Duct Ligation-induced Liver Fibrosis Research Articles

L-carnitine ameliorates bile duct ligation induced liver fibrosis via reducing the nitrosative stress in experimental animals: preclinical evidences

Bile duct ligation (BDL) has been extensively used in studying the mechanisms of fibrogenesis and anti-fibrotic drugs. Considering the liver regenerative capacity and the diverse results from BDL, the present study aimed to evaluate the protective effect of L-carnitine on bile duct ligation-induced liver fibrosis in experimental rats. Rats were randomly divided into seven groups (n = 6). The bile duct was ligated and serum aspartate transaminase (AST), alanine transaminase (ALT), total bilirubin and albumin, hepatic hydroxyproline (HP), reduced glutathione (GSH), and malondialdehyde (MDA) and cytokines were measured. iNOS expression was measured by using Western blot and finally, liver tissue was processed for histopathological analysis (H&E staining)”. The level of iNOS was increased in the control group, whereas a decrease in the level of iNOS was found in the L-carnitine treated group. In the present study, we found that bile duct ligation in rats showed an increase in body and liver weight, while treatment with carnitine showed normal body and liver weight. Serum AST, ALT, total bilirubin, HP, GSH, MDA, and cytokines were increased in bile duct ligated rats. In addition, L-carnitine treated rats showed a reduction in oxidative stress as well as inhibiting the release of cytokines in a dose-dependent manner and showed protection against bile duct ligation. The study concludes that L-carnitine has a protective effect against the liver fibrosis induced by bile duct ligation.

The ameliorative effect of niclosamide on bile duct ligation induced liver fibrosis via suppression of NOTCH and Wnt pathways

Liver fibrosis is the conjoint consequence of almost all chronic liver diseases. Cholestatic liver injury is a significant stimulus for fibrotic liver. This study was conducted to investigate the hepatoprotective effect of niclosamide as a NOTCH inhibitor and on the Wnt pathway against cholestatic liver fibrosis (CLF) which was experimentally induced by bile duct ligation (BDL). Rats were randomly divided into five main groups (6 per group): sham, BDL, BDL/niclosamide 5, BDL/niclosamide 10 and niclosamide 10 only group. Niclosamide was administered intraperitoneally (i.p.) for 4 weeks starting at the same day of surgery at doses 5 and 10 mg/kg. Liver function, cholestasis, oxidative stress, inflammation, liver fibrosis, NOTCH signaling pathway and Wnt pathway markers were assessed. Niclosamide (5 and 10 mg/kg) significantly reduced liver enzymes levels, oxidative stress, inflammation and phosphorylated signal transducer and activator of transcription3 (p-STAT3). Niclosamide (5 and 10 mg/kg) also significantly reduced NOTCH pathway (Jagged1, NOTCH2, NOTCH3, HES1, SOX9), Wnt pathway (Wnt5B, and Wnt10A), and fibrosis (transforming growth factor-beta1 (TGF-β1), alpha smooth muscle actin (α-SMA) and collagen deposition with more prominent effect of the higher dose 10 mg/kg. So, this study presents nicloamide as a promising antifibrotic agent in CLF through inhibition of NOTCH and Wnt pathways.

Human induced pluripotent stem cell–derived extracellular vesicles reduce hepatic stellate cell activation and liver fibrosis

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

Gambogic acid attenuates liver fibrosis by inhibiting the PI3K/AKT and MAPK signaling pathways via inhibiting HSP90

Gambogic acid (GA), a major ingredient of Garcinia hanburryi, is known to have diverse biological effects. The present study was designed to evaluate the anti-fibrotic effects of GA on hepatic fibrosis and reveal its underlying mechanism. We investigated the anti-fibrotic effect of GA on dimethylnitrosamine and bile duct ligation induced liver fibrosis in rats in vivo. The rat and human hepatic stellate cell lines (HSCs) lines were chose to evaluate the effect of GA in vitro. Our results indicated that GA could significantly ameliorate liver fibrosis associated with improving serum markers, decrease in extracellular matrix accumulation and HSCs activation in vivo. GA significantly inhibited the proliferation of HSC cells and induced the cell cycle arrest at the G1 phase. Moreover, GA triggered autophagy at early time point and subsequent initiates mitochondrial mediated apoptotic pathway resulting in HSC cell death. The mechanism of GA was related to inhibit heat shock protein 90 (HSP90) and degradation of the client proteins inducing PI3K/AKT and MAPK signaling pathways inhibition. This study demonstrated that GA effectively ameliorated liver fibrosis in vitro and in vivo, which provided new insights into the application of GA for liver fibrosis.

Antifibrotic effect of N-acetyl-seryl-aspartyl-lysyl-proline on bile duct ligation induced liver fibrosis in rats.

To investigate the preventive effect of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) on bile duct ligation (BDL)-induced liver fibrosis in rats. Liver fibrosis in rats was induced by BDL and AcSDKP was infused subcutaneously for 2 wk via a osmotic minipump (Alzet 2ML4) immediately after BDL operation. After scarifying, serum and liver specimens were collected. Hematoxylin and eosin staining, Sirius red staining, enzyme linked immunosorbent assay, Western blot or real-time polymerase chain reaction were used to determinate liver functions, histological alterations, collagen deposition, mRNA expression of markers for fibroblasts, transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7). When compared to model rats, chronic exogenous AcSDKP infusion suppressed profibrogenic TGF-β1 signaling, α-smooth muscle actin positivity (α-SMA), fibroblast specific protein-1 (FSP-1) staining and collagen gene expression. Col I, Col III, matrix metalloproteinase-2, tissue inhibitors of metalloproteinase-1 and tissue inhibitors of metalloproteinase-2 mRNA expressions were all significantly downregulated by AcSDKP infusion (2.02 ± 1.10 vs. 14.16 ± 6.50, 2.02 ± 0.45 vs. 10.00 ± 3.35, 2.91 ± 0.30 vs. 7.83 ± 1.10, 4.64 ± 1.25 vs. 18.52 ± 7.61, 0.46 ± 0.16 vs. 0.34 ± 0.12, respectively, P < 0.05). Chronic exogenous AcSDKP infusion attenuated BDL-induced liver injury, inflammation and fibrosis. BDL caused a remarkable increase in alanine transaminase, aspartate transaminase, total bilirubin, and prothrombin time, all of which were reduced by AcSDKP infusion. Mast cells, collagen accumulation, α-SMA, TGF-β1, FSP-1 and BMP-7 increased. The histological appearance of liver specimens was also improved. Infusion of exogenous AcSDKP attenuated BDL-induced fibrosis in the rat liver. Preservation of AcSDKP may be a useful therapeutic approach in the management of liver fibrosis.

Rifaximin Attenuates Bile Duct Ligation Induced Liver Fibrosis and Portal Hypertension Through Inhibition of the TLR4 Pathway

Rifaximin Attenuates Bile Duct Ligation Induced Liver Fibrosis and Portal Hypertension Through Inhibition of the TLR4 Pathway

Deficiency of phosphatidylinositol 3-kinase (PI3K) aggravates hepatic inflammation and fibrosis in experimental NASH but protects against bile duct ligation induced liver fibrosis

Aims: Non alcoholic steatohepatitis (NASH) leads to liver fibrosis characterized by activation of hepatic stellate cells (HSC) and enhanced collagen expression. We were able to show that PI3K, an ubiquitary heterodimeric lipid kinase, participates in HSC proliferation and collagen expression. Therefore, the aim of this study was to evaluate the role of PI3K in experimental NASH and biliary fibrosis. Methods: PI3K knockout mice (PI3K-/-, C57Bl/6 background) and C57Bl/6 wild-type control mice (WT) were allocated to 4 experimental groups (n=6) receiving either standard chow (SC) or a high fat (HF) diet according. In addition, bile duct ligation (BDL) and sham operations were performed. Results: Histological analysis revealed hepatic steatosis, inflammation and fibrosis in WT mice receiving a HF fat diet. However, these histological features of NASH were more pronounced PI3K-/- mice. Serum analysis showed elevated liver enzymes in the HF WT group, which were significantly higher in PI3K-/- mice. Hepatic mRNA expression of proinflammatory and profibrogenic genes was significantly induced in PI3K-/- HF mice compared to WT HF mice, as was expression of TLR4 and -9. Accordingly, elevated collagen I protein and alpha-smooth muscle actin (SMA) expression was evident. Furthermore, gene expression indicating oxidative stress was increased in PI3K-/- mice. In contrast, PI3K-/- mice were protected from BDL-induced liver injury indicated by lower serum transaminases, as well as decreased proinflammtory and profibrogenic gene expression, and less fibrosis. Interestingly, increased hepatic expression of PI3K p110 gamma was deteced in WT mice following BDL but not in mice with NASH. Conclusion: PI3K deficiency differently affects hepatic inflammation and fibrosis, depending on the type of injury and the predominant cell-type involved. These differences may have implications with regards to diagnosis and therapy of (fibrosis progression in) non-alcoholic liver disease.

Deficiency of phosphatidylinositol 3-kinase (PI3K) aggravates inflammation and fibrosis in experimental non alcoholic steatohepatitis but protects against bile duct ligation induced liver fibrosis in vivo

Background: Non alcoholic steatohepatitis (NASH) leads to liver fibrosis characterized by activation of hepatic stellate cells (HSC) and collagen expression. In vitro, we were able to show that PI3K participates in HSC proliferation and collagen expression. The aim of this study was to evaluate the role of PI3K in the development of experimental NASH and biliary fibrosis.

Hepatoprotective and antioxidant activity of Phaseolus trilobus, Ait on bile duct ligation induced liver fibrosis in rats

Ethnopharmacological relevance Phaseolus trilobus Ait (Fabaceae) is extensively used by tribal people of Nandurbar district (Maharashtra, India) in the treatment of Jaundice and other liver disorders. Aim of the present study was to assess the medicinal claim of Phaseolus trilobus as hepatoprotective and antioxidant. Material and methods The hepatoprotective activity of methanol and aqueous extract of Phaseolus trilobus was evaluated by bile duct ligation induced liver fibrosis and antioxidant activity was evaluated using in vitro and in vivo antioxidant models viz anti-lipid peroxidation assay, super oxide radical scavenging assay and glutathione estimation in liver. Liver function tests were carried out to detect hepatoprotective activity, which was further supported by histopathological examination. Results Methanol and aqueous extracts of Phaseolus trilobus reduced elevated level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), bilirubin and hydroxyproline significantly ( p < 0.01) in bile duct ligated Wistar rats, proving hepatoprotective activity comparable with Silymarin. Both the extracts were found to reduce the elevated levels of serum thiobarbituric acid reactive substance (TBARS) and elevate superoxide scavenging radical activity proving antioxidant activity comparable with ascorbic acid. The reduced level of glutathione was found to be elevated in liver proving antioxidant activity comparable with Silymarin. Conclusion Phaseolus trilobus posses hepatoprotective property and is effective in oxidative stress induced cholestatic hepatic injury.

578 Human Serum Amyloid P (Hsap) Inhibits Bile Duct Ligation Induced Liver Fibrosis in Mice

578 Human Serum Amyloid P (Hsap) Inhibits Bile Duct Ligation Induced Liver Fibrosis in Mice

The Effects of N-Acetylcysteine on Bile Duct Ligation–Induced Liver Fibrosis in Rats

Stellate cells are activated by free radicals, and synthesize collagen. N-acetylcysteine (NAC) is a precursor of reduced glutathione and a potent scavenger of hydroxyl radicals and has potential antifibrotic effects. We aimed to test the effects of NAC on bile duct ligation (BDL) induced liver damage in rats. Forty-seven Wistar rats were divided into 5 groups: group 1, BDL+NAC (n=10); group 2, BDL (n=10); group 3, sham+NAC (n=10); group 4, sham (n=10); and group 5, control group (n=10). NAC (50 micromol/kg per day) or saline of single doses were administered intraperitoneally for 28 days. Serum biochemical and liver oxidative stress parameters were studied. Liver collagen level was determined by the method of Lopez de Leon and Rojkind. Liver slides were stained by hematoxylin and eosin and Masson trichrome\Gomory reticulum staining. Aspartate aminotransferase (AST) and alkaline phosphatase levels in the BDL+NAC group were lower than the BDL group and were higher than the control groups (all P< .001). Malondialdehyde, luminal, and glutathione levels in group 1 were lower than the BDL group (P= .01, P= .002, and P< .001) and higher than the control groups (all P< .001). NAC had no effect on alanine aminotransferase (ALT), gammaglutamyl transferase, bilirubin, albumin, or lucigenin levels. Liver collagen levels were higher in the BDL groups (P< .001); however, NAC had no effect on the collagen levels. The BDL groups showed stage 3 fibrosis; all the control groups were normal. NAC improved some biochemical parameters (AST, alkaline phosphatase) and oxidative stress parameters (malondialdehyde, luminol, glutathione) in the BDL model. NAC was found to be effective on cholestasis-induced hepatotoxicity. However, NAC was inefficient as an antifibrotic agent within a 1-month period of administration in the BDL model.

Bone morphogenetic protein 4 mediates bile duct ligation induced liver fibrosis through activation of Smad1 and ERK1/2 in rat hepatic stellate cells

Bone morphogenetic proteins (BMPs) are the important cytokine involving in cell differentiation especially in bone morphogenesis. Hepatic stellate cells (HSCs) undergo a trans-differentiation during their activation after liver injury. Although it has been demonstrated that BMP2 and BMP4 significantly increased the abundance of smooth muscle alpha actin (alpha-SMA) in cultured HSCs, the expression of BMPs has not been examined during the activation of HSCs. In current study, we documented the expression of BMP4 in bile duct ligation (BDL) rats and HSCs in culture. We have found that the expression of BMP4 was significantly elevated in the liver of BDL rats. The increase in BMP4 protein showed two peaks during 6 weeks after BDL. The expression and phosphorylation of Smad1, ERK1/2 and p38 were also elevated after BDL. Moreover, there was a gradual elevation of BMP4 mRNA abundance during 24 days' in vitro culture of HSCs. Furthermore, BMP4 stimulated phosphorylation of Smad1 and ERK1/2 in HSCs. In conclusion, BMP4 expression was significantly increased in the liver of BDL rats and HSCs in culture. These findings indicate that BMP4 may mediate HSC activation through activation of Smad1 and ERK1/2.

Anti‐TGF‐β Strategies for the Treatment of Chronic Liver Disease

Abstract: Permanent alcohol abuse may lead to chronic liver injury with deleterious sequelae such as liver cirrhosis and hepatocellular carcinoma. Mechanisms of fibrogenesis encompass recruitment of inflammatory cells at the site of injury and cytokine mediated activation of hepatic stellate cells (HSC) with accumulation of interstitial collagens. HSC transdifferentiation and accompanying apoptosis result in destruction of liver architecture and are therefore key steps of disease progression. TGF‐β represents the main profibrogenic cytokine in liver fibrosis and other fibroproliferative disorders by inducing extracellular matrix deposition as part of the wound healing response. In parallel, TGF‐β triggers hepatocytes that are strongly responsive for this cytokine, to undergo apoptosis, thereby providing space for HSC proliferation and generation of a collagenous matrix. Anti TGF‐β approaches were established and successfully utilized for the treatment of experimental fibrogenesis. Dominant negative TGF‐β receptors (TβR), generated by fusing the Fc domain of human IgG and the N‐terminal (extracellular) fragment of TβRII (Fc:TβRII) were applied to suppress fibrosis. Similarly TGF‐β binding proteins like decorin, antagonistic cytokines such as bone morphogenetic protein‐7, hepatocyte growth factor, IL‐10, or IFN‐γ were as efficient as camostat mesilate, a protease inhibitor that possibly abrogated proteolytic activation of TGF‐β. Further, our group recently overexpressed Smad7 in bile duct ligation induced liver fibrosis and achieved efficient inhibition of intracellular TGF‐β signaling, thereby counteracting profibrogenic effects in cultured HSC and in vivo. A direct link between the effect of alcohol and TGF‐β exists through reactive oxygen species that are generated in liver cells by alcohol metabolism and represent activators of TGF‐β signaling. Thus, soluble TβRII expression reduced experimental fibrogenesis in vitro and in vivo partially by decreasing intracellular ROS and inhibiting NADH oxidase. Approaches that specifically target profibrogenic TGF‐β signaling are promising to treat alcoholic liver disease in the future. However, to ensure safety for the patients to be treated, approaches with strong specificity need to be established. Therefore, it is essential to delineate the profibrogenic actions of TGF‐β and the influence of alcohol abuse in molecular detail.