Cannabidiol alleviates carbon tetrachloride-induced liver fibrosis in mice by regulating NF-κB and PPAR-α pathways.

Liver fibrosis has become a serious public health problem that can develop into liver cirrhosis and hepatocellular carcinoma and even lead to death. Cannabidiol (CBD), which is an abundant nonpsychoactive component in the cannabis plant, exerts cytoprotective effects in many diseases and under pathological conditions. In our previous studies, CBD significantly attenuated liver injury induced by chronic and binge alcohol in a mouse model and oxidative bursts in human neutrophils. However, the effects of CBD on liver fibrosis and the underlying mechanisms still need to be further explored. A mouse liver fibrosis model was induced by carbon tetrachloride (CCl4) for 10 weeks and used to explore the protective properties of CBD and related molecular mechanisms. After the injection protocol, serum samples and livers were used for molecular biology, biochemical and pathological analyses. The results showed that CBD could effectively improve liver function and reduce liver damage and liver fibrosis progression in mice; the expression levels of transaminase and fibrotic markers were reduced, and histopathological characteristics were improved. Moreover, CBD inhibited the levels of inflammatory cytokines and reduced the protein expression levels of p-NF-κB, NF-κB, p-IκBα, p-p38 MAPK, and COX-2 but increased the expression level of PPAR-α. We found that CBD-mediated protection involves inhibiting NF-κB and activating PPAR-α. In conclusion, these results suggest that the hepatoprotective effects of CBD may be due to suppressing the inflammatory response in CCl4-induced mice and that the NF-κB and PPAR-α signaling pathways might be involved in this process.

Therapeutic benefit of oregano oil in the acute idiosyncratic hepatotoxicity induced by carbon tetrachloride in rats: Adverse effects of high dose of oreganum.

Toxicity caused by carbon tetrachloride (CCl4) can lead to serious liver injury. The aim of the study is to investigate the protective effects of oregano oil (Origanum minutiflorum extract oil) against CCl4-induced liver injury. Two doses of oregano oil were used in the experiment: a low dose (LD; 20 mg/kg) and a high dose (HD; 60 mg/kg) during 2 weeks. CCl4 caused severe liver damage, nucleolus destruction in hepatocytes and cytogenetic changes in the nucleus. Indirectly, CCl4 causes decreased protein synthesis and significantly high creatinine and urea values. Hematological disorders have been recorded, such as decreased RBC and hemoglobin concentration, increased WBC and deformability of the erythrocyte membrane. Both doses of oregano oil had protective effects. Improved protein synthesis and high globulins level, creatinine and urea were found in both groups. Cytogenetic changes in the nucleus of hepatocytes were reduced. A high dose of oregano oil had maximal protective effects for RBC, but a very weak effect on hemoglobin synthesis. Also, WBC and lymphocyte values were low. Origanum stimulates protein synthesis and recovery of hepatocytes after liver injury, reduces the deformability of the erythrocyte membrane. High doses of oregano oil decreased WBC and lymphocytes which may lead to a weakening of the immune response. However, high doses are more effective against severe platelet aggregation than low doses, suggesting an effective treatment against thrombocytosis.

TET3 boosts hepatocyte autophagy and impairs non-alcoholic fatty liver disease by increasing ENPP1 promoter hypomethylation.

BACKGROUND: Dysregulated ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family occurs in metabolic reprogramming pathological processes. Nonetheless, the epigenetic mechanisms by which ENPP family impacts NAFLD, also known as metabolic dysfunction-associated steatotic liver disease (MASLD), is poorly appreciated. METHODS: We investigated the causes and consequences of ENPP1 promoter hypomethylation may boost NAFLD using NAFLD clinical samples, as well as revealed the underlying mechanisms using high-fat diet (HFD) + carbon tetrachloride (CCl4) induced mouse model of NAFLD and FFA treatment of cultured hepatocyte. RESULTS: Herein, we report that the expression level of ENPP1 are increased in patients with NAFLD liver tissue and in mouse model of NAFLD. Hypomethylation of ENPP1, is associated with the perpetuation of hepatocyte autophagy and liver fibrosis in the NAFLD. ENPP1 hypomethylation is mediated by the DNA demethylase TET3 in NAFLD liver fibrosis and hepatocyte autophagy. Additionally, knockdown of TET3 methylated ENPP1 promoter, reduced the ENPP1 expression, ameliorated the experimental NAFLD. Mechanistically, TET3 epigenetically promoted ENPP1 expression via hypomethylation of the promoter. Knocking down TET3 can inhibit the hepatocyte autophagy but an overexpression of ENPP1 showing rescue effect. CONCLUSIONS: We describe a novel epigenetic mechanism wherein TET3 promoted ENPP1 expression through promoter hypomethylation is a critical mediator of NAFLD. Our findings provide new insight into the development of preventative measures for NAFLD.

Recruitment or activation of mast cells in the liver aggravates the accumulation of fibrosis in carbon tetrachloride-induced liver injury.

Liver diseases caused by viral infections, alcoholism, drugs, or chemical poisons are a significant health problem: Liver diseases are a leading contributor to mortality, with approximately 2 million deaths per year worldwide. Liver fibrosis, as a common liver disease characterized by excessive collagen deposition, is associated with high morbidity and mortality, and there is no effective treatment. Numerous studies have shown that the accumulation of mast cells (MCs) in the liver is closely associated with liver injury caused by a variety of factors. This study investigated the relationship between MCs and carbon tetrachloride (CCl4)-induced liver fibrosis in rats and the effects of the MC stabilizers sodium cromoglycate (SGC) and ketotifen (KET) on CCl4-induced liver fibrosis. The results showed that MCs were recruited or activated during CCl4-induced liver fibrosis. Coadministration of SCG or KET alleviated the liver fibrosis by decreasing SCF/c-kit expression, inhibiting the TGF-ß1/Smad2/3 pathway, depressing the HIF-1a/VEGF pathway, activating Nrf2/HO-1 pathway, and increasing the hepatic levels of GSH, GSH-Px, and GR, thereby reducing hepatic oxidative stress. Collectively, recruitment or activation of MCs is linked to liver fibrosis and the stabilization of MCs may provide a new approach to the prevention of liver fibrosis.

Boldine protects against carbon tetrachloride-induced chronic liver injury by regulating NF-κB signaling pathway.

Sustained liver injuries predominantly promote oxidative stress and inflammation that lead to the progression of chronic liver disease (CLD), including fibrosis, cirrhosis, and hepatocellular carcinoma. Boldine, an alkaloid isolated from Peumus boldus, has been shown to have antioxidant and anti-inflammatory effects. Currently, there is no definitive treatment option available for CLD. Therefore, we investigated the hepatoprotective effect of boldine against carbon tetrachloride (CCl4 )-induced chronic liver injury in rats. CCl4 (2 mL/kg., b.w., i.p.) was administered twice weekly for 5 weeks to induce chronic liver injury in rats. Separate groups of rats were given boldine (20 mg/kg b.w., and 40 mg/kg b.w.) and silymarin (100 mg/kg b.w.) orally, daily. Serum transaminases, lipid peroxidation, and antioxidant levels were measured, and nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (cox-2), interleukin-1 ß (IL-1ß), and α-smooth muscle actin (α-SMA) gene and protein expressions were evaluated. CCl4 administration increased liver marker enzymes of hepatotoxicity in serum and oxidative stress markers, inflammatory genes and α-smooth muscle actin expression in liver tissue. Boldine concurrent treatment suppressed CCl4 -induced elevation of transaminase levels in serum, restored enzymic and non-enzymic antioxidants, and downregulated NF-κB, TNF-α, Cox-2 and IL-1ß expressions, thereby suppressing hepatic inflammation. Boldine administration also repressed α-SMA expression. The results of this study demonstrate the antioxidant, anti-inflammatory, and antifibrotic properties of boldine, and it can be a potential therapeutic candidate in the treatment of CLD.

Therapeutic Potential of Diosgenin in Amelioration of Carbon Tetrachloride-Induced Murine Liver Injury.

Diosgenin is a sapogenin with antidiabetic, antioxidant, and anti-inflammatory properties. The current study investigated whether diosgenin could ameliorate carbon tetrachloride (CCL4)-induced liver injury. To cause liver injury, CCL4 was injected intraperitoneally twice a week for 8 weeks. Daily oral administration of diosgenin at doses of 20, 40, and 80 mg/kg was started one day before CCL4 injection and continued for 8 weeks. Finally, serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and also albumin were assessed. Catalase and superoxide dismutase (SOD) activities in addition to glutathione (GSH) and malondialdehyde (MDA) levels were also quantified in the liver homogenate and routine histological evaluation was also conducted. Elevated serum levels of liver enzymes and decreased serum level of albumin caused by CCL4 were significantly restored following diosgenin administration at doses of 40 and 80 mg/kg. Long-term administration of CCL4 increased inflammatory and apoptotic factors such as IL-1ß, caspase 3, TNF-α, and IL-6 and decreased SOD and catalase activities as well as GSH level in liver homogenates; while MDA level was increased. Treatment with diosgenin increased SOD and catalase activities and GSH levels in the liver of injured animals. In addition, liver MDA, IL-1ß, caspase 3, TNF-α, and IL-6 level or activity decreased by diosgenin treatment. Additionally, diosgenin aptly prevented aberrant liver histological changes. According to obtained results, diosgenin can dose-dependently diminish CCl4-induced liver functional deficits and histological changes in a dose-dependent manner, possibly due to its antioxidant and anti-inflammation properties, and its beneficial effect is comparable to known hepatoprotective agent silymarin.

Difference in the Mechanism of Iron Overload-Enhanced Acute Hepatotoxicity Induced by Thioacetamide and Carbon Tetrachloride in Rats.

Iron overload has been recognized as a risk factor for liver disease; however, little is known about its pathological role in the modification of liver injury. The purpose of this study is to investigate the influence of iron overload on liver injury induced by two hepatotoxicants with different pathogenesis in rats. Rats were fed a control (Cont), 0.8% high-iron (0.8% Fe), or 1% high-iron diet (1% Fe) for 4 weeks and were then administered with saline, thioacetamide (TAA), or carbon tetrachloride (CCl4). Hepatic and systemic iron overload were seen in the 0.8% and 1% Fe groups. Twenty-four hours after administration, hepatocellular necrosis induced by TAA and hepatocellular necrosis, degeneration, and vacuolation induced by CCl4, as well as serum transaminase values, were exacerbated in the 0.8% and 1% Fe groups compared to the Cont group. On the other hand, microvesicular vacuolation induced by CCl4 was decreased in 0.8% and 1% Fe groups. Hepatocellular DNA damage was increased by iron overload in both models, whereas a synergistic effect of oxidative stress by excess iron and hepatotoxicant was only present in the CCl4 model. The data showed that dietary iron overload exacerbates TAA- and CCl4-induced acute liver injury with different mechanisms.

Oleuropein, a phenolic component of Olea europaea L. ameliorates CCl4-induced liver injury in rats through the regulation of oxidative stress and inflammation.

OBJECTIVE: This study aimed to assess the hepatoprotective role of oleuropein (Olp), a phenolic compound found in olive, against carbon tetrachloride (CCl4)-induced liver damage in rats. MATERIALS AND METHODS: The research involved male albino rats, which received intraperitoneal injections of 100 mg/kg b.w. of oleuropein for 8 consecutive weeks before being subjected to carbon tetrachloride (CCl4) at a dosage of 1.0 ml/kg b.w. Changes induced by CCl4 in antioxidant and inflammatory marker levels were assessed using ELISA assay kits. Moreover, CCl4-induced liver tissue architecture alteration, fibrosis, and expression pattern of protein were evaluated by performing H&E, Sirius red, Masson trichrome, and immunohistochemistry staining. RESULTS: Increased serum transaminases and massive hepatic damage were observed by this liver toxicant. The hepatic injury was further evidenced by a significant decrease in antioxidant enzyme activity [superoxide dismutase (SOD), glutathione peroxidase (GPx), Glutathione (GSH) and Total Antioxidant Capacity (T-AOC)]. The administration of CCl4 resulted in an increased inflammatory response, which was measured by C-reactive protein, interleukin-6, as well as tumor necrosis factor-alpha. Olp as a curative regimen led to significant attenuation in the inflammatory response and oxidative/nitrosative stress. This polyphenol treatment improved the hepatic tissue architecture and decreased fibrosis. In the CCl4 treatment group, the expression pattern of IL-6 protein was high, whereas expression was decreased after Olp, as evidenced by immunohistochemistry staining. CONCLUSIONS: The study suggests that oleuropein treatment has the potential to reduce liver damage caused by CCl4 induction by inhibiting oxidative stress and inflammation and maintaining liver tissue architecture. This could make it a promising treatment option for liver pathogenesis.

A Mouse Model of Non-Alcoholic Steatohepatitis and Hepatocellular Carcinoma Induced by Western Diet and Carbon Tetrachloride.

Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease (NAFLD). Obesity is a known risk factor of NASH, which, in turn, increases the risk of developing cirrhosis (liver scarring) and hepatocellular carcinoma (HCC). In addition to being a potentially life-threatening condition, public health concerns surrounding NASH are amplified by the lack of FDA-approved treatments. Although various preclinical models reflecting both the histopathology and the pathophysiological progression of human NASH exist, most of these models are diet-based and require 6-13 months for NASH symptom manifestation. Here, we describe a simple and rapid-progression model of NASH and NASH-driven HCC in mice. Mice received a western diet equivalent (WD; i.e., a high-fat, high-fructose, and high-cholesterol diet), high-sugar water (23.1 g/L fructose and 18.9 g/L glucose), and weekly intraperitoneal injections of carbon tetrachloride (CCl4) at a dose of 0.2 µL/g of body weight. The resulting phenotype, consisting in liver fibrosis and HCC, appeared within 24 weeks of diet/treatment initiation and presented similar histological and transcriptomic features as human NASH and NASH-driven HCC, thereby supporting the adequacy of this preclinical model for the development and evaluation of drugs that can prevent or reverse these diseases.

Dietary rice bran attenuates hepatic stellate cell activation and liver fibrosis in mice through enhancing antioxidant ability.

Various endogenous and exogenous stimuli can result in an inflammatory response and collagen deposition in the liver, which affect liver function and increase the risk of developing liver cirrhosis and cancer. Rice bran, the main by-product of rice milling, contains various nutrients which possess hepatoprotective activities. In this study, we investigated the effects of rice bran on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. Mice were fed a rice-bran-containing diet (10% rice bran w/w) or a standard diet with or without an injection of 20% CCl4 to induce liver fibrosis. Our results showed that feeding a rice-bran-containing diet could alleviate CCl4-induced liver damage, collagen deposition, and expressions of fibrosis-related genes, including α-smooth muscle actin (α-SMA), collagen 1a2 (COL1A2), and transforming growth factor-ß (TGF-ß) in liver tissues. Moreover, consumption of rice bran enhanced phase II detoxification and antioxidant gene expressions, including Gsta3, Gstp1, Catalase, SOD1, SOD2, and SOD3. Treatment with γ-oryzanol, the major bioactive compound in rice bran, decreased the sensitivity of hepatic stellate cells (HSCs) to TGF-ß1-induced α-SMA, COL1A2, and phosphorylated smad2 expressions. In conclusion, a rice-bran-containing diet may have beneficial effects on liver fibrogenesis through increased antioxidant and detoxification activities. γ-Oryzanol, the major bioactive compound of rice bran, can inhibit activation of HSCs.

Serum α-SMA is a potential noninvasive biomarker of liver fibrosis.

The severity of fibrosis is central to the therapeutic course for patients with chronic liver disease; therefore, early detection of liver fibrosis is critical for timely therapeutic interventions. Liver biopsy is the gold standard for the diagnosis of liver fibrosis; however, it is contraindicated in several pathological conditions. Activated hepatic stellate cells (HSCs) are the main cells for fibrotic tissue synthesis, such as that of alpha-smooth muscle actin (α-SMA). This study aimed to determine whether serum α-SMA levels are a suitable noninvasive, sensitive, and reliable liver fibrosis marker. Fibrosis was induced in male Wistar rats via chronic CCl4 administration. Fibrosis was determined in the liver tissues by quantifying the hydroxyproline content and visualized using Masson's trichrome staining. Rats chronically administered CCl4 exhibited a progressive increment in the hepatic collagen content, as well as both hepatic and serum α-SMA levels in a time-dependent manner. Moreover, serum levels of α-SMA significantly correlated with hepatic α-SMA levels (p ≤ 0.001), as well as with the severity of liver fibrosis (p ≤ 0.001). These findings suggest that increased levels of serum α-SMA can be considered a potential reliable and noninvasive biomarker for early liver fibrosis.

Aging aggravates liver fibrosis through downregulated hepatocyte SIRT1-induced liver sinusoidal endothelial cell dysfunction.

BACKGROUND: Aging increases the susceptibility to chronic liver diseases and hastens liver fibrosis deterioration, but the underlying mechanisms remain partially understood. The aim of this study was to investigate the effect of aging and chronic liver diseases on hepatocyte Sirtuin 1 (SIRT1) and LSECs and their contribution to liver fibrosis pathogeneses. METHODS: Young (8-12 wk) and aged (18-20 mo) mice were subjected to carbon tetrachloride-induced liver fibrosis. Primary HSCs and LSECs were isolated and cocultured for in vitro experiments. Liver tissues and blood samples from healthy controls and patients with liver fibrosis were analyzed. RESULTS: Downregulated hepatocytes SIRT1 in aged mice increased high mobility group box 1 acetylation, cytoplasmic translocation, and extracellular secretion, causing LSECs dysfunction by means of the toll-like receptor 4/AK strain transforming (AKT)/endothelial nitric oxide synthase pathway, ultimately activating HSCs and increasing susceptibility to liver injury and fibrosis. Adeno-associated virus-mediated overexpression of SIRT1 in hepatocytes suppressed the abovementioned alterations and attenuated carbon tetrachloride-induced liver injury and fibrosis in liver fibrosis mice, and there were no significant differences in liver injury and fibrosis indicators between young and aged mice after SIRT1 overexpression treatment. In vitro experiments demonstrated that SIRT1 overexpression and endothelial nitric oxide synthase agonist YC-1 improved LSECs function and inhibited HSCs activation, mediated by nitric oxide. Similarly, downregulated hepatocytes SIRT1 and LSECs dysfunction were observed in the livers of aged individuals compared to young individuals and were more pronounced in aged patients with liver fibrosis. CONCLUSIONS: Aging aggravates liver fibrosis through downregulated hepatocytes SIRT1-induced LSECs dysfunction, providing a prospective curative approach for preventing and treating liver fibrosis.

Antioxidant and Protective Effect of Ocimum basilicum Seeds Extract on Renal Toxicity Induced by Carbon Tetrachloride in Rats.

Basil (Ocimum basilicum L.) is distributed worldwide and used in the food, pharmaceutical, and cosmetic industries. Most applications are for the herb basil, recently the basil seeds have also been used commercially; however, little is known about the nutritional and functional properties of the seeds. The present study aimed to investigate a possible protective effect of the methanol extract of O. basilicum seeds (MEOB), based on its phytochemical content, against kidney toxicity induced by CCl4 in adult rats. A single dose of CCl4 was used to induce oxidative stress in rats, which was demonstrated by a significant rise of serum enzyme markers. MEOB was administrated for 15 consecutive days (200 mg/kg body weight) to Wistar rats before CCl4 treatment and the effects on serum urea, creatinine, and uric acid, as well as the kidney superoxide dismutase, catalase, glutathione peroxidase, and glutathione activity and thiobarbituric acid reactive substances and protein carbonyl (PCO) levels were evaluated. In addition, histopathological examinations of kidneys were performed. In the positive control group, CCl4 induced an increase in serum biochemical parameters and triggered oxidative stress in the kidney. MEOB (200 mg/kg BW) resulted in significant reduction of CCl4-elevated levels of kidney markers, urea and creatinine, and a significant increase of uric acid compared with the CCl4-only group. In addition, MEOB pretreatment resulted in a significant reduction in lipid peroxidation and PCO levels in renal tissue compared with CCl4-exposed group. MEOB definitely could prevent the development of pathological changes in the kidneys. Overall, we conclude that MEOB is effective in protecting renal function from CCl4 toxicity.

Hepatocellular Brg1 promotes CCl4-induced liver inflammation, ECM accumulation and fibrosis in mice.

INTRODUCTION: Hepatic fibrosis is a progressive pathological process involving the exhaustion of hepatocellular regenerative capacity and ultimately leading to the development of cirrhosis and even hepatocellular carcinoma. Brg1, the core subunit of the SWI/SNF chromatin-remodeling complex, was recently identified as important for liver regeneration. This study investigates the role of Brg1 in hepatic fibrosis development. METHODS: Hepatocyte-specific Brg1 knockout mice were generated and injected with carbon tetrachloride (CCl4) for 4, 6, 8, and 12 weeks to induce liver fibrosis. Afterwards, liver fibrosis and liver damage were assessed. RESULTS: Brg1 expression was significantly increased in the fibrotic liver tissue of wild-type mice, as compared to that of untreated wild-type mice. The livers of the Brg1 knockout animals showed reduced liver inflammation, extracellular matrix accumulation, and liver fibrosis. TNF-α and NF-κB-mediated inflammatory response was reduced in Brg1 knockout animals. CONCLUSION: Brg1 promotes the progression of liver fibrosis in mice and may therefore be used as a potential therapeutic target for treating patients with liver fibrosis due to chronic injury.

Cakes fortified with papaya seeds effectively protects against CCl4-induced immunotoxicity.

Maintaining a robust immune system and safeguarding the liver from toxins are crucial for overall health. The study aimed to investigate the immunostimulant effects of papaya seed-enriched cakes (CPS) in countering carbon tetrachloride (CCl4)-induced immunocytotoxicity in rats (n = 48). The rats were divided into six groups (8 each): a control group (Group 1), rats fed cakes containing 15% papaya seeds (Group 2 - CPS), rats exposed only to CCl4 (Group 3 - CCl4), rats injected with CCl4 and administered silymarin (Group 4 - CCl4 + S), rats receiving both CCl4 and cakes with papaya seeds (Group 5 - CCl4 + CPS), and rats receiving both CCl4 and silymarin with papaya seed-enriched cakes (Group 6 - CCl4 + CPS + S). HPLC analysis of papaya seeds revealed the presence of ten polyphenol compounds, with quercetin, apigenin, and catechin identified as major flavonoids, along with pyrogallol, ellagic, and gallic acid as predominant phenolic acids. These compounds displayed potent antioxidant activity, attributed to the seeds' high total phenolic and flavonoid content. The administration of CCl4 significantly affected hematological parameters, liver enzymes, hepatic oxidative stress, levels of TNF-α, IL-6, IgG, as well as IgM. However, rats fed with CPS exhibited mitigation of CCl4-induced toxic effects on hematological parameters and hepatotoxicity. CPS consumption enhanced the antioxidant system, improved inflammatory markers, and immune parameters, restoring them to normal levels. Histopathological analysis confirmed CPS's ability to reduce CCl4-induced hepatocellular necrosis. Immunohistochemical assessment further revealed reduced immunoreactivity against cleaved caspase-3 expression and increased COX2 immunoreactivity, indicating hepatocellular regeneration in CPS. The combination of CPS and silymarin demonstrated even more notable improvements, suggesting augmented protective impacts against CCl4-induced immunosuppression and hepatotoxicity. In conclusion, CPS exhibited antioxidant properties and effectively protected against CCl4-induced immunotoxicity and hepatotoxicity, with additional benefits observed when combined with silymarin. These findings emphasize the potential health advantages of incorporating papaya seeds into food products, promoting immune system health, and safeguarding against liver damage induced by hazardous agents like CCl4.

ADAMTS12 is a stromal modulator in chronic liver disease.

Adamalysins, a family of metalloproteinases containing a disintegrin and metalloproteinases (ADAMs) and ADAM with thrombospondin motifs (ADAMTSs), belong to the matrisome and play important roles in various biological and pathological processes, such as development, immunity and cancer. Using a liver cancer dataset from the International Cancer Genome Consortium, we developed an extensive in silico screening that identified a cluster of adamalysins co-expressed in livers from patients with hepatocellular carcinoma (HCC). Within this cluster, ADAMTS12 expression was highly associated with recurrence risk and poorly differentiated HCC signatures. We showed that ADAMTS12 was expressed in the stromal cells of the tumor and adjacent fibrotic tissues of HCC patients, and more specifically in activated stellate cells. Using a mouse model of carbon tetrachloride-induced liver injury, we showed that Adamts12 was strongly and transiently expressed after a 24 h acute treatment, and that fibrosis was exacerbated in Adamts12-null mice submitted to carbon tetrachloride-induced chronic liver injury. Using the HSC-derived LX-2 cell line, we showed that silencing of ADAMTS12 resulted in profound changes of the gene expression program. In particular, genes previously reported to be induced upon HSC activation, such as PAI-1, were mostly down-regulated following ADAMTS12 knock-down. The phenotype of these cells was changed to a less differentiated state, showing an altered actin network and decreased nuclear spreading. These phenotypic changes, together with the down-regulation of PAI-1, were offset by TGF-ß treatment. The present study thus identifies ADAMTS12 as a modulator of HSC differentiation, and a new player in chronic liver disease.

Reparative and Antioxidant Effects of New Analogues of Immunomodulator Thymogen in Experimental Model of Liver Damage.

We studied the reparative and antioxidant effects of Thymogen and its new structural analogues obtained by binding amino acid D-Ala to the N- or C-end of the peptide molecule in acute toxic hepatopathy. Intragastric administration of carbon tetrachloride for 5 days caused the development of fat degeneration of hepatocytes, a decrease in catalase activity, and an increase in malondialdehyde concentration. Administration of peptides suppressed oxidative peroxidation and stimulated reparative regeneration of hepatocytes; Thymogen analogues produced more pronounced hepatotropic and antioxidant effects than Thymogen. Inclusion of D-Ala enhanced the effect of Thymogen on the processes of regeneration in hepatocytes and the antioxidant effect under conditions of acute carbon tetrachloride hepatopathy. The highest efficiency was achieved when the amino acid was added to the C-end of the molecule.

Neuroblastoma RAS viral oncogene homolog (N-RAS) deficiency aggravates liver injury and fibrosis.

Progressive hepatic damage and fibrosis are major features of chronic liver diseases of different etiology, yet the underlying molecular mechanisms remain to be fully defined. N-RAS, a member of the RAS family of small guanine nucleotide-binding proteins also encompassing the highly homologous H-RAS and K-RAS isoforms, was previously reported to modulate cell death and renal fibrosis; however, its role in liver damage and fibrogenesis remains unknown. Here, we approached this question by using N-RAS deficient (N-RAS-/-) mice and two experimental models of liver injury and fibrosis, namely carbon tetrachloride (CCl4) intoxication and bile duct ligation (BDL). In wild-type (N-RAS+/+) mice both hepatotoxic procedures augmented N-RAS expression in the liver. Compared to N-RAS+/+ counterparts, N-RAS-/- mice subjected to either CCl4 or BDL showed exacerbated liver injury and fibrosis, which was associated with enhanced hepatic stellate cell (HSC) activation and leukocyte infiltration in the damaged liver. At the molecular level, after CCl4 or BDL, N-RAS-/- livers exhibited augmented expression of necroptotic death markers along with JNK1/2 hyperactivation. In line with this, N-RAS ablation in a human hepatocytic cell line resulted in enhanced activation of JNK and necroptosis mediators in response to cell death stimuli. Of note, loss of hepatic N-RAS expression was characteristic of chronic liver disease patients with fibrosis. Collectively, our study unveils a novel role for N-RAS as a negative controller of the progression of liver injury and fibrogenesis, by critically downregulating signaling pathways leading to hepatocyte necroptosis. Furthermore, it suggests that N-RAS may be of potential clinical value as prognostic biomarker of progressive fibrotic liver damage, or as a novel therapeutic target for the treatment of chronic liver disease.

Hydroxyurea attenuates hepatic stellate cell proliferation in vitro and liver fibrogenesis in vivo.

Liver fibrosis results from excessive proliferation of, and collagen production by hepatic stellate cells (HSCs) that is caused by chronic liver injury. No drugs are available to cure liver fibrosis. Hydroxyurea is an anti-proliferative drug that is used in benign and malignant disorders. Here, we studied the effect of hydroxyurea on primary HSCs and its anti-fibrotic effect in the CCl4 mouse model of liver fibrosis. Primary rat HSCs were cultured in the absence or presence of hydroxyurea (0.1-1.0 mmol/L). CCl4 or vehicle was administered to C57BL/6/J mice for 4 weeks, with or without hydroxyurea (100 mg/kg/day) co-treatment. We used real-time cell proliferation analysis, Oil Red O (lipid droplet) staining, immunohistochemistry, Acridine Orange staining (apoptosis), Sytox green staining (necrosis), RT-qPCR, ELISA, and Western Blotting for analysis. Hydroxyurea dose-dependently suppressed lipid droplet-loss and mRNA levels of Col1α1 and Acta2 in transdifferentiating HSCs. In fully-activated HSCs, hydroxyurea dose-dependently attenuated PCNA protein levels and BrdU incorporation, but did not reverse Col1α1 and Acta2 mRNA expression. Hydroxyurea did not induce apoptosis or necrosis in HSCs or hepatocytes. Hydroxyurea suppressed accumulation of desmin-positive HSCs and hepatic collagen deposition after CCl4 treatment. CCl4 -induced regenerative hepatocyte proliferation, Col1α1 and Acta2 mRNA expression and α-SMA protein levels were not affected. This study demonstrates that hydroxyurea inhibits HSC proliferation in vitro and attenuates early development of liver fibrosis in vivo, while preserving hepatocyte regeneration after toxic insults by CCl4. Thus, hydroxyurea may have therapeutic value against liver fibrosis.

Liver fibrotic development is reduced through inflammation prevention by an adenosine derivative compound.

BACKGROUND: Liver fibrosis is a global health problem, and studying its development provides important information to address its treatment. Here, we characterized the effects of an adenosine compound (IFC-305) on preventing fibrosis and liver inflammation. METHODS: We studied the impact of IFC-305 on a carbon tetrachloride-induced liver fibrosis model in Wistar male rats at 4, 6, and 8 weeks. The effects were characterized by liver tissue histology, macrophages identification by flow cytometry with CD163+/CD11b/c+ antibodies, hepatic and plasmatic cytokine levels employing MILLIPLEX MAP and ELISA, Col1a1 and Il6 gene expression by RTqPCR, lipoperoxidation by TBARS reaction, and reactive oxygen species using 2'-7'dichlorofluorescin diacetate. RESULTS: CCl4-induced liver fibrosis and inflammation were significantly reduced in rats treated with IFC-305 at 6 and 8 weeks. In addition, we observed diminished expression of Col1a1; a decrease in the inflammatory cytokines IL-1ß, IL-6, MCP-1, TNF-α, and IL-4 a; reduction in inflammatory macrophages; inhibition of lipoperoxidation; and ROS production in Kupffer cells. CONCLUSION: This study showed that IFC-305 can inhibit liver fibrosis establishment by regulating the immune response during CCl4-induced damage. The immunomodulatory action of IFC-305 supports its use as a potential therapeutic strategy for preventing liver fibrosis.