-γ in hepatocyte EMT, we silenced ROR-γ in FL83B cells using a lentiviral short hairpin RNA (shRNA) vector. The therapeutic effect of ROR-γ silencing was investigated in a mouse model of TAA-induced fibrosis by hydrodynamic injection of plasmids. ROR-γ expression was elevated in hepatocyte cells treated with TGF-β1, and ROR-γ protein levels were elevated in the fibrotic mouse livers and human livers with cirrhosis. Knockdown of ROR-γ resulted in the attenuation of TGF-β1-induced EMT in hepatocytes. Strikingly, ROR-γ bound to ROR-specific DNA response elements (ROREs) in the promoter region of TGF-β type I receptor (Tgfbr1) and Smad2, resulting in the downregulation of Tgfbr1 and Smad2 after silencing of ROR-γ. Therapeutic delivery of shRNA against ROR-γ attenuated hepatocyte EMT and ameliorated liver fibrosis in a mouse model of TAA-induced liver fibrosis. Overall, our results suggest that ROR-γ regulates TGF-β-induced EMT in hepatocytes during liver fibrosis. We suggest that ROR-γ may become a potential therapeutic target in treating liver fibrosis. J. Cell. Biochem. 118: 2026-2036, 2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.

Topics: Animals; Antigens, CD; Cadherins; Disease Models, Animal; Epithelial-Mesenchymal Transition; Gene Expression Regulation; Hepatocytes; Humans; Liver; Liver Cirrhosis; Mice; Mice, Inbred BALB C; Nuclear Receptor Subfamily 1, Group F, Member 3; Primary Cell Culture; Promoter Regions, Genetic; Protein Binding; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA, Small Interfering; Signal Transduction; Smad2 Protein; Thioacetamide; Transforming Growth Factor beta1

Animal models provide a useful platform for developing and testing new drugs to treat liver fibrosis. Accordingly, we developed a novel automated system to evaluate liver fibrosis in rodent models. This system uses second-harmonic generation (SHG)/two-photon excited fluorescence (TPEF) microscopy to assess a total of four mouse and rat models, using chemical treatment with either thioacetamide (TAA) or carbon tetrachloride (CCl

Topics: Animals; Automation, Laboratory; Bile Ducts; Carbon Tetrachloride; Disease Models, Animal; Hydroxyproline; Ligation; Liver; Liver Cirrhosis; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; Microscopy, Fluorescence, Multiphoton; Rats, Sprague-Dawley; Rats, Wistar; Reproducibility of Results; Sensitivity and Specificity; Species Specificity; Thioacetamide

Mesenchymal stem cells (MSCs) mediate tissue repair and might be used to prevent or reduce liver fibrosis. However, little is known about the anti-fibrotic factors secreted from MSCs or their mechanisms.. Umbilical cord-derived MSCs (UCMSCs) were differentiated into hepatocyte-like cells (hpUCMSCs), medium was collected, and secretome proteins were identified and quantified using nanochip-liquid chromatography/quadrupole time-of-flight mass spectrometry. Liver fibrosis was induced in mice by intraperitoneal injection of thioacetamide or CCl. In mice with fibrosis, accumulation of extracellular matrix proteins was significantly reduced 3 days after injecting secretomes from UCMSCs, and to a greater extent from hpUCMSCs; numbers of activated HSCs that expressed the myogenic marker α-smooth muscle actin (α-SMA, encoded by ACTA2 [actin, alpha 2, smooth muscle]) were also reduced. Secretomes from UCMSCs, and to a greater extent from hpUCMSCs, reduced liver expression of multiple fibrotic factors, collagens, metalloproteinases, TGFβ, and Smad proteins in the TGFβ signaling pathways. In HSC cell lines and primary HSCs, TGFβ1-stimulated upregulation of α-SMA was significantly inhibited (and SMAD2 phosphorylation reduced) by secretomes from UCMSCs, and to a greater extent from hpUCMSCs. We identified 32 proteins in secretomes of UCMSCs that were more highly concentrated in secretomes from hpUCMSCs and inhibited TGFβ-mediated activation of HSCs. One of these, milk fat globule-EGF factor 8 (MFGE8), was a strong inhibitor of activation of human primary HSCs. We found MFGE8 to down-regulate expression of TGFβ type I receptor by binding to α. MFGE8 is an anti-fibrotic protein in MSC secretomes that strongly inhibits TGFβ signaling and reduces extracellular matrix deposition and liver fibrosis in mice.

Topics: Animals; Antigens, Surface; Carbon Tetrachloride; Cell Line; Collagen; Extracellular Matrix; Hepatic Stellate Cells; Hepatocytes; Humans; Integrin alphaVbeta3; Liver Cirrhosis; Male; Mesenchymal Stem Cells; Metabolome; Metalloproteases; Mice; Milk Proteins; Receptors, Transforming Growth Factor beta; Smad2 Protein; Thioacetamide; Transforming Growth Factor beta1

Fibrosis is the excessive accumulation of extracellular matrix components due to chronic injury, with collagens as predominant structural components. Liver fibrosis can progress to cirrhosis, which is characterized by a severe distortion of the delicate hepatic vascular architecture, the shunting of the blood supply away from hepatocytes and the resultant functional liver failure. Cirrhosis is associated with a highly increased morbidity and mortality and represents the major hard endpoint in clinical studies of chronic liver diseases. Moreover, cirrhosis is a strong cofactor of primary liver cancer. In vivo models are indispensable tools to study the cellular and molecular mechanisms of liver fibrosis and to develop specific antifibrotic therapies towards clinical translation. Here, we provide a detailed description of select optimized mouse models of liver fibrosis and state-of-the-art fibrosis readouts.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Sub-Family B Member 4; Azo Compounds; Biomarkers; Carbon Tetrachloride; Collagen; Disease Models, Animal; Disease Progression; Extracellular Matrix; Gene Expression; Histocytochemistry; Humans; Hydroxyproline; Liver; Liver Cirrhosis; Mice; Mice, Inbred C57BL; Mice, Knockout; Thioacetamide

Thioacetamide (TAA), usually used as a fungicide to control the decay of citrus products, itself is not toxic to the liver, but its intermediates are able to increase oxidative stress in livers and further cause fibrosis. Ophiocordyceps sinensis mycelium (OSM) which contains 10% polysaccharides and 0.25% adenosine decreased (P < 0.05) the lipid accumulation and increased (P < 0.05) antioxidative capacity in livers of thioacetamide (TAA) injected rats. Meanwhile, the increased (P < 0.05) liver sizes, serum alanine transaminase (AST) and aspartate transaminase (ALT) values in thioacetamide (TAA)-injected rats were ameliorated (P < 0.05) by OSM supplementation. Moreover, the levels of proinflammatory cytokines, such as the tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), were also reduced (P < 0.05). The fibrosis phenomena in pathological (Masson's trichrome and H&E stainings) and immunohistochemical [α-smooth actin (αSMA) and CD86/ED1] observations in TAA-treated rats were reduced (P < 0.05) by OSM cotreatment. The protective effect of OSM against TAA-induced liver inflammation/fibrosis may be via downregulations (P < 0.05) of TGF-β pathways and NFκB which further influenced (P < 0.05) the expressions of fibrotic and inflammatory genes (i. e., αSMA, Col1α, COX2). Therefore, OSM shows preventive effects on the development of TAA-induced hepatic fibrosis.

Topics: Actins; Alanine Transaminase; Animals; Anti-Inflammatory Agents; Antioxidants; Aspartate Aminotransferases; Cytokines; Hypocreales; Interleukin-1beta; Liver; Liver Cirrhosis; Liver Function Tests; Male; Mycelium; NF-kappa B; Oxidative Stress; Rats, Wistar; Thioacetamide; Tumor Necrosis Factor-alpha

To test a magnetic resonance (MR) scanning protocol as a noninvasive tool to determine hepatic hemodynamics and to assess the degree of liver fibrosis in an animal model of liver fibrosis and cirrhosis.. Fifty-four male Wistar rats were studied. Thirty-nine received thioacetamide (TAA) in their drinking water for either 12 or 16 weeks. MR measurements were performed using flow-sensitive 2D phase-contrast MRI and a 9.4T preclinical scanner. The following hemodynamic parameters were investigated: portal cross-sectional area, mean portal flow velocity, and portal and aortic flow volume rate. Therefore, rats (n = 46) were divided into three groups: CON (control, n = 13), FIB (fibrosis, n = 25), and CIR (cirrhosis, n = 8). Furthermore, the degree of liver fibrosis was assessed by a self-established MR score and verified by a standardized histological score (n = 48).. Portal and aortic flow parameters could be reliably detected. A significant decrease in portal flow velocity was found in FIB (FIB vs. CON: -21%, P = 0.006 and CIR vs. CON: -17%, P = 0.105) and in portal flow volume rate in FIB and CIR (FIB vs. CON: -20%, P = 0.009 and CIR vs. CON: -25%, P = 0.024). If the histological score is taken as standard, the self-established MR score enabled discrimination between healthy and diseased livers (sensitivity to identify diseased livers: 89% and specificity to identify healthy livers: 100%).. This MR scanning protocol presents a noninvasive tool to determine hepatic hemodynamics in healthy and diseased rats.. 2 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2017;46:1526-1534.

Topics: Animals; Hemodynamics; Humans; Hypertension, Portal; Image Processing, Computer-Assisted; Liver; Liver Cirrhosis; Magnetic Resonance Imaging; Male; Observer Variation; Portal Vein; Rats; Rats, Wistar; Regional Blood Flow; Thioacetamide; Water

Silymarin, a mixture of antihepatotoxic flavonolignans used in the treatment of liver diseases, and lactulose, a nonabsorbable synthetic disaccharide, were investigated to analyze their probable synergic and healing effects in a hepatic cirrhotic rat model. Liver damage was induced by the administration and subsequent withdrawal of thioacetamide. The significant decrease in liver enzymes and malondialdehyde levels confirmed the curative effects of silymarin and lactulose. In the silymarin + lactulose group, liver enzyme and malondialdehyde levels were significantly reduced compared with those in the thioacetamide group. All treatments led to liver regeneration and triggered enhanced regeneration. Silymarin and lactulose alone or in combination have potent curative effects and reduce thioacetamide-induced liver damage.

Topics: Animals; Drug Interactions; Lactulose; Lipid Peroxidation; Liver; Liver Cirrhosis; Male; Rats; Rats, Wistar; Silymarin; Thioacetamide

Liver injuries induced by carbon tetrachloride (CCL4) or thioacetamide (TAA) are dependent on cytochrome P450 2E1 (CYP2E1). CYP2A5 can be induced by TAA but not by CCL4. In this study, liver injury including fibrosis induced by CCL4 or TAA were investigated in wild-type (WT) mice and CYP2A5 knockout (cyp2a5 (-/-) ) mice as well as in CYP2E1 knockout (cyp2e1 (-/-) ) mice as a comparison. Acute and subchronic liver injuries including fibrosis were induced by CCL4 and TAA in WT mice but not in cyp2e1 (-/-) mice, confirming the indispensable role of CYP2E1 in CCL4 and TAA hepatotoxicity. WT mice and cyp2a5 (-/-) mice developed comparable acute liver injury induced by a single injection of CCL4 as well as subchronic liver injury including fibrosis induced by 1 month of repeated administration of CCL4, suggesting that CYP2A5 does not affect CCL4-induced liver injury and fibrosis. However, while 200 mg/kg TAA-induced acute liver injury was comparable in WT mice and cyp2a5 (-/-) mice, 75 and 100 mg/kg TAA-induced liver injury were more severe in cyp2a5 (-/-) mice than those found in WT mice. After multiple injections with 200 mg/kg TAA for 1 month, while subchronic liver injury as indicated by serum aminotransferases was comparable in WT mice and cyp2a5 (-/-) mice, liver fibrosis was more severe in cyp2a5 (-/-) mice than that found in WT mice. These results suggest that while both CCL4- and TAA-induced liver injuries and fibrosis are CYP2E1 dependent, under some conditions, CYP2A5 may protect against TAA-induced liver injury and fibrosis, but it does not affect CCL4 hepatotoxicity.

Topics: Alanine Transaminase; Animals; Aryl Hydrocarbon Hydroxylases; Aspartate Aminotransferases; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Cytochrome P-450 CYP2E1; Cytochrome P450 Family 2; Disease Models, Animal; Glutathione; Liver; Liver Cirrhosis; Male; Mice, Inbred C57BL; Mice, Knockout; Thioacetamide; Thiobarbituric Acid Reactive Substances

Adiponectin is an adipocyte-derived circulating protein with beneficial effects on injured livers. Adiponectin-deficient (adipo(-/-)) mice develop enhanced liver fibrosis, suggesting that adiponectin could be a therapeutic target for liver injury. In the present study, we investigated the protective role of ADP355, an adiponectin-based active short peptide, in thioacetamide (TAA)-induced acute injury and chronic liver fibrosis in mice. ADP355 remarkably reduced TAA-induced necroinflammation and liver fibrosis. ADP355 treatment increased liver glycogen, decreased serum alanine transaminase and alkaline phosphatase activity, and promoted body weight gain, hyper-proliferation and hypo-apoptosis. In addition, ADP355 administration suppressed the TAA-induced activation of hepatic stellate cells and macrophages in the liver. These were associated with the inactivation of TGF-β1/SMAD2 signaling and the promotion of AMPK and STAT3 signaling. Sensitivity of adipo(-/-) mice to chronic liver injury was decreased with ADP355. In conclusion, ADP355 could mimic adiponectin's action and may be suitable for the preclinical or clinical therapy of chronic liver injury.

Topics: Adiponectin; Animals; Anti-Inflammatory Agents; Apoptosis; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Disease Progression; Female; Hepatic Stellate Cells; Liver Cirrhosis; Liver Function Tests; Liver Regeneration; Macrophage Activation; Macrophages; Male; Mice; Mice, Knockout; Oligopeptides; Signal Transduction; STAT3 Transcription Factor; Thioacetamide

Heat shock protein 90 (Hsp90) is proposed to be involved in liver disorders. This study was conducted to test effect of 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of Hsp90, on attenuating thioacetamide induced liver fibrosis in vivo.. Four groups of Swiss albino male mice (CD-1 strain) were used as follows: control group; thioacetamide group (received 100mg/kg thioacetamide, ip injection, 3 times/week for 8 weeks); thioacetamide plus 17-AAG groups (received 100mg/kg thioacetamide, ip injection, 3 times/week for 8 weeks plus 25 or 50mg/kg 17-AAG, ip injection, 5 days/week along the last 4 weeks). Fibrosis was quantified by measuring hydroxyproline level and by morphometry and oxidative stress biomarkers were assigned. Relative hepatic mRNA expressions of α-smooth muscle actin (α-SMA), collagen-1-alpha-1 (Col1A1) and tissue inhibitor metalloproteinase-1 (TIMP-1) mRNAs were measured by RT-PCR. Levels of the apoptotic markers caspase-3, factor related apoptosis (Fas) and Hsp-90 were assigned in tissue homogenate.. 17-AAG (50mg/kg) significantly decreased fibrosis percentage significantly (p<0.001, 0.05) compared to thioaceatmide and 25mg/kg, respectively. Malondialdehyde, Hsp90, α-SMA, Col1A1 and TIMP-1 expression levels were significantly reduced (p<0.05) by the inhibitor large dose. Levels of GSH, caspase-3 and Fas were markedly (p<0.001) increased in the group received 17-AAG (50mg/kg) compared to other groups.. The Hsp90 inhibitor, 17-AAG, can attenuate thioacetamide hepatotoxicity through oxidative stress counterbalance, reducing stellate cells activity and inducing apoptosis.

Topics: Actins; Animals; Apoptosis; Benzoquinones; Caspase 3; Collagen Type I; Collagen Type I, alpha 1 Chain; fas Receptor; Glutathione; HSP90 Heat-Shock Proteins; Lactams, Macrocyclic; Liver Cirrhosis; Male; Malondialdehyde; Mice; Oxidative Stress; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1

Liver fibrosis can progress to cirrhosis and result in serious complications of liver disease. The pathogenesis of liver fibrosis involves the activation of hepatic stellate cells (HSCs), the underlying mechanisms of which are not fully known. Emerging evidence suggests that the classic histone deacetylases play a role in liver fibrosis, but the role of another subfamily of histone deacetylases, the sirtuins, in the development of hepatic fibrosis remains unknown. In this study, we found that blocking the activity of sirtuin 2 (SIRT2) by using inhibitors or shRNAs significantly suppressed fibrogenic gene expression in HSCs. We further demonstrated that inhibition of SIRT2 results in the degradation of c-MYC, which is important for HSC activation. In addition, we discovered that inhibition of SIRT2 suppresses the phosphorylation of ERK, which is critical for the stabilization of c-MYC. Moreover, we found that Sirt2 deficiency attenuates the hepatic fibrosis induced by carbon tetrachloride (CCl4) and thioacetamide (TAA). Furthermore, we showed that SIRT2, p-ERK, and c-MYC proteins are all overexpressed in human hepatic fibrotic tissues. These data suggest a critical role for the SIRT2/ERK/c-MYC axis in promoting hepatic fibrogenesis. Inhibition of the SIRT2/ERK/c-MYC axis represents a novel strategy to prevent and to potentially treat liver fibrosis and cirrhosis.

Topics: Adult; Aged; Animals; Carbon Tetrachloride; Case-Control Studies; Cell Line; Extracellular Signal-Regulated MAP Kinases; Female; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Middle Aged; Proto-Oncogene Proteins c-myc; Sirtuin 2; Thioacetamide

Piceatannol is a polyphenolic analog of resveratrol that selectively inhibits the non-receptor tyrosine kinase-Syk. This study investigates the potential ability of piceatannol to attenuate liver fibrosis and protect hepatocytes from injury. Thioacetamide was injected in adult male mice (100 mg/kg, i.p., 3 times/week) for 8 weeks. Piceatannol (1 or 5 mg/kg per day) was administered by oral gavage during the last 4 weeks. Liver function biomarkers, tissue malondialdehyde (MDA), cytokeratin-18 (CK18), hepatocyte growth factor (HGF), and interleukin-10 (IL-10) were measured. Necroinflammation, fibrosis, expression of transforming growth factor (TGF)-β1, and α-smooth muscle actin (SMA) were scored by histopathological examination and immunohistochemistry. Obtained results showed ability of piceatannol (1 mg/kg) to restore liver function and reduce inflammation. It significantly (p < 0.001) reduced MDA, CK18, TGF-β1, and α-SMA expression, and increased HGF and IL-10. It can be concluded that piceatannol at low dose can inhibit TGF-β1 induced hepatocytes apoptosis and exerts an anti-inflammatory effect attenuating fibrosis progression.

Topics: Animals; Gene Expression Regulation; Hepatocyte Growth Factor; Hepatocytes; Interleukin-10; Liver Cirrhosis; Male; Mice; Protective Agents; Stilbenes; Thioacetamide

The study evaluated the potential protective effect and underlying mechanism of Cucurbitacin E (CuE) in both thioacetamide-induced hepatic fibrosis and activated HSCs. CuE inhibited the proliferation of activated HSC/T-6 cells in a concentration- and time-dependent manner; triggered the activation of caspase-3, cleaved PARP, altered ratio of bcl-2-to-bax, and affected cytochrome C protein in a time- and concentration-dependent manner. CuE arrested activated HSCs at the G2/M phase. Furthermore, CuE reduced levels of p-Erk/MAPK and also inhibited the protein and mRNA expressions of α-SMA, TIMP-1 and collagen I in activated HSC-T6 cells. CuE inhibited PI3K and Akt phosphorylation, and reduced the levels of p-mTOR and p-P70S6K and increased the expression of p-AMPK, which is similar with AICAR and metformin. C57BL/6 mice were intraperitoneally injected with thioacetamide (TAA) for five continuous weeks (100 or 200mg/kg, three times per week) along with daily administration of CuE (5 or 10mg/kg/d) and curcumin (Cur, 20mg/kg). CuE treatments significantly reduced serum ALT/AST levels, α-SMA, TIMP-1, and collagen I protein expressions. HE, Masson trichrome, Sirius red and immunohistochemical staining also suggested that CuE could ameliorate hepatic fibrosis. Our findings suggest that CuE induces apoptosis of activated HSC and ameliorates TAA-induced hepatic fibrosis through activation of AMPK and blocking mTOR-dependent signaling pathway.

Topics: AMP-Activated Protein Kinases; Animals; Antioxidants; Cell Line, Tumor; Cell Proliferation; Dietary Supplements; Disease Models, Animal; Enzyme Activation; G2 Phase; Gene Expression Regulation; Hepatic Stellate Cells; Liver Cirrhosis; Male; Mice, Inbred C57BL; Random Allocation; Signal Transduction; Specific Pathogen-Free Organisms; Thioacetamide; TOR Serine-Threonine Kinases; Triterpenes

Demethyleneberberine (DMB) is an essential metabolite of Berberine (BBR) in vivo. Recent reports have revealed multiple novel therapeutic applications of BBR. However, the pharmacological activities of DMB remain to be elucidated. This study aimed to demonstrate the hepatoprotective and anti-fibrotic effects of DMB both in vitro and in vivo. Here we showed that DMB protects against thioacetamide (TAA)-induced hepatic fibrosis in mice and exhibits a higher safety profile as compared to BBR. Flow cytometry and Western blotting analysis showed that DMB is able to suppress the activation of hepatic stellate cells (HSCs) and induce cell apoptosis through the nuclear factor-κB (NF-κB) cascade. Immunohistochemical (IHC) and quantitative polymerase chain reaction (qPCR) analysis indicated that DMB also has inhibitory effects on collagen synthesis and is able to increase collagen degradation by blocking the transforming growth factor β 1 (TGF-β1)-Smad signaling and reducing the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMP (TIMPs). These findings indicate that DMB has the potential to attenuate hepatic fibrosis via suppressing HSC activation.

Topics: Actins; Animals; Apoptosis; Berberine; Cell Line; Collagen; Disease Models, Animal; Hepatic Stellate Cells; Liver Cirrhosis; Male; Matrix Metalloproteinases; Mice; Mice, Inbred ICR; NF-kappa B; Protective Agents; Rats; Signal Transduction; Smad Proteins; Thioacetamide; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta1

In chronic liver diseases, regardless of their etiology, the development of fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. Hepatic stellate cells (HSCs) are the main profibrogenic cells that promote the pathogenesis of liver fibrosis, and so it is important to identify the molecules that regulate HSCs activation and liver fibrosis. Niemann-Pick type C2 (NPC2) protein plays an important role in the regulation of intracellular cholesterol homeostasis by directly binding with free cholesterol. However, the roles of NPC2 in HSCs activation and liver fibrosis have not been explored in detail. Since a high-cholesterol diet exacerbates liver fibrosis progression in both rodents and humans, we propose that the expression of NPC2 affects free cholesterol metabolism and regulates HSCs activation. In this study, we found that NPC2 is decreased in both thioacetamide- and carbon tetrachloride-induced liver fibrosis tissues. In addition, NPC2 is expressed in quiescent HSCs, but its activation status is down-regulated. Knockdown of NPC2 in HSC-T6 cells resulted in marked increases in transforming growth factor-β1 (TGF-β1)-induced collagen type 1 α1 (Col1a1), α-smooth muscle actin (α-SMA) expression, and Smad2 phosphorylation. In contrast, NPC2 overexpression decreased TGF-β1-induced HSCs activation. We further demonstrated that NPC2 deficiency significantly increased the accumulation of free cholesterol in HSCs, increasing Col1a1 and α-SMA expression and activating Smad2, and leading to sensitization of HSCs to TGF-β1 activation. In contrast, overexpression of NPC2 decreased U18666A-induced free cholesterol accumulation and inhibited the subsequent HSCs activation. In conclusion, our study has demonstrated that NPC2 plays an important role in HSCs activation by regulating the accumulation of free cholesterol. NPC2 overexpression may thus represent a new treatment strategy for liver fibrosis.

Topics: Animals; Blotting, Western; Carbon Tetrachloride; Carrier Proteins; Cholesterol; Disease Models, Animal; Glycoproteins; Hepatic Stellate Cells; Humans; Immunoenzyme Techniques; Liver Cirrhosis; Mice; Mice, Inbred C57BL; Real-Time Polymerase Chain Reaction; Thioacetamide; Transforming Growth Factor beta1; Vesicular Transport Proteins

Liver steatosis is the most frequent liver disease and may further develop into non-alcoholic steatohepatitis (NASH), liver cirrhosis, and finally hepatocellular carcinoma. Adipophilin (Adp) is localized on lipid droplet membrane in cytoplasm, and its increased expression is related to development of steatosis and NASH. The relationship between M1-/M2-macrophage polarization and Adp-rich hepatocyte-consisting pseudolobules (PLs) was investigated in thioacetamide (TAA)-induced rat cirrhosis.. F344 rats were injected twice weekly with TAA (100mg/kg bodyweight) and sacrificed at post-first injection (PFI) weeks 5, 10, 15, 20, 25 and 32. Macrophage immunophenotypes and Adp-containing hepatocytes were analyzed by single immunolabeling. Adp and M1-/M2-related factors were analyzed by real -time RT-PCR.. PLs consisting exclusively of Adp-containing hepatocytes (Adp-positive) and PLs consisting of few Adp-containing hepatocytes (Adp-negative) were clearly distinguishable at PFI week 20 onwards. The numbers of M1-macrophages (reacting to CD68 and Iba1) and M2- macrophages (reacting to CD163, CD204 and Gal-3) were considerably greater in Adp-positive PLs. Expressions for both M1 (TNF-α, MCP-1, and Iba1)- and M2 (IL-4, TGF-β1, Gal-3, and Hsp25)-related factors were markedly higher in Adp-positive PLs at PFI week 25. Interestingly, MHC class II-positive macrophages/dendritic cells were increased in Adp-positive clusters/foci at the early stages at PFI weeks 5 and 10, and the level was gradually decreased thereafter.. M1-/M2-macrophages may simultaneously participate in the pathogenesis of steatosis in TAA-induced cirrhosis through M1- and M2-related factors. MHC class II cells may be responsible for steatosis at early stages, suggesting different functions from the above M1-/M2-macropahges.

Topics: Animals; Antigens, CD; Apoptosis; Cell Polarity; Cell Proliferation; Galectin 3; Gene Expression Regulation; Hepatocytes; Immunohistochemistry; Immunophenotyping; Kinetics; Liver; Liver Cirrhosis; Macrophages; Male; Perilipin-2; Rats, Inbred F344; RNA, Messenger; Thioacetamide

Vitamin D was found to be involved in liver fibrosis modulation through binding to its receptor (VDR) halting many fibrotic pathways. Targeting vitamin D-VDR axis using vitamin D analogs may represent an efficient strategy for liver fibrosis treatment . The study aims at testing the potential ability of the VDR agonist, calcipotriol, to stop fibrosis progression and/or regeneration of hepatocytes in an experimental model of liver fibrosis. Mice (CD-1) were injected with thioacetamide (TAA, 100mg/kg, i.p., 3 times/week) for 8 weeks to induce fibrosis and were treated with calcipotriol (20, 60 or 80µg/kg, i.p., daily) concurrently with TAA during the last 4 weeks. Liver function and oxidative stress biomarkers were measured by the end of the study and hepatic sections were examined for inflammation, necrosis and fibrosis percentage. Additionally, liver contents of collagen-1-alpha-1 (COL1a1), transforming growth factor (TGF)-β1 and phospho-Smad2 (Ser456/467)/Smad3 (Ser423/425) were measured. Finally, expression of TGF-β1, tissue inhibitor metalloproteinase (TIMP)-1, Smad2/3 and Smad1/5/9 were scored using immunohistochemistry techniques. Mainly, calcipotriol (80µg/kg) significantly (P<0.001) reduced fibrosis percentage and improved TAA effect on transaminases, alkaline phosphatase, COL1a1 level, malondialdehyde, albumin and reduced glutathione (GSH). It also decreased the profibrogenic cytokine TGF-β1, TIMP-1, Smad2/3, Smad1/5/9 and phospoSmad2/3 significantly (P<0.01) when compared to TAA group. Calcipotriol attenuates TAA induced liver fibrosis and can stop its progression through limiting stellate cells activity by decreasing TGF-β1 level and modulating TGF-β1/Smad signaling pathway. It also can help fibrolysis through decreasing TIMP-1 and restoring the balance between metalloproteinases and their inhibitors.

Topics: Animals; Calcitriol; Collagen Type I; Dose-Response Relationship, Drug; Liver; Liver Cirrhosis; Male; Malondialdehyde; Mice; Receptors, Calcitriol; Thioacetamide

The microRNA-29 (miR-29) family is known to suppress the activation of hepatic stellate cells (HSCs) and reversibly control liver fibrosis; however, the mechanism of how miR-29a controls liver fibrosis remains largely unknown. This study was conducted to clarify the mechanism of anti-fibrotic effect of miR-29a and to explore if miR-29a is a promising candidate for nucleic acid medicine against liver fibrosis. Two liver fibrosis murine models (carbon tetrachloride or thioacetamide) were used. MiR-29a mixed with atelocollagen was systemically administered. Hepatic fibrosis was evaluated by histological analysis and the expression levels of fibrosis-related genes. We observed that miR-29a treatment dramatically accelerated the reversion of liver fibrosis in vivo. Additionally, miR-29a regulated the mRNA expression of collagen type I alpha 1 (COL1A1) and platelet-derived growth factor C (PDGFC). We also noted that miR-29a significantly suppressed COL1A1 mRNA expression and cell viability and significantly increased caspase-9 activity (P < 0.05) in LX-2 cells. Pretreatment of miR-29a inhibited activation of LX-2 cell by transforming growth factor beta treatment. MiR-29a exhibited anti-fibrotic effect without cell toxicity in vivo and directly suppressed the expression of PDGF-related genes as well as COL1A1 and induced apoptosis of LX-2 cells. MiR-29a is a promising nucleic acid inhibitor to target liver fibrosis.

Topics: Animals; Carbon Tetrachloride; Cell Line; Cell Survival; Collagen; Collagen Type I; Collagen Type I, alpha 1 Chain; Disease Models, Animal; Gene Expression Regulation; Genetic Therapy; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Lymphokines; Mice; MicroRNAs; Platelet-Derived Growth Factor; Thioacetamide

Cirrhosis, the end-stage of hepatic fibrosis, is not only life-threatening by itself, but also a causative factor of liver cancer. Despite efforts to develop treatment for liver fibrosis, there are no approved agents as anti-fibrotic drugs to date. In the present study, we aimed to investigate the anti-fibrotic effect of the AMP-activated protein kinase (AMPK) activator, HL156A. A mouse model of thioacetamide (TAA)-induced liver fibrosis was used to examine the effect of HL156A in vivo. Mice received either TAA alone or a combination of TAA and HL156A intraperitoneally for a total duration of 6 weeks. Including HL156A during exposure to TAA significantly reduced extracellular matrix (ECM) deposition and production of the hepatic transforming growth factor-β1 (TGF-β1). Immunohistochemical analysis revealed that the activation of hepatic stellate cells and the capillarization of liver sinusoids were also diminished significantly by HL156A co-treatment. The anti-fibrotic effect of HL156A was further studied in vitro by using a rat hepatic stellate cell line, HSC-T6 cells. The induction of α-smooth muscle actin (α-SMA) by TGF-β1 treatment was reversed by HL156A, which was likely via the activation of AMPK. Moreover, HL156A showed anti-inflammatory effects on macrophages. Treatment with HL156A diminished LPS-induced activation of both Raw264.7 macrophage cells and primary cultured mouse macrophages. Taken together, these results imply that the AMPK activator HL156A inhibits hepatic fibrosis via multiple mechanisms and could be a potentially effective agent for fibrosis treatment.

Topics: Actins; Animals; Cell Line; Disease Models, Animal; Extracellular Matrix; Gene Expression Regulation; Guanidines; Hepatic Stellate Cells; Humans; Injections, Intraperitoneal; Liver Cirrhosis; Macrophages; Mice; Pyrrolidines; Rats; RAW 264.7 Cells; Thioacetamide; Transforming Growth Factor beta1

Compound-induced liver injury leading to fibrosis remains a challenge for the development of an Adverse Outcome Pathway useful for human risk assessment. Latency to detection and lack of early, systematically detectable biomarkers make it difficult to characterize the dynamic and complex intercellular interactions that occur during progressive liver injury. Here, we demonstrate the utility of bioprinted tissue constructs comprising primary hepatocytes, hepatic stellate cells, and endothelial cells to model methotrexate- and thioacetamide-induced liver injury leading to fibrosis. Repeated, low-concentration exposure to these compounds enabled the detection and differentiation of multiple modes of liver injury, including hepatocellular damage, and progressive fibrogenesis characterized by the deposition and accumulation of fibrillar collagens in patterns analogous to those described in clinical samples obtained from patients with fibrotic liver injury. Transient cytokine production and upregulation of fibrosis-associated genes ACTA2 and COL1A1 mimics hallmark features of a classic wound-healing response. A surge in proinflammatory cytokines (eg, IL-8, IL-1β) during the early culture time period is followed by concentration- and treatment-dependent alterations in immunomodulatory and chemotactic cytokines such as IL-13, IL-6, and MCP-1. These combined data provide strong proof-of-concept that 3D bioprinted liver tissues can recapitulate drug-, chemical-, and TGF-β1-induced fibrogenesis at the cellular, molecular, and histological levels and underscore the value of the model for further exploration of compound-specific fibrogenic responses. This novel system will enable a more comprehensive characterization of key attributes unique to fibrogenic agents during the onset and progression of liver injury as well as mechanistic insights, thus improving compound risk assessment.

Topics: Biomarkers; Bioprinting; Cells, Cultured; Chemical and Drug Induced Liver Injury; Coculture Techniques; Collagen; Cytokines; Dose-Response Relationship, Drug; Endothelial Cells; Feasibility Studies; Gene Expression Regulation; Hepatic Stellate Cells; Hepatocytes; Humans; Liver; Liver Cirrhosis; Methotrexate; Phenotype; Printing, Three-Dimensional; Risk Assessment; Thioacetamide; Time Factors

Hepatic inflammation drives hepatic stellate cells (HSC), resulting in liver fibrosis. The Farnesoid-X receptor (FXR) antagonizes inflammation through NF-κB inhibition. We investigated preventive and therapeutic effects of FXR agonist obeticholic acid (OCA) on hepatic inflammation and fibrosis in toxic cirrhotic rats. Cirrhosis was induced by thioacetamide (TAA) intoxication. OCA was given during or after intoxication with vehicle-treated rats as controls. At sacrifice, fibrosis, hemodynamic and biochemical parameters were assessed. HSC activation, cell turn-over, hepatic NF-κB activation, pro-inflammatory and pro-fibrotic cytokines were determined. The effect of OCA was further evaluated in isolated HSC, Kupffer cells, hepatocytes and liver sinusoidal endothelial cells (LSEC). OCA decreased hepatic inflammation and fibrogenesis during TAA-administration and reversed fibrosis in established cirrhosis. Portal pressure decreased through reduced intrahepatic vascular resistance. This was paralleled by decreased expression of pro-fibrotic cytokines (transforming growth-factor β, connective tissue growth factor, platelet-derived growth factor β-receptor) as well as markers of hepatic cell turn-over, by blunting effects of pro-inflammatory cytokines (e.g. monocyte chemo-attractant protein-1). In vitro, OCA inhibited both LSEC and Kupffer cell activation; while HSC remained unaffected. This related to NF-κB inhibition via up-regulated IκBα. In conclusion, OCA inhibits hepatic inflammation in toxic cirrhotic rats resulting in decreased HSC activation and fibrosis.

Topics: Animals; Apoptosis; Biomarkers; Cell Cycle; Cell Line; Cell Proliferation; Chenodeoxycholic Acid; Cytokines; Disease Models, Animal; Endothelial Cells; Hemodynamics; Hepatic Stellate Cells; Hepatocytes; Humans; Inflammation; Inflammation Mediators; Kupffer Cells; Lipopolysaccharides; Liver; Liver Cirrhosis; Male; Mice; NF-kappa B; NF-KappaB Inhibitor alpha; Portal Pressure; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Thioacetamide; Tumor Necrosis Factor-alpha; Up-Regulation; Vascular Resistance

It has been reported previously that the density of angiotensin II receptors is increased in the rat liver in experimentally-induced fibrosis. We hypothesized that pharmacological blockade of angiotensin receptors may produce beneficial effects in models of liver fibrosis. In this study, we used the widely used thioacetamide (TAA)-induced model of liver fibrosis (300 mg/L TAA ad libitum for 12 weeks). Rats received daily injections (i.p), lasting 4 weeks of the angiotensin II type 1 receptor antagonists, losartan 30 mg/kg (TAA + L) or telmisartan 10 mg/kg (TAA + T) and were compared to rat that received TAA alone. Chronic treatment with losartan and telmisartan was associated with a significant reduction in the activity of alkaline phosphatase, and decreased concentrations of tumor necrosis factor-alpha and transforming growth factor beta-1 compared to controls. We also found a significant reduction interleukin-6 in rats receiving telmisartan (P < 0.05) but not losartan. Both treatments increased the concentration of liver glutathione along with a concomitant decrease of GSSG compared to controls. In addition, increased paraoxonase 1 activity was observed in the serum of rats receiving telmisartan group compared to the TAA alone controls. Finally, histological evaluation of liver sections revealed losartan and telmisartan treatment was associated with reduced inflammation and liver fibrosis. Taken together, these results indicate that both telmisartan and losartan have anti-inflammatory and anti-oxidative properties in the TAA model of liver fibrosis. These finding add support to a growing body of literature indicating a potentially important role for the angiotensin system in liver fibrosis and indicate angiotensin antagonists may be useful agents for fibrosis treatment.

Topics: Alanine Transaminase; Alkaline Phosphatase; Angiotensin II Type 1 Receptor Blockers; Animals; Anti-Inflammatory Agents; Antioxidants; Aryldialkylphosphatase; Aspartate Aminotransferases; Benzimidazoles; Benzoates; Cytokines; Glutathione; Glutathione Disulfide; Liver; Liver Cirrhosis; Losartan; Male; Rats, Wistar; Telmisartan; Thioacetamide

Collagen produced during the process of liver fibrosis can induce a hepatocellular protective response through ERK1 signalling. However, the influence of T cells and associated cytokine production on this protection is unknown. In addition, athymic mice are frequently used in hepatocellular carcinoma xenograft experiments but current methods limit our ability to study the impact of liver fibrosis in this setting due to high mortality. Therefore, a mouse model of liver fibrosis lacking T cells was developed using Foxn1 nu/nu mice and progressive oral administration of thioacetamide (TAA) [0.01-0.02%] in drinking water. Fibrosis developed over a period of 16 weeks (alpha-SMA positive area: 20.0 ± 2.2%, preCol1a1 mRNA expression: 11.7 ± 4.1 fold changes, hydroxyproline content: 1041.2 ± 77μg/g of liver) at levels comparable to that of BALB/c mice that received intraperitoneal TAA injections [200 μg/g of body weight (bw)] (alpha-SMA positive area: 20.9 ± 2.9%, preCol1a1 mRNA expression: 13.1 ± 2.3 fold changes, hydroxyproline content: 931.6 ± 14.8μg/g of liver). No mortality was observed. Athymic mice showed phosphorylation of ERK1/2 during fibrogenesis (control 0.03 ± 0.01 vs 16 weeks 0.22 ± 0.06AU; P<0.05). The fibrosis-induced hepatoprotection against cytotoxic agents, as assessed histologically and by serum AST levels, was not affected by the absence of circulating T cells (anti-Fas JO2 [0.5μg/g bw] for 6h (fibrotic 4665 ± 2596 vs non-fibrotic 13953 ± 2260 U/L; P<0.05), APAP [750 mg/kg bw] for 6 hours (fibrotic 292 ± 66 U/L vs non-fibrotic 4086 ± 2205; P<0.01) and CCl4 [0.5mL/Kg bw] for 24h (fibrotic 888 ± 268 vs non-fibrotic 15673 ± 2782 U/L; P<0.001)). In conclusion, liver fibrosis can be induced in athymic Foxn1 nu/nu mice without early mortality. Liver fibrosis leads to ERK1/2 phosphorylation. Finally, circulating T lymphocytes and associated cytokines are not involved in the hepatocellular protection afforded by liver fibrosis.

Topics: Animals; Blotting, Western; Carcinogens; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Fluorescent Antibody Technique; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred BALB C; Mice, Nude; T-Lymphocytes; Thioacetamide; Transcriptome

Cirrhosis is a major health problem worldwide and new therapies are needed. Hepatic macrophages (hMø) have a pivotal role in liver fibrosis, being able to act in both its promotion and its resolution. It is well-known that mesenchymal stromal cells (MSCs) can modulate the immune/inflammatory cells. However, the effects of MSCs over hMø in the context of liver fibrosis remain unclear. We previously described evidence of the antifibrotic effects of in vivo applying MSCs, which were enhanced by forced overexpression of insulin-like growth factor 1 (AdIGF-I-MSCs). The aim of this work was to analyze the effect of MSCs on hMø behavior in the context of liver fibrosis resolution.. Fibrosis was induced in BALB/c mice by chronic administration of thioacetamide (8 weeks). In vivo gene expression analyses, in vitro experiments using hMø isolated from the nonparenchymal liver cells fraction, and in vivo experiments with depletion of Mø were performed.. One day after treatment, hMø from fibrotic livers of MSCs-treated animals showed reduced pro-inflammatory and pro-fibrogenic gene expression profiles. These shifts were more pronounced in AdIGF-I-MSCs condition. This group showed a significant upregulation in the expression of arginase-1 and a higher downregulation of iNOS expression thus suggesting decreased levels of oxidative stress. An upregulation in IGF-I and HGF expression was observed in hMø from AdIGF-I-MSCs-treated mice suggesting a restorative phenotype in these cells. Factors secreted by hMø, preconditioned with MSCs supernatant, caused a reduction in the expression levels of hepatic stellate cells pro-fibrogenic and activation markers. Interestingly, hMø depletion abrogated the therapeutic effect achieved with AdIGF-I-MSCs therapy. Expression profile analyses for cell cycle markers were performed on fibrotic livers after treatment with AdIGF-I-MSCs and showed a significant regulation in genes related to DNA synthesis and repair quality control, cell cycle progression, and DNA damage/cellular stress compatible with early induction of pro-regenerative and hepatoprotective mechanisms. Moreover, depletion of hMø abrogated such effects on the expression of the most highly regulated genes.. Our results indicate that AdIGF-I-MSCs are able to induce a pro-fibrotic to resolutive phenotype shift on hepatic macrophages, which is a key early event driving liver fibrosis amelioration.

Topics: Animals; Cells, Cultured; Down-Regulation; Gene Expression; Hepatic Stellate Cells; Hepatocyte Growth Factor; Hepatocytes; Insulin-Like Growth Factor I; Liver; Liver Cirrhosis; Macrophages; Male; Mesenchymal Stem Cells; Mice; Mice, Inbred BALB C; Thioacetamide; Up-Regulation

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cells, Cultured; Curcumin; Drug Delivery Systems; Humans; Hyaluronan Receptors; Hyaluronic Acid; Liver Cirrhosis; Male; Mice, Inbred BALB C; Nanoparticles; Polyesters; Protective Agents; Rats; Thioacetamide

Valproic acid (VPA) has been reported as inhibitor of histone deacetylases (HDACs). Several reports indicated that HDACs play a crucial role in the pathogenesis of fibrosis and hepatic stellate cell (HSC) activation. The present study was aimed to evaluate the anti-fibrotic effect of VPA against thioacetamide (TAA)-induced hepatic fibrosis and activation of the HSC in rat. VPA and TAA were administrated intraperitoneally at the dose of 400 and 200 mg/kg each at 2 days interval, respectively for a period of 6 weeks. Administration of TAA significantly increased the absolute and relative liver weight, aspartate aminotransferase and alanine aminotransferase levels, which were significantly decreased by VPA treatment as compared to TAA control. VPA treatment prevents the TAA-induced activation of HSC and decreases collagen deposition and infiltration of inflammatory cells as revealed by Sirius red and H&E staining. Interestingly, VPA co-treatment led to significantly increase the DNA damage and apoptosis in the activated HSC as compared to TAA control. Further, TAA decreased the expression of matrix metalloproteinase-2 (MMP-2), while VPA co-treatment significantly increased the expression of MMP-2 as compared to respective control. The present study clearly demonstrated that VPA treatment significantly alleviates TAA-induced activation of HSC and subsequent hepatic fibrosis.

Topics: Alanine Transaminase; Animals; Apoptosis; Aspartate Aminotransferases; Body Weight; Comet Assay; DNA Damage; Glutathione; Hepatic Stellate Cells; Lipid Peroxidation; Liver; Liver Cirrhosis; Male; Matrix Metalloproteinase 2; Microscopy, Electron, Transmission; Organ Size; Rats, Wistar; Thioacetamide; Thiobarbituric Acid Reactive Substances; Valproic Acid

We previously reported the in vitro and in vivo hepatoprotective and anti-fibrotic effects of PF2401-SF, a standardized fraction of Salvia miltiorrhiza, against acute and subacute liver injury. The aim of this study was to investigate the effect of PF2401-SF on liver fibrosis induced by thioacetamide (TAA), a chronic liver injury model (12 weeks) that closely resembles fibrosis and cirrhosis in humans. Hepatoprotective activity was indicated by low serum levels of the markers aspartate amino transferase and alanine amino transferase .In addition, compared to the TAA-group livers, the PF2401-SF-treated liver tissues showed no fibrous tissue deposition in the portal areas, hepatocyte morphology more closely resembling normal tissue morphology, and significantly reduced collagen deposition. Furthermore, downregulation of collagen 1(α) and tissue inhibitor of metalloproteinase (TIMP)1 protein and mRNA expression also supports PF2401-SF's anti-fibrotic effect. We also observed reduced expression of α-smooth muscle actin (α-SMA), an important marker of hepatic stellate cells (HSCs) activation. From these results, we conclude that PF2401-SF's anti-fibrotic mechanism in the TAA model involves reduced HSC activation, and may be mediated by downregulation of central markers of fibrosis, including collagen 1(α), TIMP1, and α-SMA.

Topics: Animals; Drugs, Chinese Herbal; Liver Cirrhosis; Male; Plant Extracts; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza; Thioacetamide; Treatment Outcome

1,25(OH)2D3, the active form of vitamin D, has an antiproliferative and antifibrotic effect on hepatic stellate cells. Our aim was to investigate the potential of 1,25(OH)2D3 to inhibit the development of liver fibrosis and to ameliorate established fibrosis in vivo. The antifibrotic effect of 1,25(OH)2D3 was investigated in a thioacetamide (TAA) model (as a preventive treatment and as a remedial treatment) and in a bile duct ligation model. In the preventive model, rats received simultaneously intraperitoneum injection of TAA and/or 1,25(OH)2D3 for 10 wk. In the remedial model, rats were treated with TAA for 10 wk and then received 1,25(OH)2D3 or saline for 8 wk. Fibrotic score was determined by Masson staining. Collagen I, α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase-1 (TIMP1), platelet-derived growth factor (PDGF), and transforming growth factor-β (TGF-β) expression were measured by Western blot analysis and real-time PCR. Hypercalemia was detected by chemistry measurements. Preventive treatment of 1,25(OH)2D3 significantly suppressed liver fibrosis both macroscopically and microscopically and significantly lowered the fibrotic score of the TAA + 1,25(OH)2D3 group compared with the TAA group. 1,25(OH)2D3 significantly inhibited expression of PDGF and TGF-β by ∼50% and suppressed the expression of collagen Iα1, TIMP1, and α-SMA by approximately three-, two-, and threefold, respectively. In contrast, 1,25(OH)2D3 was inefficient in amelioration of established liver fibrosis. Administration of 1,25(OH)2D3 to bile duct ligation rats led to a high mortality rate probably caused by hypercalcemia. We conclude that 1,25(OH)2D3 may be considered as a potential preventive treatment in an in vivo model but failed to ameliorate established cirrhosis.

Topics: Animals; Cell Proliferation; Disease Models, Animal; Fibrosis; Hepatic Stellate Cells; Liver Cirrhosis; Liver Cirrhosis, Experimental; Male; Rats, Wistar; Thioacetamide; Vitamin D

Hepatic stellate cells, the principal fibrogenic cell type in the liver, are known to express the astrocyte marker glial fibrillary acidic protein (GFAP). However, the exact role of GFAP-expressing cells in liver fibrosis remains to be elucidated. In this study, cellular properties of GFAP-expressing cells were investigated in a rat model of liver cirrhosis. Six-week-old male F344 rats were injected intraperitoneally with thioacetamide (100 mg/kg BW, twice a week) and examined at post first injection weeks 5, 10, 15, 20 and 25. Appearance of GFAP-expressing myofibroblasts peaked at week 15, associated with fibrosis progression. The majority of GFAP-expressing myofibroblasts co-expressed vimentin, desmin and alpha-smooth muscle actin. Some GFAP-positive myofibroblasts co-expressed nestin (neural stem cell marker), while a few co-expressed A3 (mesenchymal stem cell marker) and Thy-1 (immature mesenchymal cell marker). A few GFAP expressing cells underwent both mitosis and apoptosis. These results indicate that there is a dynamic participation of GFAP-expressing myofibroblasts in rat liver cirrhosis, and that they are mainly derived from hepatic stellate cells, and partly from cells in the stem cell lineage. These findings, which were shown for the first time in detail, would be useful to understand the role of GFAP-expressing myofibroblasts in the pathogenesis of chemically induced liver cirrhosis.

Topics: Animals; Cell Lineage; Disease Models, Animal; Glial Fibrillary Acidic Protein; Hepatic Stellate Cells; Immunohistochemistry; In Situ Nick-End Labeling; Liver Cirrhosis; Male; Microscopy, Confocal; Myofibroblasts; Rats; Rats, Inbred F344; Real-Time Polymerase Chain Reaction; Stem Cells; Thioacetamide

Translocation of bacteria and their products across the intestinal barrier is common in patients with liver disease, and there is evidence that experimental liver fibrosis depends on bacterial translocation. The purpose of our study was to investigate liver fibrosis in conventional and germ-free (GF) C57BL/6 mice. Chronic liver injury was induced by administration of thioacetamide (TAA) in the drinking water for 21 wk or by repeated intraperitoneal injections of carbon tetrachloride (CCl4). Increased liver fibrosis was observed in GF mice compared with conventional mice. Hepatocytes showed more toxin-induced oxidative stress and cell death. This was accompanied by increased activation of hepatic stellate cells, but hepatic mediators of inflammation were not significantly different. Similarly, a genetic model using Myd88/Trif-deficient mice, which lack downstream innate immunity signaling, had more severe fibrosis than wild-type mice. Isolated Myd88/Trif-deficient hepatocytes were more susceptible to toxin-induced cell death in culture. In conclusion, the commensal microbiota prevents fibrosis upon chronic liver injury in mice. This is the first study describing a beneficial role of the commensal microbiota in maintaining liver homeostasis and preventing liver fibrosis.

Topics: Animals; Apoptosis; Blotting, Western; Cell Proliferation; Cells, Cultured; Chromatography, Liquid; Disease Models, Animal; Hepatocytes; Humans; Immunoenzyme Techniques; Inflammation; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Microbiota; Protective Agents; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thioacetamide

Tanshinone IIA (TA) has been recently used to treat liver diseases. However, the poor water solubility and fast metabolism obstruct TA in to be used for the treatment of liver diseases. To overcome this, TA was encapsulated into globin to form nanoparticles (TA-Gb-NPs) by our self-assembling method. We evaluated their biodistribution, pharmacokinetics, targeting ability to liver and antifibrotic effects. As a result, TA-Gb-NPs had a good hepatic targeting ability and achieved higher concentration and longer retention in liver than tanshinone IIA suspension (TA-S). Compared with TA-S, TA-Gb-NPs significantly improved serum biochemical parameters in thioacetamide (TAA) induced liver fibrosis mouse model. Furthermore, histological analysis of mouse liver slices revealed that TA-Gb-NPs could markedly reduce the fibrosis scores and attenuate the progression of the hepatic fibrosis. In conclusion, the TA-Gb-NPs may be a good candidate for the treatment of hepatic fibrosis.

Topics: Abietanes; Animals; Calorimetry, Differential Scanning; Chemistry, Pharmaceutical; Drug Delivery Systems; Drug Stability; Fibrinolytic Agents; Globins; In Vitro Techniques; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred ICR; Nanoparticles; Thioacetamide; Tissue Distribution; X-Ray Diffraction

Recent studies have shown that increased expression of liver hypoxia inducible factor 2-α (HIF-2α) leads to liver inflammation and a pro-fibrotic gene expression signature. Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) is required for HIF-2α transcriptional activity and has previously been shown to regulate hepatic metabolism in mice. In these studies we examined the role of hepatocyte ARNT in the thioacetamide (TAA)-induced model of liver fibrosis.. Hepatocyte-specific ARNT-null (LARNT) mice were created using an albumin promoter-driven Cre recombinase. LARNT and floxed control (FC) littermates were placed on chow diet and received twice weekly intraperitoneal injections of 0.15mg/g body weight of TAA for 13 weeks.. TAA treated LARNT and FC mice had a similar pattern of fibrosis. Quantification of Sirius red histology staining and hydroxyproline content revealed mixed results in terms of collagen deposition in LARNT livers. There was no significant difference in hepatocyte apoptosis or proliferation, as assessed by cleaved Caspase-3 and Ki67 respectively. LARNT mice had decreased macrophage accumulation, and decreased liver mRNA expression of Col1A1, Col1A2, Col5A1, Tgfβ1, Tgfβ2, Timp1 and Timp2.. Deletion of hepatocyte ARNT leads to altered expression of collagen associated mRNA and reduced macrophage infiltration in the TAA-induced model of liver fibrosis. It appears that hepatocyte ARNT is not a requirement for initiation of liver fibrogenesis, but does regulate pro-fibrotic gene expression and macrophage accumulation.

Topics: Animals; Apoptosis; Aryl Hydrocarbon Receptor Nuclear Translocator; Cell Proliferation; Chemical and Drug Induced Liver Injury; Collagen; Cytokines; Disease Models, Animal; Gene Deletion; Gene Expression Regulation; Hepatocytes; Liver Cirrhosis; Macrophages; Male; Mice; Mice, Knockout; RNA, Messenger; Thioacetamide

Liver fibrosis represents the consequences of a sustained wound healing response to chronic liver injuries, and its progression toward cirrhosis is the major cause of liver-related morbidity and mortality worldwide. However, anti-fibrotic treatment remains an unconquered area for drug development. Accumulating evidence indicate that oxidative stress plays a critical role in liver fibrogenesis. In this study, we found that PQQ, a natural anti-oxidant present in a wide variety of human foods, exerted potent anti-fibrotic and ROS-scavenging activity in Balb/C mouse models of liver fibrosis. The antioxidant activity of PQQ was involved in the modulation of multiple steps during liver fibrogenesis, including chronic liver injury, hepatic inflammation, as well as activation of hepatic stellate cells and production of extracellular matrix. PQQ also suppressed the up-regulation of RACK1 in activated HSCs in vivo and in vitro. Our data suggest that PQQ suppresses oxidative stress and liver fibrogenesis in mice, and provide rationale for the clinical application of PQQ in the prevention and treatment of liver fibrosis.

Topics: Animals; Antioxidants; Cell Death; Cell Movement; Cell Proliferation; Cells, Cultured; Cytokines; Disease Models, Animal; Hepatic Stellate Cells; Hepatocytes; Humans; Liver Cirrhosis; Macrophages; Male; Mice; Mice, Inbred BALB C; Neuropeptides; Oxidative Stress; PQQ Cofactor; Receptors for Activated C Kinase; Thioacetamide

The clinical significance of enhancing endogenous circulating haematopoietic stem cells is becoming increasingly recognized, and the augmentation of circulating stem cells using granulocyte-colony stimulating factor (G-CSF) has led to promising preclinical and clinical results for several liver fibrotic conditions. However, this approach is largely limited by cost and the infeasibility of maintaining long-term administration. Preclinical studies have reported that StemEnhance, a mild haematopoietic stem cell mobilizer, promotes cardiac muscle regeneration and remedies the manifestation of diabetes. However, the effectiveness of StemEnhance in ameliorating liver cirrhosis has not been studied. This study is the first to evaluate the beneficial effect of StemEnhance administration in a thioacetamide-induced mouse model of liver fibrosis. StemEnhance augmented the number of peripheral CD34-positive cells, reduced hepatic fibrosis, improved histopathological changes, and induced endogenous liver proliferation. In addition, VEGF expression was up-regulated, while TNF-α expression was down-regulated in thioacetamide-induced fibrotic livers after StemEnhance intake. These data suggest that StemEnhance may be useful as a potential therapeutic candidate for liver fibrosis by inducing reparative effects via mobilization of haematopoietic stem cells.

Topics: Animals; Antigens, CD34; Bone Marrow Cells; Disease Models, Animal; Gene Expression Regulation; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cells; Humans; Liver; Liver Cirrhosis; Mice; Thioacetamide; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Cirrhosis is characterized by an excessive accumulation of extracellular matrix components including hyaluronic acid (HA) and is widely considered a preneoplastic condition for hepatocellular carcinoma (HCC). 4-Methylumbelliferone (4MU) is an inhibitor of HA synthesis and has anticancer activity in an orthotopic HCC model with underlying fibrosis. Our aim was to explore the effects of HA inhibition by 4MU orally administered on tumor microenvironment. Hepa129 tumor cells were inoculated orthotopically in C3H/HeJ male mice with fibrosis induced by thioacetamide. Mice were orally treated with 4MU. The effects of 4MU on angiogenesis were evaluated by immunostaining of CD31 and quantification of proangiogenic factors (vascular endothelial growth factor, VEGF, interleukin-6, IL-6 and C-X-C motif chemokine 12, CXCL12). IL-6 was also quantified in Hepa129 cells in vitro after treatment with 4MU. Migration of endothelial cells and tube formation were also analyzed. As a result, 4MU treatment decreases tumor growth and increased animal survival. Systemic levels of VEGF were significantly inhibited in 4MU-treated mice. Expression of CD31 was reduced after 4MU therapy in liver parenchyma in comparison with control group. In addition, mRNA expression and protein levels of IL-6 and VEGF were inhibited both in tumor tissue and in nontumoral liver parenchyma. Interestingly, IL-6 production was dramatically reduced in Kupffer cells isolated from 4MU-treated mice, and in Hepa129 cells in vitro. Besides, 4MU was able to inhibit endothelial cell migration and tube formation. In conclusion, 4MU has antitumor activity in vivo and its mechanisms of action involve an inhibition of angiogenesis and IL-6 production. 4MU is an orally available molecule with potential for HCC treatment.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Chemokine CXCL12; Endothelial Cells; Gene Expression Regulation, Neoplastic; Hymecromone; Interleukin-6; Kupffer Cells; Liver Cirrhosis; Liver Neoplasms; Male; Mice; Mice, Inbred C3H; Neovascularization, Pathologic; Platelet Endothelial Cell Adhesion Molecule-1; Signal Transduction; Survival Analysis; Thioacetamide; Vascular Endothelial Growth Factor A

Pituitary tumor-transforming gene (PTTG) is involved in multiple cellular pathways. We studied the development of liver fibrosis induced by thioacetamide (TAA) in knockout (PTTG-/-) and wildtype (PTTG+/+) mice.. Liver fibrosis in PTTG+/+ and PTTG-/- mice was induced by escalating dose TAA treatment (50-400mg/kg, i.p.) for 12 weeks and assessed by histochemistry, immunohistochemistry, liver hydroxyproline, serum fibrosis markers and fibrosis-related mRNA expression by real-time PCR determination.. Both PTTG+/+ and PTTG-/- mice treated with TAA developed signs of fibrosis and inflammatory cell infiltration. However, histological signs of bridging fibrosis and connective tissue square morphometry were significantly attenuated in mice lacking PTTG. α-SMA immunohistochemistry revealed that hepatic stellate cell activation was markedly reduced in PTTG-/- mice compared to wildtype controls. Hepatic hydroxyproline levels were significantly lower in fibrotic PTTG-/- group. The serum TNFα and hepatic TNFα mRNA expression were significantly lower in fibrotic PTTG-/- animals, as well as hepatic TGFβ and VEGF mRNA levels compared to TAA-treated wildtype controls. Serum hyaluronate and TGFβ levels were markedly elevated in fibrotic mice of both genotypes, but were not altered by the absence of PTTG.. TAA-induced fibrosis development is significantly ameliorated in PTTG-/- mice. These animals demonstrated diminished stellate cell activation, suppressed circulating serum markers of inflammation, fibrogenesis and angiogenesis. The presented findings suggest that PTTG is functionally required for hepatic fibrosis progression in an animal model of chronic liver injury. PTTG can be considered as a new important target for prevention and treatment of liver fibrosis/cirrhosis.

Topics: Animals; Biomarkers; Genes, Regulator; Hydroxyproline; Immunohistochemistry; Liver; Liver Cirrhosis; Mice; Mice, Knockout; Real-Time Polymerase Chain Reaction; Securin; Thioacetamide; Tumor Necrosis Factor-alpha

Hepatic encephalopathy (HE) is a neurological complication observed in patients with liver disease. Patients who suffer from HE present neuropsychiatric, neuromuscular and behavioral symptoms. Animal models proposed to study HE resulting from cirrhosis mimic the clinical characteristics of cirrhosis and portal hypertension, and require the administration of hepatotoxins such as thioacetamide (TAA). The aim of this study was to assess the effects of a high protein diet on motor function, anxiety and memory processes in a model of cirrhosis induced by TAA administration. In addition, we used cytochrome c-oxidase (COx) histochemistry to assess the metabolic activity of the limbic system regions. Male rats were distributed into groups: control, animals with cirrhosis, Control rats receiving a high protein diet, and animals with cirrhosis receiving a high protein diet. Results showed preserved motor function and normal anxiety levels in all the groups. The animals with cirrhosis showed an impairment in active avoidance behavior and spatial memory, regardless of the diet they received. However, the animals with cirrhosis and a high protein diet showed longer escape latencies on the spatial memory task. The model of cirrhosis presented an under-activation of the dentate gyrus and CA3 hippocampal subfields and the medial part of the medial mammillary nucleus. The results suggest that a high protein intake worsens spatial memory deficits shown by the TAA-induced model of cirrhosis. However, high protein ingestion has no influence on the COx hypoactivity associated with the model.

Topics: Analysis of Variance; Animals; Association Learning; Body Weight; Brain; Cognition Disorders; Disease Models, Animal; Electron Transport Complex IV; Exploratory Behavior; Liver; Liver Cirrhosis; Male; Maze Learning; Motor Activity; Proteins; Rats; Rats, Wistar; Thioacetamide

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in most chronic liver diseases. Nicotinamide treatment has been shown to prevent collagen accumulation and fibrogenesis in a bleomycin model of lung fibrosis. In this study, we evaluated the effects of nicotinic acid (NA) on experimental liver fibrosis and investigated its underlying mechanism.. Fibrosis was induced by chronic TAA administration and the effects of co-administration with NA for 8 weeks were evaluated, including control groups.. TAA administration induced liver fibrosis, which was prevented by nicotinic acid. NA prevented the elevation of liver enzymes and prevented hepatic glycogen depletion. Liver histopathology and hydroxyproline levels were significantly lower in the rats treated with TAA plus NA compared with TAA only. NA demonstrated antioxidant properties by restoring the redox equilibrium (lipid peroxidation and GPx levels). Western blot assays showed decreased expression levels of TGF-β and its downstream inductor CTGF. Additionally, NA prevented hepatic stellate cell activation due by blocking the expression of α-SMA. Zymography assays showed that NA decreased the activity of matrix metalloproteinases 2 and 9.. NA prevents experimental fibrosis; the mechanisms of action are associated with its antioxidant properties and the reduction in TGF-β expression. The decrease in TGF-β levels may be associated with the attenuation of the oxidative processes, thus resulting in a reduction in HSC activation and ECM deposition. The findings suggest a possible role for NA as an antifibrotic agent for liver injury.

Topics: Actins; Animals; Antioxidants; Connective Tissue Growth Factor; Glycogen; Hepatic Stellate Cells; Lipid Peroxidation; Liver; Liver Cirrhosis; Male; Matrix Metalloproteinases; Niacin; Oxidants; Rats; Rats, Wistar; Thioacetamide; Transforming Growth Factor beta

Hepatic fibrosis is a dynamic process which ultimately leads to cirrhosis in almost patients with chronic hepatic injury. However, progressive fibrosis is a reversible scarring response. Activation of hepatic stellate cells (HSCs) is the prevailing process during hepatic fibrosis. Osthole is an active component majorly contained in the fruit of Cnidium monnieri (L.) Cusson. This present study investigated the therapeutic effects of osthole on rat liver fibrosis and HSC activation.. We established the thioacetamide (TAA)-model of Sprague-Dawley (SD) rats to induce hepatic fibrosis. Rats were divided into three groups: control, TAA, and TAA + osthole (10 mg/kg). In vivo, osthole significantly reduced liver injury by diminishing levels of plasma AST and ALT, improving histological architecture, decreasing collagen and α-SMA accumulation, and improving hepatic fibrosis scores. Additionally, osthole reduced the expression of fibrosis-related genes significantly. Osthole also suppressed the production of fibrosis-related cytokines and chemokines. Moreover, nuclear translocation of p65 was significantly suppressed in osthole-treated liver. Osthole also ameliorated TAA-induced injury through reducing cellular oxidation. Osthole showed inhibitory effects in inflammation-related genes and chemokines production as well. In vitro, we assessed osthole effects in activated HSCs (HSC-T6 and LX-2). Osthole attenuated TGF-β1-induced migration and invasion in HSCs. Furthermore, osthole decreased TNF-α-triggered NF-κB activities significantly. Besides, osthole alleviated TGF-β1- or ET-1-induced HSCs contractility.. Our study demonstrated that osthole improved TAA-caused liver injury, fibrogenesis and inflammation in rats. In addition, osthole suppressed HSCs activation in vitro significantly.

Topics: Actins; Animals; Coumarins; Cytokines; Hepatic Stellate Cells; Liver Cirrhosis; Oxidation-Reduction; Rats; Rats, Sprague-Dawley; Thioacetamide

The aim of this study was to determine the effect of melatonin on thioacetamide (TAA) induced liver fibrosis in rats. The antifibrotic effects of melatonin were assessed by determining activity indirect markers of fibrosis: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), and proinflammatory cytokines: interleukin 6 (IL-6), interleukin-1beta (IL-1β), tumour necrosis factor alpha (TNF-α), transforming growth factor-beta (TGF-β) and platelet-derived growth factor (PDGF). Parameters of oxidative stress: oxidised glutathione (GSSG), reduced glutathione (GSH) and presaged activity of paraoxonase 1 (PON-1), an antioxidative enzyme were determined. Inflammatory changes and fibrosis extent were evaluated histologically. Experiments were carried out in Wistar rats. Animals were divided into 4 groups: I - controls, water ad libitum for 12 weeks, group II - TAA, 300 mg/L ad libitum for 12 weeks, III - melatonin, 10 mg/kg b.w. intraperitoneally (i.p.) daily for 4 weeks, IV - TAA, 300 mg/L ad libitum for 12 weeks followed by melatonin, 10 mg/kg/b.w. i.p. daily for 4 weeks. Results of serum determinations demonstrated significantly lower activity of AST, ALT and AP in the group receiving TAA followed by melatonin compared to the group receiving only TAA. Immunoenzymatic findings on effect of melatonin on concentration of proinflammatory cytokines confirmed these data. Biochemical examinations in liver homogenates revealed statistically significant improvement (concentration of GSH increases and concentration of GSSG decreases) in animals with TAA-induced liver damage receiving melatonin. Moreover, the activity of PON-1 toward phenyl acetate and paraoxon was increased in liver homogenates and serum in the group receiving TAA followed by melatonin compared to the TAA group without melatonin treatment. Microscopic evaluation disclosed inhibitory effects of melatonin on inflammatory changes and extent of liver fibrosis.

Topics: Animals; Aryldialkylphosphatase; Cytokines; Liver; Liver Cirrhosis; Liver Function Tests; Male; Melatonin; Oxidative Stress; Rats; Rats, Wistar; Thioacetamide

Dectin-1 is a C-type lectin receptor critical in anti-fungal immunity, but Dectin-1 has not been linked to regulation of sterile inflammation or oncogenesis. We found that Dectin-1 expression is upregulated in hepatic fibrosis and liver cancer. However, Dectin-1 deletion exacerbates liver fibro-inflammatory disease and accelerates hepatocarcinogenesis. Mechanistically, we found that Dectin-1 protects against chronic liver disease by suppressing TLR4 signaling in hepatic inflammatory and stellate cells. Accordingly, Dectin-1(-/-) mice exhibited augmented cytokine production and reduced survival in lipopolysaccharide (LPS)-mediated sepsis, whereas Dectin-1 activation was protective. We showed that Dectin-1 inhibits TLR4 signaling by mitigating TLR4 and CD14 expression, which are regulated by Dectin-1-dependent macrophage colony stimulating factor (M-CSF) expression. Our study suggests that Dectin-1 is an attractive target for experimental therapeutics in hepatic fibrosis and neoplastic transformation. More broadly, our work deciphers critical cross-talk between pattern recognition receptors and implicates a role for Dectin-1 in suppression of sterile inflammation, inflammation-induced oncogenesis, and LPS-mediated sepsis.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Chemokine CCL2; Cytokines; Diethylnitrosamine; Hepatocytes; Humans; Inflammation; Lectins, C-Type; Lipopolysaccharide Receptors; Lipopolysaccharides; Liver; Liver Cirrhosis; Liver Neoplasms; Macrophage Colony-Stimulating Factor; Mice; Mice, Inbred C57BL; Mice, Knockout; Recombinant Proteins; Sepsis; Signal Transduction; Thioacetamide; Toll-Like Receptor 4; Up-Regulation

Ammonia metabolizing enzymes, carbamyol phosphate synthetase (CPS) and glutamine synthetase (GS), are expressed in the periportal and pericentral hepatocytes, respectively. CPS and GS function complementary to ensure complete ammonia detoxification. Immunohistochemical analysis confirmed the decline of both CPS and GS in cirrhotic rat liver induced by thioacetamide (TAA). Alpha-mangostin (AM), a major derivative of xanthone from mangosteen, has been reported to possess a wide range of pharmacological properties.. To examine the preventive effects of AM on CPS and GS expression in fibrotic and cirrhotic rats induced by TAA over sixteen weeks.. Twenty-four male Wistar rats were divided into 4 groups of 6 animals each. Group 1 was for control. Group 2 wasfor pure TAA treatment. Group 3 was for pure AM administration. Group 4, prevention group, was concurrently treated with TAA and AM. Immunohistochemical technique was employed in order to elucidate the expression of CPS and GS in each animal group.. Immunohistochemical staining for CPS and GS showed an increasing decline from week eight to sixteen under pure-TAA condition. Fibrous bridgings, nodule formations, and regenerative nodules were detected. Pure-AM condition yielded strongly CPS and GS-stained hepatocytes in afashion similar to the control. Results from the prevention group showed a decreasing decline of CPS and GS immuno-reactivity from week eight to sixteen as compared to pure-TAA condition. Fewer fibrous portal-caval bridgings were observed at week eight and CPS-positive hepatocytes were found in continuous rings.. Alpha-mangostin could partially preserve the normal expression of ammonia-metabolizing enzymes under TAA-induced fibrotic and cirrhotic conditions.

Topics: Ammonia; Animals; Glutamate-Ammonia Ligase; Hepatocytes; Liver Cirrhosis; Male; Rats; Rats, Wistar; Thioacetamide; Xanthones

To determine the effects of alpha-mangostin on thioacetamide (TAA)-induced liver cirrhosis in rats.. Male Wistar rats were divided into 3 groups and treated with intraperitoneal injections of TAA (200 mg/kg) 3 times per week for per week for 8, 12 and 16 weeks, respectively. One subgroup was left untreated whereas the other two were treated either with 100 mg/kg α-mangostin or vehicle alone (80% DMSO, 20% water), which were administered intraperitoneally 3 times per weekfor a total of4 weeks. The incidence offibrotic nodules on the liver and the serum levels of the liver enzymes aspartate transaminase (AST) and alanine transaminase (ALT) were measured. Moreover the liver cirrhosis-related genes expression and p53 protein level in liver were analyzed by quantitative reverse transcription PCR and Western blot analysis, respectively.. Fibrotic nodules on the liver were formed upon treatment with TAA for 12 or 16 weeks. The nodules were then reduced by treatment with α-mangostin as compared to treatment with the vehicle DMSO. Moreover, the serum levels of the liver enzymes AST and ALT after treatment with α-mangostin decreased as compared to DMSO alone. The liver cirrhosis-related genes expression showed no significant differences, whereas the p53 protein level in liver showed that α-mangostin reduced risk of liver fibrosis through the decrease in p53 expression as compared to the TAA_DMSO treatment.. The results suggest that α-mangostin has a beneficial therapeutic effect in the TAA liver cirrhosis model. Further investigations on mechanisms of α-mangostin as therapeutic agent should be determined.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Liver Cirrhosis; Male; Rats; Rats, Wistar; Thioacetamide; Xanthones

Liver resection is a suitable option for the treatment of certain hepatic conditions, particularly hepatocarcinomas, in patients with cirrhosis. However, this disease impairs liver regeneration, which increases the risk of liver failure and postoperative death. Supportive treatments for regeneration of the remaining liver may be useful for the recovery of these patients. We demonstrated that nutritional hepatotrophic factors (NHF) is an effective regenerative stimulus for cirrhotic livers in rats subjected to partial hepatectomy (PH). The rats with thioacetamide-induced cirrhosis were subjected to PH, and they were divided into 2 groups. One group received intraperitoneal administration of NHF, and the other group received saline solution. After 12 days, biometric data, collagen content, hepatocyte regeneration (proliferation cell nuclear antigen immunochemistry), and profibrotic gene expression (Collagen-α1, matrix metalloproteinase 2, tissue inhibitor of metalloproteinase 1, and transforming growth factor beta 1) were assessed. The results indicated that the rats treated with NHF after PH had an increased liver size, a reduced amount of collagen, and a higher hepatocyte proliferation index compared with the rats that underwent PH alone. In addition, collagen-α1 gene expression was decreased in the NHF-treated rats. Thus, postoperative improvement in the liver morphology following NHF treatment may cause a significant decrease in the risk of liver failure and mortality after hepatic resection.

Topics: Alkaline Phosphatase; Analysis of Variance; Animals; Cell Proliferation; Dietary Supplements; Female; Hepatectomy; Infusions, Parenteral; Intercellular Signaling Peptides and Proteins; Liver; Liver Cirrhosis; Liver Regeneration; Organ Size; Proliferating Cell Nuclear Antigen; Random Allocation; Rats; Rats, Wistar; Thioacetamide

Considerable progress has been made in developing antifibrotic agents and other strategies to treat liver fibrosis; however, significant long-term restoration of functional liver mass has not yet been achieved. Therefore, we investigated whether transplanted hepatic stem/progenitor cells can effectively repopulate the liver with advanced fibrosis/cirrhosis. Stem/progenitor cells derived from fetal livers or mature hepatocytes from DPPIV(+) F344 rats were transplanted into DPPIV(-) rats with thioacetamide (TAA)-induced fibrosis/cirrhosis; rats were sacrificed 1, 2, or 4 months later. Liver tissues were analyzed by histochemistry, hydroxyproline determination, reverse-transcription polymerase chain reaction (RT-PCR), and immunohistochemistry. After chronic TAA administration, DPPIV(-) F344 rats exhibited progressive fibrosis, cirrhosis, and severe hepatocyte damage. Besides stellate cell activation, increased numbers of stem/progenitor cells (Dlk-1(+), AFP(+), CD133(+), Sox-9(+), FoxJ1(+)) were observed. In conjunction with partial hepatectomy (PH), transplanted stem/progenitor cells engrafted, proliferated competitively compared to host hepatocytes, differentiated into hepatocytic and biliary epithelial cells, and generated new liver mass with extensive long-term liver repopulation (40.8 ± 10.3%). Remarkably, more than 20% liver repopulation was achieved in the absence of PH, associated with reduced fibrogenic activity (e.g., expression of alpha smooth muscle actin, platelet-derived growth factor receptor β, desmin, vimentin, tissue inhibitor of metalloproteinase-1) and fibrosis (reduced collagen). Furthermore, hepatocytes can also replace liver mass with advanced fibrosis/cirrhosis, but to a lesser extent than fetal liver stem/progenitor cells.. This study is a proof of principle demonstration that transplanted epithelial stem/progenitor cells can restore injured parenchyma in a liver environment with advanced fibrosis/cirrhosis and exhibit antifibrotic effects.

Topics: Animals; Cell Differentiation; Cell Proliferation; Female; Fetal Stem Cells; Hepatocytes; Liver; Liver Cirrhosis; Liver Regeneration; Male; Pregnancy; Rats; Rats, Inbred F344; Stem Cell Transplantation; Thioacetamide

Recent studies have demonstrated that bone marrow-derived mesenchymal stem cells (BM-MSCs) can potentially revert liver fibrosis, but it is not known if preparative hepatic irradiation (HIR) contributes to the therapeutic effect of transplanted BM-MSCs. In this study, we investigate the effects of HIR on transplanted BM-MSCs in cirrhotic rats and the underlying mechanism by which mesenchymal stem cells (MSCs) relieve liver fibrosis.. The BM-MSCs from male rats were labeled with CM-Dil and injected via portal vein into two groups of thioacetamide-induced cirrhotic rats, and the controls were injected with the same volume of saline. The right hemiliver of one cirrhotic rat group was irradiated (15 Gy) 4 d before transplantation. Liver function tests and histologic experiments were performed, and the liver population of BM-MSCs was estimated.. The transplantation of MSCs alleviated liver fibrosis and reduced expression of transforming growth factor-β1, Smad2, collagen type I, and α-SMA. HIR preconditioning promoted homing and repopulation of MSCs and resulted in better treatment outcomes.. HIR preconditioning enhances the effect of BM-MSCs in improving thioacetamide-induced liver fibrosis in rats by promoting their homing and repopulation. BM-MSCs may function by inhibiting transforming growth factor-β1-Smad signaling pathway in the liver.

Topics: Animals; Bone Marrow Cells; Liver; Liver Cirrhosis; Male; Mesenchymal Stem Cell Transplantation; Rats; Rats, Sprague-Dawley; Signal Transduction; Smad Proteins; Thioacetamide; Transforming Growth Factor beta1

Conophylline (CnP) is a vinca alkaloid purified from a tropical plant and inhibits activation of pancreatic stellate cells. We investigated the effect of CnP on hepatic stellate cells (HSC) in vitro. We also examined whether CnP attenuates hepatic fibrosis in vivo.. We examined the effect of CnP on the expression of α-smooth muscle actin (α-SMA) and collagen-1, DNA synthesis and apoptosis in rat HSC and Lx-2 cells. We also examined the effect of CnP on hepatic fibrosis induced by thioacetamide (TAA).. In rat HSC and Lx-2 cells, CnP reduced the expression of α-SMA and collagen-1. CnP inhibited DNA synthesis induced by serum. CnP also promoted activation of caspase-3 and induced apoptosis as assessed by DNA ladder formation and TUNEL assay. In contrast, CnP did not induce apoptosis in AML12 cells. We then examined the effect of CnP on TAA-induced cirrhosis. In TAA-treated rats, the surface of the liver was irregular and multiple nodules were observed. Histologically, formation of pseudolobules surrounded by massive fibrous tissues was observed. When CnP was administered together with TAA, the surface of the liver was smooth and liver fibrosis was markedly inhibited. Collagen content was significantly reduced in CnP-treated liver.. Conophylline suppresses HSC and induces apoptosis in vitro. CnP also attenuates formation of the liver fibrosis induced by TAA in vivo.

Topics: Actins; Animals; Apoptosis; Blotting, Western; Caspase 3; Cell Line; Collagen Type I; DNA Replication; Hepatic Stellate Cells; Immunohistochemistry; In Situ Nick-End Labeling; Liver Cirrhosis; Rats; Thioacetamide; Vinca Alkaloids

Given the involvement of oxidative stress in liver-disease- or hepato-toxicant-induced hepatic damage and fibrosis, antioxidants are an effective preventive and therapeutic tool. The beneficial results of vitamin C, one of the physiological antioxidants, have been observed both in experimental animals and in humans. However, most of these studies have been concerned with supplementary vitamin C; the effects of under vitamin C insufficiency, which humans sometimes confront, have not been substantially investigated. In the present study, we established a vitamin C-insufficient animal model (half-to-normal serum vitamin C concentration) with gulo(-/-) mice that cannot synthesize vitamin C, and induced hepatotoxicity by means of thioacetamide (TAA) injections twice a week for 18 weeks. Additionally, we explored the direct effects of vitamin C both on immortalized human hepatic stellate LX-2 cells and on rat primary hepatic stellate cells. Vitamin C insufficiency resulted in a decreased survival rate and increased serum markers for hepatocyte damage, such as alanine aminotransferase and aspartate aminotransferase. Concomitantly, the levels of reactive oxygen species (ROS) and lipid peroxides in the liver were increased. Histological examinations of the vitamin C-insufficient liver revealed increases in collagen fiber deposition and activated-hepatic-stellate-cell number. Vitamin C, when directly applied to the LX-2 cells as well as the rat primary hepatic stellate cells, suppressed not only proliferation but hydrogen peroxide-induced collagen expression as well. In conclusion, vitamin C insufficiency exacerbated TAA-induced hepatotoxicity. These effects seem to be mainly from insufficient scavenging of ROS in the liver, and possibly in part, by directly affecting hepatic stellate cells.

Topics: Alanine Transaminase; Animals; Ascorbic Acid; Ascorbic Acid Deficiency; Aspartate Aminotransferases; Collagen; Gene Expression; Hepatic Stellate Cells; Humans; L-Gulonolactone Oxidase; Lipid Peroxidation; Liver Cirrhosis; Male; Mice; Mice, Knockout; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Thioacetamide

Hepatic stellate cell activation is a key cellular event in the development of liver fibrosis. Recently, Delta-like homolog 1 (DLK1) protein level has been shown to increase in HSC activation and serve as a new contributor to HSC activation and liver fibrosis. Curcumin, a natural yellow polyphenol, possesses therapeutic roles in many diseases including liver fibrosis and has long been used in traditional medicine. The present study was aimed to elucidate the effect of curcumin on DLK1 expression in HSCs in vitro and in vivo, which is still unknown. Our results demonstrated that curcumin reduced DLK1 expression in culture-activated HSCs and in rat model of liver fibrosis. The inhibitory effect of curcumin on DLK1 expression may be mediated in part by interruption of Shh signaling pathway, which contributes to the promotion effect of curcumin on the expression of PPAR-gamma, a key factor in inhibiting HSC activation. Our results in this study may reveal a new mechanisms through which curcumin exerts its inhibitory effect on HSC activation and liver fibrosis.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Cells, Cultured; Curcumin; Disease Models, Animal; Gene Expression; Hepatic Stellate Cells; Intercellular Signaling Peptides and Proteins; Liver Cirrhosis; Membrane Proteins; Rats; Rats, Sprague-Dawley; Thioacetamide

The current study was conducted to investigate the anti-fibrotic effect and its possible underlying mechanisms of thymoquinone (TQ) against hepatic fibrosis in vivo. TQ is the major active compound derived from the medicinal Nigella sativa. Liver fibrosis was induced in male Kunming mice by intraperitoneal injections of thioacetamide (TAA, 200 mg/kg). Mice were treated concurrently with TAA alone or TAA plus TQ (20 mg/kg or 40 mg/kg) given daily by oral gavage. Our data demonstrated that TQ treatment obviously reversed liver tissue damage compared with TAA alone group, characterized by less inflammatory infiltration and accumulation of extracellular matrix (ECM) proteins. TQ significantly attenuated TAA-induced liver fibrosis, accompanied by reduced protein and mRNA expression of α-smooth muscle actin (α-SMA), collagen-І and tissue inhibitor of metalloproteinase-1 (TIMP-1). TQ downregulated the expression of toll-like receptor 4 (TLR4) and remarkably decreased proinflammatory cytokine levels as well. TQ also significantly inhibited phosphatidylinositol 3-kinase (PI3K) phosphorylation. Furthermore, TQ enhanced the phosphorylation adenosine monophosphate-activated protein kinase (AMPK) and liver kinase B (LKB)-1. In conclusion, TQ may reduce ECM accumulation, and it may be at least regulated by phosphorylation of AMPK signaling pathways, suggesting that TQ may be a potential candidate for the therapy of hepatic fibrosis.

Topics: Actins; AMP-Activated Protein Kinases; Animals; Anti-Inflammatory Agents; Benzoquinones; Collagen Type I; Cytokines; Liver; Liver Cirrhosis; Male; Mice; Protein Serine-Threonine Kinases; Signal Transduction; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1

Activation of the renin angiotensin system resulting in stimulation of angiotensin-II (AngII) type I receptor (AT1R) is an important factor in the development of liver fibrosis. Here, we investigated the role of Janus kinase 2 (JAK2) as a newly described intracellular effector of AT1R in mediating liver fibrosis. Fibrotic liver samples from rodents and humans were compared to respective controls. Transcription, protein expression, activation, and localization of JAK2 and downstream effectors were analyzed by real-time polymerase chain reaction, western blotting, immunohistochemistry, and confocal microscopy. Experimental fibrosis was induced by bile duct ligation (BDL), CCl4 intoxication, thioacetamide intoxication or continuous AngII infusion. JAK2 was inhibited by AG490. In vitro experiments were performed with primary rodent hepatic stellate cells (HSCs), Kupffer cells (KCs), and hepatocytes as well as primary human and human-derived LX2 cells. JAK2 expression and activity were increased in experimental rodent and human liver fibrosis, specifically in myofibroblastic HSCs. AT1R stimulation in wild-type animals led to activation of HSCs and fibrosis in vivo through phosphorylation of JAK2 and subsequent RhoA/Rho-kinase activation. These effects were prevented in AT1R(-/-) mice. Pharmacological inhibition of JAK2 attenuated liver fibrosis in rodent fibrosis models. In vitro, JAK2 and downstream effectors showed increased expression and activation in activated HSCs, when compared to quiescent HSCs, KCs, and hepatocytes isolated from rodents. In primary human and LX2 cells, AG490 blocked AngII-induced profibrotic gene expression. Overexpression of JAK2 led to increased profibrotic gene expression in LX2 cells, which was blocked by AG490.. Our study substantiates the important cell-intrinsic role of JAK2 in HSCs for development of liver fibrosis. Inhibition of JAK2 might therefore offer a promising therapy for liver fibrosis.

Topics: Angiotensin II; Animals; Bile Ducts; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Hepatic Stellate Cells; Humans; Janus Kinase 2; Ligation; Liver Cirrhosis; Mice; Mice, Inbred C57BL; Mice, Knockout; Myofibroblasts; Phosphorylation; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Signal Transduction; Thioacetamide

The antifibrotic effects of traditional medicinal herb Caesalpinia sappan (CS) extract on liver fibrosis induced by thioacetamide (TAA) and the expression of transforming growth factor β1 (TGF-β1), α-smooth muscle actin (αSMA), and proliferating cell nuclear antigen (PCNA) in rats were studied. A computer-aided prediction of antioxidant and hepatoprotective activities was primarily performed with the Prediction Activity Spectra of the Substance (PASS) Program. Liver fibrosis was induced in male Sprague Dawley rats by TAA administration (0.03% w/v) in drinking water for a period of 12 weeks. Rats were divided into seven groups: control, TAA, Silymarin (SY), and CS 300 mg/kg body weight and 100 mg/kg groups. The effect of CS on liver fibrogenesis was determined by Masson's trichrome staining, immunohistochemical analysis, and western blotting. In vivo determination of hepatic antioxidant activities, cytochrome P450 2E1 (CYP2E1), and matrix metalloproteinases (MPPS) was employed. CS treatment had significantly increased hepatic antioxidant enzymes activity in the TAA-treated rats. Liver fibrosis was greatly alleviated in rats when treated with CS extract. CS treatment was noted to normalize the expression of TGF-β1, αSMA, PCNA, MMPs, and TIMP1 proteins. PASS-predicted plant activity could efficiently guide in selecting a promising pharmaceutical lead with high accuracy and required antioxidant and hepatoprotective properties.

Topics: Animals; Caesalpinia; Forecasting; Liver Cirrhosis; Male; Phytotherapy; Plant Extracts; Plant Leaves; Random Allocation; Rats; Rats, Sprague-Dawley; Thioacetamide; Treatment Outcome

Liver cirrhosis is the final consequence of a progressive fibrotic process characterized by excessive collagen deposition and destruction of the normal liver architecture. This study aimed to investigate the protective effects of curcumin, silybin-phytosome and alpha-R-lipoic acid against thioacetamide-induced cirrhosis. Male rats were allocated into five groups of which one group received saline and served as normal control. Animals from groups 2-5 were treated with thioacetamide administered intraperitoneally at a dose of 200 mg/kg 3 times per week for 7 weeks. Group 2 was left untreated while groups from 3 to 5 were given a daily oral dose of curcumin, silybin-phytosome or alpha-R-lipoic acid simultaneously with thioacetamide. Increases in hepatic levels of malondialdehyde (MDA) and protein carbonyls (Pr Co) associated with thioacetamide administration were partially blocked in those groups receiving supplements. Glutathione (GSH) depletion, collagen deposition, matrix metalloproteinase-2 (MMP-2) activity, transforming growth factor-β1 (TGF-β1) level as well as α-smooth muscle actin (α-SMA) and heat shock protein-47 (HSP-47) gene expressions were also decreased in response to supplements administration. Serological analysis of liver function and histopathological examination reinforced the results. In conclusion, the present study highlights the antioxidant and the antifibrotic potentials of these supplements against chronic liver diseases caused by ongoing hepatic damage.

Topics: Animals; Antioxidants; Biomarkers; Chemical and Drug Induced Liver Injury; Curcumin; Gene Expression Regulation; HSP47 Heat-Shock Proteins; Liver Cirrhosis; Male; Matrix Metalloproteinase 2; Oxidative Stress; Rats; Rats, Wistar; Silybin; Silymarin; Thioacetamide; Thioctic Acid; Transforming Growth Factor beta1

The accurate staging of liver fibrosis is of paramount importance to determine the state of disease progression, therapy responses, and to optimize disease treatment strategies. Non-linear optical microscopy techniques such as two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) can image the endogenous signals of tissue structures and can be used for fibrosis assessment on non-stained tissue samples. While image analysis of collagen in SHG images was consistently addressed until now, cellular and tissue information included in TPEF images, such as inflammatory and hepatic cell damage, equally important as collagen deposition imaged by SHG, remain poorly exploited to date. We address this situation by experimenting liver fibrosis quantification and scoring using a combined approach based on TPEF liver surface imaging on a Thioacetamide-induced rat model and a gradient based Bag-of-Features (BoF) image classification strategy. We report the assessed performance results and discuss the influence of specific BoF parameters to the performance of the fibrosis scoring framework.

Topics: Animals; Disease Progression; Image Processing, Computer-Assisted; Liver; Liver Cirrhosis; Male; Microscopy, Fluorescence, Multiphoton; Rats; Rats, Wistar; Thioacetamide

Resident and exudate macrophages play an important role in the development of liver cirrhosis. Ionized calcium binding adaptor molecule 1(+) (Iba1(+)) and galectin-3(+) (Gal-3(+)) macrophages regulate liver fibrosis probably through pro-inflammatory and pro-fibrotic factors. Macrophages show polarized functions in liver fibrosis; however, M1-/M2-polarization of Iba1(+) and Gal-3(+) macrophages remains obscured. This study investigated the M1-/M2-polarized properties of Iba1(+) and Gal-3(+) macrophages in chemical-induced liver cirrhosis.. Cirrhosis was induced in F344 rats by repeated injections of thioacetamide (100mg/kg BW, twice a week for 25 weeks). Liver samples were collected from post-first-injection (PFI) week 5 to 25. Macrophage immunophenotypes and myofibroblasts in the fibrous bridges (FBs) and pseudolobules (PLs) were analyzed by immunohistochemistry. Expressions of M1- and M2-related factors were analyzed with RT-PCR, separately in FBs and PLs.. Activation of myofibroblasts was most pronounced in livers at week 15. CD68(+) (M1), CD204(+) (M2), Iba1(+) and Gal-3(+) macrophages in the FBs increased gradually and peaked at week 15, consistent with the upregulation of both M1-(MCP-1, IFN-γ, IL-1β, IL-6, and TNF-α) and M2-(TGF-β1, IL-4, and IL-10) related factors. Iba1(+) and Gal-3(+) macrophages showed both M1- and M2-immunophenotypes. CD163(+) macrophages showed a persistent increase, consistent with TGF-β1 upregulation. MHC class II(+) macrophages increased in the developing fibrotic lesions, and then reduced in the advanced stage cirrhosis.. Both M1- and M2-macrophage polarizations occur during development of liver cirrhosis. Iba1(+) and Gal-3(+) macrophages participate in liver cirrhosis through production of both M1- and M2-related factors.

Topics: Animals; Calcium-Binding Proteins; Carrier Proteins; Chemokine CCL2; Extracellular Matrix Proteins; Gene Expression Profiling; Immunohistochemistry; Interferon-gamma; Interleukin-10; Interleukin-1beta; Interleukin-4; Interleukin-6; Liver; Liver Cirrhosis; Macrophages; Male; Microfilament Proteins; Myofibroblasts; Rats; Rats, Inbred F344; RNA, Messenger; Thioacetamide; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Up-Regulation

The epithelial-mesenchymal transition (EMT) of hepatocytes is a key step for hepatic fibrosis and cirrhosis. Long-term administration of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, can ameliorate hepatic fibrosis. This research aimed to examine the effect of celecoxib on the EMT of hepatocytes during the development of liver cirrhosis.. Cirrhotic liver model of rat was established by peritoneal injection of thiacetamide (TAA). Thirty-six rats were randomly assigned to control, TAA, and TAA + celecoxib groups. Hepatic expressions of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), COX-2, prostaglandin E2 (PGE2 ), matrix metalloproteinase (MMP)-2 and -9, transforming growth factor-β1 (TGF-β1), Phospho-Smad2/3, Snail1, α-smooth muscle actin (α-SMA), vimentin, collagen I, fibroblast-specific protein (FSP-1), E-cadherin and N-cadherin were quantitated. Hepatic fibrosis was assessed by the visible hepatic fibrotic areas and Ishak's scoring system.. Exposed to TAA treatment, hepatocytes underwent the process of EMT during hepatic fibrosis. Compared with those in TAA group, celecoxib significantly downregulated the hepatic expressions of TNF-α, IL-6, COX-2, PGE2 , MMP-2, MMP-9, TGF-β1, Phospho-Smad2/3, Snail1, α-SMA, FSP-1, and vimentin while greatly restoring the levels of E-cadherin. The fibrotic areas and collagen I levels of TAA + celecoxib group were much lower than those in TAA group.. Celecoxib could ameliorate hepatic fibrosis and cirrhosis in TAA-rat model through suppression of the mesenchymal biomarkers in the hepatocytes while restoring the levels of their epithelial biomarkers. The inhibitory effect of celecoxib on the EMT of hepatocytes is associated with reduction of intrahepatic inflammation, preservation of normal basement matrix, and inhibition of TGF-β1/Smad pathway.

Topics: Animals; Celecoxib; Cyclooxygenase 2 Inhibitors; Disease Models, Animal; Down-Regulation; Epithelial-Mesenchymal Transition; Hepatocytes; Liver Cirrhosis; Liver Cirrhosis, Experimental; Pyrazoles; Rats, Sprague-Dawley; Signal Transduction; Smad Proteins; Sulfonamides; Thioacetamide; Transforming Growth Factor beta1

Many researchers have focused on developing traditional herbal medicines as pharmacological medicines to treat hepatic fibrosis. In this study, we evaluated the possible mechanism of Ger-Gen-Chyn-Lian-Tang (GGCLT) on thioacetamide (TAA)-induced hepatic injury in mice.. Hepatic fibrosis mice were established by intraperitoneal injection with TAA (100 mg/kg, 3 times/week), and treated with daily oral administration of 30 mg/kg, 100 mg/kg, and 300 mg/kg of GGCLT for 6 weeks. There were 40 mice randomly assigned to control, TAA and TAA+GGCLT groups. When the experiment was completed, Masson's trichrome staining was used to measure the degree of liver fibrosis. Hepatic fibrosis molecules were assessed by Western blot and real-time polymerase chain reaction. Hepatic glutathione levels, matrix metalloproteinase (MMP-2 and MMP-9), and hydroxyproline were also measured.. Treatment with GGCLT significantly reduced the toxicity of TAA and exhibited effective hepatoprotective activity. The mechanism of the hepatoprotective effect of GGCLT is proposed to be by normalizing oxidative stress. Additionally, the data of fibrotic areas, expression of procollagen III, and MMP2 and 9 mRNA levels in the TAA+GGCLT group were much lower than those in the TAA group (p < 0.05). Furthermore, the upregulation of hepatic protein levels of nuclear factor-κB, transforming growth factor (TGF)-β receptor-1, and smooth muscle α-actin induced by TAA was significantly inhibited after GGCLT treatment.. GGCLT can efficiently ameliorate hepatic fibrosis by its inhibitory effects on the intrahepatic oxidative stress in TAA mice model. The antioxidant properties afforded by GGCLT may be attributed to its modulation on TGF-β/TGFβ receptor signaling through the downregulation of integrated signal pathways involving smooth muscle α-actin and lipid peroxidation.

Topics: Animals; Blotting, Western; Drugs, Chinese Herbal; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Oxidative Stress; Polymerase Chain Reaction; Thioacetamide

Thioacetamide (TAA) is a well-known toxicant and its long term exposure could induce liver fibrosis and cirrhosis. A liver fibrosis rat model was established by consecutive injection of TAA solution for 7 weeks. Serum and urine samples were collected weekly for NMR based metabolomic study. Clinical biochemistry of serum samples revealed liver impairment and fibrosis. Histopathological inspections disclosed severe liver fibrosis and cirrhosis formation, and pathological changes in kidney by long-term TAA administration. Orthogonal partial least squares-discriminant analysis (OPLS-DA) was applied on serum and urine samples to excavate differential metabolites associated with TAA induced impairment and explore the time-dependent metabolic event associated with this xenobiotic perturbation. Integration of metabolomics results with serum biochemical revealed several potential biomarkers for liver fibrosis (2-hydroxybutyrate, 3-hydroxybutyrate and adipate in urine, and phenylalanine, N,N-dimethyl glycine, O-acetyl glycoprotein, N-acetyl glycoprotein and choline in serum). Pathway analysis revealed disturbed pathways concerning tricarboxylic acid (TCA) cycle, pyruvate metabolism, starch and sucrose metabolism, glycolysis or gluconeogenesis, degradation of ketone bodies, butanoate metabolism, and biosynthesis of BCAAs (valine, leucine and isoleucine) and AAAs (phenylalanine, tyrosine and tryptophan). This integrative study should help to develop a systematic understanding of liver fibrosis-related diseases and their metabolic events.

Topics: Animals; Biomarkers; Discriminant Analysis; Liver; Liver Cirrhosis; Magnetic Resonance Spectroscopy; Metabolome; Metabolomics; Rats; Serum; Signal Transduction; Thioacetamide; Urine

This study investigated the hepatoprotective effects of ethanolic Andrographis paniculata leaf extract (ELAP) on thioacetamide-induced hepatotoxicity in rats. An acute toxicity study proved that ELAP is not toxic in rats. To examine the effects of ELAP in vivo, male Sprague Dawley rats were given intraperitoneal injections of vehicle 10% Tween-20, 5 mL/kg (normal control) or 200 mg/kg TAA thioacetamide (to induce liver cirrhosis) three times per week. Three additional groups were treated with thioacetamide plus daily oral silymarin (50 mg/kg) or ELAP (250 or 500 mg/kg). Liver injury was assessed using biochemical tests, macroscopic and microscopic tissue analysis, histopathology, and immunohistochemistry. In addition, HepG2 and WRL-68 cells were treated in vitro with ELAP fractions to test cytotoxicity. Rats treated with ELAP exhibited significantly lower liver/body weight ratios and smoother, more normal liver surfaces compared with the cirrhosis group. Histopathology using Hematoxylin and Eosin along with Masson's Trichrome stain showed minimal disruption of hepatic cellular structure, minor fibrotic septa, a low degree of lymphocyte infiltration, and minimal collagen deposition after ELAP treatment. Immunohistochemistry indicated that ELAP induced down regulation of proliferating cell nuclear antigen. Also, hepatic antioxidant enzymes and oxidative stress parameters in ELAP-treated rats were comparable to silymarin-treated rats. ELAP administration reduced levels of altered serum liver biomarkers. ELAP fractions were non-cytotoxic to WRL-68 cells, but possessed anti-proliferative activity on HepG2 cells, which was confirmed by a significant elevation of lactate dehydrogenase, reactive oxygen species, cell membrane permeability, cytochrome c, and caspase-8,-9, and, -3/7 activity in HepG2 cells. A reduction of mitochondrial membrane potential was also detected in ELAP-treated HepG2 cells. The hepatoprotective effect of 500 mg/kg of ELAP is proposed to result from the reduction of thioacetamide-induced toxicity, normalizing reactive oxygen species levels, inhibiting cellular proliferation, and inducing apoptosis in HepG2 cells.

Topics: Andrographis; Animals; Antioxidants; Apoptosis; Blotting, Western; Cell Proliferation; Cells, Cultured; Female; Hep G2 Cells; Humans; Immunoenzyme Techniques; Liver Cirrhosis; Liver Function Tests; Male; Oxidative Stress; Phytotherapy; Plant Extracts; Plant Leaves; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Thioacetamide

Cirrhosis is a long-term consequence of chronic hepatic injury with fibrosis. No effective therapy is currently available for decompensated cirrhosis except liver transplantation. Hence, we investigated the effect of bone marrow-derived mesenchymal stem cells (BM-MSCs) on hepatic fibrosis in a thioacetamide (TAA)-induced cirrhotic rat model.. The BM-MSCs were injected directly into the right liver lobe twice, at 6 and 8 weeks during the 12-week TAA administration, in thioacetamide (TAA)-induced cirrhotic rats model, and hepatic fibrosis was evaluated. At 12 weeks, the effect of BM-MSCs on hepatic fibrosis was analyzed histomorphologically using the Laennec fibrosis scoring system, and the collagen proportionate area was quantified. Cirrhosis-related factors, such as transforming growth factor β1 (TGF-β1), type 1 collagen (collagen-1), α-smooth muscle actin (α-SMA), and P-Smad3/Smad3 expression levels, were evaluated using real-time polymerase chain reaction and western blot assays.. According to the Laennec fibrosis scoring system, histological improvement was observed in hepatic fibrosis after BM-MSC treatment (P <0.01). The percentage of the collagen proportionate area decreased from 16.72 ± 5.51 to 5.06 ± 1.27 after BM-MSC treatment (P <0.01). The content of hepatic hydroxyproline was significantly lower in the BM-MSC treated group (46.25 ± 13.19) compared to the untreated cirrhotic group (85.81 ± 17.62; P <0.01). BM-MSC administration significantly decreased TGF-β1, collagen-1, and α-SMA expression in TAA-induced cirrhotic rats (P <0.01). We also confirmed P-Smad3/Smad3, downstream effectors of the TGF-β1 signaling pathway, and found that MSC transplantation inhibited Smad3 phosphorylation.. BM-MSC treatment attenuated hepatic fibrosis in rats with TAA-induced cirrhosis, raising the possibility of the clinical use of BM-MSCs in the treatment of cirrhosis.

Topics: Actins; Animals; Cell Differentiation; Collagen Type I; Disease Models, Animal; Glyceraldehyde-3-Phosphate Dehydrogenases; Hepatocytes; Immunohistochemistry; Immunophenotyping; Liver Cirrhosis; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Rats, Sprague-Dawley; RNA, Messenger; Thioacetamide; Transforming Growth Factor alpha; Transforming Growth Factor beta

Liver fibrosis is a complex disease in which several pathological processes, such as inflammation and angiogenesis, are closely integrated.. We hypothesised that treatment with the pharmacological agent, andrographolide (AP), which has multiple mechanisms of action, will provide a greater understanding of the role of AP during the multiple pathological processes that occur in advanced liver disease.. Liver fibrogenesis was induced in mice using thioacetamide (TAA), which was administrated for 6 weeks. Andrographolide (5, 20 or 100mg/kg) was then given once daily following TAA injection. Liver collagen was examined using hydroxyproline and α-SMA, while the inflammatory response was quantified by Western blot and RT-PCR assays. Liver angiogenesis, neutrophil infiltration and hypoxia were assessed using CD11b+, vWF and HIF-1α immunostaining. Mice with liver injuries that were treated with andrographolide showed improved inflammatory response and diminished angiogenesis and hepatic fibrosis. Andrographolide treatment inhibited liver neutrophil infiltration, while a decreased in TNF-α and COX-2 signalling indicated macrophage activation. Andrographolide decreased overall liver hypoxia, as shown by the downregulation of hypoxia-inducible cascade genes, such as VEGF. Andrographolide treatment resulted in a significant decrease in hepatic fibrogenesis, α-SMA abundance, and TGF-βR1 expression.. The present results suggest that multi-targeted therapies directed against angiogenesis, inflammation, and fibrosis should be considered for the treatment of advanced liver injury. They further suggest that andrographolide treatment may be a novel therapeutic agent for the treatment of liver disease.

Topics: Animals; Chemical and Drug Induced Liver Injury; Diterpenes; Liver; Liver Cirrhosis; Mice; Mice, Inbred BALB C; Neovascularization, Pathologic; Thioacetamide

This study aimed to compare the performance of gadoxetic acid -enhanced magnetic resonance imaging (MRI) and sonoelastography in evaluating chemopreventive effects of Sho-Saiko-To (SST) in thioacetamide (TAA)-induced early liver fibrosis in rats.. Ten of Sprague-Dawley rats receiving TAA (200 mg/kg of body weight) intraperitoneal injection were divided into three groups: Group 1 (TAA only, n = 3), Group 2 (TAA +0.25 g/kg SST, n = 4) and Group 3 (TAA+1 g/kg SST, n = 3). Core needle liver biopsy at week 2 and liver specimens after sacrifice at week 6 confirmed liver fibrosis using histological examinations, including Sirius red staining, Ishak and Metavir scoring systems. Gadoxetic acid-enhanced MRI and shear-wave sonoelastography were employed to evaluate liver fibrosis. The expression of hepatic transporter organic anion transporter 1 (Oatp1), multidrug-resistant protein 2 (Mrp2) and alpha-smooth muscle actin (α-Sma) were also analyzed in each group by immunohistochemistry (IHC) and Western blot.. According to histological grading by Sirius red staining, Ishak scores of liver fibrosis in Groups 1, 2 and 3 were 3, 2 and 1, respectively. As shown in gadoxetic acid-enhanced MRI, the ratio of relative enhancement was significantly lower in Group 1 (1.87 ± 0.21) than in Group 2 of low-dose (2.82 ± 0.25) and Group 3 of high-dose (2.72 ± 0.12) SST treatment at 10 minutes after gadoxetic acid intravenous injection (p < 0.05). Sonoelastography showed that the mean difference before and after experiments in Groups 1, 2 and 3 were 4.66 ± 0.1, 4.4 ± 0.57 and 3 ± 0.4 KPa (p < 0.1), respectively. Chemopreventive effects of SST reduced the Mrp2 protein level (p < 0.01) but not Oatp1 and α-Sma levels.. Sonoelastography and gadoxetic acid-enhanced MRI could monitor the treatment effect of SST in an animal model of early hepatic fibrosis.

Topics: Animals; Biomarkers; Contrast Media; Disease Models, Animal; Drugs, Chinese Herbal; Elasticity Imaging Techniques; Gadolinium DTPA; Immunoenzyme Techniques; Liver Cirrhosis; Liver Function Tests; Magnetic Resonance Imaging; Male; Plant Extracts; Rats; Rats, Sprague-Dawley; Thioacetamide; Tissue Distribution

The aim of this study was to elucidate the effects of zedoary turmeric oil (ZTO) on P450 activities (CYP1A2, CYP2C9, CYP2C19, CYP2B6, CYP2D6 and CYP3A4) in rats with liver cirrhosis induced by thioacetamide (TAA). For the induction of liver cirrhosis, rats were given TAA in their drinking water at a concentration of 0.03% for consecutive 5 weeks and then 0.04% for the next consecutive 5 weeks throughout the establishment of cirrhosis. Then the cirrhotic rats were ip given saline, ZTO 100, 200 and 400 mg/kg, respectively, once daily for 2 weeks. When cirrhosis model was established at week 10, all rats of five groups were administered intragastrically with 15 mg/kg phenacetin, 0.6 mg/kg tolbutamide, 15 mg/kg omeprazole, 15 mg/kg bupropion, 15 mg/kg metoprolol, and 10 mg/kg midazolam. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The degree of liver cirrhosis was assessed by HE staining. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) from the model group increased by approximately 4-fold, and a decreased level of albumin (Alb) was also observed, as compared to the control group (P < 0.05). However, ZTO was found to reverse those changes of serum levels observed in the model group, and the 200 mg/kg ZTO treatment group showed the most obvious reverse tendency with significantly decreased ALT, AST and increased Alb levels (P < 0.05). The results indicated that ZTO with the dose of 100 mg/kg could inhibit the activities of CYP450 isoforms CYP2C9 and CYP2D6 in vivo in cirrhotic rats induced by TAA, while ZTO with the dose of 400 mg/kg could induce the activity of CYP2C19 in vivo in cirrhotic rats induced by TAA. However, ZTO showed no influence on cirrhotic rat hepatic CYP1A2, CYP2B6 and CYP3A4 activity in vivo. This has certain guiding significance to clinical treatment.

Topics: Animals; Curcuma; Cytochrome P-450 Enzyme System; Disease Models, Animal; Liver; Liver Cirrhosis; Male; Plant Extracts; Rats; Rats, Sprague-Dawley; Thioacetamide

Aim of present study was to investigate the effect of NAC on experimental chronic hepatotoxicity models induced by carbon tetrachloride (CCl₄) and thioacetamide (TAA). CCl₄ toxicity was induced by administering 200 μl CCl₄ (diluted 2:3 in coconut oil)/100 g body weight, p.o., twice weekly for 8 weeks. TAA toxicity was induced by administering 150 mg/kg b. wt. of TAA i.p., twice weekly for 8 weeks. NAC treatment was started along with toxicants (CCl₄ and TAA) for 8 weeks and continued for further 4 weeks. Self reversal group was kept without any treatment for 4 weeks after completion of toxicant treatments. Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Bilirubin were measured in serum. Hydroxyproline (HP), lipid peroxidation (LPO), catalase (CAT), Glutathione peroxidase (GPx) and Glutathione (GSH) were determined in liver samples by colorimetric methods. Cytochrome P450 2E1 (CYP 450 2E1), activity was determined as hydroxylation of aniline in liver microsomes. General examination and histological analysis were also performed. Serum markers of liver damage (AST, ALT, ALP and Bilirubin) were increased by CCl₄ and TAA intoxication (p<0.001), whereas co-treatment with NAC reversed such changes (p<0.001). HP was enhanced in toxicant groups (p<0.001 in CCl₄ and TAA), but inhibited by NAC (p<0.001). LPO was increased while as GSH, CAT and GPx decreased by the administration of CCl₄ and TAA (p<0.001); co-administration of NAC restored these liver markers to normal levels (p<0.001). Biochemical determinations were corroborated by general and histological findings. Keeping in view the biochemical and histopathological studies, it was concluded that CCl₄ and TAA are strong hepatotoxic agents that produce liver fibrosis with close proximity to human etiology (micronodular cirrhosis) and NAC has a significant protective activity against CCl₄ and TAA. NAC has also been validated as a model against oxidative burden in chronic liver pathology.

Topics: Acetylcysteine; Allium; Animals; Carbon Tetrachloride; Disease Models, Animal; Liver Cirrhosis; Male; Oxidative Stress; Phytotherapy; Rats; Rats, Wistar; Sulfur; Thioacetamide

Aberrant N-glycosylation caused by altered N-acetyl glucosaminyltransferase V (GnT-V) expression is known to regulate tumor invasion and metastasis by modulating multiple cytokine signaling pathways. However, the exact role of GnT-V in the development of liver fibrosis has not been clearly defined. Here, we induced mouse liver fibrosis by ip injections of carbon tetrachloride (CCl4) or thioacetamide (TAA) and observed significant increase of hepatic GnT-V during the processes of liver fibrogenesis. Meanwhile, upregulations of GnT-V were detected in the activated hepatic stellate cells (HSCs) and injured hepatocytes. To knock down hepatic GnT-V expression, adenovirus that expressed the GnT-V siRNA was injected via the tail vein. Adenovirus-mediated delivery of GnT-V siRNA dramatically reduced the GnT-V expression in fibrotic liver and activated HSC in vivo and consequently alleviated CCl4- or TAA-induced liver fibrosis as assessed through collagen deposition and profiles of profibrogenic markers. Furthermore, knockdown of GnT-V in HSCs reduced transforming growth factor beta (TGF-β)/Smad signaling and blunted the activated HSC phenotype. The suppression of TGF-β/Smad signaling in HSCs correlated with the decrease of GnT-V-modified β1,6-branched N-glycan on TGF-β receptors. Knockdown of GnT-V also suppressed platelet-derived growth factor (PDGF)-induced HSC proliferation and migration through inhibiting PDGF/Erk signaling. Finally, we demonstrated that knockdown of GnT-V profoundly suppressed TGF-β1-induced epithelial-mesenchymal transition (EMT) in hepatocytes by morphological assessment and reversal of EMT markers. In conclusion, this study demonstrates that GnT-V is implicated in hepatotoxin-induced liver fibrosis, and targeting GnT-V may be a feasible and promising approach for treating liver fibrosis.

Topics: Actins; Alanine Transaminase; Animals; Carbon Tetrachloride; Cells, Cultured; Epithelial-Mesenchymal Transition; Glycosylation; Hepatocytes; Liver Cirrhosis; Male; Mice; Mice, Inbred ICR; Myofibroblasts; N-Acetylglucosaminyltransferases; Receptors, Transforming Growth Factor beta; Thioacetamide

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

Increased intra-hepatic resistance to portal blood flow is the primary factor leading to portal hypertension in cirrhosis. Up-regulated expression of cyclooxygenase-2 (COX-2) in the cirrhotic liver might be a potential target to ameliorate portal hypertension.. To verify the effect of celecoxib, a selective inhibitor of COX-2, on portal hypertension and the mechanisms behind it.. Cirrhotic liver model of rat was established by peritoneal injection of thiacetamide (TAA). 36 rats were randomly assigned to control, TAA and TAA+celecoxib groups. Portal pressures were measured by introduction of catheters into portal vein. Hepatic fibrosis was assessed by the visible hepatic fibrotic areas and mRNAs for collagen III and α-SMA. The neovasculature was determined by hepatic vascular areas, vascular casts and CD31 expression. Expressions of COX-2, vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2) and related signal molecules were quantitated.. Compared with TAA group, the portal pressure in TAA+celecoxib group was significantly decreased by 17.8%, p<0.01. Celecoxib treatment greatly reduced the tortuous hepatic portal venules. The data of fibrotic areas, CD31expression, mRNA levels of α-SMA and collagen III in TAA+celecoxib group were much lower than those in TAA group, p<0.01. Furthermore, the up-regulation of hepatic mRNA and protein levels of VEGF, VEGFR-2 and COX-2 induced by TAA was significantly inhibited after celecoxib treatment. The expressions of prostaglandin E2 (PGE2), phosphorylated extracellular signal-regulated kinase (p-ERK), hypoxia-inducible factor-1α (HIF-1α), and c-fos were also down-regulated after celecoxib treatment.. Long term administration of celecoxib can efficiently ameliorate portal hypertension in TAA rat model by its dual inhibitory effects on the intrahepatic fibrosis and angiogenesis. The anti-angiogenesis effect afforded by celecoxib may attribute to its modulation on VEGF/VEGFR-2 through the down-regulation of integrated signal pathways involving PGE2- HIF-1α- VEGF and p-ERK- c-fos- VEGFR-2.

Topics: Angiogenesis Inhibitors; Animals; Celecoxib; Disease Models, Animal; Hypertension, Portal; Kidney; Liver Cirrhosis; Male; Neovascularization, Pathologic; Pyrazoles; Rats; Rats, Sprague-Dawley; Signal Transduction; Sulfonamides; Thioacetamide

Recombinant vesicular stomatitis virus (VSV) shows promise for the treatment of hepatocellular carcinoma (HCC), but its safety and efficacy when administered in a setting of hepatic fibrosis, which occurs in the majority of clinical cases, is unknown. We hypothesized that VSV could provide a novel benefit to the underlying fibrosis, due to its ability to replicate and cause cell death specifically in activated hepatic stellate cells. In addition to the ability of VSV to produce a significant oncolytic response in HCC-bearing rats in the background of thioacetamide-induced hepatic fibrosis without signs of hepatotoxicity, we observed a significant downgrading of fibrosis stage, a decrease in collagen content in the liver, and modulation of gene expression in favor of fibrotic regression. Together, this work suggests that VSV is not only safe and effective for the treatment of HCC with underlying fibrosis, but it could potentially be developed for clinical application as a novel antifibrotic agent.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Hep G2 Cells; Humans; Infusions, Intra-Arterial; Liver Cirrhosis; Liver Cirrhosis, Experimental; Liver Neoplasms; Male; Oncolytic Virotherapy; Oncolytic Viruses; Rats; Rats, Inbred BUF; Thioacetamide; Vesicular stomatitis Indiana virus

To date, considerable progress has been made both in the mechanisms driving liver fibrosis and in the prevention of disease progression. Resolution of liver fibrosis is an emerging field in hepatology; yet, the mediators involved remain elusive. Earlier work from our laboratory demonstrated that the matricellular cytokine osteopontin (OPN) is pro-fibrogenic by promoting hepatic stellate cell (HSC) activation and extracellular matrix (ECM) deposition in vitro and in vivo and specifically by governing fibrillar collagen-I expression, the key pro-fibrogenic protein. Here we hypothesized that OPN could also delay the resolution of liver fibrosis by sustaining collagen-I synthesis or by preventing its degradation. To demonstrate this, wild-type (WT) and OPN-knockout (Opn(-/-)) mice were administered thioacetamide (TAA) in the drinking water for 4 months. Half of the mice were killed at 4 months to assess the extent of fibrosis at the peak of injury, and the rest of the mice were killed 2 months after TAA withdrawal to determine the rate of fibrosis resolution. Following TAA cessation, livers from Opn(-/-) mice showed no centrilobular and parenchymal necrosis along with faster ECM remodeling than WT mice. The latter was quantified by less fibrillar collagen-I immunostaining. Western blot analysis demonstrated a significant decrease in fibrillar collagen-I and in tissue inhibitor of metalloproteinase-1 (TIMP-1) in Opn(-/-) mice undergoing fibrosis resolution compared with WT mice. In conclusion, these results suggest that OPN delays liver fibrosis resolution due to sustained fibrillar collagen-I deposition; hence, inhibiting OPN could be an effective therapeutic strategy for resolving liver fibrosis.

Topics: Actins; Animals; Biomarkers; Collagen Type I; Crosses, Genetic; Disease Models, Animal; Extracellular Matrix; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Liver Regeneration; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Necrosis; Osteopontin; Protein Stability; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1

Antrodia camphorata is a unique fungus in Taiwan; the submerged fermentation product is used as the functional food for liver protection. Deep ocean water (DOW) containing rich metals and trace elements is proven to stimulate the production of functional metabolites and health function of functional fungus product in our previous study. Therefore, A. camphorata-fermented product cultured in DOW (DOW-AC) or reverse osmosis water (ROW-AC) as culture water was daily fed thioacetamide (TAA)-induced fibrosis rat for 8 weeks in order to investigate whether DOW promoted the effect of A. camphorata-fermented product on the prevention against TAA-induced liver damage and fibrosis. In the results, feeding one dose of DOW-AC prevented from TAA-induced weight loss and had more effect on inhibiting lipid peroxidation, reactive oxygen species, iNOS, and TNF-α expression than one dose of ROW-AC. Furthermore, DOW-AC also had more potent effect on protection against TAA-induced liver damage and fibrosis according to the results of H&E stain and collagen stain. However, higher liver protection of DOW-AC should be due to the fact that DOW not only increased the production of A. camphorata-fermented functional metabolites including triterpenoids, polysaccharides, flavonoids, and polyphenols but also contributed to protection against TAA-induced damage and fibrosis.

Topics: Animals; Antrodia; Biological Factors; Culture Media; Fermentation; Humans; Liver; Liver Cirrhosis; Male; Protective Agents; Rats; Rats, Sprague-Dawley; Seawater; Thioacetamide

Certain bioactive peptides are reported to be able to alleviate hepatic fibrosis. Our previous work has confirmed the hepatoprotective effect of corn peptides (CPs) that are prepared from a high protein by-product, corn gluten meal, on acute liver injury in an animal model. However, the antifibrotic activity of CPs remained to be elucidated. In this study, the hepatoprotective effect of CPs on thioacetamide (TAA)-induced liver fibrosis was tested. Results showed that CPs (100 mg/kg body weight) significantly decreased the levels of alanine transaminase/aspartate transaminase, laminin, type IV collagen, and type III collagen in serum and increased the serum albumin levels and total antioxidant capacity. Additionally, with CP treatment (100 mg/kg body weight), a significant decrease was observed in the levels of malondialdehyde, nitric oxide, hydroxyproline, transforming growth factor β1, and lactate dehydrogenase activity as well as the liver index, while the activity of superoxidedismutase was significantly increased in livers. The histological and morphological analysis showed that the hepatocyte structure in CP-treated rats was superior to that of TAA-injured rats, and inflammation and fibrosis were also ameliorated. Therefore, CPs can be used as an option for prevention and adjuvant therapy of liver fibrosis.

Topics: Animals; Antioxidants; Humans; Liver Cirrhosis; Liver Function Tests; Male; Malondialdehyde; Peptide Mapping; Peptides; Plant Proteins; Protective Agents; Rats; Rats, Wistar; Thioacetamide; Transforming Growth Factor beta1; Zea mays

Galectin-3 protein is critical to the development of liver fibrosis because galectin-3 null mice have attenuated fibrosis after liver injury. Therefore, we examined the ability of novel complex carbohydrate galectin inhibitors to treat toxin-induced fibrosis and cirrhosis. Fibrosis was induced in rats by intraperitoneal injections with thioacetamide (TAA) and groups were treated with vehicle, GR-MD-02 (galactoarabino-rhamnogalaturonan) or GM-CT-01 (galactomannan). In initial experiments, 4 weeks of treatment with GR-MD-02 following completion of 8 weeks of TAA significantly reduced collagen content by almost 50% based on Sirius red staining. Rats were then exposed to more intense and longer TAA treatment, which included either GR-MD-02 or GM-CT-01 during weeks 8 through 11. TAA rats treated with vehicle developed extensive fibrosis and pathological stage 6 Ishak fibrosis, or cirrhosis. Treatment with either GR-MD-02 (90 mg/kg ip) or GM-CT-01 (180 mg/kg ip) given once weekly during weeks 8-11 led to marked reduction in fibrosis with reduction in portal and septal galectin-3 positive macrophages and reduction in portal pressure. Vehicle-treated animals had cirrhosis whereas in the treated animals the fibrosis stage was significantly reduced, with evidence of resolved or resolving cirrhosis and reduced portal inflammation and ballooning. In this model of toxin-induced liver fibrosis, treatment with two galectin protein inhibitors with different chemical compositions significantly reduced fibrosis, reversed cirrhosis, reduced galectin-3 expressing portal and septal macrophages, and reduced portal pressure. These findings suggest a potential role of these drugs in human liver fibrosis and cirrhosis.

Topics: Animals; Apoptosis; Blotting, Western; Cell Line; Cell Proliferation; Fibrosis; Galactans; Galactose; Galectins; Humans; Liver Cirrhosis; Liver Diseases; Male; Mannans; Pectins; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Thioacetamide

Sildenafil citrate is a phosphodiesterase-5 inhibitor, approved for the treatment of erectile dysfunction. It enhances nitric-oxide-induced vasodilatation and it promotes angiogenesis. A relationship between angiogenesis and hepatic fibrosis has long been speculated, where the 2 are believed to progress together. In this study, the ability of sildenafil (10 mg·(kg body mass)(-1), orally, once daily) to prevent and also reverse liver fibrosis/cirrhosis experimentally induced by thioacetamide injection (200 mg·kg(-1), intraperitoneal (i.p.), 3 times·week(-1)) in male Sprague-Dawley rats has been investigated. Sildenafil administration, either to prevent or to reverse liver fibrosis/cirrhosis significantly improved the estimated hepatic functions, reduced hepatic hydroxyproline and, in turn, hepatic collagen content, as well as reducing serum levels of the pro-fibrogenic mediator transforming growth factor β1. In co-ordination with such improvement, fibrosis grades declined and fibrosis retracted. Herein, the observed results provide evidence for the potential therapeutic efficacy of sildenafil as an antifibrotic agent.

Topics: Animals; Collagen; Disease Progression; Hydroxyproline; Liver; Liver Cirrhosis; Liver Cirrhosis, Experimental; Male; Piperazines; Purines; Rats; Rats, Sprague-Dawley; Sildenafil Citrate; Sulfones; Thioacetamide; Transforming Growth Factor beta1

The oral administration of thioacetamide to rats induces cholangiofibrosis characterized by hyperplasia of ductules surrounded by fibrous tissue. In the present study, we examined the expression of markers of cholangiocyte and hepatocyte phenotypes in these hyperplastic ductule cells and their proliferative activity immunohistochemically. The oral administration of thioacetamide to 21-day-old male Fisher 344 rats for 12 weeks induced multiple areas of various sizes with hyperplastic ductules. The ductules consisted of two types of ductules; ductules composed of cholangiocyte-like cuboidal cells with transparent nuclei and cytoplasm, and of intestinal epithelium-like (IE-like) cells of basophilic nuclei and cytoplasm, and the transition of these two types of cells in the same ductule was sometimes observed. The cholangiocyte-like cells expressed cytokeratin (CK)-7, CK-19 and OV-6 (cholangiocyte phenotype markers) but not Hep Par-1 antigen or HNF4α (hepatocyte phenotype markers). In contrast, the IE-like cells expressed Hep Par-1 antigen and HNF4α but not CK-7, CK-19 or OV-6. The examination of Ki-67 expression showed a much higher proliferative activity for the IE-like cells compared to the cholangiocyte-like cells. The present results show that the hyperplastic ductules induced by thioacetamide are composed of IE-like cells with a high proliferative activity expressing the hepatocyte phenotype markers and of cholangiocyte-like cells with a low proliferative activity expressing the cholangiocyte phenotype markers.

Topics: Animals; Bile Ducts; Biomarkers; Cell Proliferation; Fibrosis; Hepatocytes; Hyperplasia; Immunohistochemistry; Liver Cirrhosis; Male; Phenotype; Rats; Rats, Inbred F344; Thioacetamide

This study was performed to evaluate the antifibrotic properties of coffee in a model of liver damage induced by repeated administration of thioacetamide (TAA) in male Wistar rats. In this study, cirrhosis was induced by chronic TAA administration and the effects of co-administration of conventional caffeinated coffee or decaffeinated coffee (CC, DC, respectively) for 8 weeks were evaluated. TAA administration elevated serum alkaline phosphatase (AP), γ-glutamyl transpeptidase (γ-GTP) and alanine aminotransferase (ALAT), liver lipid peroxidation, collagen content, depleted liver glycogen and glutathione peroxidase (GPx) activity. Additionally increased levels of a number of proteins were detected including transforming growth factor-beta (TGF-β), connective tissue growth factor (CTGF) and alpha-smooth muscle actin (α-SMA), and matrix metalloproteinase (MMP)-2, 9 and 13. Coffee suppressed most of the changes produced by TAA. Histopathological analysis was in agreement with biochemical and molecular findings. These results indicate that coffee attenuates experimental cirrhosis; the action mechanisms are probably associated with its antioxidant properties and mainly by its ability to block the elevation of the profibrogenic cytokine TGF-β and its downstream effector CTGF. Various components of coffee that have been related to such a favorable effect include caffeine, coffee oils kahweol, cafestol and antioxidant substances; however, no definite evidence for the role of these components has been established. These results support earlier findings suggesting a beneficial effect of coffee on the liver. However, more basic clinical studies must be performed to confirm this hypothesis.

Topics: Actins; Alanine Transaminase; Alkaline Phosphatase; Animals; Antioxidants; Coffee; Collagen; Connective Tissue Growth Factor; Disease Models, Animal; Fibrosis; gamma-Glutamyltransferase; Glutathione Peroxidase; Lipid Peroxidation; Liver; Liver Cirrhosis; Male; Matrix Metalloproteinase 13; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Rats; Rats, Wistar; Thioacetamide; Transforming Growth Factor beta

Sorafenib, which is approved for treatment of HCC, has also shown promising antifibrotic activity, and therefore refinement of its dosing requirements and window of efficacy are important goals prior to antifibrotic clinical trials.. The purpose of this study was to determine the minimal effective dose and optimal timing of sorafenib therapy in cultured human stellate cells and in rats with experimental hepatic fibrosis.. Effects of sorafenib were assessed in a human stellate cell line (LX-2). In vivo, rats were treated for 8 weeks with TAA three times per week (150 mg/kg IP), and with either PBS or sorafenib administered daily at doses of 1.25, 5 or 7 mg/kg/day gavage either at the beginning of TAA administration for 8 weeks, during weeks 4-8, or from weeks 8-12.. Sorafenib treatment significantly inhibited LX-2 proliferation by >75% (7.5 or 15 μM). Treatment with 7.5-μM sorafenib for 12 h markedly inhibited expression of TGFβ1, TIMP-1, collagen I, and MMP2 mRNAs, but not of β-PDGFR or type I TGFβR. In vivo, sorafenib significantly inhibited liver fibrosis when started concurrently with TAA and during weeks 4-8 with TAA. In contrast, there was no significant effect of sorafenib on fibrogenic gene expression or fibrosis when begun after cirrhosis was already established.. Sorafenib is anti-proliferative and antifibrotic towards human HSCs in culture, and is a potent antifibrotic agent in TAA-induced hepatic fibrosis in rats. The drug is effective at relatively low doses at the early stage of liver fibrosis, but is not effective when cirrhosis is already established.

Topics: Animals; Cells, Cultured; Dose-Response Relationship, Drug; Gene Expression Regulation; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Male; Niacinamide; Phenylurea Compounds; Rats; Rats, Sprague-Dawley; RNA; Sorafenib; Thioacetamide

The identification of the active phenolic compounds in the mixed extract of sea cucumber (Holothuria atra) body wall by high-performance liquid chromatography and an assessment of its hepatoprotective activity against thioacetamide-induced liver fibrosis in rats.. Female Swiss albino rats were divided into four groups: normal controls; oral administration of a sea cucumber mixed extract (14.4 mg/kg of body weight) on days 2, 4, and 6 weekly for 8 consecutive weeks; intoxication with thioacetamide (200 mg/kg of body weight, intraperitoneally) on days 2 and 6 weekly for 8 wk; and oral administration of a sea cucumber extract and then intoxication with thioacetamide 2 h later for 8 wk.. High-performance liquid chromatographic analysis of the sea cucumber mixed extract revealed the presence of some phenolic components, such as chlorogenic acid, pyrogallol, rutin, coumaric acid, catechin, and ascorbic acid. In vitro studies have shown that the extract has a high scavenging activity for the nitric oxide radical, a moderate iron-chelating activity, and a weak inhibitory effect of lipid peroxidation. The subchronic oral administration of sea cucumber extract to the rats did not show any toxic side effects but increased hepatic superoxide dismutase and glutathione peroxidase activities. The coadministration of sea cucumber extract and thioacetamide (protection modality) normalized serum direct bilirubin, alanine and aspartate aminotransferases, hepatic malondialdehyde, and hydroxyproline concentrations and antioxidant enzyme activities. In addition, the histologic examination of liver sections from the protection group that were stained with hematoxylin and eosin showed substantial attenuation of the degenerative cellular changes and regressions in liver fibrosis and necrosis induced by the thioacetamide intoxication.. Sea cucumber mixed extract contains physiologically active phenolic compounds with antioxidant activity, which afforded a potential hepatoprotective activity against thioacetamide-induced liver injury in a rat model.

Topics: Animals; Antioxidants; Female; Glutathione Peroxidase; Holothuria; Lipid Peroxidation; Liver; Liver Cirrhosis; Phenols; Rats; Superoxide Dismutase; Thioacetamide

Transplantation of bone marrow mesenchymal stem cells (BM-MSCs) has been considered as an alternative therapy, replacing liver transplantation in clinical trials, to treat liver cirrhosis, an irreversible disease that may eventually lead to liver cancer development. However, low survival rate of the BM-MSCs leading to unsatisfactory efficacy remains a major concern. Gender differences have been suggested in BM-MSCs therapeutic application, but the effect of the androgen receptor (AR), a key factor in male sexual phenotype, in this application is not clear. Using two liver cirrhosis mouse models induced by CCl4 or thioacetamide, we showed that targeting AR in the BM-MSCs improved their self-renewal and migration potentials and increased paracrine effects to exert anti-inflammatory and anti-fibrotic actions to enhance liver repair. Mechanism dissection studies suggested that knocking out AR in BM-MSCs led to improved self-renewal and migration by alteration of the signaling of epidermal growth factor receptor and matrix metalloproteinase 9 and resulted in suppression of infiltrating macrophages and hepatic stellate cell activation through modulation of interleukin (IL)1R/IL1Ra signaling. Therapeutic approaches using either AR/small interfering RNA or the AR degradation enhancer, ASC-J9, to target AR in BM-MSCs all led to increased efficacy for liver repair.. Targeting AR, a key factor in male sexual phenotype, in BM-MSCs improves transplantation therapeutic efficacy for treating liver fibrosis.

Topics: Animals; Carbon Tetrachloride; Disease Models, Animal; ErbB Receptors; Female; Liver Cirrhosis; Male; Matrix Metalloproteinase 9; Mesenchymal Stem Cell Transplantation; Mice; Mice, Knockout; Phenotype; Receptors, Androgen; Receptors, Interleukin-1; RNA, Small Interfering; Signal Transduction; Thioacetamide; Treatment Outcome

To examine whether the administration of atorvastatin and rosuvastatin would prevent experimentally-induced hepatic cirrhosis in rats.. Liver cirrhosis was induced by injections of thioacetamide (TAA). Rats were treated concurrently with TAA alone or TAA and either atorvastatin (1,10 and 20 mg/kg) or rosuvastatin (1, 2.5, 5, 10 and 20 mg/kg) given daily by nasogastric gavage.. Liver fibrosis and hepatic hydroxyproline content, in the TAA-treated group was significantly higher than those of the controls [11.5 ± 3.2 vs 2.6 ± 0.6 mg/g protein (P = 0.02)]. There were no differences in serum aminotransferase levels in the TAA controls compared to all the groups treated concomitantly by statins. Both statins used in our study did not prevent liver fibrosis or reduce portal hypertension, and had no effect on hepatic oxidative stress. Accordingly, the hepatic level of malondialdehyde was not lower in those groups treated by TAA + statins compared to TAA only. In vitro studies, using the BrdU method have shown that atorvastatin had no effect of hepatic stellate cells proliferation. Nevertheless, statin treatment was not associated with worsening of liver damage, portal hypertension or survival rate.. Atorvastatin or rosuvastatin did not inhibit TAA-induced liver cirrhosis or oxidative stress in rats. Whether statins may have therapeutic applications in hepatic fibrosis due to other etiologies deserve further investigation.

Topics: Animals; Atorvastatin; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Fluorobenzenes; Hepatic Stellate Cells; Heptanoic Acids; Hypertension, Portal; In Vitro Techniques; Incidence; Injections; Liver Cirrhosis; Liver Cirrhosis, Experimental; Male; Oxidative Stress; Pyrimidines; Pyrroles; Rats; Rats, Wistar; Rosuvastatin Calcium; Sulfonamides; Thioacetamide

Considerable evidence has demonstrated that transforming growth factor β (TGF-β) plays a key role in hepatic fibrosis, the final common pathway for a variety of chronic liver diseases leading to liver insufficiency. Although a few studies have reported that blocking TGF-β with soluble receptors or siRNA can prevent the progression of hepatic fibrosis, as yet no evidence has been provided that TGF-β antagonism can improve pre-existing hepatic fibrosis. The aim of this study was to examine the effects of a murine neutralizing TGF-β monoclonal antibody (1D11), in a rat model of thioacetamide (TAA)-induced hepatic fibrosis. TAA administration for 8 weeks induced extensive hepatic fibrosis, whereupon 1D11 dosing was initiated and maintained for 8 additional weeks. Comparing the extent of fibrosis at two time points, pre- and post-1D11 dosing, we observed a profound regression of tissue injury and fibrosis upon treatment, as reflected by a reduction of collagen deposition to a level significantly less than that observed before 1D11 dosing. Hepatic TGF-β1 mRNA, tissue hydroxyproline, and plasminogen activator inhibitor 1 (PAI-1) levels were significantly elevated at the end of the 8 week TAA treatment. Vehicle and antibody control groups demonstrated progressive injury through 16 weeks, whereas those animals treated for 8 weeks with 1D11 showed striking improvement in histologic and molecular endpoints. During the course of tissue injury, TAA also induced cholangiocarcinomas. At the end of study, the number and area of cholangiocarcinomas were significantly diminished in rats receiving 1D11 as compared to control groups, presumably by the marked reduction of supporting fibrosis/stroma. The present study demonstrates that 1D11 can reverse pre-existing hepatic fibrosis induced by extended dosing of TAA. The regression of fibrosis was accompanied by a marked reduction in concomitantly developed cholangiocarcinomas. These data provide evidence that therapeutic dosing of a TGF-β antagonist can diminish and potentially reverse hepatic fibrosis and also reduce the number and size of attendant cholangiocarcinomas.

Topics: Animals; Antibodies, Monoclonal, Murine-Derived; Cholangiocarcinoma; Humans; Liver; Liver Cirrhosis; Male; Mice; Molecular Targeted Therapy; Plasminogen Activator Inhibitor 1; Rats; Signal Transduction; Thioacetamide; Transforming Growth Factor beta

The aim of this study was to investigate if armepavine (Arm, C₁₉H₂₃O₃N) could exert inhibitory effects against hepatic fibrosis in rats. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with tumour necrosis factor-α (TNF-α) to evaluate the inhibitory effects of Arm. Rats were injected with thioacetamide (TAA; 300 mg/kg, intraperitoneally) thrice a week for 4 weeks to induce hepatic fibrosis, with Arm (3 or 10 mg/kg) given by gavage twice a day. Liver sections were taken for western blotting, fibrosis scoring and immunofluorescence staining. Arm (1-10 µm) concentration-dependently attenuated TNF-α-stimulated: (i) protein expressions of α-smooth muscle actin (α-SMA), collagen type I and angiopoietin-1; (ii) H₂O₂ production; and (iii) NF-κB, JunD and C/EBPß (cytidine-cytidine-adenosine-adenosine-thymidine (CCAAT)/enhancer binding protein-ß (EBPß)) nuclear translocations in HSC-T6 cells. In vivo Arm treatment significantly reduced plasma aspartate transaminase and alanine transaminase levels, hepatic α-SMA expression and collagen contents, and fibrosis scores of TAA-injected rats. Moreover, Arm treatment decreased α-SMA- and NF-κB-positive cells in immunohistochemical staining, and mRNA expression levels of IL-6, TGF-ß1, TIMP-1, col1α2, iNOS and ICAM-1 genes, but up-regulated the metallothionein gene in the livers of TAA-injected rats. Our results indicated that Arm exerted both in vitro and in vivo antifibrotic effects in rats, with inhibition of NF-κB, JunD and C/EBPß pathways.

Topics: Actins; Active Transport, Cell Nucleus; Alanine Transaminase; Angiopoietin-1; Animals; Aspartate Aminotransferases; Benzylisoquinolines; Blotting, Western; CCAAT-Enhancer-Binding Protein-beta; Collagen Type I; Cytokines; Dose-Response Relationship, Drug; Fluorescent Antibody Technique; Hepatic Stellate Cells; Hydrogen Peroxide; Liver Cirrhosis; Male; Metallothionein; NF-kappa B; Phytotherapy; Rats; Rats, Sprague-Dawley; Thioacetamide; Transcription Factors; Transcription, Genetic

Oxidative stress plays a critical role in liver fibrogenesis. Reactive oxygen species (ROS) stimulate hepatic stellate cells (HSCs), and ROS-mediated increases in calcium influx further increase ROS production. Azelnidipine is a calcium blocker that has been shown to have antioxidant effects in endothelial cells and cardiomyocytes. Therefore, we evaluated the anti-fibrotic and antioxidative effects of azelnidipine on liver fibrosis.. We used TGF-β1-activated LX-2 cells (a human HSC line) and mouse models of fibrosis induced by treatment with either carbon tetrachloride (CCl(4) ) or thioacetamide (TAA).. Azelnidipine inhibited TGF-β1 and angiotensin II (Ang II)-activated α1(I) collagen mRNA expression in HSCs. Furthermore, TGF-β1- and Ang II-induced oxidative stress and TGF-β1-induced p38 and JNK phosphorylation were reduced in HSCs treated with azelnidipine. Azelnidipine significantly decreased inflammatory cell infiltration, pro-fibrotic gene expressions, HSC activation, lipid peroxidation, oxidative DNA damage and fibrosis in the livers of CCl(4) - or TAA-treated mice. Finally, azelnidipine prevented a decrease in the expression of some antioxidant enzymes and accelerated regression of liver fibrosis in CCl(4) -treated mice.. Azelnidipine inhibited TGF-β1- and Ang II-induced HSC activation in vitro and attenuated CCl(4) - and TAA-induced liver fibrosis, and it accelerated regression of CCl(4) -induced liver fibrosis in mice. The anti-fibrotic mechanism of azelnidipine against CCl(4) -induced liver fibrosis in mice may have been due an increased level of antioxidant defence. As azelnidipine is widely used in clinical practice without serious adverse effects, it may provide an effective new strategy for anti-fibrotic therapy.

Topics: Angiotensin II; Animals; Antioxidants; Azetidinecarboxylic Acid; Calcium; Calcium Channel Blockers; Carbon Tetrachloride; Cell Line; Cell Survival; Collagen Type I; Collagen Type I, alpha 1 Chain; Dihydropyridines; Disease Models, Animal; Gene Expression Regulation; Humans; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Reactive Oxygen Species; RNA, Messenger; Thioacetamide; Transforming Growth Factor beta1

A key feature in the pathogenesis of liver fibrosis is fibrillar Collagen-I deposition; yet, mediators that could be key therapeutic targets remain elusive. We hypothesized that osteopontin (OPN), an extracellular matrix (ECM) cytokine expressed in hepatic stellate cells (HSCs), could drive fibrogenesis by modulating the HSC pro-fibrogenic phenotype and Collagen-I expression. Recombinant OPN (rOPN) up-regulated Collagen-I protein in primary HSCs in a transforming growth factor beta (TGFβ)-independent fashion, whereas it down-regulated matrix metalloprotease-13 (MMP13), thus favoring scarring. rOPN activated primary HSCs, confirmed by increased α-smooth muscle actin (αSMA) expression and enhanced their invasive and wound-healing potential. HSCs isolated from wild-type (WT) mice were more profibrogenic than those from OPN knockout (Opn(-/-)) mice and infection of primary HSCs with an Ad-OPN increased Collagen-I, indicating correlation between both proteins. OPN induction of Collagen-I occurred via integrin α(v)β(3) engagement and activation of the phosphoinositide 3-kinase/phosphorylated Akt/nuclear factor kappa B (PI3K/pAkt/NFκB)-signaling pathway, whereas cluster of differentiation 44 (CD44) binding and mammalian target of rapamycin/70-kDa ribosomal protein S6 kinase (mTOR/p70S6K) were not involved. Neutralization of integrin α(v) β(3) prevented the OPN-mediated activation of the PI3K/pAkt/NFκB-signaling cascade and Collagen-I up-regulation. Likewise, inhibition of PI3K and NFκB blocked the OPN-mediated Collagen-I increase. Hepatitis C Virus (HCV) cirrhotic patients showed coinduction of Collagen-I and cleaved OPN compared to healthy individuals. Acute and chronic liver injury by CCl(4) injection or thioacetamide (TAA) treatment elevated OPN expression. Reactive oxygen species up-regulated OPN in vitro and in vivo and antioxidants prevented this effect. Transgenic mice overexpressing OPN in hepatocytes (Opn(HEP) Tg) mice developed spontaneous liver fibrosis compared to WT mice. Last, chronic CCl(4) injection and TAA treatment caused more liver fibrosis to WT than to Opn(-/-) mice and the reverse occurred in Opn(HEP) Tg mice.. OPN emerges as a key cytokine within the ECM protein network driving the increase in Collagen-I protein contributing to scarring and liver fibrosis.

Topics: Animals; Carbon Tetrachloride; Collagen Type I; Hepatic Stellate Cells; Humans; Integrin alphaVbeta3; Liver Cirrhosis; Male; Mice; Mice, Knockout; NF-kappa B; Osteopontin; Oxidative Stress; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction; Thioacetamide; Up-Regulation

Implantation of bone-marrow-derived MSCs (mesenchymal stem cells) has emerged as a potential treatment modality for liver failure, but in vivo differentiation of MSCs into functioning hepatocytes and its therapeutic effects have not yet been determined. We investigated MSC differentiation process in a rat model of TAA (thioacetamide)-induced liver cirrhosis. Male Sprague-Dawley rats were administered 0.04% TAA-containing water for 8 weeks, MSCs were injected into the spleen for transsplenic migration into the liver, and liver tissues were examined over 3 weeks. Ingestion of TAA for 8 weeks induced micronodular liver cirrhosis in 93% of rats. Injected MSCs were diffusely engrafted in the liver parenchyma, differentiated into CK19 (cytokeratin 19)- and thy1-positive oval cells and later into albumin-producing hepatocyte-like cells. MSC engraftment rate per slice was measured as 1.0-1.6%. MSC injection resulted in apoptosis of hepatic stellate cells and resultant resolution of fibrosis, but did not cause apoptosis of hepatocytes. Injection of MSCs treated with HGF (hepatocyte growth factor) in vitro for 2 weeks, which became CD90-negative and CK18-positive, resulted in chronological advancement of hepatogenic cellular differentiation by 2 weeks and decrease in anti-fibrotic activity. Early differentiation of MSCs to progenitor oval cells and hepatocytes results in various therapeutic effects, including repair of damaged hepatocytes, intracellular glycogen restoration and resolution of fibrosis. Thus, these results support that the in vivo hepatogenic differentiation of MSCs is related to the beneficial effects of MSCs rather than the differentiated hepatocytes themselves.

Topics: Animals; Cell Differentiation; Hepatocytes; Liver; Liver Cirrhosis; Male; Mesenchymal Stem Cells; Rats; Rats, Sprague-Dawley; Thioacetamide

Cirrhotic patients commonly have a liver zinc deficiency, which may aggravate liver fibrosis due to the lack of antioxidative effects of zinc. This study examined the ability of polaprezinc, N-(3-aminopropionyl)-l-histidinato zinc, to prevent fibrosis in a rat model of thioacetamide (TAA)-induced hepatic fibrosis.. Liver cirrhosis was induced by orally administering TAA for 20 weeks. The rats were cotreated with one of the following for the last 10 weeks of TAA treatment: (1) polaprezinc (50 or 200mg/kg/day); (2) l-carnosine (155 mg/kg/day), which contained equal amounts of l-carnosine as 200mg/kg/day polaprezinc; (3) zinc sulfate (112 mg/kg/day) or (4) zinc-l-aspartic complex (317.8 mg/kg/day). Both zinc supplementations contained equal amounts of zinc as high-dose polaprezinc.. Hepatic zinc levels fell significantly in rats treated with TAA for 20 weeks. Cotreating with high-dose polaprezinc and zinc-l-aspartic complex for 10 weeks prevented hepatic zinc loss. Hepatic hydroxyproline and tissue inhibitor of metalloproteinases-1 (TIMP-1) were significantly higher in rats treated with TAA for 20 weeks than 10 weeks, whereas polaprezinc prevented changes in these fibrosis markers and reduced hepatic transforming growth factor-β1 protein concentration, macroscopic and histologic changes. TAA caused oxidative stress-related changes in the liver that were prevented by high-dose polaprezinc and partially by zinc-l-aspartic complex. Treatment with l-carnosine, low-dose polaprezinc or zinc sulfate for 10 weeks did not affect liver fibrosis progression or oxidative stress-related changes.. Polaprezinc may prevent ongoing fibrosis by preventing zinc depletion, oxidative stress and fibrosis markers in cirrhotic livers.

Topics: Animals; Antioxidants; Carnosine; Liver Cirrhosis; Male; Organometallic Compounds; Oxidative Stress; Rats; Rats, Wistar; Thioacetamide; Zinc; Zinc Compounds

We previously demonstrated that kaerophyllin, a lignan, isolated from a widely used traditional Chinese herb, Bupleurum scorzonerifolium, leading to the inhibition of hepatic stellate cells (HSCs) activation in vitro. This current study evaluated the in vivo role of kaerophyllin in protecting the liver against injury and fibrogenesis caused by thioacetamide (TAA) in rats and further explored the underlying mechanisms.. Liver fibrosis in Sprague-Dawley rats was induced by intraperitoneal injection of TAA (200 mg/kg) twice per week for 6 weeks. Animals were divided into five groups: vehicle control, TAA control, TAA + low dose kaerophyllin, TAA + high dose kaerophyllin and TAA + curcumin groups. Kaerophyllin (10 or 30 mg/kg) or curcumin (150 mg/mL) was given by gavage twice per day consecutively for 4 weeks starting 2 weeks after TAA injection. Rat HSCs were used to investigate the anti-inflammatory role of kaerophyllin against tumour necrosis factor α (TNF-α) in vitro. Peroxisome proliferator-activated receptor-γ (PPAR-γ) expression was knocked down in rat HSCs using PPAR-γ small interfering RNAs.. Kaerophyllin significantly protected liver from injury by reducing serum aspartate transaminase and alanine transaminase levels and by improving the histological architecture and fibrosis score. In addition, kaerophyllin suppressed inflammation by reducing the mRNA of TNF-α, interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) genes. In HSCs, kaerophyllin elevated PPAR-γ activity and reduced TNF-α-stimulated mRNA levels of intracellular adhesion molecule-1 (ICAM-1), MCP-1 and IL-1β genes, which were reversed by small interfering RNA knockdown of PPAR-γ gene.. Our results demonstrated that kaerophyllin protected the rat liver from TAA-caused injury and fibrogenesis by suppressing hepatic inflammation and inhibiting HSC activation, possibly through upregulation of PPAR-γ expression.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Carcinogens; Drugs, Chinese Herbal; Hepatic Stellate Cells; Lignans; Liver; Liver Cirrhosis; Male; Plant Extracts; Plant Roots; Rats; Rats, Sprague-Dawley; Thioacetamide

Collagen I deposition contributes to liver fibrosis, yet little is known about other factors that mediate this process. Fibromodulin is a liver proteoglycan that regulates extracellular matrix organization and is induced by fibrogenic stimuli. We propose that fibromodulin contributes to the pathogenesis of fibrosis by regulating the fibrogenic phenotype of hepatic stellate cells (HSCs).. We analyzed liver samples from patients with hepatitis C-associated cirrhosis and healthy individuals (controls). We used a coculture model to study interactions among rat HSCs, hepatocytes, and sinusoidal endothelial cells. We induced fibrosis in livers of wild-type and Fmod(-/-) mice by bile duct ligation, injection of CCl(4), or administration of thioacetamide.. Liver samples from patients with cirrhosis had higher levels of fibromodulin messenger RNA and protein than controls. Bile duct ligation, CCl(4), and thioacetamide each increased levels of fibromodulin protein in wild-type mice. HSCs, hepatocytes, and sinusoidal endothelial cells produced and secreted fibromodulin. Infection of HSCs with an adenovirus that expressed fibromodulin increased expression of collagen I and α-smooth muscle actin, indicating increased activation of HSCs and fibrogenic potential. Recombinant fibromodulin promoted proliferation, migration, and invasion of HSCs, contributing to their fibrogenic activity. Fibromodulin was sensitive to reactive oxygen species. HepG2 cells that express cytochrome P450 2E1 produced fibromodulin, and HSCs increased fibromodulin production in response to pro-oxidants. In mice, administration of an antioxidant prevented the increase in fibromodulin in response to CCl(4). Coculture of hepatocytes or sinusoidal endothelial cells with HSCs increased the levels of reactive oxygen species in the culture medium, along with collagen I and fibromodulin proteins; this increase was prevented by catalase. Fibromodulin bound to collagen I, but the binding did not prevent collagen I degradation by matrix metalloproteinase 13. Bile duct ligation caused liver fibrosis in wild-type but not Fmod(-/-) mice.. Fibromodulin levels are increased in livers of patients with cirrhosis. Hepatic fibromodulin activates HSCs and promotes collagen I deposition, which leads to liver fibrosis in mice.

Topics: Actins; Animals; Antioxidants; Carbon Tetrachloride; Case-Control Studies; Cell Movement; Cell Proliferation; Chemical and Drug Induced Liver Injury; Coculture Techniques; Collagen Type I; Endothelial Cells; Extracellular Matrix Proteins; Fibromodulin; Hep G2 Cells; Hepatic Stellate Cells; Hepatitis C; Hepatocytes; Humans; Liver; Liver Cirrhosis; Liver Cirrhosis, Biliary; Liver Cirrhosis, Experimental; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxidative Stress; Proteoglycans; Rats; RNA, Messenger; Thioacetamide; Time Factors; Transfection; Up-Regulation

Inflammation and hepatic stellate cell (HSC) activation are the most crucial steps in the formation of hepatic fibrosis. Hepatocytes damaged by viral or bacterial infection, alcohol or toxic chemicals initiate an inflammatory response that activates collagen production by HSCs. Recent studies indicate curcumin has liver-protective effects due to its anti-inflammatory, antioxidant and anticancer activities; however, the mechanisms are not well understood. In this study, we show that curcumin protected against hepatic fibrosis in BALB/c mice in vivo by inhibiting HSC activation, inflammatory responses and inducing apoptosis of damaged hepatocytes. Using the thioacetamide (TAA)-induced hepatic fibrosis animal model, we found that curcumin treatment up-regulated P53 protein expression and Bax messenger RNA (mRNA) expression and down-regulated Bcl-2 mRNA expression. Together, these responses increased hepatocyte sensitivity to TAA-induced cytotoxicity and forced the damaged cells to undergo apoptosis. Enhancing the tendency of damaged hepatocytes to undergo apoptosis may be the protective mechanism whereby curcumin suppresses inflammatory responses and hepatic fibrogenesis. These results provide a novel insight into the cause of hepatic fibrosis and the cytoprotective effects curcumin has on hepatic fibrosis suppression.

Topics: Animals; Antineoplastic Agents; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Cell Line; Cell Proliferation; Curcumin; DNA Damage; Gene Expression Regulation; Hepatic Stellate Cells; Hepatocytes; In Situ Nick-End Labeling; Inflammation; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred BALB C; RNA, Messenger; Thioacetamide; Tumor Suppressor Protein p53

The proliferative response of hepatocytes in vivo can be induced by two mechanisms: severe damage to hepatic tissue results in regenerative growth and so-called primary hepatocyte mitogens can initiate liver cell proliferation without preceding loss of parenchyma. The regulation of the two responses is quite different. The decreased regenerative response of cirrhotic/fibrotic liver is well known, and is a severe obstacle to surgery of the diseased liver. In the present experiments we investigated the efficiency of a primary hepatocyte mitogen 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOB) on two different liver cirrhosis/fibrosis models in mice induced by chronic administration of CCl(4) and thioacetamide respectively. BrdU incorporation and cyclin A expression established clearly that there is a reduced but still powerful mitogenic response of the fibrotic livers. Therefore, primary hepatocyte mitogens appear to be suitable to be used to rescue the regenerative response of cirrhotic livers.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Biomarkers; Bromodeoxyuridine; Carbon Tetrachloride; Cell Proliferation; Cyclin A; Cytochrome P450 Family 2; Disease Models, Animal; Gene Expression; Hepatocytes; Hyperplasia; Liver; Liver Cirrhosis; Liver Regeneration; Male; Mice; Mice, Inbred C57BL; Pyridines; Steroid Hydroxylases; Thioacetamide; Transforming Growth Factor beta

The purpose of the study was to investigate the anti-fibrotic effect and the potential mechanisms of action of betulinic acid (BA) against hepatic fibrosis in vivo and in vitro. BA is an active compound isolated from the bark of the birch tree Betula spp. (Betulaceae). Liver fibrosis was induced by intraperitoneal injections of thioacetamide (TAA, 200mg/kg) twice weekly for 6weeks in Wistar rats. The administration of BA (20 or 50mg/kg) was started following TAA injections and was continued for 6 or 8weeks to evaluate both the preventive and the protective effects. BA demonstrated great efficacy in preventing and curing hepatic fibrosis via attenuating the TAA-mediated increases in liver tissue hydroxyproline and α-smooth muscle actin (α-SMA). In vitro, BA effectively decreased the HSC-T6 cell viability induced by TNF-α and showed low toxicity in normal human chang liver cells. Moreover, BA significantly attenuated the expression of α-SMA and tissue inhibitor of metalloproteinase-1 (TIMP-1) and increased the levels of matrix metalloprotease (MMP)-13. BA also inhibited the expression of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and the activation of nuclear factor-κB (NF-κB) in a time-dependent manner. This study provides evidence that BA exerts a significant anti-fibrosis effect by modulating the TLR4/MyD88/NF-κB signaling pathway.

Topics: Actins; Animals; Betulinic Acid; Blotting, Western; Cell Line; Cell Survival; Hydroxyproline; Immunohistochemistry; Liver Cirrhosis; Liver Cirrhosis, Experimental; Male; Matrix Metalloproteinase 13; Myeloid Differentiation Factor 88; NF-kappa B; Pentacyclic Triterpenes; Protein Transport; Random Allocation; Rats; Rats, Wistar; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1; Toll-Like Receptor 4; Triterpenes

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a cytoprotective agent in several organ systems but its roles in liver fibrosis are unclear. We studied the roles of HB-EGF in experimental liver fibrosis in mice and during hepatic stellate cell (HSC) activation. Thioacetamide (TAA; 100 mg/kg) was administered by intraperitoneal injection three times a week for 4 weeks to wild-type HB-EGF(+/+) or HB-EGF-null (HB-EGF(-/-)) male mice. Livers were examined for histology and expression of key fibrotic markers. Primary cultured HSCs isolated from untreated HB-EGF(+/+) or HB-EGF(-/-) mice were examined for fibrotic markers and/or cell migration either during culture-induced activation or after exogenous HB-EGF (100 ng/ml) treatment. TAA induced liver fibrosis in both HB-EGF(+/+) and HB-EGF(-/-) mice. Hepatic HB-EGF expression was decreased in TAA-treated HB-EGF(+/+) mice by 37.6% (P<0.05) as compared with animals receiving saline alone. HB-EGF(-/-) mice treated with TAA showed increased hepatic α-smooth muscle actin-positive cells and collagen deposition, and, as compared with HB-EGF(+/+) mice, TAA-stimulated hepatic mRNA levels in HB-EGF(-/-) mice were, respectively, 2.1-, 1.7-, 1.8-, 2.2-, 1.2- or 3.3-fold greater for α-smooth muscle actin, α1 chain of collagen I or III (COL1A1 or COL3A1), transforming growth factor-β1, connective tissue growth factor or tissue inhibitor of metalloproteinase-1 (P<0.05). HB-EGF expression was detectable in primary cultured HSCs from HB-EGF(+/+) mice. Both endogenous and exogenous HB-EGF inhibited HSC activation in primary culture, and HB-EGF enhanced HSC migration. These findings suggest that HB-EGF gene knockout in mice increases susceptibility to chronic TAA-induced hepatic fibrosis and that HB-EGF expression or action is associated with suppression of fibrogenic pathways in HSCs.

Topics: Actins; Animals; Carbon Tetrachloride; Collagen Type I; Collagen Type III; Connective Tissue Growth Factor; Heparin-binding EGF-like Growth Factor; Hepatic Stellate Cells; Humans; Intercellular Signaling Peptides and Proteins; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta1

To optimize a compound prescription for treatment of liver fibrosis with an improved therapeutic effect and low toxicity.. In rat models of liver fibrosis induced by thioacetamide (TAA), the optimized prescription was screened based on a uniform design with 2-factor 5-level table using Uniform Design 3.0 software and tested using liver content of Hyp as the screening index. To verify the efficacy of the optimized prescription, the rat models of liver fibrosis were randomized into normal control group, model group, colchicine group and optimized prescription group, and the changes of hepatic Hyp content, serum HA, ALT, AST, and ALB levels, and the pathology liver fibrosis were observed after corresponding treatments.. The optimized prescription, which contained 70 mg/kg glycyrrhizin and 70 mg/kg matrine, showed a significant therapeutic effect against liver fibrosis in rats (Plt;0.05), and the effect was equivalent to that of colchicine (P>0.05).. Uniform design is a valuable method in prescription optimization. The optimized compound prescription of matrine and glycyrrhizin has a significant effect in inhibiting liver fibrosis.

Topics: Alkaloids; Animals; Drug Therapy, Combination; Female; Glycyrrhizic Acid; Liver Cirrhosis; Male; Matrines; Phytotherapy; Quinolizines; Rats; Rats, Sprague-Dawley; Thioacetamide

Oxidative stress contributes to liver fibrosis through the activation of hepatic stellate cells. In a cell-based screening study, a class of imidazolium salts demonstrates anti-fibrogenic properties. Little is known on imidazolium salt mechanistic effects. We investigated the anti-fibrogenic effect of one of the imidazolium salts, 1,3-bisbenzylimidazoliumbromide (DBZIM), in a chronic mouse model of liver fibrosis and evaluated the mechanism of this treatment.. Liver fibrosis was induced in mice by oral feeding of thioacetamide for 16 weeks. DBZIM was administered weekly, starting on the first day or 12 weeks from the day of thioacetamide administration. Hepatic function, histology and oxidative stress were examined. Expression of key inflammatory molecules and the molecular mechanism of DBZIM were assessed in hepatic stellate cells.. DBZIM decreased the fibrogenic response in thioacetamide-mice as measured by collagen deposition and α-smooth muscle actin expression (P<0.01). DBZIM improved liver function and reduced both oxidative damage and inflammation (P<0.01). Most importantly, our findings report the discovery that astrocyte elevated gene-1, involved in tumour progression, was up-regulated in thioacetamide-mice and DBZIM modulated astrocyte elevated gene-1 and NF-κB expression.. These findings indicate DBZIM is a potent therapeutic agent for the treatment of liver fibrosis.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Actins; Analysis of Variance; Animals; Cell Movement; Cells, Cultured; Collagen Type I; Collagen Type I, alpha 1 Chain; Cyclooxygenase 2; Deoxyguanosine; Gene Expression; Hepatic Stellate Cells; Imidazoles; Inflammation; Liver Cirrhosis; Male; Membrane Proteins; Mice; Nerve Tissue Proteins; NF-kappa B; Oxidative Stress; RNA-Binding Proteins; Superoxide Dismutase; Thioacetamide; Up-Regulation

Parthenolide (PT), a sesquiterpene lactone derived from the plant feverfew, has pro-apoptotic activity in a number of cancer cell types. We assessed whether PT induces the apoptosis of hepatic stellate cells (HCSs) and examined its effects on hepatic fibrosis in an in vivo model. The effects of PT on rat HSCs were investigated in relation to cell growth inhibition, apoptosis, NF-κB binding activity, intracellular reactive oxygen species (ROS) generation, and glutathione (GSH) levels. In addition, the anti-fibrotic effects of PT were investigated in a thioacetamide-treated rat model. PT induced growth inhibition and apoptosis in HSCs, as evidenced by cell growth inhibition and apoptosis assays. PT increased the expression of Bax proteins during apoptosis, but decreased the expression of Bcl-2 and Bcl-X(L) proteins. PT also induced a reduction in mitochondrial membrane potential, poly(ADP-ribose) polymerase cleavage, and caspase-3 activation. PT inhibited TNF-α-stimulated NF-κB binding activity in HSCs. The pro-apoptotic activity of PT in HSCs was associated with increased intracellular oxidative stress as evidenced by increased intracellular ROS levels and depleted intracellular GSH levels. Furthermore, PT ameliorated hepatic fibrosis significantly in a thioacetamide- treated rat model. In conclusion, PT exhibited pro-apoptotic effects in rat HSCs and ameliorated hepatic fibrosis in a thioacetamide-induced rat model.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Gene Expression; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Membrane Potential, Mitochondrial; NF-kappa B; Oxidative Stress; Rats; Reactive Oxygen Species; Sesquiterpenes; Thioacetamide; Tumor Necrosis Factor-alpha

Coffee intake has been inversely related to the incidence of liver diseases, although there are controversies on whether these beneficial effects on human health are because of caffeine or other specific components in this popular beverage. Thus, this study evaluated the protective effects of coffee or caffeine intake on liver injury induced by repeated thioacetamide (TAA) administration in male Wistar rats. Rats were randomized into five groups: one untreated group (G1) and four groups (G2-G5) treated with the hepatotoxicant TAA (200 mg/kg b.w., i.p.) twice a week for 8 weeks. Concomitantly, rats received tap water (G1 and G2), conventional coffee (G3), decaffeinated coffee (G4) or 0.1% caffeine (G5). After 8 weeks of treatment, rats were killed and blood and liver samples were collected. Conventional and decaffeinated coffee and caffeine intake significantly reduced serum levels of alanine aminotransferase (ALT) (p < 0.001) and oxidized glutathione (p < 0.05), fibrosis/inflammation scores (p < 0.001), collagen volume fraction (p < 0.01) and transforming growth factor β-1 (TGF-β1) protein expression (p ≤ 0.001) in the liver from TAA-treated groups. In addition, conventional coffee and caffeine intake significantly reduced proliferating cellular nuclear antigen (PCNA) S-phase indexes (p < 0.001), but only conventional coffee reduced cleaved caspase-3 indexes (p < 0.001), active metalloproteinase 2 (p ≤ 0.004) and the number of glutathione S-transferase placental form (GST-P)-positive preneoplastic lesions (p < 0.05) in the liver from TAA-treated groups. In conclusion, conventional coffee and 0.1% caffeine intake presented better beneficial effects than decaffeinated coffee against liver injury induced by TAA in male Wistar rats.

Topics: Animals; Caffeine; Caspase 3; Chemical and Drug Induced Liver Injury; Coffee; Collagen; Food Handling; Glutathione; Liver; Liver Cirrhosis; Male; Matrix Metalloproteinase 2; Oxidation-Reduction; Precancerous Conditions; Proliferating Cell Nuclear Antigen; Random Allocation; Rats; Rats, Wistar; Thioacetamide; Transforming Growth Factor beta1

Chronic liver disease promotes hepatocellular injury involving apoptosis and triggers compensatory regeneration that leads to the activation of quiescent stellate cells in the liver. The deposition of extracellular matrix from activated myofibroblasts promotes hepatic fibrosis and the progression to cirrhosis with deleterious effects on liver physiology. The role of apoptosis signaling pathways in the development of fibrosis remains undefined. The aim of the current study was to determine the involvement of the caspase-8 homologue cellular FLICE-inhibitory protein (cFLIP) during the initiation and progression of fibrosis. Liver injury and fibrosis from carbon tetrachloride (CCl(4)) and thioacetamide (TAA) were examined in mice exhibiting a hepatocyte-specific deletion of cFLIP (flip(-/-)). Acute liver injury from CCl(4) and TAA were enhanced in flip(-/-) mice. This was accompanied by increased activation of caspase-3 and -9, pronounced phosphorylation of JNK, and decreased phosphorylation of Erk. Deletion of the cJun NH(2)-terminal kinase 2 (JNK2) in flip(-/-) mice protected from injury. Hepatic fibrosis was increased at baseline in 12-wk-old flip(-/-) mice, and progression of fibrosis from TAA was accelerated compared with the wild type. In conclusion, deletion of cFLIP in hepatocytes leads to increased fibrosis and accelerated fibrosis progression. This is accompanied by increased injury involving the activation of caspases and JNK2. Thus predisposition to liver injury involving increased hepatocellular apoptosis is a critical mediator of accelerated fibrogenesis, and prevention of liver injury will be a most important measure for patients with chronic liver disease.

Topics: Animals; Apoptosis; Carbon Tetrachloride; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 3; Caspase 9; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Disease Progression; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Genotype; Hepatocytes; Liver; Liver Cirrhosis; Male; Mice; Mice, Knockout; Mitogen-Activated Protein Kinase 9; Phenotype; Phosphorylation; Signal Transduction; Thioacetamide; Time Factors

Adipose-derived stem cells (ADSCs) are easy to harvest and have the ability for self-renewal and to differentiate into various cell types, including those of the hepatic lineage. Studies on the use of ADSCs for liver transplantation are, however, limited. The objective of this study was to investigate the feasibility of using human ADSCs and to better understand their mechanism of action for the repair of liver damage in a thioacetamide (TAA)-induced model of chronic liver damage in the rat. To induce liver damage, 200 mg/kg TAA was injected intraperitoneally into Wistar rats every 3 days for 60 days. For cell therapy, 1 × 10(6) human ADSCs suspended in 300 μl of phosphate-buffered saline were transplanted into each experimental rat by direct liver injection. Immunohistochemistry showed that the transplanted ADSCs differentiated into albumin- and α-fetoprotein-secreting liver-like cells 1 week after transplantation. In addition, liver function recovered significantly, as determined by biochemical analyses that analyzed total bilirubin, prothrombin time, and albumin levels. The Metavir score, derived from histopathological analysis, also showed a significant decrease in liver fibrosis and inflammatory activity after ADSC transplantation. Finally, we found a reduction in the expression of α-smooth muscle actin, a marker of hepatic stellate cells, which produce collagen fiber, and an increase in the expression of matrix metalloproteinase-9, which degrades collagen fiber, after ADSC transplantation. These findings are consistent with abrogation of liver fibrosis in the ADSC therapy group. Consequently, these results suggest that ADSC transplantation may facilitate recovery from chronic liver damage and thus may have clinical applications.

Topics: Actins; Adipose Tissue; Adult; Aged; alpha-Fetoproteins; Animals; Cells, Cultured; Female; Hepatic Stellate Cells; Humans; Immunohistochemistry; Liver; Liver Cirrhosis; Male; Matrix Metalloproteinase 9; Rats; Rats, Wistar; Recovery of Function; Stem Cell Transplantation; Stem Cells; Thioacetamide

The induction of prolonged choline-deprivation (CD) in rats receiving thioacetamide (TAA) is an experimental approach of mild hepatotoxicity that could resemble commonly presented cases in clinical practice (in which states of malnutrition and/or alcoholism are complicated by the development of other liver-associated diseases).. The present study aimed to investigate the time-dependent effects of a 30-, a 60- and a 90-day dietary CD and/or TAA administration on the adult rat liver histopathology and the serum markers of hepatic functional integrity.. Rats were divided into four main groups: (a) control, (b) CD, (c) TAA and (d) CD + TAA. Dietary CD was provoked through the administration of choline-deficient diet, while TAA administration was performed ad libitum through the drinking water (300 mg/l of drinking water).. Histological examination of the CD + TAA liver sections revealed micro- and macro-vesicular steatosis with degeneration and primary fibrosis at day 30, to extensive steatosis and fibrosis at day 90. Steatosis was mostly of the macrovesicular type, involving all zones of the lobule, while inflammatory infiltrate consisted of foci of acute and chronic inflammatory cells randomly distributed in the lobule. These changes were accompanied by gradually increasing mitotic activity, as well as by a constantly high alpha-smooth muscle actin immunohistochemical staining. The determination of hepatocellular injury markers such as the serum enzyme levels' of alanine aminotransferase and aspartate aminotransferase demonstrated a decrease at day 30 (they returned to control levels at days 60 and 90). However, the determination of those serum enzymes used for the assessment of cholestatic liver injury (gamma-glutamyltransferase, alkaline phosphatase) revealed a constant (time-independent) statistically-significant increase versus control values.. Long-term combined dietary CD and TAA administration could be a more realistic experimental approach to human liver diseases involving severe steatosis, fibrosis, stellate cell activation and significant regenerative hepatocellular response.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Chemical and Drug Induced Liver Injury; Choline; Choline Deficiency; Diet; Fatty Liver; Hepatic Stellate Cells; Liver Cirrhosis; Male; Rats; Thioacetamide

The purpose of this study is to investigate the effect of Ginkgo biloba leaves extract on experimental liver fibrosis induced by thioacetamide (TAA) in male albino mice. The experimental mice were divided into four groups. The mice of the first group were served as control. The experimental animals of the second group were given 150 mg/kg body weight of TAA by intraperitoneal injection, twice weekly, for 9 weeks. The mice of the third group were exposed to TAA and supplemented with G. biloba leaves extract. The animals of the fourth group were supplemented with G. biloba leaves extract. The levels of plasma alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, triglycerides, cholesterol, and low-density lipoprotein cholesterol were statistically increased while the levels of plasma total protein, albumin, glucose, and high-density lipoprotein cholesterol were significantly decreased. The levels of liver superoxide dismutase, glutathione, glycogen and total protein were notably declined, whereas the level of total lipid was increased in mice of the second group. Furthermore, microscopic examination of liver sections from mice treated with TAA showed an abnormal morphology characterized by nodular transformations in liver parenchyma which surrounded by fibrous septa. Administration of G. biloba leaves extract reduced extent and development of fibrous septa, liver cells change, and biochemical alterations in mice exposed to TAA. This study showed that G. biloba leaves extract has a potential activity against TAA-induced liver fibrosis and suggested that the chemical constituents of G. biloba are effective in modulation of oxidative stress induced by TAA.

Topics: Animals; Ginkgo biloba; Liver Cirrhosis; Male; Mice; Plant Extracts; Plant Leaves; Thioacetamide; Treatment Outcome

Progressive hepatic fibrosis is the eventual cause of liver cirrhosis. Doppler ultrasound has been used to detect hemodynamic changes that are known to be present during the pre-cirrhotic stages of hepatic fibrogenesis. However, the relationship between the Doppler ultrasound parameters and the impairment of the liver function has not been fully investigated. The purpose of this study was to explore the hepatic function reserve and its relationship with the hepatic hemodynamics in a rabbit model of liver fibrosis using Doppler ultrasound.. A prospective study was performed. Sixty healthy New Zealand rabbits were included in this study. Eleven of them served as controls and were normally fed and provided with water drink; the rest of 49 rabbits that served as fibrosis group were normally fed but provided with 1.2 g/L of thioacetamide to create liver fibrosis model. Doppler measurements were performed in the portal trunk, proper hepatic artery and proper splenic artery. The hepatic circulation index (HCI) was calculated. Hepatic function reverse was evaluated by measuring the indocyanine green clearance and retention rate at 15 min (ICG R15) test. Portal venous pressure (PVP) was measured using the portal vein punctuation equipment.. HCI was significantly decreased and PVP increased in the advanced fibrotic stage (F4) compared to mild and moderate fibrotic stage (F1-3), respectively (p<0.05). PVP and ICG R15 in the fibrotic group were significantly higher than that in the control group (ICG: 0.209±0.086 vs. 0.093±0.023, p<0.01). Within the fibrotic groups, PVP was higher in advanced fibrotic stage (F4) than those in mild (F1-2) or moderate (F3) fibrotic stages (p<0.05). Both HCI and PVP correlated well with ICG R15 (r = -0.890, and r = 0.780, p <0.01).. Hepatic function reserve closely relates to the hepatic hemodynamics in the rabbit model of liver fibrosis. Doppler Ultrasound could be reliably used to assess the hepatic function reserve and hemodynamic changes in different stages of liver fibrosis.

Topics: Animals; Disease Models, Animal; Hemodynamics; Hepatic Artery; Indocyanine Green; Liver; Liver Circulation; Liver Cirrhosis; Liver Function Tests; Portal Pressure; Portal Vein; Rabbits; Severity of Illness Index; Splenic Artery; Thioacetamide; Ultrasonography

Hepatic encephalopathy (HE) is a syndrome observed in patients with liver dysfunction such as hepatitis and cirrhosis, and is characterized by cognitive impairment, personality changes, and a depressed level of consciousness. The detailed mechanism underlying the pathogenesis of HE remains unclear. In the present study, our aim was to establish an animal model for HE with cirrhosis. Therefore, we carried out behavioral and biochemical analysis of cirrhotic rats after treatment with thioacetamide (TAA) for 20 weeks. The rats subjected to chronic TAA treatment (TAA rats) showed reduction of cognitive scores in the novel object recognition test (NOR), and a decrease in immobility and an increase in swimming in the forced swim test (FST). In biochemical analyses, the TAA rats exhibited elevated blood levels of ammonia, and increased metabolic activities of serotonergic and noradrenergic neurons in the brain, while the levels of Glu and GABA were not affected. Post-oral treatment of lactulose, a clinically utilized drug for HE, effectively reduced the elevated blood ammonia levels, and restored the reduced cognitive scores and the decreased immobility, without any effects on neurotransmitter contents in the brain, compared with the control. These results indicated lactulose-restorable memory disturbance and irritated mood in the TAA rats. In other words, rats treated chronically with TAA are a potential model for cirrhosis-HE, and the combination of NOR and FST in TAA rats may be useful as a simple assay system for the screening and development of anti-HE agents.

Topics: Animals; Behavior, Animal; Biogenic Monoamines; Brain; Chronic Disease; Disease Models, Animal; Hepatic Encephalopathy; Liver Cirrhosis; Neurotransmitter Agents; Rats; Rats, Wistar; Thioacetamide; Time Factors

The aim of this study was to evaluate and compare the effects of imatinib and nilotinib to that of silymarin on established liver fibrosis and oxidative stress in a thioacetamide (TAA) rat model. Male Wistar rats received intraperitoneal (i.p.) injections of TAA (150mg/kg, twice weekly) for 12weeks. Daily treatments with imatinib (10mg/kg), nilotinib (10mg/kg), and silymarin (100mg/kg) were administered orally during the last 4weeks of TAA-administration. At the end of the study, hepatic damage was evaluated by analysis of liver function tests in serum. Hepatic histopathology and collagen content were employed to quantify liver fibrosis. Hepatic oxidative stress was assessed by measuring malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), total nitrate/nitrite (NOx), and reduced glutathione (GSH) contents, as well as myeloperoxidase (MPO) and superoxide dismutase (SOD) activities. Nilotinib, silymarin and, to a lesser extent, imatinib treatments ameliorated TAA-induced hepatic oxidative stress and damage as indicated by hepatic MDA, 4-HNE, NOx, GSH, MPO and SOD levels, as well as liver function tests. Hepatic histopathology results revealed that nilotinib, imatinib, and silymarin treatments decreased the mean score of fibrosis in TAA-treated rats by 24, 14, and 3%, respectively. However, nilotinib and silymarin, but not imatinib, treatments decreased hepatic collagen content in TAA-treated rats by 17 and 36%, respectively. In conclusion, we demonstrated for the first time that nilotinib not only protected against hepatic oxidative stress, but also slowed down liver fibrosis progression. Thus, we provide the first evidence that nilotinib might be a promising anti-fibrotic drug.

Topics: Administration, Oral; Animals; Benzamides; Disease Models, Animal; Disease Progression; Imatinib Mesylate; Liver Cirrhosis; Liver Function Tests; Male; Oxidative Stress; Piperazines; Protein Kinase Inhibitors; Pyrimidines; Rats; Rats, Wistar; Silymarin; Thioacetamide

Characteristics of thioacetamide (TAA)-induced liver cirrhosis in rat was observed for 120 days after TAA withdrawal as part of the radiobiological study of partial liver irradiation on TAA-induced cirrhotic rats. The natural process focused on cirrhosis and regeneration was recorded as a baseline condition for the interpretation of the outcome of the partial liver irradiation study. Cirrhosis in rats was successfully induced by drinking 0.03% TAA water orally for 29 weeks with a modeling rate of 96%. After establishment of the cirrhosis model, the rats were observed for 120 days upon TAA withdrawal to investigate the dynamic changes of cirrhosis and regeneration. The following characteristics were observed: (1) Histological changes; (2) Liver functions; (3) Cirrhosis: trichrome stain, quantification of hydroxyproline in hydrolysed liver tissue and TGF-β1; (4) Liver regeneration: liver index, hepatocyte mitotic index (MI), hepatocyte proliferation index (PI) by flow cytometry, PCNA labeling index (LI) by IHC and expression of PCNA mRNA; and (5) Growth factors: serum HGF, VEGF, TGF-α, and IL-6. After TAA withdrawal, gradual improvement in liver functions was noted with decreases of ALT, AST, and ALP, and increase of PA. The resolution of cirrhosis was evident by histological improvement with attenuation of collagen fiber and decrease of TGF-β1 IHC index, and also decrease of trichrome stain and hydroxyproline content. However, cirrhosis was still existed on 120 days after TAA withdrawal. Significant deceleration of liver regeneration was demonstrated with TAA withdrawal, evidenced by decrease of MI and PI, reduced expression of PCNA mRNA and PCNA LI. In conclusion, upon TAA withdrawal hepatic cirrhosis was continuously resolved, but persisted up to 120 days, and liver regeneration was significantly decelerated.

Topics: Animals; Body Weight; Collagen; Disease Models, Animal; Hydroxyproline; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Interleukin-6; Liver; Liver Cirrhosis; Liver Function Tests; Liver Regeneration; Male; Proliferating Cell Nuclear Antigen; Rats; Rats, Wistar; Staining and Labeling; Thioacetamide; Transforming Growth Factor beta1

Following liver injury, the wound-healing process is characterized by hepatic stellate cell (HSC) activation from the quiescent fat-storing phenotype to a highly proliferative myofibroblast-like phenotype. Snail1 is a transcription factor best known for its ability to trigger epithelial-mesenchymal transition, to influence mesoderm formation during embryonic development, and to favor cell survival. In this study, we evaluated the expression of Snail1 in experimental and human liver fibrosis and analyzed its role in the HSC transdifferentiation process. Liver samples from patients with liver fibrosis and from mice treated by either carbon tetrachloride (CCl(4)) or thioacetamide (TAA) were evaluated for mRNA expression of Snail1. The transcription factor expression was investigated by immunostaining and real-time quantitative RT-PCR (qRT-PCR) on in vitro and in vivo activated murine HSC. Snail1 knockdown studies on cultured HSC and on CCl(4)-treated mice were performed by adenoviral delivery of short-hairpin RNA; activation-related genes were quantitated by real-time qRT-PCR and Western blotting. Snail1 mRNA expression resulted upregulated in murine experimental models of liver injury and in human hepatic fibrosis. In vitro studies showed that Snail1 is expressed by HSC and that its transcription is augmented in in vitro and in vivo activated HSC compared with quiescent HSC. At the protein level, we could observe the nuclear translocation of Snail1 in activated HSC. Snail1 knockdown resulted in the downregulation of activation-related genes both in vitro and in vivo. Our data support a role for Snail1 transcription factor in the hepatic wound-healing response and its involvement in the HSC transdifferentiation process.

Topics: Acute Disease; Adult; Aged; Animals; Blotting, Western; Carbon Tetrachloride; Cell Transdifferentiation; Cells, Cultured; Chronic Disease; Gene Silencing; Hepatic Stellate Cells; Humans; Immunologic Techniques; Liver; Liver Cirrhosis; Mice; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; Snail Family Transcription Factors; Thioacetamide; Transcription Factors; Up-Regulation; Wound Healing

Several lines of evidence suggest that innate immunity plays a key role in hepatic fibrogenesis. To clarify the role of natural killer (NK) T cells in hepatic inflammation and fibrogenesis, we here investigated xenobiotics-induced liver injury and subsequent fibrogenesis in mice lacking mature NKT cells caused by genetic disruption of the CD1d molecule.. Male CD1d-knockout (KO) and wild-type (WT) mice were given repeated intraperitoneal injections of thioacetamide (TAA, 3times/week; 0.1-0.2mg/g BW) for up to 9 weeks, or a single intraperitoneal injection of CCl(4) (1 μl/g). Liver histology was evaluated, and expression levels of cytokines and matrix-related genes in the liver were quantitatively measured by real-time reverse transcription-polymerase chain reaction (RT-PCR).. Mortality following repeated injections of TAA was prevented almost completely in CD1d-KO mice. TAA-induced inflammatory responses and hepatocellular damage were markedly ameliorated in CD1d-KO mice. TAA-induced expression of smooth muscle α-actin (SMA) and transforming growth factor (TGF)β1 mRNA in the liver were also prevented largely in CD1d-KO mice. In fact, CD1d-KO mice developed minimal hepatic fibrosis after 9-weeks of administration of TAA, which caused overt bridging fibrosis in WT mice. Indeed, TAA-induced increases in α1(I)procollagen (COL1A1) and tissue inhibitor of matrix metalloproteinase (TIMP)-1 mRNA were blunted significantly in CD1d-KO mice. Similarly, acute CCl(4)-induced hepatic injury and subsequent profibrogenic responses were also reduced significantly in CD1d-KO mice.. These findings clearly indicated that CD1d-restricted NKT cells contribute to xenobiotics-induced hepatic inflammation, hepatocellular damage, and subsequent profibrogenic responses in the liver.

Topics: Animals; Antigens, CD1d; Base Sequence; Cell Death; Chemical and Drug Induced Liver Injury; Collagen Type I; Collagen Type I, alpha 1 Chain; Hepatocytes; Immunity, Innate; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Natural Killer T-Cells; RNA, Messenger; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1

Amomum xanthoides is a well-known traditional herbal medicine mainly for diverse digestive system disorders in Asia for a long time. In the present study, we investigate the effects and action mechanism of methanol fraction of Amomum xanthoides (MFAX) on thioacetamide (TAA)-induced liver fibrosis in rat model.. TAA (200mg/kg, ip on twice a week for 14 weeks) treated rats were orally administered with MFAX (25, 50 or 100mg/kg) once a day from the 7th week until 14th week.. Significantly elevated serum bilirubin, liver tissue hydroxyproline and malondialdehyde (MDA) in liver fibrosis were ameliorated by MFAX treatment. Further, MFAX treatment attenuated the reactive oxygen species (ROS) levels and restored glutathione (GSH) content and glutathione-peroxidase (GPx) activity. Histopathological data showed that MFAX treatment inhibited collagen accumulation and activation of hepatocyte stellate cells (HSCs) in the liver tissue. Compared to the TAA group, activation of inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), platelet-derived growth factor beta (PDGF-β) mRNAs and the level of pro-fibrotic cytokines PDGF-β and connective tissue growth factor (CTGF) in the liver tissue were attenuated in MFAX treated groups.. The above evidences collectively indicate that MFAX is a potential herb which can be used as an anti-hepatofibrotic remedy.

Topics: Amomum; Animals; Antioxidants; Base Sequence; Body Weight; Cells, Cultured; Chromatography, Thin Layer; DNA Primers; Glutathione; Immunohistochemistry; Liver Cirrhosis; Malondialdehyde; Methanol; Organ Size; Plant Extracts; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Thioacetamide

In this study, we elucidated the mechanism by which adiponectin modulates hepatic stellate cell activation and fibrogenesis. Adiponectin-overexpressing transgenic mice receiving thioacetamide were resistant to fibrosis, compared with controls. In contrast, adiponectin-null animals developed severe fibrosis. Expression of collagen α1(I) and α-smooth muscle actin (α-SMA) mRNAs were significantly lower in adiponectin-overexpressing mice, compared with controls. In wild-type stellate cells exposed to a lentivirus encoding adiponectin, expression of peroxisome proliferator-activated receptor-γ (PPARγ), SREBP1c, and CEBPα mRNAs was significantly increased (3.2-, 4.1-, and 2.2-fold, respectively; n = 3; P < 0.05, adiponectin virus versus control), consistent with possible activation of an adipogenic transcriptional program. Troglitazone, a PPARγ agonist, strongly suppressed up-regulation of collagen α1(I) and α-SMA mRNA in stellate cells isolated from wild-type mice; however, stellate cells from adiponectin-null animals failed to respond to troglitazone. Furthermore, in isolated stellate cells in which PPARγ was depleted using an adenovirus-Cre-recombinase system and in which adiponectin was also overexpressed, collagen α1(I) and α-SMA were significantly inhibited. We conclude that the PPARγ effect on stellate cell activation and the fibrogenic cascade appears to be adiponectin-dependent; however, the inhibitory effect of adiponectin on stellate cell activation was not dependent on PPARγ, suggesting the presence of PPARγ-dependent as well as independent pathways in stellate cells.

Topics: Adiponectin; Alanine Transaminase; Animals; Aspartate Aminotransferases; Body Weight; Disease Susceptibility; Gene Deletion; Hepatic Stellate Cells; Ligands; Liver; Liver Cirrhosis; Mice; Mice, Transgenic; Phenotype; PPAR gamma; Thioacetamide

The objective of the present study is to find out the quantitative relationship between progression of liver fibrosis and the levels of certain serum markers using mathematic model. We provide the sparse logistic regression by using smoothly clipped absolute deviation (SCAD) penalized function to diagnose the liver fibrosis in rats. Not only does it give a sparse solution with high accuracy, it also provides the users with the precise probabilities of classification with the class information. In the simulative case and the experiment case, the proposed method is comparable to the stepwise linear discriminant analysis (SLDA) and the sparse logistic regression with least absolute shrinkage and selection operator (LASSO) penalty, by using receiver operating characteristic (ROC) with bayesian bootstrap estimating area under the curve (AUC) diagnostic sensitivity for selected variable. Results show that the new approach provides a good correlation between the serum marker levels and the liver fibrosis induced by thioacetamide (TAA) in rats. Meanwhile, this approach might also be used in predicting the development of liver cirrhosis.

Topics: Animals; Bayes Theorem; Biomarkers; Disease Models, Animal; Disease Progression; Likelihood Functions; Liver Cirrhosis; Logistic Models; Male; Models, Statistical; Probability; Rats; ROC Curve; Thioacetamide

Thioacetamide (TAA) is a potent hepatotoxicant and has been widely used to develop experimental liver fibrosis/cirrhosis models. Although the liver toxicity of TAA has been extensively studied, little is known about its potential influence on UDP-glucuronosyltransferases (UGTs) associated with the development of liver fibrosis. The study presented here aimed to uncover the regulation patterns of UGTs in TAA-induced liver fibrosis of rats. Potential counteracting effects of hepatoprotective agents were also determined. TAA treatment for 8 weeks induced a significant transcriptional up-regulation of the major UGT isoforms, including UGT1A1, UGT1A6, and UGT2B1, accompanied with the dramatic elevations of most typical serum biomarkers of liver function and fibrosis scores. Upon TAA intoxication, the mRNA and protein levels of the major UGT isoforms were increased to 1.5- to 2.5-fold and 2.5- to 3.3-fold of that of the normal control, respectively. The hepatoprotective agents Schisandra spp. lignans extract and dimethyl diphenyl bicarboxylate could largely abolish TAA-induced up-regulation of all three UGT isoforms. However, enzyme activities of UGTs remained unchanged after TAA treatment. The dissociation of protein expression and enzyme activity could possibly be attributed to the inactivating effects of TAA, upon a NADPH-dependent bioactivation, on UGTs. This study suggests that the transcriptional up-regulation of UGTs may be an alternative mechanism of their preserved activities in liver fibrosis/cirrhosis.

Topics: Animals; Biomarkers; Dioxoles; Glucuronosyltransferase; Lignans; Liver; Liver Cirrhosis; Male; Protective Agents; Protein Isoforms; Rats; Rats, Sprague-Dawley; RNA, Messenger; Schisandra; Thioacetamide; Transcriptional Activation; Up-Regulation

Hepatic stellate cells (HSCs) are key participants in liver fibrosis development. 1,25(OH)(2)D(3), the active form of vitamin D, has antiproliferative properties and antifibrotic potential, as well as a role in extracellular matrix and matrix metalloproteinase (MMP) regulation in renal and lung fibrosis. Little is known about the role of 1,25(OH)(2)D(3) in liver and its involvement in liver fibrosis. Therefore, we investigated the antiproliferative and antifibrotic effects of 1,25(OH)(2)D(3) in primary cultured HSCs and in a rat model of liver fibrosis induced by thioacetamide (TAA).. Primary HSCs were isolated from rats' livers and treated with 1,25(OH)(2)D(3). Proliferation was examined by bromodeoxyuridine. Vitamin D receptor (VDR) expression and several fibrotic markers were detected by western blot analysis and real-time PCR. Collagen Iα1 and MMP-9 promoter activity were measured by luciferase assay. MMP-9 enzymatic activity was investigated by zymography. VDR silencing was performed by sh-RNA. An in vivo study was performed on TAA-induced liver fibrosis model in rats treated with or without 1,25(OH)(2)D(3). The fibrotic score and collagen deposition were determined by Masson and by Sirius red staining.. While VDR was highly expressed in quiescent HSCs, its expression decreased up to 40% during activation. Addition of 1,25(OH)(2)D(3) to activated HSCs stimulated VDR expression. 1,25(OH)(2)D(3) suppressed HSC proliferation and cyclin D1 expression by ~50% and tissue inhibitor of metalloproteinase 1 (TIMP-1) by 60% and led to a 40% downregulation of collagen Iα1 expression. Moreover, 1,25(OH)(2)D(3) increased MMP-9 activity by 30%. Silencing VDR by sh-RNA demonstrated that suppression of cyclin D1 and collagen Iα1 protein expression was VDR dependent. Treatment with 1,25(OH)(2)D(3) significantly reduced extracellular matrix deposition and lowered the fibrotic score in TAA-induced liver fibrosis.. 1,25(OH)(2)D(3) has antiproliferative and antifibrotic effects on liver fibrosis in in vitro and in vivo models and may be considered as having potential therapeutic value.

Topics: Animals; Blotting, Western; Cell Proliferation; Collagen Type I; Disease Models, Animal; Hepatic Stellate Cells; Liver Cirrhosis; Male; Matrix Metalloproteinase 9; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; Thioacetamide; Vitamin D

Our previous study has already confirmed a promising anti-fibrotic activity especially for nilotinib; when given at a daily dose of 10 mg/kg during the last 4 weeks of thioacetamide (TAA)-induced liver fibrosis for 12 weeks in rats. Therefore, this study was carried out to compare the prophylactic potential of low dose of nilotinib to that of its predecessor, imatinib, and a clinically relevant dose of the standard hepatoprotective treatment, silymarin, in TAA-intoxication. Male Wistar rats received intraperitoneal injections of TAA (150 mg/kg, twice weekly) for 8 weeks, as well as oral treatments with imatinib (5 mg/kg/day), nilotinib (5 mg/kg/day) and silymarin (50 mg/kg/day) from the first day of TAA-intoxication. At the end of the study, chronic hepatic injury was evaluated by analysis of liver function tests in serum. Hepatic oxidative stress was assessed by measuring malondialdehyde, 4-hydroxynonenal, total nitrate/nitrite and reduced glutathione contents, as well as myeloperoxidase and superoxide dismutase activities. Hepatic fibrosis was evaluated by histopathology and collagen content. Our results suggest that the prophylactic potential of nilotinib (5 mg/kg/day), imatinib (5mg/kg/day) and silymarin (50 mg/kg/day) in TAA-intoxication for 8 weeks is lower than the late treatments of nilotinib (10 mg/kg/day), imatinib (10mg/kg/day) and silymarin (100 mg/kg/day) during the last 4 weeks of TAA-intoxication for 12 weeks in rats. Taken together, this study suggests that nilotinib may have higher anti-fibrotic activity when administered at a significant stage of fibrosis as a result of impairment of its metabolism in the fibrotic livers.

Topics: Alanine Transaminase; Alkaline Phosphatase; Animals; Aspartate Aminotransferases; Benzamides; Bilirubin; Collagen; Cytoprotection; Dose-Response Relationship, Drug; gamma-Glutamyltransferase; Imatinib Mesylate; L-Lactate Dehydrogenase; Liver; Liver Cirrhosis; Male; Oxidative Stress; Piperazines; Pyrimidines; Rats; Rats, Wistar; Serum Albumin; Silymarin; Thioacetamide; Time Factors

Liver fibrosis represents a process of healing and scarring in response to chronic liver injury. Following injury, an acute inflammation response takes place resulting in moderate cell necrosis and extracellular matrix damage. Melittin, the major bioactive component in the venom of honey bee Apis mellifera, is a 26-residue amphipathic peptide with well-known cytolytic, antimicrobial and proinflammatory properties. However, the molecular mechanisms responsible for the anti-inflammatory activity of melittin have not been elucidated in liver fibrosis. We investigated whether melittin ameliorates liver inflammation and fibrosis in thioacetamide (TAA)-induced liver fibrosis. Two groups of mice were treated with TAA (200 mg/L, in drinking water), one of the groups of mice was co-treated with melittin (0.1 mg/kg) for 12 weeks while the other was not. Hepatic stellate cells (HSCs) were cultured with tumor necrosis factor α in the absence or presence of melittin. Melittin suppresses the expression of proinflammatory cytokines through the nuclear factor (NF)-κB signaling pathway. Moreover, melittin reduces the activity of HSCs in vitro, and decreases the expression of fibrotic gene responses in TAA-induced liver fibrosis. Taken together, melittin prevents TAA-induced liver fibrosis by inhibiting liver inflammation and fibrosis, the mechanism of which is the interruption of the NF-κB signaling pathway. These results suggest that melittin could be an effective agent for preventing liver fibrosis.

Topics: Animals; Bee Venoms; Hepatic Stellate Cells; Liver; Liver Cirrhosis; Melitten; Mice; Mice, Inbred C57BL; NF-kappa B; Rats; Signal Transduction; Thioacetamide; Tumor Necrosis Factor-alpha

Hepatic fibrosis is a widespread alteration in the liver that primarily consists of increased collagen deposition in the tissue. The aim of the present study was to evaluate the protective effects of poly-phytocompound EH-1501 containing small amounts of silymarin but also other potentially effective substances on thioacetamide (TTA)-induced liver fibrosis and to elucidate the mechanisms underlying these protective effects in rats. Forty rats were randomly divided into four groups. Liver fibrosis was induced by intraperitoneal injection of 200 mg/kg body weight. TAA dissolved in saline was administered thrice a week, for 8 weeks. Groups 1 (normal healthy control) and 2 (liver injury model) received water for 8 weeks or silymarin (50 mg/kg p.o. daily) for 8 weeks (group 3) or a poly-phytocompound EH-1501 (containing grape leaf, wild strawberry, dandelion and milk thistle, EuroHealth, Italy) (200 mg/kg, daily respectively) for 8 weeks (group 4). Biochemistry and serum fibrosis markers were AST, ALT, GGT, bilirubin, thiobarbituric acid reactive substances (TBARs), hyaluronic acid and type IV collagen 7s. Liver tissue was used to assay glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD), TBARs, hydroxyproline and gene expression of collagen alpha1 (col alpha1) and transforming growth factor-beta1 (TGF-beta1). Silymarine and EH-1501 were equally effective in reducing serum markers of liver damage and fibrosis as well as oxidative stress. However, as compared to silymarine, EH-1501 was significantly more effective in improving tissue level of GPx while decreasing TBARs and hydroproline content (p < 0.05). When looking at gene expression of col alpha1 and TGF-beta1, EH-1501 showed a significantly higher degree of gene down-regulation as compared to silymarine (p < 0.05). Taken altogether, these data suggest that a natural antioxidant-containing phytocompound EH-1501 exerts an effective hepatoprotective property in experimental chronic fibrotizing liver injury to a significantly higher degree than silymarin.

Topics: Animals; Disease Models, Animal; Glutathione Peroxidase; Hydroxyproline; Lipid Peroxidation; Liver; Liver Cirrhosis; Male; Oxidative Stress; Phytotherapy; Plant Extracts; Rats; Rats, Wistar; Thioacetamide; Thiobarbituric Acid Reactive Substances

The aquaporin (AQP) water channel is expected to play a decisive role of hyponatremia and water retention in cirrhotic patients. Despite the importance of the water channel, however, previous findings vary widely when it concerns AQP2 of the kidneys in subjects with cirrhosis. The purpose of this study was to investigate the expression of AQP2 in the distal renal tubule in cirrhosis, and the presence of the nitric oxide-AQP2 signaling pathway as a possible vasopressin-aquaporin-independent pathway. Sixty male Wister rats were assigned to six groups: (1) control; (2) TAA (thioacetamide); (3) TAA with nitric oxide donor; (4) TAA with nitric oxide inhibitor; (5) TAA with HMG CoA reductase inhibitor; (6) TAA with tetrahydrobiopterin. Immunohistochemical staining for AQP2, real-time polymerase chain reaction (PCR) for AQP2 and 3, citrulline assay, and renal cGMP concentration were measured. The AQP2-positivity of cirrhotic rats were higher than the controls (P < 0.05). The AQP2-positivity decreased in the nitric oxide donor group, but the proportion rose back up when the subjects were injected with the nitric oxide inhibitor (P < 0.05). The expression of AQP2 and AQP3 mRNA was also found to show an increase in the cirrhotic group as compared with the normal controls (P < 0.05). The cirrhotic group administered with nitric oxide donor showed a significant decline in the expression of the mRNA. The control group's cGMP concentration was lower than that of the cirrhotic group (P < 0.05), but a comparison of the two groups injected with nitric oxide modulators, such as statin and BH4, did not show significant differences in the cGMP concentration level. The expression of AQP2 of the kidneys increased in the cirrhotic rats. AQP2 had relations to the activity changes of nitric oxide synthetase.

Topics: Analysis of Variance; Animals; Aquaporin 2; Biopterins; Cyclic GMP; Disease Models, Animal; Immunoenzyme Techniques; Isosorbide Dinitrate; Kidney; Liver Cirrhosis; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Random Allocation; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Simvastatin; Statistics, Nonparametric; Thioacetamide

The present study was aimed at assessing the effects of Ganoderma lucidum extract (GLE) on an established liver fibrosis model with reference to the previously reported hepatoprotective effect of GLE against CCl(4)-induced fibrosis in rats. Repeated administration of thioacetamide (TAA) for 12 weeks to mice induced liver fibrosis. Treatment with GLE after the induction of liver fibrosis decreased the hepatic hydroxyproline content and improved liver histology. RT-qPCR analysis showed that GLE treatment reduced the mRNA expression of collagen (alpha1)(I), smooth muscle alpha-actin, tissue inhibitor of metalloproteinase 1 and metalloproteinase-13. In addition, the TAA-induced decrease in total collagenase activity was reversed by GLE treatment. In conclusion, oral administration of GLE reversed TAA-induced liver fibrosis, the mechanism of which might be related to the enhancement of collagenase activity.

Topics: Animals; Body Weight; Collagenases; Drug Evaluation, Preclinical; Hydroxyproline; Liver; Liver Cirrhosis; Male; Medicine, Chinese Traditional; Mice; Mice, Inbred BALB C; Organ Size; Reishi; Reverse Transcriptase Polymerase Chain Reaction; Thioacetamide

Statin has antifibrotic efficacy in human fibrosing diseases, such as pulmonary and renal fibrosis, and is therefore implicated in hepatic fibrosis. However, statin can also activate protein kinase C (PKC), which augments hepatic fibrogenesis and is thereby likely to reduce the antifibrotic efficacy of statin. This study was designed to explore the hypothesis that simultaneous treatment with statin and PKC inhibitor may synergistically enhance antifibrotic efficacy in hepatic fibrosis. Hepatic fibrosis models were established in BALB/c mice by intraperitoneal injection of carbon tetrachloride or thioacetamide for 6 wk. Pravastatin and enzastaurin (PKC inhibitor) were administered by gavage for 5 wk. Cellular apoptosis was explored using 4',6-diamidino-2-phenylindole or terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end-labeling (TUNEL) staining and immunoblot analysis. Hepatic fibrosis and hepatic stellate cell (HSC) activation were assessed by morphometric analysis of histological findings and immunohistochemistry for alpha-smooth muscle actin. In vitro, the addition of PKC inhibitor significantly increased statin-induced LX-2 cell apoptosis by enhancing the activation of mitochondrial apoptotic signals. TUNEL-positive HSCs were significantly increased in mice treated with statin + PKC inhibitor compared with those in control or single compound-treated mice. The percentage of area occupied by activated HSCs and the extent of collagen deposition were significantly decreased in mice treated with statin + PKC inhibitor compared with those in control or statin-treated mice. In conclusion, simultaneous treatment with statin and PKC inhibitor synergistically enhanced the antifibrotic efficacy in both in vitro and in vivo models of hepatic fibrosis and may therefore have therapeutic implication for reducing hepatic fibrosis.

Topics: Animals; Apoptosis; Body Weight; Carbon Tetrachloride; Cell Line, Transformed; Disease Models, Animal; Drug Synergism; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Indoles; Liver Cirrhosis; Male; Mice; Mice, Inbred BALB C; Mitochondria; Organ Size; Pravastatin; Protein Kinase C; Protein Kinase Inhibitors; Thioacetamide

Carnosine (beta-alanyl-L-histidine) is a dipeptide with antioxidant properties. Oxidative stress has been proposed to be involved in thioacetamide (TAA)-induced liver cirrhosis in rats, that is similar to human disease. In this study we aimed to investigate the role of carnosine on the development of TAA-induced cirrhosis. 200mg TAA/kg body weight has been given i.p. twice a week for three months to female wistar rats. Another group received same dose of TAA in the same pattern plus 2g carnosine/L of drinking water for three months. TAA administration resulted in hepatic fibrosis, significant increases in plasma transaminase activities as well as hepatic hydroxyproline and lipid peroxide levels, while liver glutathione (GSH) and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) protein expressions and activities decreased. Carnosine was found to behave as an antioxidant reducing malondialdehyde (MDA) and diene conjugate (DC) levels although it was not effective on increased transaminase activities and decreased antioxidants. It also did not affect the histopathological changes observed in TAA group. Thus our findings indicate that carnosine appears to attenuate peroxidation as an antioxidant itself but does not seem to prevent the development of TAA-induced cirrhotic process.

Topics: Alanine Transaminase; Animals; Antioxidants; Aspartate Aminotransferases; Carnosine; Female; Glutathione; Glutathione Peroxidase; Humans; Liver; Liver Cirrhosis; Malondialdehyde; Oxidative Stress; Rats; Rats, Wistar; Superoxide Dismutase; Thioacetamide

Panax notoginseng saponins have recently been reported to suppress liver fibrosis. Since ginsenoside-Rg1 is the most abundant component of P.notoginseng saponins, we investigated the effect of ginsenoside-Rg1 on experimental liver fibrosis in rats. Histological analysis revealed that ginsenoside-Rg1 significantly improved the extent of liver fibrosis in rats induced by thioacetamide. Ginsenoside-Rg1 markedly suppressed the serum levels of fibrotic markers and hepatic hydroxyproline content in rats treated with thioacetamide. Ginsenoside-Rg1 also reduced the serum levels of alanine transaminase, aspartate transaminase and alkaline phosphatase. Finally, ginsenoside-Rg1 attenuated the levels of thiobarbituric acid reactive substances in livers of rats treated by thioacetamide. In cultured hepatic stellate cells, ginsenoside-Rg1 markedly inhibited cell proliferation, activation and formation of reactive oxygen species stimulated by platelet-derived growth factor-BB (PDGF-BB). Additionally, ginsenoside-Rg1 down-regulated the expression of PDGF receptor-beta by reducing the nuclear factor-kappaB activity, which was required for the gene expression. These results suggest that ginsenoside-Rg1, which exhibits its antioxidant and antifibrotic properties, may be of potential therapeutic value in protecting the liver fibrosis.

Topics: Animals; Cells, Cultured; Drugs, Chinese Herbal; Ginsenosides; Hepatic Stellate Cells; Liver Cirrhosis; Panax notoginseng; Rats; Rats, Sprague-Dawley; Thioacetamide

Hepatic fibrogenesis, a complex process that involves a marked accumulation of extracellular matrix components, activation of cells capable of producing matrix materials, cytokine release, and tissue remodeling, is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The MMP-TIMP balance can regulate liver fibrogenesis. The aim of this study was to evaluate the expression patterns of MMPs and TIMPs during thioacetamide (TAA)-induced liver fibrogenesis. Chronic liver injury was induced with TAA (200 mg/kg i.p.) for 4 or 7 weeks in male Sprague-Dawley rats. Hepatic injury and fibrosis were assessed by hematoxylin-eosin (H&E) staining, and collagen deposition was confirmed by Sirius Red staining. The level of hepatic injury was quantified by serological analysis. The transcriptional and translational levels of alpha-smooth muscle actin (alpha-SMA), MMPs, and TIMPs in the liver were measured by Western blotting, RT-PCR, and immunohistochemistry. MMP, TIMP, and alpha-SMA were observed along fibrotic septa and portal spaces around the lobules. TAA treatment increased transcription of both MMPs and TIMPs, but only TIMPs showed increased translation. The dominant expression of TIMPs may regulate the function of MMPs to maintain liver fibrosis induced by TAA.

Topics: Animals; Collagen; Extracellular Matrix; Liver Cirrhosis; Male; Matrix Metalloproteinases; Rats; Rats, Sprague-Dawley; Thioacetamide; Tissue Inhibitor of Metalloproteinases

Anoectochilus formosanus is used in traditional folk medicine as an hepatoprotective agent. The purpose of this study was to investigate the effects of a standardized aqueous extract of A. formosanus (SAEAF) on thioacetamide (TAA)-induced liver fibrosis. An in vitro study showed that the inhibitive effect of kinsenoside, a major component of SAEAF, on tumor necrosis factor alpha (TNF-alpha) secretion from Kupffer cells might be derived at least partly from downregulation of LPS-receptor Toll-like receptor 4 (TLR4) signaling. Hepatic fibrosis was produced by TAA (200 mg/kg, i.p.) 3 times per week for 12 weeks. Mice in the three TAA groups were treated daily with distilled water and SAEAF (1.0, 0.2 g/kg) via gastrogavage throughout the experimental period. The mice that received the SAEAF treatment had significantly reduced plasma alanine aminotransferase activity, relative liver weights, and hepatic hydroxyproline contents. A histological examination also confirmed that SAEAF reduced the degree of fibrosis caused by TAA treatment. RT-PCR analysis showed that SAEAF treatment reduced mRNA expression of collagen (alpha1)(I), lipopolysaccharide-binding protein, CD14, TLR4, and TNF receptor 1. An immunohistochemical examination also indicated that SAEAF reduced the number of CD68-positive cells (macrophages). In conclusion, oral administration of SAEAF significantly reduced TAA-induced hepatic fibrosis in mice, probably through inhibition of hepatic Kupffer cell activation.

Topics: Acute-Phase Proteins; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Carrier Proteins; Down-Regulation; Hydroxyproline; Kupffer Cells; Lipopolysaccharides; Liver; Liver Cirrhosis; Male; Medicine, Traditional; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Thioacetamide; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

The mechanisms underlying intrahepatic vasoconstriction are not fully elucidated. Here we investigated the Kupffer cell (KC)-dependent increase in portal pressure by way of actions of vasoconstrictive cysteinyl leukotrienes (Cys-LTs). Liver cirrhosis was induced in rats by bile duct ligation (BDL for 4 weeks; controls: sham-operation) and thioacetamide application (18 weeks). Infusion of leukotriene (LT) C(4) or LTD(4) in isolated perfused livers (20 nM, BDL and sham) demonstrated that LTC(4) is a more relevant vasoconstrictor. In BDL animals the Cys-LT(1) receptor inhibitor montelukast (1 microM) reduced the maximal portal perfusion pressure following LTC(4) or LTD(4) infusion. The infusion of LTC(4) or D(4) in vivo (15 microg/kg b.w.) confirmed LTC(4) as the more relevant vasoconstrictor. Activation of KCs with zymosan (150 microg/mL) in isolated perfused BDL livers increased the portal perfusion pressure markedly, which was attenuated by LT receptor blockade (Ly171883, 20 microM). Cys-LTs in the effluent perfusate increased with KC activation but less with additional blockade of KCs with gadolinium chloride (10 mg/kg body weight, 48 and 24 hours pretreatment). KCs were isolated from normal rat livers and activated with zymosan or lipopolysaccharide at different timepoints. This resulted in an increase in Cys-LT production that was not influenced by preincubation with montelukast (1 microM). Infusion of LTC(4) (20 nM) and the thromboxane analog U46619 (0.1 microM) further enhanced portal pressure, indicating additive effects. Treatment with montelukast for 10 days resulted in an impressive reduction in the basal portal pressure and an attenuation of the KC-dependent increase in portal pressure.. Activation of isolated KCs produced Cys-LTs. Infusion of Cys-LTs increased portal pressure and, vice versa, treatment with montelukast reduced portal pressure in rat liver cirrhosis. Therefore, montelukast may be of therapeutic benefit for patients with portal hypertension.

Topics: Acetates; Animals; Cyclopropanes; Hypertension, Portal; Kupffer Cells; Leukotriene Antagonists; Leukotrienes; Ligation; Liver; Liver Cirrhosis; Male; Quinolines; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; Sulfides; Thioacetamide; Thromboxane A2

Non invasive approaches will likely be increasing utilized to assess liver fibrosis. This work provides a new non invasive index to predict liver fibrosis induced in mice.. Fibrosis was generated by thioacetamide (TAA), chronic intake of ethanol, or infection with S. mansoni in 240 mice. Both progression and regression of fibrosis (after treatment with silymarin and/or praziquantel) were monitored. The following methods were employed: (i) The METAVIR system was utilized to grade and stage liver inflammation and fibosis; (ii) Determination of hepatic hydroxyproline and collagen; and (iii) Derivation of a new hepatic fibrosis index from the induced changes, and its prospective validation in a group of 70 mice.. The index is composed of 4 serum variable including total proteins, gamma-GT, bilirubin and reduced glutathione (GSH), measured in diseased, treated and normal mice. These parameters were highly correlated with both the histological stage and the grade. They were combined in a logarithmic formula, which non-invasively scores the severity of liver fibrosis through a range (0 to 2), starting with healthy liver (corresponding to stage 0) to advanced fibrosis (corresponding stage 3).Receiver operating characteristic curves (ROC) for the accuracy of the index to predict the histological stages demonstrated that the areas under the curve (AUC) were 0.954, 0.979 and 0.99 for index values corresponding to histological stages 1, 2 and 3, respectively. Also, the index was correlated with stage and grade, (0.947 and 0.859, respectively). The cut off values that cover the range between stages 0-1, 1-2 and 2-3 are 0.4, 1.12 and 1.79, respectively. The results in the validation group confirmed the accuracy of the test. The AUROC was 0.869 and there was good correlation with the stage of fibrosis and grade of inflammation.. The index fulfils the basic criteria of non-invasive marker of liver fibrosis since it is liver-specific, easy to implement, reliable, and inexpensive. It proved to be accurate in discriminating precirrhotic stages.

Topics: Animals; Bilirubin; Biomarkers; Biopsy; Collagen; Disease Models, Animal; Ethanol; gamma-Glutamyltransferase; Glutathione; Hydroxyproline; Liver; Liver Cirrhosis; Mice; Mice, Inbred Strains; Schistosoma mansoni; Schistosomiasis mansoni; Severity of Illness Index; Thioacetamide

There is currently no effective treatment method available for liver fibrosis. We therefore evaluated the use of Wharton's jelly stem cells (WJSCs; the major umbilical cord stem cell population) to treat chemically induced liver fibrosis via intraperitoneal injection of thioacetamide. WJSCs were transplanted into liver-damaged rats via the portal vein and the treatment was evaluated by assessing serum biochemistry and histopathology. Transplanted WJSCs were distributed in the fibrotic area and around blood vessels, and hepatic recovery was accelerated. Serum prothrombin time significantly recovered, and serum albumin also improved at 21 days posttransplantation; collagen accumulation also decreased at 14 days. Thus, human WJSCs promoted recovery after chronic liver damage. Using immunohistochemical analyses, we determined that transplanted WJSCs produce albumin, hepatocyte growth factor (HGF), and metalloproteinase (MMP) after transplantation to chemically injured liver, indicating that WJSC may help to decrease liver collagen and thus may be useful for treating liver fibrosis.

Topics: Animals; Hepatocyte Growth Factor; Humans; Liver Cirrhosis; Male; Metalloproteases; Rats; Rats, Wistar; Serum Albumin; Stem Cell Transplantation; Stem Cells; Thioacetamide; Umbilical Cord

The normal liver is characterized by immunologic tolerance. Primary mediators of hepatic immune tolerance are liver sinusoidal endothelial cells (LSECs). LSECs block adaptive immunogenic responses to Ag and induce the generation of T regulatory cells. Hepatic fibrosis is characterized by both intense intrahepatic inflammation and altered hepatic immunity. We postulated that, in liver fibrosis, a reversal of LSEC function from tolerogenic to proinflammatory and immunogenic may contribute to both the heightened inflammatory milieu and altered intrahepatic immunity. We found that, after fibrotic liver injury from hepatotoxins, LSECs become highly proinflammatory and secrete an array of cytokines and chemokines. In addition, LSECs gain enhanced capacity to capture Ag and induce T cell proliferation. Similarly, unlike LSECs in normal livers, in fibrosis, LSECs do not veto dendritic cell priming of T cells. Furthermore, whereas in normal livers, LSECs are active in the generation of T regulatory cells, in hepatic fibrosis LSECs induce an immunogenic T cell phenotype capable of enhancing endogenous CTLs and generating potent de novo CTL responses. Moreover, depletion of LSECs from fibrotic liver cultures mitigates the proinflammatory milieu characteristic of hepatic fibrosis. Our findings offer a critical understanding of the role of LSECs in modulating intrahepatic immunity and inflammation in fibro-inflammatory liver disease.

Topics: Animals; Antigens; Carbon Tetrachloride; Cell Proliferation; Chemokines; Cytokines; Dendritic Cells; Endothelial Cells; Flow Cytometry; Inflammation Mediators; Liver; Liver Cirrhosis; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Thioacetamide

Liver cirrhosis and hepatocellular carcinomas are two major causes of morbidity and mortality worldwide, and can synergistically interact to expedite the tumor progression. How fibrosis promotes the hepatoma growth remains completely unexplained. Using an in situ murine hepatoma model together with fibrosis induction by thioacetamide (TAA), the hepatoma growth and the immune factors in the fibrotic liver were analyzed. We found that TAA-fibrosis induction enhanced hepatoma cell growth in the liver and increased the mortality of hepatoma-bearing mice. The tumor-infiltrating CD4(+) or CD8(+) T cells are downregulated by fibrosis induction. The Foxp3(+) regulatory T cells (Treg) cells were induced. We conclude that fibrosis induction causes further immunosuppression, in which Treg cells exert a downregulation effect on the antitumor immunity.

Topics: Animals; Carcinogens; Carcinoma, Hepatocellular; CD4-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Forkhead Transcription Factors; Immune Tolerance; Liver Cirrhosis; Liver Neoplasms, Experimental; Lymphocyte Count; Male; Mice; Mice, Inbred BALB C; Neoplasms; T-Lymphocytes, Regulatory; Thioacetamide; Tumor Escape

The liver is the major site of pigment epithelium-derived factor (PEDF) synthesis. Recent evidence suggests a protective role of PEDF in liver cirrhosis. In the present study, immunohistochemical analyses revealed lower PEDF levels in liver tissues of patients with cirrhosis and in animals with chemically induced liver fibrosis. Delivery of the PEDF gene into liver cells produced local PEDF synthesis and ameliorated liver fibrosis in animals treated with either carbon tetrachloride or thioacetamide. In addition, suppression of peroxisome proliferator-activated receptor gamma expression, as well as nuclear translocation of nuclear factor-kappa B was found in hepatic stellate cells (HSCs) from fibrotic livers, and both changes were reversed by PEDF gene delivery. In culture-activated HSCs, PEDF, through the induction of peroxisome proliferator-activated receptor gamma, reduced the activity of nuclear factor-kappa B and prevented the nuclear localization of JunD. In conclusion, our observations that PEDF levels are reduced during liver cirrhosis and that PEDF gene delivery ameliorates cirrhosis suggest that PEDF is an intrinsic protector against liver cirrhosis. Direct inactivation of HSCs and the induction of apoptosis of activated HSCs may be two of the mechanisms by which PEDF suppresses liver cirrhosis.

Topics: Animals; Apoptosis; Blotting, Western; Carbon Tetrachloride; Cell Proliferation; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Eye Proteins; Fluorescent Antibody Technique; Hepatic Stellate Cells; Humans; Immunoenzyme Techniques; Immunoprecipitation; Intrinsic Factor; Liver; Liver Cirrhosis; Mice; Mice, Inbred BALB C; Nerve Growth Factors; NF-kappa B; PPAR gamma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Serpins; Signal Transduction; Thioacetamide

Insulin-like growth factor binding protein-related protein 1 (IGFBPrP1) can activate hepatic stellate cells and increase extracellular matrix (ECM) in vitro. However, the effects of IGFBPrP1 in mice with hepatic fibrosis, and the mechanisms of these effects, are currently unknown. We aim to address these issues in this study.. Intraperitoneal injection of thioacetamide (TAA) is a classic method for establishing a mouse model of hepatic fibrosis. Using this model, we administered anti-IGFBPrP1 antibody, again via intraperitoneal injection. The morphological changes of liver fibrosis were observed with both HE and Masson stainning. The immunohistochemical assays and Western blotting were used to measure changes in IGFBPrP1, α-smooth muscle actin (α-SMA) and ECM in liver tissues, and the expression of transforming growth factor-β1 (TGF-β1) and Smad3. Data were statistically analyzed using one-way analysis of variance (ANOVA), the SNK-q test for inter-group differences.. The Masson staining analysis showed that compared with normal control group, content of collagen fiber in TAA5w group was significantly increased (P < 0.01), and it was significantly decreased in TAA5w/aIGFBPrP1 group compared with in TAA5w group (P < 0.01). The expression of hepatic IGFBPrP1, α-SMA, TGF-β1, Smad3, collagen I and fibronectin (FN) was significantly up-regulated in the TAA5w group (P < 0.01). Anti-IGFBPrP1 treatment reversed these changes (P < 0.01).. IGFBPrP1 plays an important role in the development of hepatic fibrosis. Anti-IGFBPrP1 prevents fibrosis in mice by suppressing the activation of hepatic stellate cells, inhibiting the synthesis of major components of the ECM (namely, collagen I and FN). The mechanism for this suppression of fibrosis is associated with the TGF-β1/Smad3 signaling pathways.

Topics: Actins; Animals; Antibodies; Blotting, Western; Immunohistochemistry; Insulin-Like Growth Factor Binding Proteins; Liver Cirrhosis; Male; Mice; Thioacetamide

Hepatic encephalopathy is a neurological complication observed in patients with liver disease. Subjects with hepatic encephalopathy can develop memory alterations. In order to investigate brain oxidative metabolism in an animal model of chronic cirrhosis and its modification after spatial working memory task, we determined the neural metabolic activity of several brain limbic system regions by cytochrome oxidase (COx) histochemistry and assessed the spatial working memory in the Morris water maze of rats with cirrhosis by administration of thioacetamide. This COx histochemistry was done in cirrhotic and control rats under basal conditions and after the spatial working memory task. The histochemical results showed differences in basal COx activity between control and cirrhotic rats in hippocampal and thalamic regions. In cirrhotic rats basal COx activity was increased in the CA1 and CA3 areas of the hippocampus and reduced in the anterodorsal and anteroventral thalamic nuclei. We found impaired spatial working memory in animals with cirrhosis. These animals showed absence of metabolic activation of the CA3 hippocampal subfield and the lateral mammillary nucleus and disturbance of COx activity in the medial mammillary nucleus and the anteroventral thalamus. These findings suggest that cirrhotic rats show spatial working memory deficits that could be related to the alteration of metabolic activity of neural regions thought to be involved in the processing of spatial memories.

Topics: Analysis of Variance; Animals; Brain; Disease Models, Animal; Electron Transport Complex IV; Exploratory Behavior; Hippocampus; Immunohistochemistry; Liver Cirrhosis; Male; Mammillary Bodies; Maze Learning; Memory; Neurons; Rats; Rats, Wistar; Space Perception; Spatial Behavior; Thalamus; Thioacetamide

People with hepatic insufficiency can develop hepatic encephalopathy (HE), a complex neuropsychological syndrome covering a wide range of neurological and cognitive and motor alterations. The cognitive deficits include disturbances in intellectual functions such as memory and learning. In spite of its high prevalence in western societies, the causes of HE have not yet been clearly established. For this reason, experimental models of HE are used to study this condition. In this work, two experimental models were used, one Type B HE (portacaval shunt) and the other Type C HE (cirrhosis by intoxication with thioacetamide), to evaluate its effect on two tasks of associative learning: two-way active avoidance and step-through passive avoidance. The results show an impediment both in acquisition and retention of active avoidance in both models of HE. However, in passive avoidance, only the rats with portacaval shunt presented a memory deficit for the aversive event. In our opinion, these results can be explained by alterations in the neurotransmission system presented by animals with hepatic insufficiency, which are mainly caused by a rise in cerebral histamine and a dysfunction of the glutamatergic system.

Topics: Animals; Association Learning; Avoidance Learning; Cognition; Disease Models, Animal; Hepatic Encephalopathy; Liver Cirrhosis; Male; Memory; Portacaval Shunt, Surgical; Rats; Rats, Wistar; Thioacetamide

Leptin signaling is involved in T-cell polarization and is required for profibrotic function of hepatic stellate cells (HSCs). Leptin-deficient ob/ob mice do not develop liver fibrosis despite the presence of severe long-standing steatohepatitis. Here, we blocked leptin signaling with our recently generated mouse leptin antagonist (MLA), and examined the effects on chronic liver fibrosis in vivo using the chronic thioacetamide (TAA) fibrosis model, and in vitro using freshly-isolated primary HSCs. In the chronic TAA fibrosis model, leptin administration was associated with significantly enhanced liver disease and a 100% 5-week to 8-week mortality rate, while administration or coadministration of MLA markedly improved survival, attenuated liver fibrosis, and reduced interferon gamma (IFN-gamma) levels. No significant changes in weight, serum cholesterol, or triglycerides were noted. In vitro administration of rat leptin antagonist (RLA), either alone or with leptin, to rat primary HSCs reduced leptin-stimulated effects such as increased expression of alpha-smooth muscle actin (alpha-SMA), and activation of alpha1 procollagen promoter.. Inhibition of leptin-enhanced hepatic fibrosis may hold promise as a future antifibrotic therapeutic modality.

Topics: Animals; Female; Hepatic Stellate Cells; Hormone Antagonists; Leptin; Liver; Liver Cirrhosis; Mice; Models, Animal; Platelet-Derived Growth Factor; Rats; Signal Transduction; Thioacetamide

Liver fibrosis results from sustained wound healing response to chronic liver injury. Liver cirrhosis, the end stage of the fibrotic process, is characterized by disruption of the entire liver architecture and reduced hepatocyte regenerative ability. Hepatic stimulator substance (HSS) is a liver-specific growth factor triggering hepatocyte proliferation in vitro and in vivo. Previous studies have indicated the involvement of HSS in animal models of acute liver injury. The aim of the present study was to investigate the involvement of HSS in the process of fibrosis and cirrhosis induction. Liver fibrosis and cirrhosis were induced in rats by thioacetamide (TAA) administration (300 mg/l) in the drinking water for 3 months, and animals were killed at 0, 1, 2, and 3 months of treatment. TAA administration resulted in progressively increasing liver fibrosis, leading to the onset of cirrhosis at the end of the experimental time. HSS was continuously produced during the course of fibrosis and cirrhosis induction, peaking at the 2nd month of TAA treatment, coinciding with markers of hepatic proliferative capacity, as thymidine kinase activity and DNA biosynthesis. Significantly reduced HSS activity was noted in cirrhotic liver (3rd month). In this case, the exogenous HSS administration during the 3rd month of TAA treatment suppressed the onset of liver cirrhosis, stimulating the hepatic regenerative capacity. Our data indicate the active participation of HSS in the process of fibrosis and cirrhosis induction post-TAA treatment in rats, suggesting also the beneficial effect of HSS treatment against cirrhosis induction with future possible clinical implications.

Topics: Animals; Intercellular Signaling Peptides and Proteins; Liver Cirrhosis; Liver Regeneration; Male; Peptides; Rats; Rats, Wistar; Thioacetamide

Dominant-negative transforming growth factor beta type II receptor (TbetaRIIDeltacyt) is a protein that blocks transforming growth factor (TGF-beta) signaling. Because the consequences of blocking TGF-beta have not been completely elucidated in liver fibrosis, we analysed the effects of adenoviral delivery of TbetaRIIDeltacyt on profibrogenic genes and matrix metalloproteinase (MMP) proteins, as well as on TGF-beta signal repressor SKI-like oncogene (SnoN), in cultured hepatic stellate cells (HSCs) and in a rat model of liver fibrosis.. To induce liver fibrosis, rats were treated with thioacetamide for 7 weeks and administrated once with Ad-TbetaRIIDeltacyt or Ad-betagal through the iliac vein. Fibrosis was measured by morphometric analysis. We evaluated SnoN by western blot, immunocytochemistry and immunohistochemistry; MMP activity was determined by zymography and profibrogenic gene expression by the real-time reverse transcriptase-polymerase chain reaction in cultured HSCs and liver tissue.. Profibrogenic gene expression of collagen alpha1 (I), TGF-beta1, platelet-derived growth factor-B, plasminogen activator inhibitor (PAI)-1, tissue inhibitor of matrix metalloproteinase-1 and MMP-2 was down-regulated; whereas MMP-3 was over-expressed in response to Ad-TbetaRIIDeltacyt in HSCs. Moreover, zymography assays corroborated MMP-2 and MMP-3 changes in activity. Surprisingly, anti-TGF-beta molecular intervention increased nuclear SnoN in HSCs. In vivo, Ad-TbetaRIIDeltacyt reduced liver fibrosis, increased nuclear SnoN in sinusoidal cells, and also produced significant suppression in collagen alpha1 (I), TGF-beta1, PAI-1, MMP-2 and over-expression in MMP-3 in thioacetamide-intoxicated animals.. The results obtained in the present study suggest that the molecular mechanism for the blocking effects of Ad-TbetaRIIDeltacyt in TGF-beta signaling acts via up-regulation of the transcriptional repressor SnoN, which antagonizes TGF-beta signaling (TGF-beta/Smad-pathway inhibitor). Consequently, profibrogenic genes are down-regulated.

Topics: Adenoviridae; Animals; Body Weight; Cells, Cultured; Gene Transfer Techniques; Hepatic Stellate Cells; Intracellular Signaling Peptides and Proteins; Liver Cirrhosis; Matrix Metalloproteinase 2; Matrix Metalloproteinase 3; Oncogene Proteins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Rats; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Repressor Proteins; Signal Transduction; Thioacetamide; Transduction, Genetic; Transgenes; Up-Regulation

Interleukin-1 (IL-1) is rapidly expressed in response to tissue damage; however, its role in coordinating the progression from injury to fibrogenesis is not fully understood. Liver fibrosis is a consequence of the activation of hepatic stellate cells (HSCs), which reside within the extracellular matrix (ECM) of subsinusoids. We have hypothesized that, among the hepatic inflammatory cytokines, IL-1 may directly activate HSCs through autocrine signaling and stimulate the matrix metalloproteinases (MMPs) produced by HSCs within the space of Disse, resulting in liver fibrogenesis. In this study, we first established a temporal relationship between IL-1, MMPs, HSC activation, and early fibrosis. The roles of IL-1 and MMP-9 in HSC activation and fibrogenesis were determined by mice deficient of these genes. After liver injury, IL-1, MMP-9, and MMP-13 levels were found to be elevated before the onset of HSC activation and fibrogenesis. IL-1 receptor-deficient mice exhibited ameliorated liver damage and reduced fibrogenesis. Similarly, advanced fibrosis, as determined by type-I and -III collagen mRNA expression and fibrotic septa, was partially attenuated by the deficiency of IL-1. In the early phase of liver injury, the MMP-9, MMP-13, and TIMP-1 expression correlated well with IL-1 levels. In injured livers, MMP-9 was predominantly colocalized to desmin-positive cells, suggesting that HSCs are MMP-producing cells in vivo. MMP-9-deficient mice were partially protected from liver injury and HSC activation. Thus IL-1 is an important participant, along with other cytokines, and controls the progression from liver injury to fibrogenesis through activation of HSCs in vivo.

Topics: Actins; Alanine Transaminase; Animals; Collagen Type I; Collagen Type III; Gene Expression; Hepatic Stellate Cells; Interleukin-1; Interleukin-1alpha; Interleukin-1beta; Liver; Liver Cirrhosis; Male; Matrix Metalloproteinase 13; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Knockout; Receptors, Interleukin-1; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2; Transforming Growth Factor beta

The inflammasome is a cytoplasmic multiprotein complex that has recently been identified in immune cells as an important sensor of signals released by cellular injury and death. Analogous to immune cells, hepatic stellate cells (HSC) also respond to cellular injury and death. Our aim was to establish whether inflammasome components were present in HSC and could regulate HSC functionality. Monosodium urate (MSU) crystals (100 microg/ml) were used to experimentally induce inflammasome activation in LX-2 and primary mouse HSC. Twenty-four hours later primary mouse HSC were stained with alpha-smooth muscle actin and visualized by confocal microscopy, and TGF-beta and collagen1 mRNA expression was quantified. LX-2 cells were further cultured with or without MSU crystals for 24 h in a transwell chemotaxis assay with PDGF as the chemoattractant. We also examined inhibition of calcium (Ca(2+)) signaling in LX-2 cells treated with or without MSU crystals using caged inositol 1,4,5-triphosphate (IP(3)). Finally, we confirmed an important role of the inflammasome in experimental liver fibrosis by the injection of carbon tetrachloride (CCl(4)) or thioacetamide (TAA) in wild-type mice and mice lacking components of the inflammasome. Components of the inflammasome are expressed in LX-2 cells and primary HSC. MSU crystals induced upregulation of TGF-beta and collagen1 mRNA and actin reorganization in HSCs from wild-type mice but not mice lacking inflammasome components. MSU crystals inhibited the release of Ca(2+) via IP(3) in LX-2 cells and also inhibited PDGF-induced chemotaxis. Mice lacking the inflammasome-sensing and adaptor molecules, NLRP3 and apoptosis-associated speck-like protein containing CARD, had reduced CCl(4) and TAA-induced liver fibrosis. We concluded that inflammasome components are present in HSC, can regulate a variety of HSC functions, and are required for the development of liver fibrosis.

Topics: Actins; Animals; Apoptosis Regulatory Proteins; Calcium Signaling; Carbon Tetrachloride; CARD Signaling Adaptor Proteins; Carrier Proteins; Cell Line, Transformed; Chemotaxis; Collagen Type I; Cytoskeletal Proteins; Gene Expression; Hepatic Stellate Cells; Humans; Inflammation; Inositol 1,4,5-Trisphosphate; Liver; Liver Cirrhosis; Mice; Mice, Inbred C57BL; Mice, Knockout; NLR Family, Pyrin Domain-Containing 3 Protein; Platelet-Derived Growth Factor; Thioacetamide; Transforming Growth Factor beta; Uric Acid

Our research group investigates whether human mononuclear cells isolated from umbilical cord blood (HUCBM cells) might be valuable in hepatic regenerative medicine. We recently demonstrated that HUCBM cell transplantation improves histological alterations and function of the liver in rats with acute liver damage induced by D-galactosamine. In the present study, HUCBM cells were transplanted into rats with thioacetamide (TAA)-induced liver cirrhosis, an experimental model that generates an intense fibrosis and mimics the histological and biochemical alterations found in the human disease. HUCBM transplantation had no effect on hepatic histology of cirrhotic animals. In contrast, analysis of plasma albumin and total bilirubin, liver damage markers, revealed a harmful effect of HUCBM cell transplantation in our experimental model of liver cirrhosis. Significantly higher plasma urea concentrations, marker of renal function, were observed in the cirrhotic and control rats intraportally injected with HUCBM cells than in those not receiving this therapy. Histological study revealed tubular and glomerular lesions in kidneys of cirrhotic animals transplanted with HUCBM cells. The glomeruli appeared ischemic, and the tubules showed a severe involvement that included peripheral asymmetric vacuolization and disappearance of the tubular lumen. Taken together, the histological and biochemical data suggest that the cirrhotic rats subjected to HUCBM cell therapy developed a hepatorenal syndrome.

Topics: Alanine Transaminase; Albumins; Animals; Aspartate Aminotransferases; Bilirubin; Biomarkers; Cord Blood Stem Cell Transplantation; Disease Models, Animal; Fetal Blood; Hepatorenal Syndrome; Humans; Kidney; Kidney Tubules; Leukocyte Common Antigens; Liver Cirrhosis; Male; Rats; Rats, Wistar; Thioacetamide; Urea

The vitronectin receptor integrin alphavbeta3 promotes angiogenesis by mediating migration and proliferation of endothelial cells, but also drives fibrogenic activation of hepatic stellate cells (HSCs) in vitro. Expecting antifibrotic synergism, we studied the effect of alphavbeta3 inhibition in two in vivo models of liver fibrogenesis. Liver fibrosis was induced in rats by way of bile duct ligation (BDL) for 6 weeks or thioacetamide (TAA) injections for 12 weeks. A specific alphavbeta3 (alphavbeta5) inhibitor (Cilengitide) was given intraperitoneally twice daily at 15 mg/kg during BDL or after TAA administration. Liver collagen was determined as hydroxyproline, and gene expression was quantified by way of quantitative polymerase chain reaction. Liver angiogenesis, macrophage infiltration, and hypoxia were assessed by way of CD31, CD68 and hypoxia-inducible factor-1alpha immunostaining. Cilengitide decreased overall vessel formation. This was significant in portal areas of BDL and septal areas of TAA fibrotic rats and was associated with a significant increase of liver collagen by 31% (BDL) and 27% (TAA), and up-regulation of profibrogenic genes and matrix metalloproteinase-13. Treatment increased gamma glutamyl transpeptidase in both models, while other serum markers remained unchanged. alphavbeta3 inhibition resulted in mild liver hypoxia, as evidenced by up-regulation of hypoxia-inducible genes. Liver infiltration by macrophages/Kupffer cells was not affected, although increases in tumor necrosis factor alpha, interleukin-18, and cyclooxygenase-2 messenger RNA indicated modest macrophage activation.. Specific inhibition of integrin alphavbeta3 (alphavbeta5) in vivo decreased angiogenesis but worsened biliary (BDL) and septal (TAA) fibrosis, despite its antifibrogenic effect on HSCs in vitro. Angiogenesis inhibitors should be used with caution in patients with hepatic fibrosis.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Cell Movement; Cell Proliferation; Disease Models, Animal; Endothelium, Vascular; gamma-Glutamyltransferase; Hepatic Stellate Cells; Hypoxia-Inducible Factor 1, alpha Subunit; Integrin alphaVbeta3; Ligation; Liver; Liver Cirrhosis; Male; Neovascularization, Physiologic; Platelet Endothelial Cell Adhesion Molecule-1; Rats; Rats, Wistar; Snake Venoms; Thioacetamide

Solanum nigrum (Solanaceae) has been used in traditional folk medicine for its hepatoprotective agent. The purpose of this study was to investigate the effects of Solanum nigrum extract (SNE) on thioacetamide (TAA)-induced liver fibrosis in mice.. Hepatic fibrosis was produced by TAA (0.2 g/kg, i.p.) three times a week for 12 weeks. Mice in the three TAA groups were treated daily with distilled water and SNE (0.2 or 1.0 g/kg) via gastrogavage throughout the experimental period.. SNE reduced the hepatic hydroxyproline and alpha-smooth muscle actin protein levels of TAA-treated mice. SNE inhibited TAA-induced collagene (alpha1)(I) and transforming growth factor-beta1 (TGF-beta1) mRNA levels in the liver. Histological examination also confirmed that SNE reduced the degree of fibrosis caused by TAA treatment.. Oral administration of SNE significantly reduces TAA-induced hepatic fibrosis in mice, probably through the reduction of TGF-beta1 secretion.

Topics: Actins; Administration, Oral; Animals; Collagen Type I; Dose-Response Relationship, Drug; Hydroxyproline; Liver Cirrhosis; Male; Medicine, Traditional; Mice; Mice, Inbred BALB C; Plant Extracts; Solanum nigrum; Thioacetamide; Transforming Growth Factor beta1

Liver diseases and regeneration are associated with hemodynamic changes denoting pathological alterations. Determining and monitoring physiological and pathological liver changes is essential for diagnostic and therapeutic objectives. Our aim was to determine the feasibility of functional magnetic resonance imaging (fMRI) during hypercapnia and hyperoxia for monitoring liver pathology. Liver fMRI images were acquired in rodents following acute bleeding, partial hepatectomy, and fibrosis. Results were quantitated and confirmed by histology. Changes induced by hyperoxia and hypercapnia following hemorrhage significantly correlated with the percentage of blood loss, reflecting lower liver perfusion and diminished vessel responsiveness to gas saturation. Hepatectomy resulted in an early decline in signal intensity changes due to hyperoxia, suggesting a decrease in liver perfusion and blood content. Following hepatectomy, signal intensity changes due to hypercapnia increased, signifying a change in liver perfusion from a mainly portal to a more arterial source. Two weeks after induction of fibrosis, signal intensity changes due to hypercapnia became much lower and those due to hyperoxia were much higher than those in normal livers, reflecting the increased perfusion due to the inflammatory process as confirmed by histologic analysis. With fibrosis progression, signal intensity changes induced by hypercapnia and hyperoxia were gradually attenuated, indicating structural and functional alterations of the liver vasculature during fibrosis.. In various liver pathologies, fMRI response to hypercapnia and hyperoxia is sensitive to changes in liver hemodynamic status involved in hepatic damage or recovery; thus, this technique may offer an additional noninvasive diagnostic tool for evaluation and follow-up of liver diseases by means of examining perfusion-related alterations.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Sub-Family B Member 4; Disease Models, Animal; Hemorrhage; Hepatectomy; Hypercapnia; Hyperoxia; Liver; Liver Cirrhosis; Magnetic Resonance Imaging; Male; Mice; Mice, Knockout; Models, Biological; Rats; Rats, Sprague-Dawley; Thioacetamide

Brain tissue of patients diagnosed with hepatic encephalopathy exhibits cellular morphological changes that could be associated with memory impairment. The mammillary nuclei, located in the posterior part of the hypothalamus, are important for spatial memory formation. This work aimed to assess spatial reference memory and cellular changes in the mammillary nuclei of cirrhotic rats. Spatial reference memory of Wistar rats with thioacetamide-induced cirrhosis was assessed in the Morris water maze. Total cell number of neurons and glial cells and volume of the mammillary nuclei were quantified by stereology. Neuronal and astrocytic nuclear volume in mammillary nuclei and CA1 dorsal hippocampal subfield were assessed by nucleator probe. Cirrhotic rats showed an impaired spatial reference memory in comparison with control animals. Total number of neurons and glial cells were unaltered. In the medial mammillary nucleus (MMn), glial fibrillary acidic protein-immunoreactive astrocytes decreased in the cirrhotic group while the lateral part was unaffected. The medial part of the MMn was larger in the cirrhotic group. The cirrhotic rats showed morphometric cellular changes characterised by an increased neuronal and astrocytic nuclear volume in all the mammillary nuclei and CA1 hippocampal region. These findings suggest that cirrhotic rats show spatial memory impairment that could be linked to astrocytes and neuronal impairment in mammillary nuclei and hippocampus.

Topics: Animals; Astrocytes; Cell Count; Disease Models, Animal; Glial Fibrillary Acidic Protein; Hepatic Encephalopathy; Liver Cirrhosis; Male; Mammillary Bodies; Maze Learning; Memory; Neurons; Organ Size; Rats; Rats, Wistar; Space Perception; Spatial Behavior; Thioacetamide

The pathophysiological mechanisms of thioacetamide (TAA)-induced hepatic fibrogenesis are not yet fully understood. In particular, the role of hepatic stellate cells (HSCs) remains unclear. We therefore examined proliferation and transdifferentiation of HSC as well as the underlying molecular mechanisms in TAA-induced fibrosis. Hepatic fibrogenesis was induced in mice by addition of TAA to drinking water. Liver damage was determined by assessment of alanine aminotransferase and aspartate aminotransferase levels, and measurement of collagen deposition. Additionally, expression patterns of alpha-smooth muscle actin, glial fibrillary acidic protein (GFAP, specific hepatic biomarker for HSC), cysteine- and glycine-rich protein 2 (CRP2, specific marker of HSC transdifferentiation), tissue inhibitor of metalloproteinases-1, matrix metalloproteinase-9 (MMP-9), interleukins (IL-1beta, IL-6), platelet-derived growth factors (PDGF-B, PDGF-D) , tumor necrosis factor (TNF)-alpha, and (transforming growth factor (TGF)-beta1 were assessed by real-time PCR. Transcription of GFAP and CRP2 were transiently upregulated during TAA-induced fibrogenesis (punctum maxima (p.m.) week 10 for GFAP and week 14 for CRP2). Similar transient expression patterns were demonstrated for IL-1beta, IL-6, TGF-beta1, and PDGF-B (p.m. week 12) whereas TNF-alpha and PDGF-D continuously increased with ongoing liver injury. In particular, not only neutrophil granulocytes, but also macrophages and leukocytes served as a major source for MMP-9 expression. GFAP and CRP2 expression patterns demonstrated transiently increased HSC-activation during TAA-induced hepatic fibrogenesis. The rate of increase of transcription of GFAP correlated best with PDGF-B, whereas CRP2 levels correlated with PDGF-B, PDGF-D, and IL-1beta expression. This study demonstrates for the first time that transiently increased activation patterns of HSC are observed in toxically induced hepatic fibrosis. Thus, TAA in drinking water is an effective and elegant model to induce reproducible states of liver fibrosis without parenchymal damage in mice.

Topics: Animals; Cell Differentiation; Cell Proliferation; Cytokines; Disease Models, Animal; Glial Fibrillary Acidic Protein; Hepatocytes; LIM Domain Proteins; Liver; Liver Cirrhosis; Lymphokines; Mice; Muscle Proteins; Nerve Tissue Proteins; Nuclear Proteins; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Thioacetamide

Establishing an easy and reproducible model for hepatic fibrosis is absolutely necessary for research on liver reperfusion injury. We compared the characteristics of several hepatic cirrhosis models in terms of the degree of fibrosis, reproducibility, histologic characteristics, and success rate to achieve sufficient fibrosis. In mice & rats, we administered three different hepatotoxic drugs (thioacetamide, dimethylnitrosamine, and carbon tetrachloride [CCl4]) through two different routes (oral feeding and intraperitoneal injection). The animals fed thioacetamide exhibited little fibrosis; rather, more inflammatory cells infiltrated into periportal areas with bile duct proliferation. The livers from hosts administered dimethylnitrosamine showed greater early injury and severe inflammatory reactions in the peritoneal cavity. The liver showed a marked degree of piecemeal necrosis with limited fibrosis. The mice administered a 50% solution of CCl4 (2 mL/kg orally) tolerated the entire induction period of 12 weeks. The degree of fibrosis correlated well with the duration of induction. Livers from hosts administered CCl4 orally twice a week for 10 weeks was the most effective to achieve sufficient fibrosis and greatest reproducibility with acceptable animal survival.

Topics: Animals; Carbon Tetrachloride Poisoning; Dimethylnitrosamine; Disease Models, Animal; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Rats; Rats, Sprague-Dawley; Thioacetamide

Methacetin is thought to be a good substrate for the evaluation of different cytochrome P450 enzymatic systems of liver microsomes because of its rapid metabolism and lack of toxicity in small doses. Recent studies indicate that a methacetin breath test may be a non-invasive alternative for the evaluation of liver function since it correlates well with the severity of liver damage. It may also discriminate between different stages of liver cirrhosis and correlates with the Child-Pugh score. The application of this test in experimental liver damage in animal models has not yet been examined. This study aimed to evaluate the efficacy of the (13)C-methacetin breath test in assessing the extent of hepatic injury in models of acute liver failure, liver cirrhosis, and fatty liver in rats.. Absorption of methacetin given per os or intraperitoneally in normal rats was evaluated. The association between liver mass and (13)C-methacetin breath test results was assessed in a 70% hepatectomy rat model. Fulminant hepatic failure was induced by three consecutive intraperitoneal injections of thioacetamide, 300 mg/kg, at 24 h intervals. For induction of liver cirrhosis, rats were given intraperitoneal injections of thioacetamide, 200 mg/kg, twice a week for 12 weeks. A methionine-choline deficient diet was used for the induction of fatty liver. Rats were analyzed for (13)C-methacetin by BreathID (MBID) using molecular correlation spectrometry. BreathID continuously sampled the animal's breath for 60 min and displayed the results on the BreathID screen in real-time.. Methacetin was absorbed well irrespective of the administration method in normal rats. Liver mass was associated with peak amplitude, complete percent dose recovery (CPDR) at 30 and 60 min and MBID peak time. A high degree of association was also demonstrated with MBID results in acute hepatitis (peak amplitude, 19.6 +/- 3.4 vs 6.3 +/- 1.63.4; CPDR30, 6.0 +/- 3.3 vs 1.2 +/- 0.5; CPDR60, 13.3 +/- 4.5 vs 3.2 +/- 1.4; and peak time, 31.0 +/- 14.9 vs 46.9 +/- 10.8 min) and liver cirrhosis (peak amplitude, 24.4 +/- 2.3 vs 15.6 +/- 6.4; CPDR30, 7.9 +/- 1.2 vs 2.7 +/- 1.0; CPDR60, 17.8 +/- 2.6 vs 8.8 +/- 2.1; and peak time, 30.2 +/- 1.5 vs 59.6 +/- 14.5 min), but not with grade of liver steatosis.. Methacetin is well absorbed and exclusively metabolized in the liver. MBID is a sensitive test and may be a useful tool for the evaluation of functional liver mass in animal models of acute liver failure and cirrhosis. However, MBID could not distinguish between fatty liver and normal liver in rats.

Topics: Acetamides; Administration, Oral; Animals; Breath Tests; Carbon Isotopes; Choline Deficiency; Disease Models, Animal; Fatty Liver; Hepatectomy; Injections, Intraperitoneal; Liver; Liver Cirrhosis; Liver Failure, Acute; Liver Function Tests; Male; Methionine; Organ Size; Predictive Value of Tests; Rats; Rats, Wistar; Severity of Illness Index; Thioacetamide; Time Factors

Hepatocellular carcinoma (HCC) is one of the common cancers worldwide, caused by Hepatitis C virus (HCV) and hepatotoxins. Here we report the development of HCC in wild type as well as HCV core protein (HCP)-transgenic zebrafish upon treatment with a hepatotoxin, thioacetamide (TAA). Two-fold accelerated HCC development could be achieved in the TAA-treated transgenic fish, that is, the progression of the disease in TAA-treated wild type zebrafish developed HCC in 12 weeks whereas that of HCP-transgenic zebrafish shortened the HCC progression to 6 weeks. Histopathological observation showed the specific pathological features of HCC. The HCC progression was confirmed through RT-PCR that revealed an up and down regulation of different marker genes at various stages of HCC progression such as, steatohepatitis, fibrosis and HCC. Moreover, HCV core protein expressed in the HCP-transgenic zebrafish and TAA synergistically accelerate the HCC development. It must be mentioned that, this is the first report revealing HCV core protein along with TAA to induce HCC in zebrafish, particularly, in a short period of time comparing to mice model. As zebrafish has already been considered as a good human disease model and in this context, this HCC-zebrafish model may serve as a powerful preclinical platform to study the molecular events in hepatocarcinogenesis, therapeutic strategies and for evaluating chemoprevention strategies in HCC.

Topics: Animals; Animals, Genetically Modified; Carcinoma, Hepatocellular; Chemical and Drug Induced Liver Injury; DNA Primers; Fatty Liver; Hepacivirus; Liver; Liver Cirrhosis; Liver Diseases; Liver Neoplasms; Microscopy, Confocal; Reverse Transcriptase Polymerase Chain Reaction; Thioacetamide; Viral Core Proteins; Zebrafish

Stimulation of nitric oxide (NO) synthesis similar to the application of NO donors could be of benefit in liver fibrosis. Many authors believe that activation of NO synthesis by pharmacological agents is promising in the treatment of liver fibrosis. However, there is considerable controversy in understanding the role of NO in fibrogenesis and fibrolysis. The aims of our study were to evaluate the effects of L-arginine, as an NO metabolic precursor, and those of NO synthase (NOS) inhibitors, L-nitroarginine methyl ester (L-NAME) and aminoguanidine (AG) in rats with thioacetamide (TAA)-induced liver fibrosis reversal.. Male Wistar rats, 230-240 g, received TAA (200 mg kg(-1), intraperitoneally) twice a week for 3 months. Liver resolution was simulated by withdrawal of TAA administration. Thereafter the animals were subdivided into five groups and treated by intragastric intubation with: L-arginine (100 and 300 mg kg(-1)); L-NAME as an inhibitor of both constitutively expressed NOS (eNOS) and inducible NOS (iNOS) (20 mg kg(-1)), AG as a specific inhibitor of iNOS (100 mg kg(-1)) or placebo. The severity of liver fibrosis was assessed by morphometric evaluation of liver slides stained with Azan-Mallory, hydroxyproline (Hyp) determination and mRNA steady state levels of collagen I, transforming growth factor (TGF)-beta1, metalloproteinases (MMP)-13, -14, tissue inhibitor of MMP (TIMP)-1 and plasminogen activator inhibitor (PAI)-1 were quantified by real time PCR. The activities of serum marker enzyme, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase, were measured.. TAA treatment during 3 months induced micronodular liver fibrosis with a pronounced deposition of collagen fibres. L-Arginine did not affect this deposition nor did it affect both relative and total liver hydroxyproline content. Both NOS inhibitors significantly increased the square of the liver connective tissue stained by Azan-Mallory and the above parameters characterizing liver hydroxyproline content. Both NOS inhibitors up-regulated procollagen alpha1 (I), MMP-13, TIMP-1 and PAI-1 mRNA expression. The AG effects were more pronounced. than those of L-NAME. AG treatment also increased mRNA expression of TGF-beta1 and PAI-1.. Both NOS inhibitors developed a clear pro-fibrotic effect in the liver. Aminoguanidine was more fibrotic than L-NAME. Our data suggest a significant anti-fibrotic role for iNOS rather than for eNOS. L-Arginine did not show any anti-fibrotic properties in the TAA-model used.

Topics: Animals; Arginine; Disease Models, Animal; Enzyme Inhibitors; Liver; Liver Cirrhosis; Liver Regeneration; Male; Nitric Oxide Synthase Type II; Rats; Rats, Wistar; Thioacetamide

Rats with liver cirrhosis, evoked by chronic administration of thioacetamide (TAA), consumed voluntarily more alcohol than their healthy counterparts. Seeking the mechanisms underlying this phenomenon, the opioid system was screened for involvement and alterations. In vivo, the influence of chronically administered Naloxone and Naltrexone, non-specific opioid receptor antagonists, on alcohol intake was examined in free choice tests between 10% alcohol and tap water and ex vivo receptor binding studies were performed on cerebral membrane preparations.. TAA rats, selected for the study, had confirmed liver insufficiency: their plasma bilirubin concentrations were about 3 times higher, the prothrombin time was 50% longer and they consumed voluntarily 3 times more alcohol than the control animals. The drugs were given s.c. for five days, at the beginning of the dark phase of a 24h cycle, in a daily dose of 10 mg per kg body mass. Throughout the treatment, the rats were kept individually in metabolic cages with a free access to water, alcohol solution and food. Feed and fluid consumption, as well as the urine outputs, were recorded on the 2h, 4h, 6h and 24h after the drug administration. The mu opioid ligand - [(3)H]-(D-Ala(2), -N-MePhe(4), Glyol(5)) Enkephalin was used to obtain binding characteristics of the control and TAA rat brain membranes.. The drugs, if modified drinking behaviours, they did it transiently; alcohol, water and thus the total fluid intake by the cirrhotic and control rats was significantly less after 2h - 6h from either naloxone or naltrexone administration. Both drugs decreased general fluid consumption as such rather than the consumption of alcohol only, as observed from the recordings related to TAA rats. The binding data: K(d) of 2.62 +/- 0.98 nM and B(max) of 43.71 +/- 6.12 fmol/mg protein for cirrhotic rats, versus K(d) of 4.63 +/- 1.98 nM and B(max) 95.61 +/- 18.33 fmol/mg protein for the control ones, suggest that while the affinity of radioligand to cerebral mu receptors was similar for the two groups, there was a lower density of those receptors in the cirrhotic rats.. The results indicate some disturbances in the opioid system in cirrhotic rats. However, the low response to opioid therapy suggests that the opioid system may have only be partly involved in the development of the observed increased alcohol drinking in the rats with liver cirrhosis.

Topics: Alcohol Drinking; Animals; Behavior, Animal; Bilirubin; Brain; Disease Models, Animal; Drinking; Liver Cirrhosis; Male; Naloxone; Naltrexone; Narcotic Antagonists; Prothrombin Time; Radioligand Assay; Rats; Rats, Wistar; Receptors, Opioid, mu; Thioacetamide; Time Factors

Metabonomics has already been used to discriminate different pathological states in biological fields. The metabolic profiles of chronic experimental fibrosis and cirrhosis induction in rats were investigated using (1)H NMR spectroscopy of liver extracts and serum combined with pattern recognition techniques. Rats were continuously administered with thioacetamide (TAA) in the drinking water (300 mg TAA/L), and sacrificed on 1st, 2nd, and 3rd month of treatment. (1)H NMR spectra of aqueous and lipid liver extracts, together with serum were subjected to Principal Component Analysis (PCA). Liver portions were also subjected to histopathological examination and biochemical determination of malondialdehyde (MDA). Liver fibrosis and cirrhosis were progressively induced in TAA-treated rats, verified by the histopathological examination and the alterations of MDA levels. TAA administration revealed a number of changes in the (1)H NMR spectra compared to control samples. The performance of PCA in liver extracts and serum, discriminated the control samples from the fibrotic and cirrhotic ones. Metabolic alterations revealed in NMR spectra during experimental liver fibrosis and cirrhosis induction, characterize the stage of fibrosis and could be illustrated by subsequent PCA of the spectra. Additionally, the PCA plots of the serum samples presented marked clustering during fibrosis progression and could be extended in clinical diagnosis for the management of cirrhotic patients.

Topics: Animals; Carcinogens; Disease Models, Animal; Disease Progression; Lipid Peroxidation; Liver; Liver Cirrhosis; Liver Extracts; Magnetic Resonance Spectroscopy; Male; Malondialdehyde; Metabolic Networks and Pathways; Principal Component Analysis; Rats; Rats, Wistar; Thioacetamide; Time Factors

Liver cirrhosis, which is caused by the accumulation of extracellular matrix materials, is a serious clinical problem that can progress to hepatic failure. Transforming growth factor-beta (TGFbeta) plays a pivotal role in extracellular matrix production, but bone morphogenetic protein (BMP)-7, a member of the TGFbeta superfamily, can antagonise the fibrogenic activity of TGFbeta.. In this study, we examined whether adenovirus-mediated overexpression of BMP-7 (Ad-BMP-7) antagonised the effect of TGFbeta in vitro and in vivo.. In primary cultured rat stellate cells and the LX-2 human stellate cell line, induction of BMP-7 by Ad-BMP-7 infection decreased the expression of collagen 1A2 mRNA and smooth muscle alpha-actin in the presence or absence of TGFbeta, via Smad 1/5/8 phosphorylation. BMP-7 triggered the mRNA expression of inhibitors of differentiation 2 (Id2) in LX-2. Although endogenous expression of BMP-7 was hardly detectable, Smad1 and Id2 overexpression increased BMP-7 expression in LX-2. A liver fibrosis model was induced by the repetitive intraperitoneal injection of thioacetamide (200 mg/kg body weight) twice per week for up to 7 weeks. In rats administered Ad-BMP-7 via the tail vein, hydroxyproline content and the areas stained by Sirius red dye in the liver were significantly reduced compared to controls. Ad-Id2 also reduced fibrosis.. These data demonstrate that BMP-7, Smad 1/5/8 and Ids interact to antagonise hepatic fibrogenesis.

Topics: Actins; Adenoviridae; Animals; Apoptosis Regulatory Proteins; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Carrier Proteins; Cells, Cultured; Collagen Type I; Gene Expression; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Hepatocytes; Humans; Inhibitor of Differentiation Protein 2; Liver Cirrhosis; Male; Mice; Mice, Transgenic; Mitochondrial Proteins; Rats; Rats, Wistar; RNA, Messenger; Signal Transduction; Thioacetamide; Transforming Growth Factor beta

Hepatic fibrosis is associated with the activation of stellate cells (HSCs), the major source of extracellular matrix (ECM) proteins. Transforming growth factor-beta (TGF-beta), signaling via Smad3, is the most profibrogenic cytokine and the major promoter of ECM synthesis. Halofuginone, an inhibitor of liver fibrosis, inhibits TGF-beta-dependent Smad3 phosphorylation in human HSCs in culture. We have used transcriptional profiling to evaluate the effect of halofuginone on gene expression during the progression of thioacetamide (TAA)-induced liver fibrosis in the rat and have focused on genes that are associated with TGF-beta. TAA treatment causes alterations in the expression of 7% of liver genes. Halofuginone treatment prevents the changes in the expression of 41% of these genes and results in the inhibition of HSC activation and collagen synthesis. During the early stages of the disease, halofuginone affects genes involved in alcohol, lipid, protein, and phosphate metabolism and cell adhesion and, at later stages, in the cell cycle (cell development, differentiation, cell proliferation, and apoptosis). The activation of TGF-beta-dependent genes, such as tartrate-resistant acid phosphatase, its putative substrate osteopontin, stellate cell activation-association protein, and fibrillin-1, during chemically induced fibrosis is prevented by halofuginone. This study thus highlights the role of TGF-beta signaling in liver fibrosis and especially its potential for pharmacological intervention. Halofuginone, which has demonstrated efficacy and tolerance in animals and humans, could become an effective and novel therapy for liver fibrosis.

Topics: Acid Phosphatase; Animals; Antineoplastic Agents; Cluster Analysis; Cytoglobin; Disease Progression; Fibrillin-1; Fibrillins; Gene Expression Profiling; Gene Expression Regulation; Globins; Hepatocytes; Humans; Isoenzymes; Liver Cirrhosis; Male; Microfilament Proteins; Nuclear Proteins; Osteopontin; Phosphorylation; Piperidines; Quinazolinones; Rats; Rats, Wistar; Signal Transduction; Smad2 Protein; Smad3 Protein; Substrate Specificity; Tartrate-Resistant Acid Phosphatase; Thioacetamide; Transforming Growth Factor beta

We conducted a rat cirrhosis and recovery model, on the basis of which proteomics was used to audit liver resolution from cirrhosis.. Micronodular cirrhosis was established using Sprague-Dawley rats fed thioacetamide, and spontaneous recovery from cirrhosis was acquired after thioacetamide withdrawal.. Over the course of a 2-, 3-, and 6-week recovery, macronodular cirrhosis, uneven liver surface, and nearly normal liver surface were acquired, respectively. Specific liver enzymes, hepatitis activity index, hepatocytes apoptosis index, number of activated Kupffer cells and hepatic stellate cells, and area of fibrosis bands consistently peaked at the end of thioacetamide administration and decreased progressively during the recovery period. mRNA expression of proinflammatory cytokines and proapoptotic molecules peaked around the end of thioacetamide administration and decreased thereafter. Using two-dimensional gel electrophoresis, the seven most upregulated and six most downregulated protein spots were analyzed by matrix-assisted laser desorption/ionization time-of-flight. Of these, GST-P2 and its isoforms, GST-alpha and GST-M, were chosen for further validation using immunohistochemistry. Expression of GST-P peaked at the 2-week recovery, whereas GST-alpha and GST-M remained at strong levels at the 6-week recovery.. The mechanism of resolution from cirrhosis can be extensively investigated using the presented model which, for example, showed GST isoforms performing their roles at different time phases.

Topics: Amino Acid Sequence; Animals; Apoptosis; Cytokines; Disease Models, Animal; Electrophoresis, Gel, Two-Dimensional; Glutathione Transferase; Immunohistochemistry; Liver; Liver Cirrhosis; Male; Molecular Sequence Data; Proteomics; Rats; Rats, Sprague-Dawley; Recovery of Function; RNA, Messenger; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thioacetamide

Due to the loss of cell-cell and cell-matrix interactions, cell culture models poorly mimic the in vivo situation. Therefore, we tested the applicability of precision-cut liver slices (PCLS) to study the early activation of the two main liver fibrogenic cell subpopulations: hepatic stellate cells (HSC) and portal fibroblasts (PF). PCLS were treated with thioacetamide or acetaminophen to induce HSC activation. In PCLS culture, both were able to trigger centrolobular lesion and HSC activation as observed in vivo. However, thioacetamide also presented a toxic effect on portal tract cells. In this PCLS model of centrolobular lesion, the antioxidant N-acetylcysteine was able to prevent acetaminophen-induced injury. To induce a specific activation of PF, PCLS were treated with epidermal growth factor or beta-oestradiol. As in vivo, epidermal growth factor and beta-oestradiol induced bile duct epithelial cell proliferation accompanied by PF activation; however, beta-oestradiol also triggers sinusoidal cell proliferation. We demonstrated that treatments usually used in vivo to induce liver fibrosis allow, in cultured PCLS, the specific activation of the two main liver fibrogenic cell subpopulations, making this model very useful to study the mechanisms involved in early fibrogenic cell activation.

Topics: Acetaminophen; Acetylcysteine; Animal Use Alternatives; Animals; Antioxidants; Bile Ducts, Intrahepatic; Cell Survival; Disease Models, Animal; Drug Antagonism; Epidermal Growth Factor; Estradiol; Fibroblasts; Hepatocytes; Kupffer Cells; Liver; Liver Cirrhosis; Male; Necrosis; Organ Culture Techniques; Portal System; Rats; Rats, Wistar; Thioacetamide

Pretreatment with a low dose of bacterial endotoxin (lipopolysaccharide, LPS) caused the reduction of cytochrome P450 (CYP) enzymes and inflammatory factors which are capable of protecting the liver from a lethal LPS challenge. However, the effects of LPS pretreatment on the expression of transforming growth factor beta1 (TGFbeta1) and leptin in thioacetamide (TAA)-induced liver fibrosis remain unknown. In this study, Sprague-Dawley rats were pretreated intraperitoneally with LPS (5 mg/kg body weight) for 24 h, and subsequently treated with TAA (200 mg/kg body weight/ 3 days) for 1 month to examine the effects of LPS on TAA-injured rats. LPS pretreatment was associated with lower granulation and lower (P < 0.05) GOT/GPT than in TAA-injured rats. The LPS-pretreated group had less collagen (Sirius red histochemical staining). Semiquantitative RT-PCR showed that the levels of collagen 3 and TGFbeta1 mRNAs were lower (P < 0.05) in the liver of LPS-pretreated rats than in TAA-injured rats. TGFbetaRI mRNA in the liver of LPS-pretreated rats exceeded (P < 0.05) that in TAA-injured rats. LPS pretreatment reduced the leptin content (Western blot) below that of TAA-injured rats. These results imply that LPS pretreatment (endotoxin tolerance) alleviates the TAA-induced liver fibrosis of rats by reducing TGFbeta1 and leptin content.

Topics: Animals; Collagen Type III; Down-Regulation; Histocytochemistry; Immunohistochemistry; Leptin; Lipopolysaccharides; Liver Cirrhosis; Male; Rats; Rats, Sprague-Dawley; RNA, Messenger; Thioacetamide; Transforming Growth Factor beta1

Toona sinensis Roem (TS) leaf tea as a health food for the improvement of blood sugar and hypertension has been demonstrated. Thioacetamide (TAA), a hepatotoxin, causes the progression of liver fibrosis. In this study, we tested the effects of TS leaf on TAA-induced liver injury. TAA (200mg/kg Bwt/3 days, i.p.) treated rats were orally administrated with TS leaf extract (1g/kg Bwt/10 days) three times. After 30 days treatment, the morphological data showed that TS leaf extract given to TAA-treated rats had less liver fibrosis. The GOT/GPT, collagen 1 and collagen 3 mRNAs of livers in TAA-treated rats were elevated when compared to normal rats. The improvements of GOT/GPT, collagen 1 and collagen 3 mRNAs were shown in the TS leaf extract given to TAA-treated rats. TS leaf extract given to TAA-treated rats showed higher levels of cytochrome P450 (1A1, 2A and reductase) than those of TAA-treated rats. Compared to the TAA-treated group, TGFbeta1 mRNA (RT-PCR) was decreased with an increase of TGFbetaR1 protein (western blot) in the TS leaf extract given to TAA-treated rats. The decreased tendency of FGFR2 was found in the TS leaf extract given to TAA-treated rats. The result implies that TS leaf possesses beneficial effects on liver injury through increments of detoxification and the metabolic pathway.

Topics: Animals; Collagen; Cytochrome P-450 Enzyme System; Gene Expression Regulation; Liver; Liver Cirrhosis; Male; Meliaceae; Plant Extracts; Plant Leaves; Rats; Rats, Sprague-Dawley; RNA, Messenger; Thioacetamide; Transforming Growth Factor beta1

We investigated whether endothelial progenitor cell (EPC) transplantation could reduce established liver fibrosis and promote hepatic regeneration by isolating rat EPCs from bone marrow cells.. Recipient rats were injected intraperitoneally with carbon tetrachloride (CCl(4)) twice weekly for 6 weeks before initial administration of EPCs. CCl(4) was then readministered twice weekly for 4 more weeks, and EPC transplantation was carried out for these same 4 weeks.. At 7 days in culture, the cells expressed Thy-1, CD31, CD133, Flt-1, Flk-1, and Tie-2, suggesting an immature endothelial lineage. Immunohistochemical analyses showed fluorescent-labeled, transplantation EPCs were incorporated into the portal tracts and fibrous septa. Single and multiple EPC transplantation rats had reduced liver fibrosis, with decreased alpha2-(I)-procollagen, fibronectin, transforming growth factor-beta, and alpha-smooth muscle actin-positive cells. Film in situ zymographic analysis revealed strong gelatinolytic activity in the periportal area, in accordance with EPC location. Real-time polymerase chain reaction analysis of multiple EPC-transplantation livers showed significantly increased messenger RNA levels of matrix metalloproteinase (MMP)-2, -9 and -13, whereas tissue inhibitor of metalloproteinase-1 expression was significantly reduced. Expression of hepatocyte growth factor, transforming growth factor-alpha, epidermal growth factor, and vascular endothelial growth factor was increased in multiple EPC-transplantation livers, while hepatocyte proliferation increased. Transaminase, total bilirubin, total protein, and albumin levels were maintained in EPC-transplantation rats, significantly improving survival rates.. We conclude that single or repeated EPC transplantation halts established liver fibrosis in rats by suppressing activated hepatic stellate cells, increasing matrix metalloproteinase activity, and regulating hepatocyte proliferation.

Topics: Animals; Bone Marrow Cells; Carbon Tetrachloride; Cell Division; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Graft Survival; Hepatocytes; In Situ Hybridization, Fluorescence; Liver Cirrhosis; Liver Regeneration; Male; Matrix Metalloproteinases; Rats; Rats, Wistar; RNA, Messenger; Stem Cell Transplantation; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1; Up-Regulation

Selenium reduction in cirrhosis is frequently reported. The known beneficial effect of selenium supplementation on cirrhosis is probably obtained from nutritionally selenium-deficient subjects. Whether selenium supplementation truly improves cirrhosis in general needs additional experimental investigation. Thioacetamide was used to induce cirrhosis in selenium-adequate and -deficient mice. Selenoenzyme activity and selenium content were measured and the influence of selenium supplementation was evaluated. In Se-adequate mice, thioacetamide-mediated rapid onset of hepatic oxidative stress resulted in an increase in thioredoxin reductase activity and a decrease in both glutathione peroxidase activity and selenium content. The inverse activity of selenoenzymes (i.e. TrxR activity goes up and GPx activity goes down) was persistent and mute to selenium supplementation during the progress of cirrhosis; accordingly, cirrhosis was not improved by selenium supplementation in any period. On the other hand, selenium supplementation to selenium-deficient mice always more efficiently increased hepatic glutathione peroxidase activity and selenium content compared with those treated with thioacetamide, indicating that thioacetamide impairs the liver bioavailability of selenium. Although thioacetamide profoundly affects hepatic selenium status in selenium-adequate mice, selenium supplementation does not modify the changes. Selenium supplementation to cirrhotic subjects with a background of nutritional selenium deficiency can improve selenium status but cannot restore hepatic glutathione peroxidase and selenium to normal levels.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Biological Availability; Diet; Dietary Supplements; Glutathione Peroxidase; Liver; Liver Cirrhosis; Male; Mice; Oxidative Stress; Selenium; Thioacetamide; Thioredoxin-Disulfide Reductase; Thioredoxins

Efficacy of a herbal product of E. officinalis (fruit) (EO) has been evaluated against carbon tetrachloride (CCl4) and thioacetamide (TAA) induced changes in rat liver. Chronic treatment of CCl4 and TAA revealed abnormal histopathology indicative of pre-fibrogenic events. EO reversed such alterations with significant regenerative changes suggestive of its preventive role in prefibrogenesis of liver.

Topics: Animals; Carbon Tetrachloride; Carcinogens; Drug Evaluation, Preclinical; Liver; Liver Cirrhosis; Phyllanthus emblica; Plant Extracts; Rats; Rats, Wistar; Thioacetamide; Toxins, Biological

To examine the differences in the responses of normal and cirrhotic livers to partial hepatectomy in relation to the factors influencing liver regeneration.. Cirrhosis was induced in rats by administration of thioacetamide. Untreated rats were used as controls. The control rats as well as the cirrhotic rats were subjected to 70% partial hepatectomy. At different time points after hepatectomy, the livers were collected and the levels of cytokines, growth factors and cell cycle proteins were analyzed.. After hepatectomy, the cirrhotic remnant expressed significantly lower levels of cyclin D1, its kinase partner, cdk4, and cyclin E as compared to the controls up to 72 h post hepatectomy. Significantly lower levels of cyclin A and cdk2 were also observed while the cdk inhibitor, p27 was significantly higher. In addition, the cirrhotic group had lower IL-6 levels than the control group at all time points up to 72 h following resection.. The data from our study shows that impaired liver regeneration in cirrhotic remnants is associated with low expression of cyclins and cdks. This might be the consequence of the low IL-6 levels in cirrhotic liver remnant which would in turn influence the actions of transcription factors that regulate genes involved in cell proliferation and metabolic homeostasis during the regeneration process.

Topics: Animals; Blotting, Western; Cell Cycle Proteins; Cell Proliferation; Cytokines; Gene Expression Regulation; Growth Substances; Hepatectomy; Homeostasis; Liver; Liver Cirrhosis; Liver Regeneration; Male; Rats; Rats, Inbred WF; Reverse Transcriptase Polymerase Chain Reaction; Thioacetamide

Cirrhotic animal models are vital to investigate complications of chronic liver disease. We chronologically characterized the effect of thioacetamide, administrated orally and adapted weekly to weight changes, focusing on the optimal moment to obtain all typical features of portal hypertension and cirrhosis.. Male Wistar rats, 200-250 g, were intoxicated for 6, 12 or 18 weeks (n = 8 per group), respectively, and compared with age-matched controls (n = 4 per group). An in-situ perfusion model was used to evaluate intrahepatic resistance and endothelial function. Splanchnic blood flow and portosystemic shunting were assessed by a perivascular flow probe.. Rats intoxicated for 6 or 12 weeks had no mortality and histologically showed hepatitis and advanced fibrosis, respectively. At 18 weeks, mortality was 16% (on a total of 56 animals) and only at that moment all animals showed homogenous macronodular cirrhosis with signs of high-grade hepatocellular dysplasia. Portal hypertension was present at 12 weeks (11 +/- 0.4 vs. 5.9 +/- 0.4 mmHg, P < 0.001), but was not associated with the hyperdynamic state until 18 weeks (12.1 +/- 0.8 vs. 5.6 +/- 0.5 mmHg, P < 0.001). At this latter time-point, we also observed increased intrahepatic resistance associated with endothelial dysfunction, hyperresponsiveness to vasoconstrictors, splanchnic hyperaemia and portosystemic shunting. These alterations were associated with increased systemic levels of nitrate/nitrite and thromboxane A(2).. Thioacetamide, adapted to weekly weight changes, leads to a homogenous, reproducible model of cirrhosis in the rat in 18 weeks, which is associated with all the typical characteristics of portal hypertension, including endothelial dysfunction.

Topics: Administration, Oral; Analysis of Variance; Animals; Carcinogens; Hemodynamics; Hypertension, Portal; Liver; Liver Cirrhosis; Male; Models, Animal; Nitrates; Nitric Oxide Synthase; Rats; Rats, Wistar; Thioacetamide; Thromboxane B2

Exenatide is a 39 amino acid incretin mimetic for the treatment of type 2 diabetes, with glucoregulatory activity similar to glucagon-like peptide-1 (GLP-1). Exenatide is a poor substrate for the major route of GLP-1 degradation by dipeptidyl peptidase-IV, and displays enhanced pharmacokinetics and in vivo potency in rats relative to GLP-1. The kidney appears to be the major route of exenatide elimination in the rat. We further investigated the putative sites of exenatide degradation and excretion, and identified primary degradants. Plasma exenatide concentrations were elevated and sustained in renal-ligated rats, when compared to sham-operated controls. By contrast, exenatide elimination and degradation was not affected in rat models of hepatic dysfunction. In vitro, four primary cleavage sites after amino acids (AA)-15, -21, -22 and -34 were identified when exenatide was degraded by mouse kidney membranes. The primary cleavage sites of exenatide degradation by rat kidney membranes were after AA-14, -15, -21, and -22. In rabbit, monkey, and human, the primary cleavage sites were after AA-21 and -22. Exenatide was almost completely degraded into peptide fragments <3 AA by the kidney membranes of the species tested. The rates of exenatide degradation by rabbit, monkey and human kidney membranes in vitro were at least 15-fold slower than mouse and rat membranes. Exenatide (1-14), (1-15), (1-22), and (23-39) were not active as either agonists or antagonists to exenatide in vitro. Exenatide (15-39) and (16-39) had moderate-to-weak antagonist activity compared with the known antagonist, exenatide (9-39). In conclusion, the kidney appears to be the primary route of elimination and degradation of exenatide.

Topics: Animals; Chemical and Drug Induced Liver Injury; Chromatography, Liquid; Exenatide; Galactosamine; In Vitro Techniques; Kidney; Liver Cirrhosis; Male; Mass Spectrometry; Mice; Mice, Inbred Strains; Peptides; Rats; Rats, Sprague-Dawley; Thioacetamide; Venoms

The induction of liver fibrosis is difficult in mice. Here, we intended to improve fibrosis induction by combination of thioacetamide (TAA) injections and ethanol (EtOH) feeding and to characterize features of liver damage in this model. Most experimental therapeutic studies are performed in mice without pre-damaged livers.. C3H mice were injected three times/week (0.15 mg/g body weight) and fed with EtOH. Tissue and serum samples were collected and analysed.. Portal fibrosis was verified by van Gieson staining showing a mild fibrosis (score F2) in TAA-treated mice and liver fibrosis (score F4) in the combination group using TAA/EtOH. Consonant with the histological results, the fibrosis marker MMP-2 and alpha 1 procollagen (I) were elevated at week 10 and 15 after treatment initiation in the combination group, whereas tissue protective proteinase, TIMP-1, was 18.5-fold increased only at week 10 but normalized until week 15. Fibrosis development was associated with elevated ICAM-1 expression.. Taken together, TAA/EtOH application was suitable to induce liver fibrosis characterized by typical bio-markers in C3H/He.

Topics: Animals; Biomarkers; Disease Models, Animal; Ethanol; Gene Expression Regulation; Intercellular Adhesion Molecule-1; Liver; Liver Cirrhosis; Matrix Metalloproteinase 2; Mice; Mice, Inbred C3H; Procollagen; Thioacetamide; Tissue Inhibitor of Metalloproteinase-1; Up-Regulation; Vascular Endothelial Growth Factor A

1. Adenosine is a potent endogenous regulator of inflammation and tissue repair. Adenosine, which is released from injured and hypoxic tissue or in response to toxins and medications, may induce pulmonary fibrosis in mice, presumably via interaction with a specific adenosine receptor. We therefore determined whether adenosine and its receptors contribute to the pathogenesis of hepatic fibrosis. 2. As in other tissues and cell types, adenosine is released in vitro in response to the fibrogenic stimuli ethanol (40 mg dl(-1)) and methotrexate (100 nM). 3. Adenosine A(2A) receptors are expressed on rat and human hepatic stellate cell lines and adenosine A(2A) receptor occupancy promotes collagen production by these cells. Liver sections from mice treated with the hepatotoxins carbon tetrachloride (CCl(4)) (0.05 ml in oil, 50 : 50 v : v, subcutaneously) and thioacetamide (100 mg kg(-1) in PBS, intraperitoneally) released more adenosine than those from untreated mice when cultured ex vivo. 4. Adenosine A(2A) receptor-deficient, but not wild-type or A(3) receptor-deficient, mice are protected from development of hepatic fibrosis following CCl(4) or thioacetamide exposure. 5. Similarly, caffeine (50 mg kg(-1) day(-1), po), a nonselective adenosine receptor antagonist, and ZM241385 (25 mg kg(-1) bid), a more selective antagonist of the adenosine A(2A) receptor, diminished hepatic fibrosis in wild-type mice exposed to either CCl(4) or thioacetamide. 6. These results demonstrate that hepatic adenosine A(2A) receptors play an active role in the pathogenesis of hepatic fibrosis, and suggest a novel therapeutic target in the treatment and prevention of hepatic cirrhosis.

Topics: Adenosine; Adenosine A2 Receptor Agonists; Animals; Blotting, Western; Caffeine; Carbon Tetrachloride; Cell Line; Chromatography, High Pressure Liquid; Ethanol; Hepatocytes; Humans; Liver Cirrhosis; Matrix Metalloproteinases, Membrane-Associated; Methotrexate; Mice; Mice, Inbred C57BL; Rats; Receptor, Adenosine A2A; Reverse Transcriptase Polymerase Chain Reaction; Thioacetamide; Triazines; Triazoles

Liver fibrosis results from accumulation of extracellular matrix components and is associated with many chronic hepatic diseases. There is to date no specific therapy for this disease, and patients receive treatment for its associated complications. Specific progenitor cells, known as oval cells, are present in the liver. As oval cells express markers such as CD34, they are thought to arise from a hematopoietic precursor. The aim of this work was to investigate whether transplantation of hematopoietic CD34(+) stem cells could improve hepatic fibrosis by their differentiation into hepatocytes.. CD34(+) stem cells from human umbilical cord blood were purified, transduced with a lentiviral vector containing the green fluorescent protein (GFP) gene and injected via portal vein into rats with liver cirrhosis induced by the 4-month administration of thioacetamide. Rats were killed 15 and 60 days post-transplantation.. Up to 37% and 22% fluorescent cells were observed in the blood of control and cirrhotic rats, respectively, at 15 days post-transplantation. At 60 days post-transplantation, however, fluorescent cells were completely absent from the blood. Fluorescence was not detected in liver sections at either 15 or 60 days post-transplantation. Polymerase chain-reaction study to detect the GFP gene ruled out silencing of the transgene.. These results suggest that the transplanted cells did not engraft in the liver and were eliminated from the rats.

Topics: Animals; Antigens, CD34; Cell Line; Cell Separation; Cells, Cultured; Cord Blood Stem Cell Transplantation; Humans; Liver Cirrhosis; Male; Rats; Rats, Wistar; Stem Cell Transplantation; Thioacetamide; Transplantation, Heterologous

To investigate the effects of cyclosporine A (CsA) on thioacetamide (TAA)-induced liver injury.. CsA was co-administrated (7.5 microg/kg body weight per day, i.p.) into rat to investigate the role of CsA on TAA-(200 mg/kg body weight per 3 d for 30 d, i.p.)induced liver injury.. The data show that TAA caused liver fibrosis in rat after 30 d of treatment. CsA alleviates the morphological changes of TAA-induced fibrosis in rat liver. The blood glutamyl oxaloacetic transaminase (GOT)/glutamyl pyruvic transaminase (GPT) in the TAA-injury group is elevated compared to that of the normal rat. Compared with the TAA-injury group, the blood GOT/GPT and TGFbeta1 (by RT-PCR analysis) are reduced in the CsA plus TAA-treated rat. The level of the transforming growth factor receptor I (TGFbeta-R1) in the CsA plus TAA-treated group shows higher than that in the TAA only group, but shows a lower level of the fibroblast growth factor receptor 4 (FGFR4) in the CsA plus TAA-treated group, when using the Western blot analysis. After immunostaining of the frozen section, TGFbeta-R1 and FGFR4 are more concentrated in rat liver after CsA plus TAA injury.. This result suggests that CsA has an alleviated effect on TAA-induced liver injury by increasing the multidrug resistance P-glycoprotein and could be through the regulation of TGFbeta-R1 and FGFR4.

Topics: Activin Receptors, Type I; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Blotting, Western; Cyclosporine; Immunohistochemistry; Immunosuppressive Agents; Liver Cirrhosis; Male; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Rats; Receptor, Fibroblast Growth Factor, Type 4; Receptor, Transforming Growth Factor-beta Type I; Receptors, Fibroblast Growth Factor; Receptors, Transforming Growth Factor beta; Thioacetamide

To investigate the pharmacological effects of rice flavone (5,4'-dihydroxy-3',5'-dimethoxy-7-O-beta-D-glucopyranosyloxy-flavone, RF) separated from panicle-differentiating to flowing rice on rat experimental hepatic injury.. Models of rat acute hepatic injury induced by carbon tetrachloride (CCl(4)) administration, rat hepatic fibrosis induced by thioacetamide, injury of primary cultured rat hepatocytes induced by CCl(4), respectively, were established. After treated with RF, content of serum alanine transaminase (ALT), aspartate aminotransferase (AST) and albumin (Alb), hyaluronic acid (HA), the activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and hydroxyproline (Hyp) were measured and liver tissue was observed pathologically by hematoxylin-eosin (HE) staining. Effects of RF on pathological changes, function index, enzyme of scavenging free radicals and blood rheology were evaluated.. In model of rat acute hepatic injury induced by CCl(4), RF can significantly decrease the contents of serum ALT, AST, increase the content of Alb, improve the dropsy and fat denaturalization of hepatocytes. In model of rat hepatic fibrosis induced by thioacetamide, RF can inhibit the increase of HA, Hyp and whole blood viscosity, and improve the activities of GSH-Px and SOD, and inauricular microcirculation.. RF has apparent protective effects on hepatic injury by increasing activity of GSH-Px and SOD, scavenging free radicals produced by CCl(4), reducing blood viscosity, and improving microcirculation and blood supply.

Topics: Acute Disease; Animals; Carbon Tetrachloride; Cells, Cultured; Disease Models, Animal; Flavones; Hepatocytes; Liver Cirrhosis; Male; Oryza; Phytotherapy; Plant Extracts; Rats; Rats, Sprague-Dawley; Thioacetamide

In healthy rats, we recently showed that reduced intrahepatic portal blood flow leads to activation of hepatic adenosine receptors and a nerve-induced decrease in urine production. We hypothesize that the impaired urine excretion in liver cirrhosis is related to an increase in intrahepatic adenosine.. Anesthetized normal and thioacetamide-induced cirrhotic rats were instrumented for the measurement of urine flow, hepatic portal venous blood flow, and renal arterial blood flow. 8-Phenyltheophylline was used to block adenosine receptors.. Compared to normal rats, cirrhotic rats had a lower baseline urine flow (P<0.05). In both normal and cirrhotic rats, intraportal but not intravenous administration of 8-phenyltheophylline increased urine flow. Saline overload in normal rats increased urine flow (from 6.8+/-0.6 to 42.2+/-4.6 microlmin(-1)) and this ability was impaired in cirrhotic rats (from 3.9+/-0.4 to 6.2+/-0.9 microlmin(-1)). Intraportal, but not intravenous, administration of 8-phenyltheophylline partially restored the renal ability to excrete the saline load.. Impaired renal ability to excrete urine in liver cirrhosis is related to the activation of intrahepatic adenosine receptors, and this is consistent with our previous data showing renal regulation through a hepatorenal neural mechanism activated by intrahepatic adenosine.

Topics: Adenosine; Animals; Female; Injections, Intravenous; Kidney; Liver; Liver Circulation; Liver Cirrhosis; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Sodium; Theophylline; Thioacetamide; Urine

A hydroalcoholic (50%) extract of Emblica officinalis (fruit) (EO-50) reduced the severity of hepatic fibrosis induced by carbon tetrachloride (CCl4) and thioacetamide (TAA). Improved liver function was observed by measuring the levels of aspartate aminotransaminase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and bilirubin in serum. Hepatic parameters monitored were the levels of glutathione (GSH), lipid peroxidation (LPO) and hydroxyproline and the activities of catalase, glutathione peroxidase (GPx), Na+,K+-ATPase and cytochrome P450 (CYP 450 2E1) (aniline hydroxylation). The results suggested that EO-50 effectively reversed profibrogenic events possibly due to its promising antioxidative activity.

Topics: Alanine Transaminase; Alkaline Phosphatase; Animals; Aspartate Aminotransferases; Carbon Tetrachloride; Catalase; Cytochrome P-450 CYP2E1; Glutathione; Glutathione Peroxidase; Lipid Peroxidation; Liver; Liver Cirrhosis; Male; Phyllanthus emblica; Plant Extracts; Rats; Rats, Wistar; Sodium-Potassium-Exchanging ATPase; Thioacetamide

The mechanisms responsible for impaired regenerative ability after hepatic resection observed in chronic liver disease are not fully understood. We have examined the relationships between an altered expression of cell cycle-related proteins in regenerating liver after partial hepatectomy and the impaired regenerative process observed in fibrotic and cirrhotic rats.. We performed 70% partial hepatectomy in both control and porcine serum-induced fibrotic rats, and 45% partial hepatectomy in thioacetamide-induced cirrhotic rats because of the high mortality associated with 70% partial hepatectomy. Liver regeneration was monitored by proliferating cell nuclear antigen labeling index and the expression of G1 regulatory cell cycle-related proteins was determined by immunoblot analysis.. Compared with controls, hepatocyte DNA synthesis, and induction of cyclin D1 and p21(CIP1) proteins were delayed but not suppressed in porcine serum-induced fibrotic rats and markedly inhibited in thioacetamide-induced cirrhotic rats. p27(KIP1) protein levels were unaffected by partial hepatectomy and did not differ among all three groups.. Two distinct rat models of liver fibrosis and cirrhosis showed markedly different proliferative responses after partial hepatectomy. The delay or failure of cyclin D1 induction, but not the increase of p21(CIP1) or p27(KIP1) might be responsible for their impaired liver regeneration.

Topics: Animals; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; DNA; Hepatectomy; Liver; Liver Cirrhosis; Liver Regeneration; Male; Proliferating Cell Nuclear Antigen; Rats; Rats, Inbred F344; Serum; Thioacetamide

The liver plays a central role in insulin-like growth factor (IGF) homeostasis providing the majority of circulating IGF-I and some of its binding proteins (IGFBPs). In liver cirrhosis the IGF axis is severely disturbed, and these alterations are associated with reduced IGF-I, IGFBP-3 but elevated IGFBP-1 serum levels.. By Northern blotting and in situ hybridization (ISH), hepatic expression of IGF-I and of IGFBP was studied in a rat model of liver cirrhosis induced by thioacetamide.. ISH revealed a homogeneous distribution of IGFBP-1, IGFBP-4 and IGF-I mRNA over hepatic parenchyma in normal and cirrhotic liver. Fibrous septa of cirrhotic liver were IGFBP-1 mRNA negative, whereas IGFBP-4 and IGF-I transcripts were detected in single cells. In normal liver, IGFBP-3 mRNA was distributed within nonparenchymal cells of the hepatic lobule and in the wall of the portal vein. In cirrhotic liver, IGFBP-3 transcripts were abundant in mesenchymal cells of fibrous tissue. IGFBP-3 mRNA expression was also prominent in cells at the septal-nodular interface most likely representing monocyte infiltration. IGFBP-3 mRNA expression was reduced in nonparenchymal liver cells located more distantly from the septal-nodular interface in the cirrhotic nodule that correlated with reduced IGFBP-3 mRNA expression observed in Kupffer cells (KC) and sinusoidal endothelial cells (SEC) isolated from macronodular cirrhotic livers.. Cirrhosis is accompanied by an altered spatial expression of IGFBP-3 in liver tissue, which is characterized by decreased levels of IGFBP-3 mRNA in KC and SEC, but elevated IGFBP-3 expression in myofibroblast-like cells and inflammatory infiltrate.

Topics: Animals; Blotting, Northern; Endothelial Cells; Female; Hepatocytes; In Situ Hybridization; Insulin-Like Growth Factor Binding Protein 1; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor Binding Protein 4; Insulin-Like Growth Factor I; Kupffer Cells; Liver Cirrhosis; Rats; Rats, Wistar; RNA, Messenger; Thioacetamide

Hepatic fibrosis represents a process of healing and scarring in response to chronic liver injury. Interleukin-10 (IL-10) is a cytokine that downregulates the proinflammatory response and has a modulatory effect on hepatic fibrogenesis. The aim of this study was to investigate whether IL-10 gene therapy possesses anti-hepatic fibrogenesis in mice. Liver fibrosis was induced by long-term thioacetamide administration in mice. Human IL-10 expression plasmid was delivered via electroporation after liver fibrosis established. IL-10 gene therapy reversed hepatic fibrosis and prevented cell apoptosis in a thioacetamide-treated liver. RT-PCR revealed IL-10 gene therapy to reduce liver transforming growth factor-beta1, tumor necrosis factor-alpha, collagen alpha1, cell adhesion molecule, and tissue inhibitors of metalloproteinase mRNA upregulation. Following gene transfer, the activation of alpha-smooth muscle actin and cyclooxygenase-2 was significantly attenuated. In brief, IL-10 gene therapy might be an effective therapeutic reagent for liver fibrosis with potential future clinical applications.

Topics: Animals; Base Sequence; Blotting, Western; Cyclooxygenase 2; DNA Primers; Genetic Therapy; Immunohistochemistry; In Situ Nick-End Labeling; Interleukin-10; Liver Cirrhosis; Male; Mice; Mice, Inbred ICR; Prostaglandin-Endoperoxide Synthases; Reverse Transcriptase Polymerase Chain Reaction; Thioacetamide

Common bile duct ligation (CBDL) triggers a molecular cascade resulting in the hepatopulmonary syndrome (HPS). Both increased hepatic endothelin-1 (ET-1) production and pulmonary vascular ET(B) receptor expression with stimulation of endothelial nitric oxide synthase and TNF-alpha mediated inducible nitric oxide synthase and heme oxygenase-1 expression in pulmonary intravascular macrophages occur. Whether biliary cirrhosis is unique in triggering ET-1 and TNF-alpha alterations and HPS is unknown. We evaluated for HPS in rat prehepatic portal hypertension [partial portal vein ligation (PVL)], biliary (CBDL) and nonbiliary [thioacetamide treatment (TAA)] cirrhosis, and assessed ET-1 infusion in normal and PVL animals. Control, PVL, CBDL, TAA-treated, and ET-1-infused PVL animals had ET-1 and TNF-alpha levels measured and underwent molecular and physiological evaluation for HPS. HPS developed only in biliary cirrhosis in association with increased plasma ET-1 and TNF-alpha levels and the development of established molecular changes in the pulmonary microvasculature. In contrast, PVL did not increase ET-1 or TNF-alpha levels and TAA treatment increased TNF-alpha levels alone, and neither resulted in the full development of molecular or physiological changes of HPS despite portal pressure increases similar to those after CBDL. Exogenous ET-1 increased TNF-alpha levels and triggered HPS after PVL. Combination of ET-1 and TNF-alpha overproduction is unique to biliary cirrhosis and associated with experimental HPS. ET-1 infusion increases TNF-alpha levels and triggers HPS in prehepatic portal hypertension. ET-1 and TNF-alpha interact to trigger pulmonary microvascular changes in experimental HPS.

Topics: Animals; Endothelin-1; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hepatopulmonary Syndrome; Hypertension, Portal; Ligation; Liver Cirrhosis; Liver Cirrhosis, Biliary; Lung; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Portal Vein; Rats; Rats, Sprague-Dawley; Thioacetamide; Tumor Necrosis Factor-alpha

Oxidative stress is a major pathogenetic factor in hepatic fibrosis. Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor which is known to affect oxidative stress and PPARalpha ligands may have rescue effects on hepatic fibrosis. We tested this hypothesis using rat thioacetamide (TAA) models of liver cirrhosis. Rats were given intraperitoneal injection of TAA and treated with a diet containing one of the two PPARalpha ligands, Wy-14,643 (WY) or fenofibrate. WY treatment dramatically reduced hepatic fibrosis and also prevented the inhibition catalase of mRNA expression caused by TAA. Correspondingly, catalase activity increased in the TAA+WY group but decreased in the control TAA group. The antifibrotic action of fenofibrate in the TAA model was comparable with that of WY. PPARalpha ligands have an antifibrotic action in the rat TAA model of liver cirrhosis, probably due to an antioxidant effect of enhanced catalase expression and activity in the liver.

Topics: Animals; Antioxidants; Blotting, Northern; Catalase; Densitometry; Fenofibrate; Fibrosis; Hydrogen Peroxide; Ligands; Liver; Liver Cirrhosis; Male; Models, Biological; Oxidative Stress; Peroxisome Proliferators; PPAR alpha; Pyrimidines; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Superoxide Dismutase; Thioacetamide

Thioacetamide (TAA) administration is an established technique for generating rat models of liver fibrosis and cirrhosis. Oxidative stress is believed to be involved as TAA-induced liver fibrosis is initiated by thioacetamide S-oxide, which is derived from the biotransformation of TAA by the microsomal flavine-adenine dinucleotide (FAD)-containing monooxygense (FMO) and cytochrome P450 systems. A two-dimensional gel electrophoresis-mass spectrometry approach was applied to analyze the protein profiles of livers of rats administered with sublethal doses of TAA for 3, 6 and 10 weeks respectively. With this approach, 59 protein spots whose expression levels changed significantly upon TAA administration were identified, including three novel proteins. These proteins were then sorted according to their common biochemical properties and functions, so that pathways involved in the pathogenesis of rat liver fibrosis due to TAA-induced toxicity could be elucidated. As a result, it was found that TAA-administration down-regulated the enzymes of the primary metabolic pathways such as fatty acid beta-oxidation, branched chain amino acids and methionine breakdown. This phenomenon is suggestive of the depletion of succinyl-CoA which affects heme and iron metabolism. Up-regulated proteins, on the other hand, are related to oxidative stress and lipid peroxidation. Finally, these proteomics data and the data obtained from the scientific literature were integrated into an "overview model" for TAA-induced liver cirrhosis. This model could now serve as a useful resource for researchers working in the same area.

Topics: Animals; Down-Regulation; Fatty Acids; Ferritins; Fibrosis; Glutathione; Heme; Humans; Image Processing, Computer-Assisted; Iron; Lipid Peroxidation; Liver; Liver Cirrhosis; Methionine; Oxidative Stress; Oxygenases; Proteomics; Rats; Rats, Wistar; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thioacetamide; Time Factors; Trypsin; 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

During hepatic fibrogenesis, the hepatic extracellular matrix changes to fibrillar collagens types I and III, and cirrhosis is believed to produce an irreversible scar. In this study, we investigated whether gene delivery of human matrix metalloproteinase-1, which degrades collagens types I and type III, would attenuate established hepatic fibrosis in the rat, induced by either thioacetamide or bile duct ligation.. Hepatic fibrosis induced by thioacetamide for 7 weeks was persistent for at least 2 months, even after discontinuation of the treatment. The rats were infected once with a recombinant adenovirus, Ad5MMP-1, into which human pro-human matrix metalloproteinase-1 complementary DNA was packaged, or with a control adenovirus, Ad5LacZ.. In Ad5MMP-1-infected, but not in Ad5LacZ-infected, rats, the fibrosis was dramatically attenuated at 2 weeks after the infection. It is interesting to note that the number of activated hepatic stellate cells was also decreased in Ad5MMP-1-infected rats. Moreover, disorganization of the hepatic trabecula, heterogeneity in the size of hepatocytes, and increased dried liver weight were observed only in Ad5MMP-1-treated rats, suggesting that human matrix metalloproteinase-1 stimulated hepatocyte proliferation, which was confirmed by bromodeoxyuridine staining. After 4 weeks, the proliferative effect of human matrix metalloproteinase-1 almost disappeared, but the hepatic fibrosis remained attenuated, whereas the fibrosis in Ad5LacZ-treated rats persisted. Furthermore, the administration of Ad5MMP-1, but not Ad5LacZ, decreased type I collagen and generated a small collagen fragment in hepatic fibrosis induced by bile duct ligation.. Our findings show that transient human matrix metalloproteinase-1 overexpression in the liver effectively attenuates established fibrosis and induces hepatocyte proliferation.

Topics: Adenoviridae; Animals; Cell Division; Genetic Therapy; Genetic Vectors; Hepatocytes; Humans; Liver; Liver Cirrhosis; Male; Matrix Metalloproteinase 1; Prodrugs; Rats; Rats, Sprague-Dawley; Recombination, Genetic; Thioacetamide; Time Factors

Endothelin (ET)-1 contributes to hepatic ischemia and reperfusion (HIR) injury in normal liver. This study was conducted to clarify the role of ET-1 in HIR injury in cirrhotic state.. Using thioacetamide-induced cirrhotic rats with spontaneous portosystemic shunt, we determined the changes in plasma aspartate aminotransferase (AST) levels, plasma and hepatic ET-1 values, 7-day survival rates, and hepatic oxygen saturation (SO(2)) by time-resolved spectroscopy as an indicator of hepatic microcirculation under intermittent or continuous total hepatic ischemia with subsequent partial hepatectomy.. Hepatic ET-1 levels in cirrhotic rats were significantly higher than those in noncirrhotic rats. Plasma and hepatic ET-1 levels at 1, 3 and 6 h of reperfusion after intermittent hepatic ischemia were significantly lower than those after continuous hepatic ischemia. In cirrhotic animals subjected to intermittent hepatic ischemia, the elevation of plasma AST levels at 1, 3 and 6 h of reperfusion and the decline in hepatic SO(2) at the end of 60-min hepatic ischemia and after reperfusion were significantly suppressed when compared with those subjected to continuous hepatic ischemia. Pretreatment with a nonselective endothelin receptor antagonist in continuous hepatic ischemia significantly ameliorated plasma AST levels and hepatic SO(2) values with less hepatic sinusoidal congestion, resulting in an improvement in the 7-day survival rate.. Continuous hepatic ischemia in the cirrhotic liver has disadvantages relating to microcirculatory derangement with more ET-1 production in partial hepatectomy. In liver surgery, pharmacological regulation of ET-1 production may lead to attenuation of reperfusion injuries for ischemically damaged cirrhotic liver.

Topics: Animals; Aspartate Aminotransferases; Endothelin-1; Hepatectomy; Liver; Liver Cirrhosis; Male; Microcirculation; Oxygen; Portasystemic Shunt, Surgical; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Survival Rate; Thioacetamide

Hepatic fibrosis involves excess deposition of extracellular connective tissue of which collagen type I fibers form the predominant component. Left untreated it develops into cirrhosis, often linked with hepatocellular carcinoma. Owing to the fact that cirrhotic liver regeneration is impaired, resection of hepatocellular carcinoma associated with cirrhosis is questionable. The aim of the present study was to determine the potential of halofuginone, a collagen type I inhibitor, in improving liver regeneration in cirrhotic rats.. Partial hepatectomy (70%) was performed in thioacetamide-induced cirrhotic rats fed a halofuginone-containing diet. Liver regeneration was monitored by mass and proliferating cell nuclear antigen. The Ishak staging system and hydroxyproline content were used to evaluate the level of fibrosis.. Halofuginone administered prior to and following partial hepatectomy did not inhibit normal liver regeneration despite the reduced levels of collagen type I mRNA. When given to rats with established fibrosis, it caused a significant reduction in alpha smooth muscle actin, TIMP-2, collagen type I gene expression and collagen deposition. Such animals demonstrated improved capacity for regeneration.. Halofuginone may prove useful in improving survival of patients with hepatocellular carcinoma and cirrhosis undergoing surgical resection.

Topics: Animals; Collagen Type I; Extracellular Matrix; Liver Cirrhosis; Liver Regeneration; Male; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Rats; Rats, Sprague-Dawley; Thioacetamide

Dietary nucleotides reportedly promote functionality and repair in fibrotic liver. Liver fibrosis is characterized by an excessive accumulation of extracellular matrix components, which lead to the impairment of the hepatic function. The aim of this work was to evaluate the influence of dietary nucleotides on liver fibrosis induced by thioacetamide and to elucidate the mechanism by which nucleotides exert their protective effects. Rats consumed ad libitum 300 mg/L thioacetamide in drinking water and were pair-fed diets with (group TN) or without nucleotides (group TS) for 4 mo. Liver histology and extracellular matrix components, liver collagenase and prolyl 4-hydroxylase activities, and tissue inhibitor of metalloproteinases-1 were assessed. The degree of fibrosis was lower in group TN than in group TS. Group TN had lower hepatic concentration of hydroxyproline (P < 0.05), collagen type I (P = 0.12) and type III (P = 0.20), fibronectin (P = 0.05), laminin (P = 0.11) and desmin (P = 0.07), higher collagenolytic activity (P < 0.05), lower prolyl 4-hydroxylase activity (P < 0.05) and lower prolyl 4-hydroxylase (P = 0.10) and tissue inhibitor of metalloproteinase-1 (P = 0.06) expression than group TS. Moreover, expression of tissue inhibitor of the metalloproteinases-1 gene was lower in group TN than in group TS (P < 0.05). These data indicate that the reduction of liver fibrosis in nucleotide-supplemented rats may rely on the enhancement of collagenase activity and the reduction of collagen content and maturation.

Topics: Animals; Collagenases; Female; Liver Cirrhosis; Nucleotides; Rats; Rats, Wistar; Thioacetamide

Repeated administration of thioacetamide (TA), either intraperitoneally or in drinking water, produced liver cirrhosis in normal Sprague-Dawley rats (SDR) with significant histological alterations similar to those observed in human cirrhosis. In the present study, we evaluated the ability of TA to induce liver cirrhosis in mutant Nagase analbuminaemic SDR. Thioacetamide was administered either intraperitoneally up to 4 months or in drinking water up to 6 months to normal and to Nagase analbuminaemic SDR. Nagase analbuminaemic rats (NAR) were also administered TA in drinking water up to 10 months. Liver cirrhosis development was determined by macroscopic and microscopic analysis. In contrast to normal SDR, no histological characteristics of cirrhosis could be observed in NAR submitted to a 4 or 6 months treatment with TA. Such failure to induce cirrhosis in Nagase rats was confirmed even after prolonged TA administration in drinking water for up to 10 months. In contrast, fibrosis and cholangiolar proliferation occurred in the 10-month TA-treated analbuminaemic rats, suggesting that the mechanisms involved in cirrhosis induction are different from those involved in fibrosis development and carcinogenesis. It is unlikely that the protective effect against TA-induced cirrhosis observed in analbuminaemic rats is related to the absence of albumin in this rat strain, since a co-administration of TA with albumin in analbuminaemic rats did not restore the potential for TA to induce cirrhosis in this rat strain. In conclusion, the fact that induction of cirrhosis by TA is prevented in the inherently hyperlipidaemic and hypercholesterolaemic analbuminaemic rats could be considered for potential application in the treatment of clinical cirrhosis.

Topics: Administration, Oral; Animals; Bile Ducts, Intrahepatic; Cell Division; Cholangitis; Disease Models, Animal; Hypercholesterolemia; Injections, Intraperitoneal; Liver Cirrhosis; Rats; Rats, Mutant Strains; Rats, Sprague-Dawley; Serum Albumin; Thioacetamide; Water Supply

Hepatotoxin-induced rat models of liver cirrhosis are limited by the wide heterogeneity of cirrhosis produced. The present study developed a modified, reliable, and reproducible technique by which hepatic and systemic responses to thioacetamide during induction of cirrhosis were monitored by weekly weight changes.. Male Wistar rats (200-230 g) were divided into three groups. Group 1 (n=20) received continuous administration of 0.03% (w/v) thioacetamide in the drinking water for 12 weeks. Group 2 (n=20) received the same concentration of 0.03% thioacetamide as an initial concentration that was modified according to weekly weight changes in response to thioacetamide during the induction of cirrhosis. Group 3 (n=6) received normal water and served as controls.. Mortality of Group 1 was 30% and the production of cirrhosis was only 45%. In contrast, there were no deaths in Group 2 and well-developed macronodular cirrhosis was found in 90% of the rats which was associated with significant portal hypertension, as indicated by increased portal venous pressure (13.6+/-0.4 vs. 9.1+/-0.3 mmHg, cirrhotic vs. control, respectively, P<0.01, Student's unpaired t-test).. Variations in responses to thioacetamide can be easily monitored by weekly weight changes to reduce mortality to zero and simultaneously increase the production and quality of cirrhosis induced in rats.

Topics: Animals; Body Weight; Disease Models, Animal; Hypertension, Portal; Liver; Liver Cirrhosis; Male; Rats; Rats, Wistar; Reproducibility of Results; Splenomegaly; Thioacetamide

The aim of our study was to investigate the effect of carbon dioxide pneumoperitoneum on systemic and splanchnic hemodynamics in cirrhotic rats.. Sprague-Dawley rats (n = 80) were used in this study. Liver cirrhosis was induced by thioacetamide administration intraperitoneally (200 mg/kg body weight, twice a week for 16 weeks). The radioactive microsphere method was used to measure systemic and regional hemodynamic parameters before, 1 h after the start, and 1 h after the release of pneumoperitoneum.. Splanchnic blood flow and cardiac index were significantly depressed during pneumoperitoneum in liver cirrhosis and control groups, but no significant differences were seen between the two groups. In both groups, portal venous inflow decreased and hepatic arterial blood flow increased significantly during pneumoperitoneum. However, during pneumoperitoneum, total hepatic blood flow as a percentage of its value before pneumoperitoneum was lower in cirrhotic rats (71.0%) than in control rats (91.9%) (p <0.05, Mann-Whitney U-test).. Carbon dioxide pneumoperitoneum markedly decreases total hepatic blood flow in cirrhotic rats due to the impaired hepatic arterial buffer response. Liver function should be carefully controlled in cirrhotic patients after laparoscopic surgery with pneumoperitoneum.

Topics: Animals; Blood Pressure; Carbon Dioxide; Heart Rate; Hemodynamics; Laparoscopy; Liver; Liver Cirrhosis; Male; Pneumoperitoneum, Artificial; Portal Vein; Rats; Rats, Inbred Strains; Rats, Sprague-Dawley; Regional Blood Flow; Splanchnic Circulation; Thioacetamide

In this study, we investigated hepatic fibrogenesis caused by long-term thioacetamide (TAA) administration in ob/ob mice, a naturally occurring leptin deficient animal. In the lean littermates, prominent hepatic fibrosis, as well as positive staining for alpha smooth muscle actin (alpha-SMA), was induced by treatment with TAA (200 microg/g, IP, 3 times per week) for 4 to 8 weeks as expected. In sharp contrast, almost no hepatic fibrosis developed in ob/ob mice given the equivalent doses of TAA, where specific staining for alpha-SMA barely was detected. Induction of alpha1(I) procollagen mRNA caused by TAA also was prevented in ob/ob mice almost completely. Further, transforming growth factor beta (TGF-beta) mRNA was increased in the liver after TAA treatment for 4 weeks in lean littermates, which also was prevented in ob/ob mice. Interestingly, fibrotic septa in the hepatic lobules, as well as increases in alpha1(I) procollagen mRNA, was observed in ob/ob mice, when they were injected with recombinant murine leptin (1 microg/g daily) in combination with TAA treatment. Leptin per se did not cause any fibrotic changes in the liver in ob/ob mice. These findings clearly indicated that leptin deficiency is responsible for the resistance to TAA-induced profibrogenic responses in ob/ob mice. In conclusion, leptin appears to promote profibrogenic responses in the liver, in part, by up-regulation of TGF-beta.

Topics: Actins; Animals; Cells, Cultured; Female; Gene Expression; Immunohistochemistry; Leptin; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Mice, Obese; Procollagen; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thioacetamide; Transforming Growth Factor beta

To examine the property of hepatocellular carcinoma (HCC), rat HCC cells were implanted into normal and cirrhotic rat livers. Implanted HCC grew much more progressively in cirrhotic livers than in normal livers. Kupffer cells were decreased profoundly in cirrhotic livers, resulting in markedly impaired phagocytic activity. Furthermore, production of Kupffer cell-related cytokines, such as interleukin-1 beta and tumor necrosis factor-alpha, was decreased profoundly in cirrhotic livers. Our results indicate that liver cirrhosis is a prominent promoting factor in HCC progression, and that markedly depressed Kupffer cell activity may play a role in augmented HCC progression in cirrhotic livers.

Topics: Animals; Carcinoma, Hepatocellular; Disease Models, Animal; Disease Progression; Interleukin-1; Kupffer Cells; Liver; Liver Cirrhosis; Liver Neoplasms; Male; Phagocytosis; Precancerous Conditions; Rats; Rats, Sprague-Dawley; Thioacetamide; Tumor Necrosis Factor-alpha

Hepatic fibrosis is associated with activation of hepatic stellate cells (HSC), the major source of the extracellular matrix (ECM) proteins. The predominant ECM protein synthesized by the HSC is collagen type I. We evaluated the effect of halofuginone-an inhibitor of collagen synthesis-on thioacetamide (TAA)-induced liver fibrosis in rats. In the control rats the HSC did not express smooth muscle actin, collagen type I gene, or tissue inhibitor of metalloproteinases-2 (TIMP-2), suggesting that they were in their quiescent state. When treated with TAA, the livers displayed large fibrous septa, which were populated by smooth muscle actin-positive cells expressing high levels of the collagen alpha1(I) gene and containing high levels of TIMP-2, all of which are characteristic of advanced fibrosis. Halofuginone given orally before fibrosis induction prevented the activation of most of the stellate cells and the remaining cells expressed low levels of collagen alpha1(I) gene, resulting in low levels of collagen. The level of TIMP-2 was almost the same as in the control livers. When given to rats with established fibrosis, halofuginone caused almost complete resolution of the fibrotic condition. The levels of collagen, collagen alpha1(I) gene expression, TIMP-2 content, and smooth muscle actin-positive cells were as in the control rats. Halofuginone inhibited the proliferation of other cell types of the fibrotic liver in vivo and inhibited collagen production and collagen alpha1(I) gene expression in the SV40-immortalized rat HSC-T6 cells in vitro. These results suggest that halofuginone may become an effective and novel mode of therapy in the treatment of liver fibrosis.

Topics: Animals; Cell Division; Liver; Liver Cirrhosis; Male; Piperidines; Protein Synthesis Inhibitors; Quinazolines; Quinazolinones; Rats; Rats, Wistar; Thioacetamide

Protein kinase C (PKC) plays a key role in the alteration of signal transduction in the liver, which may contribute to the development of liver cirrhosis. The aim of the present study was to examine the subcellular redistribution of PKC isozymes in rat liver cirrhosis, which is induced by two different cirrhotic chemical agents, carbon tetrachloride (CCl4) and thioacetamide (TAA).. Thioacetamide and CCl4 were administered to rats for 8 and 30 weeks, respectively before rats were killed and autopsies performed at 9, 20 and 30 weeks later. The TAA induced a fibrotic pattern in the liver that differed from that produced by CCl4, notably in the formation of fibrous connective tissue and the proliferation of bile ductule cells. Cholangiofibrosis and clear-cell foci were also observed in TAA-treated rats at 30 weeks. Histological examination revealed that severe cirrhotic changes were present 9 weeks after the commencement of CCl4 treatment and 30 weeks after TAA treatment.. When the subcellular redistribution of PKC isozymes (PKCalpha, -beta1, -delta, and -epsilon) was examined, all the PKC isozymes in CCl4-treated rats were found to be translocated to the membrane fraction, which may mean PKC activation, and then downregulated by proteolytic degradation after 9 weeks of treatment, which coincided with peak cirrhotic changes. All rats treated with CCl4 recovered to the control level after 20 weeks of treatment. In the case of TAA-treated rats, PKC isozymes were translocated to the particulate fraction of the liver after 9 weeks of treatment and this persisted in most of the rats for the duration of the experiment.. From these results, it would appear that PKC translocation preceded morphologic changes, and that an altered subcellular distribution of the PKC isozyme may be associated with the response to liver damage and carcinogenesis.

Topics: Animals; Carbon Tetrachloride; Collagen; Enzyme Activation; Immunoblotting; Liver Cirrhosis; Male; Protein Kinase C; Rats; Rats, Inbred F344; Signal Transduction; Thioacetamide

To analyze the aberrant expression of cell cycle-related proteins and their biological significance in relation to cirrhosis, we compared the cirrhotic patterns induced by two different types of cirrhotic agents, CCl4 and thioacetamide (TAA) in rats. CCl4 or TAA treatment was given to rats for 8 or 30 weeks, respectively, and the livers were removed at 9, 20, and 30 weeks after the experiment began. The TAA-induced fibrotic pattern was different from the CCl4-induced one, in terms of the formation of fibrous connective tissue and the proliferation of bile ductule cells. Cholangiofibrosis and clear cell foci were also observed in TAA-treated rats at 30 weeks. Histological examination revealed severe cirrhotic changes at 9 weeks in CCl4-treated rats and at 30 weeks in TAA-treated rats. Immunoblotting for cyclin D1, E, A, B, and proliferating cell nuclear antigen (PCNA) and their counterpart protein kinases (CDK2, 4, and CDC2) showed significant overexpression in rats with severely cirrhotic livers. The p53 tumor suppressor protein increased dramatically in the CCl4-treated group, while it was not detected in the livers of TAA-treated rats. Upregulation of p21WAF1, a CDK inhibitory protein, was detected in TAA-treated rats, but not in CCl4-treated rats. Immunohistochemical data for cyclin D1, E, and PCNA were well correlated with immunoblotting data; these proteins were increased in hepatocytes surrounding the cirrhotic lesions, suggesting that hepatocyte regeneration is correlated with cell cycle-related protein expression in cirrhotic liver. In the TAA-treated rats, the expression of these proteins was increased both in hepatocytes and in ductule cells. Our data suggest that liver cirrhosis induced by CCl4 or TAA is associated with alterations in cell cycle-related proteins, and that the expression of these proteins is responsible for hepatocyte regeneration in the damaged liver and may be involved in liver carcinogenesis.

Topics: Animals; Carbon Tetrachloride; Cell Cycle Proteins; Disease Models, Animal; Liver Cirrhosis; Male; Rats; Rats, Inbred F344; Thioacetamide

To clarify the sequential changes in pRB and p16 during different stages of hepatocarcinogenesis such as fibrosis, cirrhosis, hepatocellular adenoma (HCA), and hepatocellular carcinoma (HCC), male Fischer 344 rats were singly injected with diethylnitrosamine (DEN), immediately followed with phenobarbital for 1 wk and then thioacetamide (TAA) for 39 wk in drinking water. Rats were killed at 9, 20, 30, and 40 wk after DEN initiation and changes of pRB level, p16 gene hypermethylation, and in vivo gankyrin expression were examined. Histologic examination showed stepwise appearances of fibrosis, cirrhosis, HCA, and HCC at weeks 9, 20, 30, and 40, respectively. Hypermethylation of p16 exon 1 was not found until HCA but appeared in 50% of the rats with HCC accompanied by complete loss of its mRNA expression. The amount of glutathione S-transferase--gankyrin bound to pRB and pRB degradation in the liver depended on the concentration of gankyrin and incubation time. Gankyrin expression preceded pRB degradation in liver cirrhosis. In conclusion, gankyrin expression induced in liver fibrosis accelerated the degradation of pRB during liver cirrhosis, and inactivation of p16 exon 1 by DNA hypermethylation occurred during the progression of tumor cells to poorly differentiated HCC. Inactivation of pRB and/or p16 resulted in complete loss of regulation in the cell-division cycle during early and late stages, respectively, of hepatocarcinogenesis. Mol. Carcinog. 30:138--150, 2001.

Topics: Animals; Base Sequence; Cocarcinogenesis; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; Cyclins; Diethylnitrosamine; DNA Methylation; Exons; Genes, Retinoblastoma; Liver Cirrhosis; Liver Neoplasms, Experimental; Male; Molecular Sequence Data; Oncogene Proteins; Polymerase Chain Reaction; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Rats; Rats, Inbred F344; Thioacetamide

It is known that lidocaine is rapidly metabolized by the hepatic cytochrome P-450 system to form monoethylglycinexylidide (MEGX), its primary metabolite. We analyzed serum MEGX levels experimentally and clinically by fluorescent polarization immunoassay to reassess preoperative liver microsome functions.. Liver cirrhosis was produced in rats by intra-abdominal injection of thioacetamide. MEGX, indocyanine green test (ICG), and liver biochemical variables were measured periodically. Then, survival rates were assessed after the rats received a 70% hepatectomy.. MEGX levels were measured in various human patients with chronic hepatitis or liver cirrhosis who underwent hepatectomy. Serum MEGX levels significantly dropped and ICG levels significantly rose with macroscopic and histologic progression of liver cirrhosis in rats. The MEGX levels correlated closely with albumin levels and ICG. Preoperative MEGX and ICG levels of the mortal group of rats differed significantly from those of the survival group with 70% hepatectomy. Furthermore, 100% of the rats with MEGX levels above 40 ng/ml and ICG levels below 1.0%. In the clinical study, MEGX levels were significantly lower in patients with chronic hepatitis or liver cirrhosis than in healthy volunteers and correlated significantly with liver function tests such as albumin, Fischer's ratio, prothrombin time, hepaplastin and ICG. A significant difference was found in MEGX levels between patients receiving lobectomy and those receiving subsegmentectomy or partial hepatectomy. All patients tolerated their operations. Our data indicate that the MEGX test combined with ICG test and Child-Pugh classification is a better predictor of residual liver reserve capacity, and the analysis of hepatic MEGX formation might prove useful for rapid and reliable assessment liver function and choice of surgical treatment.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Bilirubin; Disease Models, Animal; Hepatectomy; Hepatitis; Hepatitis C, Chronic; Humans; Indicators and Reagents; Indocyanine Green; Lidocaine; Liver; Liver Cirrhosis; Liver Cirrhosis, Experimental; Liver Function Tests; Male; Organic Chemicals; Prothrombin Time; Rats; Rats, Wistar; Regression Analysis; Serum Albumin; Thioacetamide

Thioacetamide (TAA) administration (0.3 g/l of tap water for a period of 3 months) to rats resulted in hepatic cirrhosis as assessed by biochemical and histopathological findings. This treatment caused an increase in the levels of malondialdehyde (MDA) and diene conjugates (DCs) and a decrease in the levels of glutathione (GSH), vitamin E, vitamin C and the activities of glutathione peroxidase (GSH-Px) in the liver of rats. Superoxide dismutase (SOD) activities were unchanged. Taurine (2% w/w, added to the chow diet) was administered together with TAA (0.3 g/l of drinking water) for 3 months. Taurine was found to decrease TAA-induced hepatic lipid peroxidation and to increase TAA-depleted vitamin E levels and GSH-Px activities. Histopathological findings also suggested that taurine has an inhibitive effect on TAA-induced hepatic cirrhosis. These results indicate that taurine treatment has a protective effect against TAA-induced liver cirrhosis by decreasing oxidative stress.

Topics: Administration, Oral; Animals; Carcinogens; Glutathione; Liver; Liver Cirrhosis; Male; Malondialdehyde; Oxidative Stress; Rats; Rats, Wistar; Superoxide Dismutase; Taurine; Thioacetamide

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

Reactive oxygen species and oxidative stress were implicated in hepatic stellate cell activation and liver fibrosis. The aim of the present study was to examine whether the administration of free radical scavengers in vivo would prevent experimentally-induced hepatic cirrhosis in rats.. Cirrhosis was induced by administration of thioacetamide (TAA; 200 mg/kg, i.p.) twice/week, for 12 weeks. Rats were treated concurrently with either dimethylsulfoxide (DMSO; 4 g/kg, s.c. or p.o.) or dimethylthiourea (DMTU; 200 mg/kg i.p.) three times a week.. Liver fibrosis (histopathological score, spleen weight, and hepatic hydroxyproline) was abolished in rats treated with TAA and either DMSO or DMTU (P < 0.001). Accordingly, the hepatic expression of alpha smooth muscle actin, tissue inhibitor of metalloproteinase 2 and collagen alpha1 (I) gene were inhibited. The hepatic level of methane-sulfinic acid (produced by the interaction of DMSO with hydroxyl radicals) was increased in rats treated with TAA + DMSO (P = 0.0005) and decreased after pretreatment of these rats with DMTU (P = 0.008). However, the hepatic levels of malondialdehyde, lipid peroxides and protein carbonyls were not lower in the DMSO- and DMTU-treated groups.. The administration of free radical scavengers prevented the development of TAA-induced liver cirrhosis probably associated with decreased oxidative stress.

Topics: Animals; Collagen Type I; Dimethyl Sulfoxide; Free Radical Scavengers; Gene Expression; Glutathione Peroxidase; Hydroxyl Radical; Hydroxyproline; Liver; Liver Cirrhosis; Male; Rats; Rats, Wistar; Spleen; Sulfinic Acids; Superoxide Dismutase; Thioacetamide

Liver cirrhosis is an inveterate disease accompanying fibrosis, hepatocyte damage, and liver dysfunction. In this study, the therapeutic effects of recombinant human hepatocyte growth factor (rhHGF) on liver cirrhosis were examined in rats administered thioacetamide (TAA). Repeated administration of TAA for 10 weeks to rats induced liver cirrhosis with collagen nodes and pseudo-lobe generation, a condition that was pathologically similar to that in humans. Administration of rhHGF after the formation of liver cirrhosis markedly decreased the incidence of pathological fibrosis and the degree of fibrosis as measured by a computed image analysis system. Continuous administration of rhHGF by infusion pump was more effective than bolus administration. Northern blotting analysis showed that rhHGF reduced mRNA levels of procollagen alpha2(I), alpha1(IV), and transforming growth factor-beta1 (TGF-beta1) that were stimulated in the TAA-treated liver. The labeling index of hepatocytes increased following administration of rhHGF in this model. These observations suggest that the pathological recession of liver fibrosis is the result of the reduction of TGF-beta1 and collagen synthesis and, in part, of the stimulation of mitosis of hepatocytes directly by rhHGF and indirectly by TGF-beta1 reduction in the cirrhotic liver. These results demonstrate the usefulness of rhHGF as a therapeutic agent in liver cirrhosis.

Topics: Animals; Collagen; Disease Models, Animal; Hepatocyte Growth Factor; Liver; Liver Cirrhosis; Male; Rats; Rats, Sprague-Dawley; RNA, Messenger; Thioacetamide; Transforming Growth Factor beta

This study was undertaken in order to investigate the effect of zinc (Zn) administration on induction of Zn-binding metallothionein in rat liver with thioacetamide-induced cirrhosis, and the localization of metallothionein in the liver. Normal and cirrhotic rats received intraperitoneal injections with or without Zn. Subsequently, metal analyses, purification of metallothionein by gel filtration and immunohistochemical assessments of metallothionein were carried out. Although in Zn-injected cirrhotic rats, the Zn contents in the liver and plasma increased significantly depending upon the dose of Zn, the Zn contents in the liver and plasma of the cirrhotic rats were lower than those of normal rats after the same dose of Zn. The results of gel filtration also showed that the levels of Zn-metallothionein in the cirrhotic liver were reduced in comparison with those of the normal liver. By the immunohistochemical method, the presence of metallothionein in the parenchymal areas but not in the fibrotic areas of the cirrhotic liver was confirmed. These results suggested that the induced metallothionein was only located in the parenchymal areas. The metallothionein induced in the parenchymal areas was considered to play a role in protecting the parenchymal cells against the progression of fibrosis, because metallothionein has been thought to be involved in the cellular defense against oxidative stress.

Topics: Animals; Copper; Immunohistochemistry; Liver; Liver Cirrhosis; Male; Metallothionein; Rats; Rats, Wistar; Thioacetamide; Zinc

Long term administration of thioacetamide (0.03% in tap water) results in a characteristic lesion in rat liver, which corresponds to cirrhosis-like patterns of micronodular cirrhosis type after treatment over 3 months. During its development a reproducible temporal course of biochemical and morphological changes can be recognized. After withdrawal of the toxic agent this lesion persists for about 2 months. Then the cirrhosis-like alterations recede and a proliferation of bile ducts predominates, which is associated with increasing portal fibrosis altering the pattern and relatively enhancing the total collagen content of the liver. Considering these peculiarities, the TAA-model is suitable for investigations into connective tissue metabolism in the fibrotic liver and cirrhosis-like patterns. Search for and test of therapeutic principles should be done during TAA-administration (prophylactic agents) or within 2 months after withdrawal of toxic agents (therapeutics).

Topics: Acetamides; Acetylglucosaminidase; Animals; Disease Models, Animal; Female; Hydroxyproline; Liver; Liver Cirrhosis; Microbial Collagenase; Rats; Rats, Inbred Strains; Thioacetamide; Time Factors

When either diazepam or imipramine hydrochloride was administered orally to rats with thioacetamide-induced hepatic cirrhosis, the biliary and faecal elimination of metabolites was significantly decreased compared with that in normal animals. However, renal excretion of metabolites of diazepam or imipramine was increased in the liver-damaged rats. Experiments in vitro showed that liver homogenates from cirrhotic rats metabolized diazepam or imipramine hydrochloride in qualitatively and quantitatively similar ways to those from normal rats. Clearance of radioactivity from the blood following i.v. administration of either diazepam or imipramine hydrochloride was prolonged in animals with experimental cirrhosis.

Topics: Animals; Bile; Carbon Radioisotopes; Diazepam; Glucuronates; Glucuronidase; Imipramine; Liver Cirrhosis; Male; Rats; Thioacetamide

The effect of prolonged application of thioacetamide (TAA) on structural and functional properties of isolated liver mitochondria from rats was studied. Mitochondria prepared either from micronodularly cirrhotic livers (TAA application for 3 months) or from macronodularly cirrhotic ones (TAA application for 6 months) exhibited loss of respiratory control due to increased state 4 respiration and decreased state 3 respiration. The mitochondrial content of cytochromes aa3 and b was lowered only after 6 months exposure to TAA. The Mg2+ content of mitochondria from macronodularly cirrhotic livers was reduced to 50%. Mitochondria isolated from rats chronically treated with TAA exhibited lowered Ca2+ uptake rates and a decreased Ca2+ retention time. The analysis of mitochondrial phospholipid fatty acids revealed marked alterations in phosphatidylcholine and cardiolipin leading to a decreased 20:4/18:2 ratio. The functional and structural alterations of the liver mitochondria from chronically treated rats remained unaffected for at least 2 weeks after cessation of TAA application.

Topics: Acetamides; Administration, Oral; Animals; Calcium; Fatty Acids; Female; Liver Cirrhosis; Magnesium; Mitochondria, Liver; Oxygen Consumption; Phospholipids; Rats; Rats, Inbred Strains; Spectrophotometry, Atomic; Thioacetamide

Experimental liver injury was induced to test rats with a daily injection of thioacetamide (ThAA). The doses used for intraperitoneal administrations were 50 mg/kg body weight. The loss of body weight in the 3-week test period was 15%. In the liver there was seen progressive changes which displayed cell necrosis and regeneration. The influence of ThAA on rat blood and serum was checked using standard biochemical assays consisting of the percentage of blood obtainable and the serum/blood ratio and analysis of serum alanine transferase, serum alkaline phosphatase, serum creatinine, and hydroxyproline in the acute, subacute, chronic, and necrotic stage of liver injury. With ThAA injections, the stimulated rate of glycosaminoglycan synthesis had its association to the serum calcium content. It decreased continuously as function of traumatization time. In the 3-week test period, histological findings show in the alveolar bone, around the teeth, when under occlusal stress and ThAA traumatization, the distinct decrease in osteoblastic activity and less osteoids indicating thus the decreased formation of new bone. Conspicuous osteoclastic resorption was also seen in the same area.

Topics: Acetamides; Alveolar Process; Animals; Bite Force; Blood; Body Weight; Dental Occlusion; Enzymes; Liver; Liver Cirrhosis; Male; Rats; Stress, Physiological; Thioacetamide

Intraperitoneally-administered thioacetamide to rats produces the incorporation of 3H2O into the liver lipids during eight days, diminishing thereafter together with a decrease in 32P incorporation to phosphatidyl choline. Contrarily, the sphingomyelin incorporated 32P increases by the effect of the toxic action of the drug, while the 3H binding decreases. The cirrhosis-inducing agent possibly produces a shunt of the common CDP-choline precursor to the biosynthesis of sphingomyelin with detriment to the synthesis of phosphatidyl choline. The role of the unsaturation of fatty acids by thioacetamide is considered.

Topics: Acetamides; Animals; Lipid Metabolism; Liver; Liver Cirrhosis; Male; Phosphatidylcholines; Phospholipids; Rats; Rats, Inbred Strains; Sphingomyelins; Thioacetamide

Topics: Acetamides; Animals; Colloids; Gold; Golgi Apparatus; Kupffer Cells; Liver Cirrhosis; Lysosomes; Rabbits; Splenectomy; Thioacetamide; Trypan Blue

Thioacetamide-fed rats developed cirrhosis with portal hypertension (P portal=23.0 +/- 5.2 cm H2O, controls: 14.4 +/- 1.0 cm H2O). The PO2 of liver tissue was markedly reduced in cirrhosis (PO2=7.6 +/- 3.4 torr, controls 22.3 +/- 5.8 torr), and the aortal pH was significantly lower as well. No correlation was found between portal hypertension, development of large--nodular cirrhosis, and ascites.

Topics: Animals; Ascites; Hypertension, Portal; Liver; Liver Cirrhosis; Oxygen Consumption; Partial Pressure; Rats; Thioacetamide

The application of the Platinum-Multiwire Surface electrode for local oxygen determination permits the determination of microcirculatory changes in the normal and cirrhotic liver. The normal liver is capable of compensating for portal blood deprivation whereas in the cirrhotic liver shunting of portal blood leads to a striking hypoxia of the liver tissue. Lack of oxygen can be prevented by means of arterialization, but despite oxygen adaptation at the time of operation uncontrollable hyperoxygenation can occur as well.

Topics: Animals; Assisted Circulation; Liver; Liver Cirrhosis; Male; Microcirculation; Oxygen; Oxygen Consumption; Partial Pressure; Portacaval Shunt, Surgical; Rats; Thioacetamide

Topics: Acetamides; Amino Acids; Animals; Collagen; Disease Models, Animal; Hydroxyproline; Liver; Liver Cirrhosis; Rats; Thioacetamide

In the tumorous phase of TAA-induced cirrhosis of the liver comparative studies on the mitotic index, on the frequency of binucleated liver cells and on nuclear volume showed approximately similar values in the small pseudolobuli and in several hepatomas. Obviously "minimum deviation hepatomas" had developed. Decrease of ploidy characteristic for hepatomas was observed. In thioacetamide hepatomas the phenomenon of quadrupling of the cell nuclei due to dysfunctional nuclear edema occurred whereas the normal nuclei of liver cells can only double in volume in all phases of thioacetamide-intoxication. Particular karyometric aspects of the nuclear-nucleolar metabolism of hepatoma cells following thioacetamide intoxication are discussed.

Topics: Animals; Carcinoma, Hepatocellular; Cell Nucleus; Karyometry; Liver Cirrhosis; Liver Neoplasms; Rats; Thioacetamide

Liver microsomes of rats poisoned with thioacetamide show a significant reduction of cytochrome P-450. Consequently, oxidative reactions of drug metabolism and the estrogen 2-hydroxylase are diminished. Enhancement of microsomal transformation of estradiol to estrone and 16alpha-hydroxyestrone is observed after treatment of rats with thioacetamide, due to diminished metabolism of estradiol by the alternative oxidation at C-2. Estriol formation is reduced by thioacetamide pretreatment. These changes in estrogen breakdown closely correlate with those observed in humans suffering from cirrhosis of the liver. It is concluded that the thioacetamide poisoned rat should be an experimental model suitable for studying estrogen metabolism in liver injury.

Topics: Acetamides; Aminopyrine N-Demethylase; Aniline Hydroxylase; Animals; Chemical and Drug Induced Liver Injury; Cytochrome P-450 Enzyme System; Disease Models, Animal; Estradiol; Estriol; Estrogens; Estrone; Liver Cirrhosis; Microsomes, Liver; Rats; Steroid Hydroxylases; Thioacetamide

Topics: Animals; Carcinoma, Hepatocellular; Chemical and Drug Induced Liver Injury; Endoplasmic Reticulum; Glycogen; Hyperplasia; Liver; Liver Cirrhosis; Liver Neoplasms; Male; Microscopy, Electron; Rats; Ribosomes; Thioacetamide; Time Factors; Water

Topics: Acetamides; Animals; Chemical and Drug Induced Liver Injury; Endoplasmic Reticulum; Hepatectomy; Karyometry; Liver Cirrhosis; Liver Regeneration; Male; Methods; Mitosis; Photometry; Rats; RNA; Thioacetamide

Topics: Animals; Anti-Inflammatory Agents; Collagen; Connective Tissue; Disease Models, Animal; Liver; Liver Cirrhosis; Penicillamine; Rats; Thioacetamide

Topics: Acetamides; Animals; Carcinoma, Hepatocellular; Cell Nucleus; Chemical and Drug Induced Liver Injury; Hepatectomy; Liver; Liver Cirrhosis; Liver Neoplasms; Liver Regeneration; Mitosis; Neoplasms, Experimental; Precancerous Conditions; Rats; Thioacetamide

Topics: Agar; Animals; Biopsy; Carbon Tetrachloride Poisoning; Chemical and Drug Induced Liver Injury; Electrophoresis; Gels; Hepatitis; Histocytochemistry; Humans; Liver Cirrhosis; Liver Diseases; Proteins; Rabbits; Rats; Thioacetamide

Topics: Amides; Antifungal Agents; Chemical and Drug Induced Liver Injury; Hepatitis; Liver Cirrhosis; Rats; Research; Sulfhydryl Compounds; Thioacetamide; Toxicology

Topics: Amides; Cell Nucleolus; Cell Nucleus; Chemical and Drug Induced Liver Injury; Hepatitis; Liver; Liver Cirrhosis; Pathology; Pharmacology; Rats; Research; Sulfhydryl Compounds; Thioacetamide; Toxicology

Topics: Amides; Animals; Connective Tissue; Liver Cirrhosis; Liver Cirrhosis, Experimental; Rats; Research; Sulfhydryl Compounds; Thioacetamide

Topics: Amides; Animals; Liver Cirrhosis; Liver Cirrhosis, Experimental; Mineral Waters; Rats; Thioacetamide; Water; Water Supply

Topics: Acetates; Amides; Animals; Connective Tissue; Liver Cirrhosis; Liver Cirrhosis, Experimental; Liver Diseases; Rats; Thioacetamide

Topics: Acetates; Amides; Animals; Liver Cirrhosis; Liver Cirrhosis, Experimental; Rats; Thioacetamide

Topics: Acetates; Animals; Liver Cirrhosis; Liver Cirrhosis, Experimental; Rats; Thioacetamide