GW501516-activated PPARβ/δ promotes liver fibrosis via p38-JNK MAPK-induced hepatic stellate cell proliferation

Background

After liver injury, the repair process comprises activation and proliferation of hepatic stellate cells (HSCs), which produce extracellular matrix (ECM) proteins. Peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is highly expressed in these cells, but its function in liver repair remains incompletely understood. This study investigated whether activation of PPARβ/δ with the ligand GW501516 influenced the fibrotic response to injury from chronic carbon tetrachloride (CCl₄) treatment in mice. Wild type and PPARβ/δ-null mice were treated with CCl₄ alone or CCl₄ co-administered with GW501516. To unveil mechanisms underlying the PPARβ/δ-dependent effects, we analyzed the proliferative response of human LX-2 HSCs to GW501516 in the presence or absence of PPARβ/δ.

Results

We found that GW501516 treatment enhanced the fibrotic response. Compared to the other experimental groups, CCl₄/GW501516-treated wild type mice exhibited increased expression of various profibrotic and pro-inflammatory genes, such as those involved in extracellular matrix deposition and macrophage recruitment. Importantly, compared to healthy liver, hepatic fibrotic tissues from alcoholic patients showed increased expression of several PPAR target genes, including phosphoinositide-dependent kinase-1, transforming growth factor beta-1, and monocyte chemoattractant protein-1. GW501516 stimulated HSC proliferation that caused enhanced fibrotic and inflammatory responses, by increasing the phosphorylation of p38 and c-Jun N-terminal kinases through the phosphoinositide-3 kinase/protein kinase-C alpha/beta mixed lineage kinase-3 pathway.

Conclusions

This study clarified the mechanism underlying GW501516-dependent promotion of hepatic repair by stimulating proliferation of HSCs via the p38 and JNK MAPK pathways.     

Hepatocyte Produced Matrix Metalloproteinases Are Regulated by CD147 in Liver Fibrogenesis

Background

The classical paradigm of liver injury asserts that hepatic stellate cells (HSC) produce, remodel and turnover the abnormal extracellular matrix (ECM) of fibrosis via matrix metalloproteinases (MMPs). In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis [1]. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC.

Methods

Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by in-situ zymography. The role of CD147 was studied in-vitro with siRNA to CD147 in hepatocytes and in-vivo in mice with CCl₄ induced liver injury using ãCD147 antibody intervention.

Results

In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14) increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, in-vivo α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-β and α-SMA expression in CCl₄ treated mice compared to controls.

Conclusion

We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo. Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be targeted by future anti-fibrogenic agents.     

The Role of Interferon-γ Inducible Protein-10 in a Mouse Model of Acute Liver Injury Post Induced Pluripotent Stem Cells Transplantation

Background

Liver injuries are important medical problems that require effective therapy. Stem cell or hepatocyte transplantation has the potential to restore function of the damaged liver and ameliorate injury. However, the regulatory factors crucial for the repair and regeneration after cell transplantation have not been fully characterized. Our study investigated the effects and the expression of the regulatory factors in mouse models of acute liver injury either transplanted with the induced pluripotent stem cells (iPS) or the hepatocytes that differentiated from iPS cells (iHL).

Methods/Principal Findings

Mice received CCl₄ injection and were randomized to receive vehicle, iPS, or iHL transfusions vial tail veins and were observed for 24, 48 or 72 hours. The group of mice with iPS transplantation performed better than the group of mice receiving iHL in reducing the serum alanine aminotransferase, aspartate aminotransferase, and liver necrosis areas at 24 hours after CCl₄ injury. Moreover, iPS significantly increased the numbers of proliferating hepatocytes at 48 hours. Cytokine array identified that chemokine IP-10 could be the potential regulatory factor that ameliorates liver injury. Further studies revealed that iPS secreted IP-10 in vitro and transfusion of iPS increased IP-10 protein and mRNA expressions in the injured livers in vivo. The primary hepatocytes and non-parenchyma cells were isolated from normal and injured livers. Hepatocytes from injured livers that received iPS treatment expressed more IP-10 mRNA than their non-hepatocyte counter-parts. In addition, animal studies revealed that administration of recombinant IP-10 (rIP-10) effectively reduced liver injuries while IP-10-neutralizing antibody attenuated the protective effects of iPS and decreased hepatocyte proliferation. Both iPS and rIP-10 significantly reduced the 72-hour mortality rate in mice that received multiple CCl₄-injuries.

Conclusions/Significance

These findings suggested that IP-10 may have an important regulatory role in facilitating the repair and regeneration of injured liver after iPS transplantation.     

Graptopetalum Paraguayense Ameliorates Chemical-Induced Rat Hepatic Fibrosis In Vivo and Inactivates Stellate Cells and Kupffer Cells In Vitro

Background

Graptopetalum paraguayense (GP) is a folk herbal medicine with hepatoprotective effects that is used in Taiwan. The aim of this study was to evaluate the hepatoprotective and antifibrotic effects of GP on experimental hepatic fibrosis in both dimethylnitrosamine (DMN)- and carbon tetrachloride (CCl₄)-induced liver injury rats.

Methods

Hepatic fibrosis-induced rats were fed with the methanolic extract of GP (MGP) by oral administration every day. Immunohistochemistry, biochemical assays, and Western blot analysis were performed. The effects of MGP on the expression of fibrotic markers and cytokines in the primary cultured hepatic stellate cells (HSCs) and Kupffer cells, respectively, were evaluated.

Results

Oral administration of MGP significantly alleviated DMN- or CCl₄-induced liver inflammation and fibrosis. High levels of alanine transaminase, aspartate transaminase, bilirubin, prothrombin activity and mortality rates also decreased in rats treated with MGP. There were significantly decreased hydroxyproline levels in therapeutic rats compared with those of the liver-damaged rats. Collagen I and alpha smooth muscle actin (α-SMA) expression were all reduced by incubation with MGP in primary cultured rat HSCs. Furthermore, MGP induced apoptotic cell death in activated HSCs. MGP also suppressed lipopolysaccharide-stimulated rat Kupffer cell activation by decreasing nitric oxide, tumor necrosis factor-α and interleukin-6 production, and increasing interleukin-10 expression.

Conclusions

The results show that the administration of MGP attenuated toxin-induced hepatic damage and fibrosis in vivo and inhibited HSC and Kupffer cell activation in vitro, suggesting that MGP might be a promising complementary or alternative therapeutic agent for liver inflammation and fibrosis.     

Overexpression of c-myc in hepatocytes promotes activation of hepatic stellate cells and facilitates the onset of liver fibrosis

BackgroundLiver fibrosis is a consequence of chronic liver injury and can further progress to hepatocellular carcinoma (HCC). Fibrogenesis involves activation of hepatic stellate cells (HSC) and proliferation of hepatocytes upon liver injury. HCC is frequently associated with overexpression of the proto-oncogene c-myc. However, the impact of c-myc for initiating pathological precursor stages such as liver fibrosis is poorly characterized. In the present study we thus investigated the impact of c-myc for liver fibrogenesis.MethodsExpression of c-myc was measured in biopsies of patients with liver fibrosis of different etiologies by quantitative real-time PCR (qPCR). Primary HSC were isolated from mice with transgenic overexpression of c-myc in hepatocytes (alb-myc^tg) and wildtype (WT) controls and investigated for markers of cell cycle progression and fibrosis by qPCR and immunofluorescence microscopy. Liver fibrosis in WT and alb-myc^tg mice was induced by repetitive CCl₄ treatment.ResultsWe detected strong up-regulation of hepatic c-myc in patients with advanced liver fibrosis. In return, overexpression of c-myc in alb-myc^tg mice resulted in increased liver collagen deposition and induction of α-smooth-muscle-actin indicating HSC activation. Primary HSC derived from alb-myc^tg mice showed enhanced proliferation and accelerated transdifferentiation into myofibroblasts in vitro. Accordingly, fibrosis initiation in vivo after chronic CCl₄ treatment was accelerated in alb-myc^tg mice compared to controls.ConclusionOverexpression of c-myc is a novel marker of liver fibrosis in man and mice. We conclude that chronic induction of c-myc especially in hepatocytes has the potential to prime resident HSC for activation, proliferation and myofibroblast differentiation.     

Decursin attenuates hepatic fibrogenesis through interrupting TGF-beta-mediated NAD(P)H oxidase activation and Smad signaling in vivo and in vitro

Aims

We studied that a potent antifibrotic effect of decursin on in vivo liver damage model and the mechanism in inhibiting which transforming growth factor (TGF)-β1-induced human hepatic stellate cells (HSCs) activation.

Main methods

Liver injury was induced in vivo by intraperitoneal injection of carbon tetrachloride (CCl₄) with or without decursin for 4 weeks in mice. Human hepatic stellate cell line, an immortalized human HSC line, was used in in vitro assay system. The effects of decursin on HSC activation were measured by analyzing the expression of α-smooth muscle actin (α-SMA) and collagen I in liver tissue and human HSCs.

Key findings

Decursin treatment significantly reduced the ratio of liver/body weight, α-SMA activation, and type I collagen overexpression in CCl₄ treated mice liver. The elevated serum levels, including ALT, AST, and ALP, were also decreased by decursin treatment. Treatment of decursin markedly proved the generation of reactive oxygen species, NAD(P)H oxidase (NOX) protein (1, 2, and 4) upregulation, NOX activity, and superoxide anion production in HSCs by TGF-β1. It also significantly reduced TGF-β1-induced Smad 2/3 phosphorylation, nuclear translocation of Smad 4, and association of Smad 2/3–Smad 4 complex. Consistent with in vitro results, decursin treatment effectively blocked the levels of NOX protein, and Smad 2/3 phosphorylation in injured mice liver.

Significance

Decursin blocked CCl₄-induced liver fibrosis and inhibited TGF-β1-mediated HSC activation in vitro. These data demonstrated that decursin exhibited hepatoprotective effects on experimental fibrosis, potentially by inhibiting the TGF-β1 induced NOX activation and Smad signaling.     

Regulator of G-Protein Signaling-5 Is a Marker of Hepatic Stellate Cells and Expression Mediates Response to Liver Injury

Liver fibrosis is mediated by hepatic stellate cells (HSCs), which respond to a variety of cytokine and growth factors to moderate the response to injury and create extracellular matrix at the site of injury. G-protein coupled receptor (GPCR)-mediated signaling, via endothelin-1 (ET-1) and angiotensin II (AngII), increases HSC contraction, migration and fibrogenesis. Regulator of G-protein signaling-5 (RGS5), an inhibitor of vasoactive GPCR agonists, functions to control GPCR-mediated contraction and hypertrophy in pericytes and smooth muscle cells (SMCs). Therefore we hypothesized that RGS5 controls GPCR signaling in activated HSCs in the context of liver injury. In this study, we localize RGS5 to the HSCs and demonstrate that Rgs5 expression is regulated during carbon tetrachloride (CCl₄)-induced acute and chronic liver injury in Rgs5^LacZ/LacZ reporter mice. Furthermore, CCl₄ treated RGS5-null mice develop increased hepatocyte damage and fibrosis in response to CCl₄ and have increased expression of markers of HSC activation. Knockdown of Rgs5 enhances ET-1-mediated signaling in HSCs in vitro. Taken together, we demonstrate that RGS5 is a critical regulator of GPCR signaling in HSCs and regulates HSC activation and fibrogenesis in liver injury.     

Inhibitory Effect of Gallic Acid on CCl4-Mediated Liver Fibrosis in Mice

The aim of this study was to investigate the effect of gallic acid (GA) on liver fibrosis induced by carbon tetrachloride (CCl₄). Male BALB/c mice were randomly divided into four groups: normal control group (group A), CCl₄-induced liver injury control group (group B), and CCl₄ induction with GA of low dose (5 mg/kg) and high dose (15 mg/kg) treatment group (group C and group D). GA was intra-gastric given for mice once a day after 2 weeks of CCl₄ induction. Animals were killed at the eighth week. Degrees of fibrosis and collagen percentage were measured. Hyaluronic acid (HA), type IV collagen (cIV), malondialdehyde (MDA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (γ-GT) were determined. Expression of matrix metalloproteinases-2 (MMP-2) and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) mRNA levels were examined by RT-PCR. Western blotting was carried out to evaluate the changes of MMP-2 protein. HE and VG stainings showed GA in a dose-dependent manner improved significantly the fibrosis condition in CCl₄-injured mice (P < 0.05 or P < 0.01). Also, the concentrations of HA, cIV, and MDA, as well as the serum levels of ALT, AST, and γ-GT were markedly reduced by GA (P < 0.05 or P < 0.01), and decreases in MMP-2, TIMP-1 mRNA, and MMP-2 protein were observed as well (P < 0.05 or P < 0.01). GA could exert protective effect on liver injury and reduce liver fibrosis induced by CCl₄ in mice, which might be through the inhibition of hepatic stellate cell activity.     

Adiponectin Reduces Hepatic Stellate Cell Migration by Promoting Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Secretion

Hepatic stellate cells (HSC) are central players in liver fibrosis that when activated, proliferate, migrate to sites of liver injury, and secrete extracellular matrix. Obesity, a known risk factor for liver fibrosis is associated with reduced levels of adiponectin, a protein that inhibits liver fibrosis in vivo and limits HSC proliferation and migration in vitro. Adiponectin-mediated activation of adenosine monophosphate-activated kinase (AMPK) inhibits HSC proliferation, but the mechanism by which it limits HSC migration to sites of injury is unknown. Here we sought to elucidate how adiponectin regulates HSC motility. Primary rat HSCs were isolated and treated with adiponectin in migration assays. The in vivo actions of adiponectin were examined by treating mice with carbon tetrachloride for 12 weeks and then injecting them with adiponectin. Cell and tissue samples were collected and analyzed for gene expression, signaling, and histology. Serum from patients with liver fibrosis was examined for adiponectin and tissue inhibitor of metalloproteinase-1 (TIMP-1) protein. Adiponectin administration into mice increased TIMP-1 gene and protein expression. In cultured HSCs, adiponectin promoted TIMP-1 expression and through binding of TIMP-1 to the CD63/β1-integrin complex reduced phosphorylation of focal adhesion kinase to limit HSC migration. In mice with liver fibrosis, adiponectin had similar effects and limited focal adhesion kinase phosphorylation. Finally, in patients with advanced fibrosis, there was a positive correlation between serum adiponectin and TIMP-1 levels. In sum, these data show that adiponectin stimulates TIMP-1 secretion by HSCs to retard their migration and contributes to the anti-fibrotic effects of adiponectin.     

TNFR1-mediated signaling is important to induce the improvement of liver fibrosis by bone marrow cell infusion

The importance of TNF-α signals mediated by tumor necrosis factor receptor type 1 (TNFR1) in inflammation and fibrosis induced by carbon tetrachloride (CCl₄), and in post-injury liver regeneration including a GFP/CCl₄ model developed as a liver repair model by bone marrow cell (BMC) infusion, was investigated. In mice in which TNFR1 was suppressed by antagonist administration or by knockout, liver fibrosis induced by CCl₄ was significantly decreased. In these mice, intrahepatic macrophage infiltration and TGF-β1 expression were reduced and stellate cell activity was decreased; however, expression of MMP-9 was also decreased. With GFP-positive BMC (TNFR1 wild-type, WT) infusion in these mice, fibrosis proliferation, including host endogenous intrahepatic macrophage infiltration, TGF-β1 expression and stellate cell activity, increased significantly. There was no significant increase of MMP-9 expression. In this study, TNFR1 in hosts had a promoting effect on CCl₄-induced hepatotoxicity and fibrosis, whereas BMC infusion in TNFR1 knockout mice enhanced host-derived intrahepatic inflammation and fibrosis proliferation. These findings differed from those in WT recipient mice, in which improvement in inflammation and fibrosis with BMC infusion had previously been reported. TNFR1-mediated signaling might be important to induce the improvement of liver fibrosis by bone marrow cell infusion.     

The Protective Roles of IL‐6 Trans‐Signaling Regulated by ADAM9 on the Liver in Carbon Tetrachloride‐Induced Liver Injury in Mice

Our study was undertaken to evaluate the important role that a disintegrin and metalloproteinase 9 (ADAM9) regulates IL‐6 trans‐signaling in carbon tetrachloride (CCl₄)‐induced liver injury in mice. Mice were divided into four groups. Each group respectively received mineral oil injection, CCl₄ injection, anti‐ADAM9 monoclonal antibody (mAb) pretreatment and CCl₄ injection, anti‐ADAM9 mAb and recombinant mouse ADAM9 molecules pretreatment with CCl₄ injection. Our results showed that anti‐ADAM9 mAb pretreatment significantly aggravated liver injury, inhibited IL‐6 trans‐signaling, which led to downregulation of proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), upregulation of Caspase3, cytochrome P450 2E1 (CYP2E1), and hepatocytes apoptosis at 24 h after CCl₄ injection. Recombinant ADAM9 molecules pretreatment reversed the impact of anti‐ADAM9 mAb pretreatment in mice. In conclusion, our study suggested that ADAM9 could regulate the hepatocytes proliferation, apoptosis, angiogenesis, and CYP2E1 expression by activating IL‐6 trans‐signaling and play important protective roles during CCl₄‐induced liver injury in mice.     

Brivanib Attenuates Hepatic Fibrosis In Vivo and Stellate Cell Activation In Vitro by Inhibition of FGF,VEGF and PDGF Signaling

MethodsIn vivo, we induced liver fibrosis by bile duct ligation (BDL), chronic carbon tetrachloride (CCl₄), and chronic thioacetamide (TAA) administration. Liver fibrosis was examined by immunohistochemistry and Western immunoblotting. In vitro, we used LX-2 human hepatic stellate cells (HSCs) to assess the effect of brivanib on stellate cell proliferation and activation.ResultsAfter in vivo induction with BDL, CCl₄, and TAA, mice treated with brivanib showed reduced liver fibrosis and decreased expression of collagen Iα1 and α-smooth muscle actin in the liver. In vitro, brivanib decreased proliferation of HSCs induced by platelet-derived growth factor (PDGF), VEGF, and FGF. Brivanib also decreased stellate cell viability and inhibited PDGFBB-induced phosphorylation of its cognate receptor.ConclusionBrivanib reduces liver fibrosis in three different animal models and decreases human hepatic stellate cell activation. Brivanib may represent a novel therapeutic approach to treatment of liver fibrosis and prevention of liver cancer.     

HMGB1 induced endothelial to mesenchymal transition in liver fibrosis: The key regulation of early growth response factor 1

BackgroundLiver fibrosis has been the focus and difficulty of medical research in the world and its concrete pathogenesis remains unclear. This study aims to observe the high-mobility group box 1 (HMGB1)-induced hepatic endothelial to mesenchymal transition (EndoMT) during the development of hepatic fibrosis, and further to explore the crucial involvement of Egr1 in this process.MethodsCarbon tetrachloride (CCl₄), diosbulbin B (DB), N-acetyl-p-aminophenol (APAP) and bile duct ligation (BDL) were used to induce liver fibrosis in mice. Serum HMGB1 content, the occurrence of EndoMT and the production of extracellular matrix (ECM) in vitro and in vivo were detected by Western-blot.ResultsThe elevated serum HMGB1 content, the occurrence of EndoMT, the production of ECM and the activation of Egr1 were observed in mice with liver fibrosis induced by CCl₄, DB, APAP or BDL. HMGB1 induced EndoMT and ECM production in human hepatic sinusoidal endothelial cells (HHSECs), and then HHSECs lost the ability to inhibit the activation of hepatic stellate cells (HSCs). The hepatic deposition of collagen, the increased serum HMGB1 content and hepatic EndoMT were further aggravated in Egr1 knockout mice. Natural compound silymarin attenuated liver fibrosis in mice induced by CCl₄ via increasing Egr1 nuclear accumulation, decreasing serum HMGB1 content and inhibiting hepatic EndoMT.ConclusionEgr1 regulated the expression of HMGB1 that induced hepatic EndoMT, which plays an important role in the development of liver fibrosis.General significance:This study provides a novel therapeutic strategy for the treatment of liver fibrosis in clinic.     

Protective effects of lipocalin-2 (LCN2) in acute liver injury suggest a novel function in liver homeostasis

Lipocalin-2 is expressed under pernicious conditions such as intoxication, infection, inflammation and other forms of cellular stress. Experimental liver injury induces rapid and sustained LCN2 production by injured hepatocytes. However, the precise biological function of LCN2 in liver is still unknown. In this study, LCN2^?/? mice were exposed to short term application of CCl₄, lipopolysaccharide and Concanavalin A, or subjected to bile duct ligation. Subsequent injuries were assessed by liver function analysis, qRT-PCR for chemokine and cytokine expression, liver tissue Western blot, histology and TUNEL assay. Serum LCN2 levels from patients suffering from liver disease were assessed and evaluated. Acute CCl₄ intoxication showed increased liver damage in LCN2^?/? mice indicated by higher levels of aminotransferases, and increased expression of inflammatory cytokines and chemokines such as IL-1β, IL-6, TNF-α and MCP-1/CCL2, resulting in sustained activation of STAT1, STAT3 and JNK pathways. Hepatocytes of LCN2^?/? mice showed lipid droplet accumulation and increased apoptosis. Hepatocyte apoptosis was confirmed in the Concanavalin A and lipopolysaccharide models. In chronic models (4 weeks bile duct ligation or 8 weeks CCl₄ application), LCN2^?/? mice showed slightly increased fibrosis compared to controls. Interestingly, serum LCN2 levels in diseased human livers were significantly higher compared to controls, but no differences were observed between cirrhotic and non-cirrhotic patients. Upregulation of LCN2 is a reliable indicator of liver damage and has significant hepato-protective effect in acute liver injury. LCN2 levels provide no correlation to the degree of liver fibrosis but show significant positive correlation to inflammation instead.     

Sulforaphane ameliorates ethanol plus carbon tetrachloride-induced liver fibrosis in mice through the Nrf2-mediated antioxidant response and acetaldehyde metabolization with inhibition of the LPS/TLR4 signaling pathway

Alcoholic liver disease (ALD)-related fibrosis results from a variety of mechanisms including the accumulation of acetaldehyde, reactive oxygen species, and hepatic overload of endogenous lipopolysaccharide (LPS). Alcohol cessation is the therapeutic mainstay for patients with all stages of ALD, whereas pharmacological strategies for liver fibrosis have not been established. Sulforaphane, a phytochemical found in cruciferous vegetables, activates nuclear factor erythroid 2-related factor 2 (Nrf2) and exerts anticancer, antidiabetic, and antimicrobial effects; however, few studies investigated its efficacy in the development of ALD-related fibrosis. Herein, we investigated the effect of sulforaphane on acetaldehyde metabolism and liver fibrosis in HepaRG and LX-2 cells, human hepatoma and hepatic stellate cell lines, respectively, as well as in a mouse model of alcoholic liver fibrosis induced by ethanol plus carbon tetrachloride (EtOH/CCl₄). Sulforaphane treatment induced the activity of acetaldehyde-metabolizing mitochondrial aldehyde dehydrogenase in HepaRG cells and suppressed the acetaldehyde-induced proliferation and profibrogenic activity in LX-2 cells with upregulation of Nrf2-regulated antioxidant genes, including HMOX1, NQO1, and GSTM3. Moreover, sulforaphane attenuated the LPS/toll-like receptor 4-mediated sensitization to transforming growth factor-β with downregulation of NADPH oxidase 1 (NOX1) and NOX4. In EtOH/CCl₄-treated mice, oral sulforaphane administration augmented hepatic acetaldehyde metabolism. Additionally, sulforaphane significantly inhibited Kupffer cell infiltration and fibrosis, decreased fat accumulation and lipid peroxidation, and induced Nrf2-regulated antioxidant response genes in EtOH/CCl₄-treated mice. Furthermore, sulforaphane treatment blunted hepatic exposure of gut-derived LPS and suppressed hepatic toll-like receptor 4 signaling pathway. Taken together, these results suggest sulforaphane as a novel therapeutic strategy in ALD-related liver fibrosis.     

Selective deletion of hepatocyte platelet-derived growth factor receptor α and development of liver fibrosis in mice

Background

Platelet-derived growth factor receptor α (PDGFRα) expression is increased in activated hepatic stellate cells (HSCs) in cirrhotic liver, while normal hepatocytes express PDGFRα at a negligible level. However, cancerous hepatocytes may show upregulation of PDGFRα, and hepatocellular carcinoma is preceded by chronic liver injury. The role of PDGFRα in non-cancerous hepatocytes and liver fibrosis is unclear. We hypothesized that upon liver injury, PDGFRα in insulted hepatocytes contributes to liver fibrosis by facilitating intercellular crosstalk between hepatocytes and HSCs.

Methods

Hepatocytes were isolated from normal and thioacetamide (TAA)-induced cirrhotic livers for assessment of PDGFRα expression. Conditional knock-out (KO) C57BL/6 mice, in which PDGFRα was selectively deleted in hepatocytes, were generated. Liver fibrosis was induced by injecting TAA for 8?weeks. Hep3B cells were transfected with a small interfering RNA (siRNA) (PDGFRα or control) and co-cultured with LX2 cells.

Results

PDGFRα expression was increased in hepatocytes from fibrotic livers compared to normal livers. Conditional PDGFRα KO mice had attenuated TAA-induced liver fibrosis with decreased HSC activation and proliferation. Immunoblot analyses revealed decreased expression of phospho-p44/42 MAPK in TAA-treated KO mice; these mice also showed almost complete suppression of the upregulation of mouse double minute 2. Although KO mice exhibited increased expression of transforming growth factor (TGF)-β and Smad2/3, this was compensated for by increased expression of inhibitory Smad7. LX2 cells co-cultured with PDGFRα siRNA-infected Hep3B cells showed decreased PDGFRα, α smooth muscle actin, collagen α1(I), TGFβ, and Smad2/3 expression. LX2/PDGFRα-deleted hepatocyte co-culture medium showed decreased PDGF-BB and PDGF-CC levels.

Conclusions

Deletion of PDGFRα in hepatocytes attenuated the upregulation of PDGFRα in HSCs after TAA treatment, resulting in decreased liver fibrosis and HSC activation. This suggests that in the event of chronic liver injury, PDGFRα in hepatocytes plays an important role in liver fibrosis by affecting PDGFRα expression in HSCs.

     

Distinct roles of ecto-nucleoside triphosphate diphosphohydrolase-2 (NTPDase2) in liver regeneration and fibrosis

Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) are cell surface-located transmembrane ecto-enzymes of the CD39 superfamily which regulate inflammation and tissue repair by catalyzing the phosphohydrolysis of extracellular nucleotides and modulating purinergic signaling. In the liver, NTPDase2 is reportedly expressed on portal fibroblasts, but its functional role in regulating tissue regeneration and fibrosis is incompletely understood. Here, we studied the role of NTPDase2 in several models of liver injury using global knockout mice. Liver regeneration and severity of fibrosis were analyzed at different time points after exposure to carbon tetrachloride (CCl₄) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or partial hepatectomy in C57BL/6 wild-type and globally NTPDase2-deficient (Entpd2 null) mice. After chronic CCl₄ intoxication, Entpd2 null mice exhibit significantly more severe liver fibrosis, as assessed by collagen content and histology. In contrast, deletion of NTPDase2 does not have a substantial effect on biliary-type fibrosis in the setting of DDC feeding. In injured livers, NTPDase2 expression extends from the portal areas to fibrotic septae in pan-lobular (CCl₄-induced) liver fibrosis; the same pattern was observed, albeit to a lesser extent in biliary-type (DDC-induced) fibrosis. Liver regeneration after partial hepatectomy is not substantively impaired in global Entpd2 null mice. NTPDase2 protects from liver fibrosis resulting from hepatocellular injury induced by CCl₄. In contrast, Entpd2 deletion does not significantly impact fibrosis secondary to DDC injury or liver regeneration after partial hepatectomy. Our observations highlight mechanisms relating to purinergic signaling in the liver and indicate possible therapeutic avenues and new cellular targets to test in the management of hepatic fibrosis.