Chronic Carbon Tetrachloride Administration Research Articles

Interleukin-17 programs liver progenitor cell transformation into cancer stem cells through miR-122 downregulation with increased risk of primary liver cancer initiation.

Chronic inflammation is a key component in the development of virtually all types of primary liver cancers. However, how chronic inflammation potentiates or even may initiate liver parenchymal cell transformation remains unclear. Cancer stem cells (CSCs) represent an exciting target for novel anticancer therapeutic strategies in several types of cancers and were also described in primary liver cancers as tumor initiating cells. Recently, we reported a key role of Interleukin (IL)-17 in Liver Progenitor Cell (LPC) accumulation in preneoplastic cirrhotic livers. In this study, we evidenced in vitro, that long-term stimulation of LPCs with IL-17 led to their transformation into CSCs. Indeed, they acquired CSC-marker expression, and self-renewal properties, showed by their increased capacity to form spheroids. The miRNome analysis revealed that long-term IL-17 treatment of LPCs led to a 90% decrease in miR-122 expression. In a model using immunodeficient mice, ectopic engraftment of LPCs in an IL-17-enriched environment led to tumor occurrence with an aggressive phenotype. Contrastingly, in a murine model of hepatocellular carcinoma induced by a unique injection of diethyl-nitrosamine associated with chronic administration of carbon tetrachloride, IL-17-deficiency or anti-IL-17 therapy protected mice from liver tumor growth. In conclusion, we showed that a chronic exposure of LPCs to IL-17 cytokine promotes their transformation into CSCs. In addition, we demonstrated that IL-17-neutralizing strategies limit CSC occurrence and liver tumor progression through miR-122 restored-expression.

Antagonizing STK25 Signaling Suppresses the Development of Hepatocellular Carcinoma Through Targeting Metabolic, Inflammatory, and Pro-Oncogenic Pathways.

Background & AimsHepatocellular carcinoma (HCC) is one of the most fatal and fastest-growing cancers. Recently, nonalcoholic steatohepatitis (NASH) has been recognized as a major catalyst for HCC. Thus, additional research is critically needed to identify mechanisms involved in NASH-induced hepatocarcinogenesis, to advance the prevention and treatment of NASH-driven HCC. Because the sterile 20-type kinase serine/threonine kinase 25 (STK25) exacerbates NASH-related phenotypes, we investigated its role in HCC development and aggravation in this study.MethodsHepatocarcinogenesis was induced in the context of NASH in Stk25 knockout and wild-type mice by combining chemical procarcinogens and a dietary challenge. In the first cohort, a single injection of diethylnitrosamine was combined with a high-fat diet-feeding. In the second cohort, chronic administration of carbon tetrachloride was combined with a choline-deficient L-amino-acid–defined diet. To study the cell-autonomous mode of action of STK25, we silenced this target in the human hepatocarcinoma cell line HepG2 by small interfering RNA.ResultsIn both mouse models of NASH-driven HCC, the livers from Stk25-/- mice showed a markedly lower tumor burden compared with wild-type controls. We also found that genetic depletion of STK25 in mice suppressed liver tumor growth through reduced hepatocellular apoptosis and decreased compensatory proliferation, by a mechanism that involves protection against hepatic lipotoxicity and inactivation of STAT3, ERK1/2, and p38 signaling. Consistently, silencing of STK25 suppressed proliferation, apoptosis, migration, and invasion in HepG2 cells, which was accompanied by lower expression of the markers of epithelial–mesenchymal transition and autophagic flux.ConclusionsThis study provides evidence that antagonizing STK25 signaling hinders the development of NASH-related HCC and provides an impetus for further analysis of STK25 as a therapeutic target for NASH-induced HCC treatment in human beings.

No Zones Left Behind: Democratic Hepatocytes Contribute to Liver Homeostasis and Repair.

Despite minimal turnover, liver cells possess immense regenerative capacity. Some studies suggest existence of a hepatocyte subset with such unique capabilities. However, in the current issue of Cell Stem Cell, three independent studies (Chen etal., 2020; Matsumoto etal., 2020, and Sun etal., 2020) demonstrate an equitable homeostatic and reparative potential of all hepatocytes, irrespective of their lobular location or ploidy status.

Metformin prevents liver tumourigenesis by attenuating fibrosis in a transgenic mouse model of hepatocellular carcinoma.

Metformin is a hypoglycaemic agent used to treat type 2 diabetes mellitus (DM2) patients, with a broad safety profile. Since previous epidemiological studies had shown that the incidence of hepatocellular carcinoma (HCC) decreased significantly in metformin treated DM2 patients, we hypothesised that intervention with metformin could reduce the risk of neoplastic transformation of hepatocytes. HCC is the most common primary liver malignancy and it generally originates in a background of liver fibrosis and cirrhosis. In the present study, we took advantage of a transgenic mouse (TG221) characterized by microRNA-221 overexpression, with cirrhotic liver background induced by chronic administration of carbon tetrachloride (CCl4). This mouse model develops fibrosis, cirrhosis and liver tumours that become visible in 100% of mice at 5-6 months of age. Our results demonstrated that metformin intervention improves liver function, inhibits hepatic stellate cell (HSC) activation, reduces liver fibrosis, depletes lipid accumulation in hepatocytes, halts progression to decompensated cirrhosis and abrogates development HCC in CCl4 challenged transgenic mouse model. The study establishes the rationale for investigating metformin in cirrhotic patients regardless of concomitant DM2 status.

Stevia prevents experimental cirrhosis by reducing hepatic myofibroblasts and modulating molecular profibrotic pathways.

The aims of the present study were to investigate the capacity of stevia leaves to prevent experimental cirrhosis induced by chronic administration of carbon tetrachloride (CCl4 ) in rats and to explore the action mechanism involved. Liver cirrhosis was established by CCl4 treatment (400mg/kg i.p. three times a week for 12weeks); stevia powder was administered (100mg/kg by gavage daily) during the CCl4 treatment. Serum markers of liver damage and hydroxyproline were evaluated and histopathological analyses were carried out. The profibrotic pathways were analyzed by western blot and immunohistochemistry. We found for the first time that stevia cotreatment prevented the elevation of serum markers of necrosis and cholestasis and the occurrence of liver fibrosis. It is worth noting that stevia downregulated several profibrogenic pathways, including the reduction of hepatic myofibroblasts and decreased matrix metalloproteinase (MMP)2 and MMP13 expression, thereby blocking the liberation of transforming growth factor-β from the extracellular matrix. Notably, stevia reduced the phosphorylation of pSmad3L, the most profibrogenic and mitogenic Smad, by inhibiting the activation of c-Jun N-terminal kinase and extracellular signal-regulated kinase. Interestingly, Smad7, an important antifibrotic molecule, was upregulated by stevia treatment in cirrhotic rats. These multitarget mechanisms led to the prevention of experimental cirrhosis. Because stevia possesses a reasonable safety profile, our results indicate that it could be useful in the clinical setting to treat chronic liver diseases.

Macrophage autophagy protects against liver fibrosis in mice

Autophagy is a lysosomal degradation pathway of cellular components that displays antiinflammatory properties in macrophages. Macrophages are critically involved in chronic liver injury by releasing mediators that promote hepatocyte apoptosis, contribute to inflammatory cell recruitment and activation of hepatic fibrogenic cells. Here, we investigated whether macrophage autophagy may protect against chronic liver injury. Experiments were performed in mice with mutations in the autophagy gene Atg5 in the myeloid lineage (Atg5fl/fl LysM-Cre mice, referred to as atg5−/−) and their wild-type (Atg5fl/fl, referred to as WT) littermates. Liver fibrosis was induced by repeated intraperitoneal injection of carbon tetrachloride. In vitro studies were performed in cultures or co-cultures of peritoneal macrophages with hepatic myofibroblasts. As compared to WT littermates, atg5−/− mice exposed to chronic carbon tetrachloride administration displayed higher hepatic levels of IL1A and IL1B and enhanced inflammatory cell recruitment associated with exacerbated liver injury. In addition, atg5−/− mice were more susceptible to liver fibrosis, as shown by enhanced matrix and fibrogenic cell accumulation. Macrophages from atg5−/− mice secreted higher levels of reactive oxygen species (ROS)-induced IL1A and IL1B. Moreover, hepatic myofibroblasts exposed to the conditioned medium of macrophages from atg5−/− mice showed increased profibrogenic gene expression; this effect was blunted when neutralizing IL1A and IL1B in the conditioned medium of atg5−/− macrophages. Finally, administration of recombinant IL1RN (interleukin 1 receptor antagonist) to carbon tetrachloride-exposed atg5−/− mice blunted liver injury and fibrosis, identifying IL1A/B as central mediators in the deleterious effects of macrophage autophagy invalidation. These results uncover macrophage autophagy as a novel antiinflammatory pathway regulating liver fibrosis.

Deficiency of BPOZ2 Decreases Liver Fibrosis After Chronic Carbon Tetrachloride Administration in Mice.

Bood POZ containing gene type 2 (BPOZ2), a Broad-Complex, Tramtrack, and Bric a brac domain containing protein, is an adaptor protein for the E3 ubiquitin ligase scaffold protein CUL3. It plays an important role in acute carbon tetrachloride (CCl4)-induced liver injury and regeneration in mice. In this study, we investigated the role of BPOZ2 in the process of liver fibrosis induced by chronic CCl4 treatment. The results indicate that BPOZ2 deficiency decreases sustained activation of hepatic stellate cells, attenuates collagen αI(I) and tissue inhibitor of matrix metalloprotease 1 expression, and decreases liver fibrosis after repeated CCl4 administration. These findings suggest BPOZ2 as a new therapeutic target for the prevention and treatment of hepatic fibrosis in chronic liver disease.

Liver Progenitor Cells Yield Functional Hepatocytes in Response to Chronic Liver Injury in Mice

Self-renewal of mature hepatocytes promotes homeostasis and regeneration of adult liver. However, recent studies have indicated that liver progenitor cells (LPC) could give rise to hepatic epithelial cells during normal turnover of the liver and after acute injury. We investigated the capacity of LPC to differentiate into hepatocytes in vivo and contribute to liver regeneration. We performed lineage tracing experiments, using mice that express tamoxifen-inducible Cre recombinase under control of osteopontin regulatory region crossed with yelow fluorescent protein reporter mice, to follow the fate of LPC and biliary cells. Adult mice received partial (two-thirds) hepatectomy, acute or chronic administration of carbon tetrachloride (CCl(4)), choline-deficient diet supplemented with ethionine, or 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet. LPC and/or biliary cells generated 0.78% and 2.45% of hepatocytes during and upon recovery of mice from liver injury, respectively. Repopulation efficiency by LPC and/or biliary cells increased when extracellular matrix and laminin deposition were reduced. The newly formed hepatocytes integrated into hepatic cords, formed biliary canaliculi, expressed hepato-specific enzymes, accumulated glycogen, and proliferated in response to partial hepatectomy, as neighboring native hepatocytes. By contrast, LPC did not contribute to hepatocyte regeneration during normal liver homeostasis, in response to surgical or toxic loss of liver mass, during chronic liver injury (CCl(4)-induced), or during ductular reactions. LPC or biliary cells terminally differentiate into functional hepatocytes in mice with liver injury.

Macrophage therapy for murine liver fibrosis recruits host effector cells improving fibrosis, regeneration, and function

Clinical studies of bone marrow (BM) cell therapy for liver cirrhosis are under way but the mechanisms of benefit remain undefined. Cells of the monocyte-macrophage lineage have key roles in the development and resolution of liver fibrosis. Therefore, we tested the therapeutic effects of these cells on murine liver fibrosis. Advanced liver fibrosis was induced in female mice by chronic administration of carbon tetrachloride. Unmanipulated, syngeneic macrophages, their specific BM precursors, or unfractionated BM cells were delivered during liver injury. Mediators of inflammation, fibrosis, and regeneration were measured. Donor cells were tracked by sex-mismatch and green fluorescent protein expression. BM-derived macrophage (BMM) delivery resulted in early chemokine up-regulation with hepatic recruitment of endogenous macrophages and neutrophils. These cells delivered matrix metalloproteinases-13 and -9, respectively, into the hepatic scar. The effector cell infiltrate was accompanied by increased levels of the antiinflammatory cytokine interleukin 10. A reduction in hepatic myofibroblasts was followed by reduced fibrosis detected 4 weeks after macrophage infusion. Serum albumin levels were elevated at this time. Up- regulation of the liver progenitor cell mitogen tumor necrosis factor-like weak inducer of apoptosis (TWEAK) preceded expansion of the progenitor cell compartment. Increased expression of colony stimulating factor-1, insulin-like growth factor-1, and vascular endothelial growth factor also followed BMM delivery. In contrast to the effects of differentiated macrophages, liver fibrosis was not significantly altered by the application of macrophage precursors and was exacerbated by whole BM. Macrophage cell therapy improves clinically relevant parameters in experimental chronic liver injury. Paracrine signaling to endogenous cells amplifies the effect. The benefits from this single, defined cell type suggest clinical potential.

Tissue Transglutaminase Does Not Affect Fibrotic Matrix Stability or Regression of Liver Fibrosis in Mice

The ubiquitous cross-linking enzyme tissue transglutaminase (TG2) has been implicated in irreversible collagen stabilization in liver fibrosis, although functional evidence is lacking. We studied the contribution of TG2 to hepatic fibrotic matrix stability, as well as liver fibrosis progression and regression in TG2-deficient mice. Advanced liver fibrosis was induced by carbon tetrachloride or thioacetamide in TG2(-/-) mice and their wild-type littermates to study fibrosis progression and its spontaneous regression for up to 36 weeks. Pattern and extent of fibrosis were analyzed by histology and hepatic hydroxyproline quantification. Dynamic changes in hepatic matrix cross-linking were assessed by stepwise collagen extraction. Expression of 7 TGs and fibrosis-related genes was determined by quantitative reverse-transcription polymerase chain reaction. Transglutaminase activity was increased in fibrosis, and the level of TG2 messenger RNA correlated with the expression of fibrosis-related genes. Biochemical analysis revealed progressive collagen stabilization, with an up to 6-fold increase in the highly cross-linked, pepsin-insoluble fraction (26%). In TG2(-/-) mice, hepatic TG activity was significantly decreased, but chronic administration of carbon tetrachloride or thioacetamide led to a comparable extent and pattern of liver fibrosis, as in wild-type mice. In TG2(-/-) mice, the composition of hepatic collagen fractions and levels of fibrosis-related transcripts were unchanged, and fibrosis reversal was not facilitated. TG2 and TG activity are up-regulated during hepatic fibrosis progression, but do not contribute to fibrogenesis or stabilization of the collagen matrix. TG2 deletion does not promote regression of liver fibrosis. TG2-independent collagen cross-linking is a remarkable feature of progressing hepatic fibrosis and represents an important therapeutic target for liver fibrosis.

Hepatic Fibrosis Is Enhanced and Accompanied by Robust Oval Cell Activation after Chronic Carbon Tetrachloride Administration to Egr-1-Deficient Mice

The transcription factor early growth response (Egr)-1 regulates the expression of genes required for execution of the wound healing response. Multiple cycles of injury, coupled to incomplete wound healing, lead to fibrosis. Therefore, we hypothesized that Egr-1 is required for the development of hepatic fibrosis. To test this hypothesis, we exposed wild-type and egr-1(-/-) mice to acute or chronic carbon tetrachloride (CCl(4)). Acute CCl(4) exposure established a profibrotic milieu in the liver, including activation of hepatic stellate cells as well as expression of type 1 collagen genes and tissue inhibitor of matrix metalloproteinase 1 in both wild-type and egr-1(-/-) mice. This response was exacerbated in egr-1(-/-) mice. After chronic CCl(4) exposure, hepatic fibrosis was established in both genotypes; however, the fibrotic response was profoundly worsened in Egr-1-deficient mice. Importantly, enhanced fibrosis in egr-1(-/-) mice was accompanied by a robust activation of the oval cell response, suggesting more severe liver injury and/or reduced hepatocyte proliferation when compared with wild-type mice. Hepatic expression of genes indicative of oval cell activation, as well as the number of cells expressing A6, a mouse oval cell marker, was greater in egr-1(-/-) mice. Taken together, these data reveal novel roles for Egr-1 as a negative regulator of both CCl(4)-induced hepatic fibrosis and the oval cell response.

Dietary olive oil prevents carbon tetrachloride-induced hepatic fibrosis in mice

The specific purpose of this study was to investigate the effects of dietary olive oil on hepatic fibrosis induced by chronic administration of carbon tetrachloride (CCl(4)) in the mouse. In addition, the effects of oleic acid, a major component of olive oil, on activation of hepatic stellate cells (HSCs) were investigated in vitro. Mice were fed liquid diets containing either corn oil (control, AIN-93) or olive oil (6.25 g/L) throughout experiments. Animals were treated with CCl(4) for 4 weeks intraperitoneally. The mRNA expression of TGF-beta1 and collagen 1alpha2 (col1alpha2) in the liver was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). The HSCs were isolated from mice, and co-cultured with either oleic acid (100 microM) or linoleic acid (100 microM) for 2 days. The expression of alpha-smooth muscle actin (alpha-SMA) was assessed by immunohistochemistry. In addition, the production of hydroxyproline was determined. Serum alanine aminotransferase levels and the mRNA expression of TGF-beta and collalpha2 were significantly reduced by treatment of olive oil. Dietary olive oil blunted the expression of alpha-SMA in the liverand liver injury and hepatic fibrosis were prevented by treatment of olive oil. The number of alpha-SMA positive cells was significantly lower in HSCs co-cultured with oleic acid than in those co-cultured with linoleic acid. Concentration of hydroxyproline in culture medium was significantly lower in cells co-cultured with oleic acid than in the control. Dietary olive oil prevents CCl(4)-induced tissue injury and fibrosis in the liver. Since oleic acid inhibited activation of HSCs, oleic acid may play a key role on this mechanism.

Deficiency of nicotinamide adenine dinucleotide phosphate, reduced form oxidase enhances hepatocellular injury but attenuates fibrosis after chronic carbon tetrachloride administration†

Reactive oxygen species (ROS) activate hepatic stellate cells and enhance fibrogenesis. This study determined the role of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase deficiency in the development of hepatocellular necrosis, inflammation, and apoptosis in relation to fibrosis produced by chronic carbon tetrachloride (CCl(4)) administration. Wild-type (WT) mice or mice with deficiency of the gp91(phox) subunit of NADPH complex (gp91(phox(-/-) )) were subjected to biweekly CCl(4) injections over 8 weeks, whereas controls were given isovolumetric injections of olive oil. Serum aspartate aminotransferase (AST) was higher after CCl(4) administration in gp91(phox(-/-) ) than in WT mice, correlating with increased necrosis on liver histology. By contrast, more hepatocyte apoptosis was found after CCl(4) in the WT than in the gp91(phox(-/-) ) mice, which was associated with changes in components of the mitochondrial pathway of apoptosis, namely, an increase in the pro-apoptotic BAX protein in the WT, but not in the gp91(phox(-/-) ) mice and also a lower cytosolic cytochrome c in the gp91(phox(-/-) ) mice. There were fewer stellate cells and less fibrosis after CCl(4) in the gp91(phox(-/-) ) as compared with the WT mice. The increase in alpha(1)(I) collagen messenger RNA (mRNA), however, was greater after CCl(4) in the gp91(phox(-/-) ) mice. Matrix metalloproteinase-2 (MMP-2) and MMP-9 mRNA increased more in the gp91(phox(-/-) ) than in WT mice after CCl(4.) Tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2 increased after CCl(4) only in the gp91(phox(-/-) ) mice. Decreased hepatic fibrosis after chronic CCl(4) administration in mice with NADPH oxidase deficiency occurs in the setting of greater necrosis and inflammation but decreased apoptosis.

Quantitative profiling of phosphatidylcholine and phosphatidylethanolamine in a steatosis/fibrosis model of rat liver by nanoflow liquid chromatography/tandem mass spectrometry

Quantitative analysis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was carried out using a steatosis/fibrosis model of rat livers, which were induced by a chronic administration of carbon tetrachloride (CCl 4). Intact phospholipid mixtures from each liver sample (from rats fed with 0.5 mL CCl 4/kg three times a week for 30, 60, and 90 days) were analyzed by nanoflow liquid chromatography–electrospray ionization tandem mass spectrometry (nLC/ESI/MS/MS), and identifications of 37 PC and 19 PE species were made by collision-induced dissociation. The quantitative analysis utilized a multiple standard addition method with an internal standard, and the relationship between the MS peak intensities of different PC species and their carbon chain length was included for calibration. It was found that the total amount of PC and PE species decreased significantly with administration of CCl 4. While concentrations of most PC and PE species decreased to a great extent, three PEs and seven PCs were up-regulated more than twofold.

Reduced liver injury in the interleukin‐6 knockout mice by chronic carbon tetrachloride administration

Interleukin-6 has been involved in restoration of liver function after partial hepatectomy and toxic liver injury. However, normal liver regeneration in interleukin-6 knockout mice has also been reported. The aim of this work was to investigate the effect of interleukin-6 deficiency on liver injury and its regeneration in a model of long term carbon tetrachloride (CCl4) administration. Serum and whole livers from wild type and interleukin-6 knockout mice treated with carbon tetrachloride (0.25 mL kg(-1)) twice a week were obtained after 4, 6 and 8 weeks (n = 4-6). Sections were assessed for liver regeneration, liver injury and hepatocyte apoptosis whereas sera were assayed for aminotransferase levels. Nuclear extracts and total liver lysates were assayed for transcription factor activation and apoptosis related proteins, respectively. When compared to wild type, interleukin-6 knockout mice showed reduced liver damage scores, lower aminotransferase levels and diminished apoptosis, as well as reduced nuclear factor kappa B activation. Although the level of active protein was lower, activation of signal transducer and activator of transcription 3 still takes place in knockout mice. Furthermore, liver regeneration measured by bromodeoxyuridine incorporation showed no differences between wild type and knockout animals after 6 and 8 weeks of treatment. Compared to the wild type mice liver regeneration after chronic treatment with carbon tetrachloride proceeds at a slower rate in interleukin-6 deficient mice. However, this low recovery rate is accompanied by a reduction not only in hepatocyte apoptosis, but also in activation of nuclear factor kappa B and liver injury.

Lack of inducible nitric oxide synthase leads to increased hepatic apoptosis and decreased fibrosis in mice after chronic carbon tetrachloride administration

The role of nitric oxide (NO) in liver injury and fibrosis is unclear. The purpose of this study was to determine whether inducible NO synthase deficiency (iNOS(-/-)) affects liver injury and fibrosis produced in mice by chronic carbon tetrachloride (CCl(4)) administration. Wild-type (WT) or iNOS(-/-) mice were subjected to biweekly CCl(4) injections over 8 weeks, whereas controls were given isovolumetric injections of olive oil. Serum aminotransferases were lower after CCl(4) in the iNOS(-/-) than in the WT mice, which correlated with decreased necrosis on liver histology. There was increased apoptosis, a lower number of stellate cells, and a lesser degree of fibrosis after CCl(4) in the iNOS(-/-) as compared with the WT mice. alpha(1)(I) collagen messenger RNA (mRNA) was markedly increased after CCl(4) in the WT and to a significantly lesser extent in the iNOS(-/-) mice. Liver matrix metalloproteinase-9 (MMP-9) mRNA and MMP-2 mRNA were increased more in the WT than in the iNOS(-/-) mice after CCl(4). Also tissue inhibitor metalloproteinase 1 (TIMP-1) mRNA was increased to a much greater extent in the WT than in the iNOS(-/-) mice after CCl(4) (P < 0.05). However, MMP-9 and TIMP-1 protein, determined by western blot, were similarly increased after CCl(4) in both groups of mice. NO protects against CCl(4)-induced apoptosis. In the absence of iNOS, there is decreased necrosis, increased apoptosis, and reduced liver fibrosis.

Genistein Modifies Liver Fibrosis and Improves Liver Function by Inducing uPA Expression and Proteolytic Activity in CCl4-Treated Rats

Aim: To evaluate the effect of genistein on the fibrosis and matrix degradation caused by experimentally induced fibrosis in rats. Methods: Hepatic fibrosis was brought about by chronic administration of carbon tetrachloride to rats. To evaluate the effect of genistein on liver fibrosis and function, total collagen content and proteolytic activity in the liver were quantified. Urokinase-type plasminogen activator (uPA) expression during experimental fibrosis was localized by immunohistochemistry. Histopathological changes were evaluated using light and electron microscopy. Results: Animals with fibrosis and treated with genistein showed an important reduction (73%) in hepatic collagen content as well as an improvement in liver function (p < 0.001). Genistein increased the capacity of the liver to degrade type I collagen and Matrigel (3.1- and 3.7-fold, respectively; p < 0.001) in animals with liver fibrosis. Genistein increased the number of uPA-immunoreactive cells. The increase in the uPA expression correlated with an increase in proteolytic activity. Histological analysis revealed a reduction in the number of fiber septa in pericentral and perisinusoidal areas. Transmission electron micrographs of livers from animals with fibrosis and treated with genistein showed a reduction in the number of hepatic stellate cells activated and a smaller number of collagen fibers. Conclusion: Genistein is able to improve the liver after injury and fibrosis induced by chronic administration of carbon tetrachloride. This finding suggests that genistein has antifibrogenic potential and could therefore be useful for treating chronic liver disease.

Differential gene expression profiles in the steatosis/fibrosis model of rat liver by chronic administration of carbon tetrachloride

Global gene expression profile was analyzed by microarray analysis of rat liver RNA after chronic carbon tetrachloride (CCl 4) administration. Rats received 0.5 ml CCl 4/kg three times a week, and the liver samples were obtained after 0, 30, 60, and 90 days of injection. Histopathologic studies of liver tissues enabled the classification of the CCl 4 effect into mild and severe fatty liver/steatosis (30 and 60 days, respectively) and fibrosis/cirrhosis (90 days) stages. The expression levels of 4900 clones on a custom rat gene microarray were analyzed and the results were confirmed by semi-quantitative RT-PCR. Four hundred thirty-eight clones were differentially expressed with more than a 1.625-fold difference (which equals 0.7 in log2 scale) at one or more time points. Multiple genes involved in lipid metabolism and ribosome biogenesis showed differential transcript levels upon chronic CCl 4 administration, which was previously seen in acute rat model as well. In addition, a total of 149 clones were identified as fibrosis/cirrhosis-specific genes by either fold changes or Significance Analysis of Microarrays. In conclusion, we report microarray analysis results in rat liver upon chronic CCl 4 administration with a full chronological profile that not only covered fatty liver/steatosis but also later points of fibrosis/cirrhosis. These data will provide the insight of specific gene expression profiles that is implicated in the multistep process of fatty liver/steatosis and fibrosis/cirrhosis after chronic hepatotoxin exposure.

Influence of leptin in the development of hepatic fibrosis produced in mice by Schistosoma mansoni infection and by chronic carbon tetrachloride administration

Background/Aims: Leptin, a product of the obese ( ob) gene is present in activated stellate cells. This study investigated whether leptin is essential for the development of hepatic fibrosis caused by various agents. Methods: Control and ob/ ob mice were infected with Schistosoma mansoni or were administered chronic carbon tetrachloride to cause hepatic fibrosis. Results: Fibrosis developed in both ob/ ob and control mice. However, the amount of histologically detectable fibrosis and the increase in liver hydroxyproline content was significantly greater in both models of fibrosis for treated controls than for treated ob/ ob mice. Fibrosis was associated with higher secretion of TGFβ1 from spleen cells of treated control than treated ob/ ob mice. Chronic leptin administration in ob/ ob mice infected with Schistosoma mansoni resulted in an increase in the amount of fibrosis caused by Schistosoma mansoni, eliminating any significant differences in the amount of fibrosis between infected ob/ ob mice and control mice. It also eliminated any significant difference in TGFβ1 secretion between the infected ob/ ob and infected control mice. Conclusions: This study shows that leptin deficiency decreases but does not eliminate hepatic fibrosis produced by Schistosoma mansoni and carbon tetrachloride administration. The effect of leptin in potentiating fibrogenesis is most likely mediated by TGFβ1.

Degeneration of skeletal muscle fibers in the rat administrated carbon tetrachloride: similar histological findings of the muscle in a 64-year-old patient of LC with muscle cramp

It is well known that painful muscle cramps occur frequently in patients with advanced liver cirrhosis (LC). Although many studies discuss the pathophysiological causes of these muscle cramps in various conditions, the results are not clear as far as the cause associated to LC is concerned. In order to investigate the cause of muscle cramps in LC, we examined the histological findings of skeletal muscle fibers in LC rat model and in a patient with LC. LC ( n=9) was induced in rats by chronic carbon tetrachloride administration. The histological findings of skeletal muscle tissues from the lower leg in LC rats and those of the upper arm in a patient with LC were compared. The degenerated muscle fibers and centronucleus in LC rats were similar to the opaque fibers frequently observed in myotonic dystrophy with severe muscle rigidity in patients with LC. In conclusion, results of this study suggest that one of the causes for muscle cramps in patients with LC is due to skeletal muscle fiber degeneration. Therefore, histological observation of skeletal muscle fibers should be considered in the treatment of painful muscle cramps.