Murine Model Of Liver Fibrosis Research Articles

γδT Cells Suppress Liver Fibrosis via Strong Cytolysis and Enhanced NK Cell-Mediated Cytotoxicity Against Hepatic Stellate Cells.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins, resulting from maladaptive wound healing responses to chronic liver injury. γδT cells are important in chronic liver injury pathogenesis and subsequent liver fibrosis; however, their role and underlying mechanisms are not fully understood. The present study aims to assess whether γδT cells contribute to liver fibrosis regression. Using a carbon tetrachloride (CCl4)-induced murine model of liver fibrosis in wild-type (WT) and γδT cell deficient (TCRδ−/−) mice, we demonstrated that γδT cells protected against liver fibrosis and exhibited strong cytotoxicity against activated hepatic stellate cells (HSCs). Further study show that chronic liver inflammation promoted hepatic γδT cells to express NKp46, which contribute to the direct killing of activated HSCs by γδT cells. Moreover, we identified that an IFNγ-producing γδT cell subset (γδT1) cells exhibited stronger cytotoxicity against activated HSCs than the IL-17-producing subset (γδT17) cells upon chronic liver injury. In addition, γδT cells promoted the anti-fibrotic ability of conventional natural killer (cNK) cells and liver-resident NK (lrNK) cells by enhancing their cytotoxicity against activated HSCs. The cell crosstalk between γδT and NK cells was shown to depend partly on co-stimulatory receptor 4-1BB (CD137) engagement. In conclusion, our data confirmed the protective effects of γδT cells, especially the γδT1 subset, by directly killing activated HSCs and increasing NK cell-mediated cytotoxicity against activated HSCs in CCl4-induced liver fibrosis, which suggest valuable therapeutic targets to treat liver fibrosis.

The therapeutic effects of interleukin 10 gene-modified bone marrow-derived dendritic cells in a murine model of liver fibrosis

Objective: To investigate the therapeutic effects and mechanisms of interleukin-10 (IL-10) gene-modified dendritic cells (DC-IL-10) in mice with liver fibrosis. Methods: DC-IL-10 was constructed in vitro, the phenotype and function of which were evaluated by flow cytometry. BALB/c mice were treated with intraperitoneal injection of carbon tetrachloride (CCl4) to establish liver fibrotic model. DC-IL-10 was administrated via tail vein. Animals were divided into 4 groups including normal dendritic cell(DC) control, liver fibrosis only, negative lentiviral transfection DC (DC-mock) and DC-IL-10. Liver function, cytokine secretion, T lymphocyte differentiation and liver histomorphology were tested. Real-time PCR and western blot were used to analyze the effect of DC-IL-10 on Wnt/β-catenin signaling pathway and its role in liver fibrosis. Results: When compared with DC control and DC-mock, the expression of DC-IL-10 surface stimulating molecules (major histocompatibity complex-Ⅱ, CD(80), CD(86)) were significantly decreased (F=14.708, 22.503, 12.595, respectively, all P<0.05), and DC-IL-10 significantly inhibited T lymphocyte proliferation (F=50.295, P<0.05). When compared with liver fibrosis group, serum alanine aminotransferase and aspartate transaminase were decreased in DC-IL-10 treated group (all P<0.05), other parameters including inflammatory factors (tumor necrosis factor α, IL-6, IL-1β) reduced (all P <0.05), the proportion of regulatory T cells (Treg) increased (F=6.742, P<0.05), pathological damage improved, the expression of Wnt3a, α-SMA and β-catenin mRNA and protein significantly reduced in DC-IL-10 treatment group (all P<0.001) . Conclusions: DC-IL-10 induces elevation of Treg for immune tolerance, as well as inhibition of inflammatory response, block of Wnt/β-catenin signaling pathway, which translates into improvement of liver fibrosis.

Activation of autophagy is required for Oroxylin A to alleviate carbon tetrachloride-induced liver fibrosis and hepatic stellate cell activation

Liver fibrosis is a reversible pathophysiological process correlated with intense repair and cicatrization mechanisms, and its end-stage cirrhosis is responsible for high morbidity and mortality worldwide. Interestingly, the use of natural products as a realistic option for the treatment of liver fibrosis has broadly been accepted. Oroxylin A, a safe and natural product, shows a wide range of pharmacological activities such as anti-inflammatory, anti-oxidant, and anti-tumor properties. However, the effects of Oroxylin A on liver fibrosis remain poorly understood. In the present study, we sought to determine the effect of Oroxylin A on carbon tetrachloride (CCl4)-induced liver fibrosis, and to further examine the molecular mechanisms. We found that treatment with Oroxylin A markedly decreased the level of liver injury markers, alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT), in a dose dependent manner. Moreover, Oroxylin A treatment remarkably inhibited extracellular matrix (ECM) deposition, and significantly down-regulated the mRNA and protein expression of liver fibrosis markers including α1(I)collagen, fibronectin, alpha-smooth muscle actin (α-SMA), PDGF-βR, and TGF-βR1 in CCl4-induced murine model of liver fibrosis. Furthermore, experimental results in vitro showed that Oroxylin A treatment reduced the mRNA and protein expression of HSC activation markers, α-SMA, desmin, α1 (I) collagen, fibronectin, TGF-β, and TNF-α, in a dose dependent manner. Attractively, Oroxylin A treatment also markedly up-regulated the expression of autophagy makers, LC3-B, Atg3, Atg4, Atg5, Beclin1/Atg6, Atg7, Atg9, ATG12, and Atg14, and apparently reduced the expression of autophagy substrate p62 in both CCl4-induced murine model of liver fibrosis and PDGF-BB-treated HSCs. Importantly, inhibition of autophagy by specific inhibitor 3-methyladenine (3-MA) completely abolished Oroxylin A-induced anti-fibrosis effect, indicating that activation of autophagy was required for Oroxylin A to alleviate liver fibrosis. Overall, these results provide novel implications to reveal the molecular mechanism of Oroxylin A-induced anti-fibrosis properties, by which points to the possibility of using Oroxylin A for the treatment of liver fibrosis.

Antifibrotic Effect of Marine Ovothiol in an In Vivo Model of Liver Fibrosis.

Liver fibrosis is a complex process caused by chronic hepatic injury, which leads to an excessive increase in extracellular matrix protein accumulation and fibrogenesis. Several natural products, including sulfur-containing compounds, have been investigated for their antifibrotic effects; however, the molecular mechanisms underpinning their action are partially still obscure. In this study, we have investigated for the first time the effect of ovothiol A, π-methyl-5-thiohistidine, isolated from sea urchin eggs on an in vivo murine model of liver fibrosis. Mice were intraperitoneally injected with carbon tetrachloride (CCl4) to induce liver fibrosis and treated with ovothiol A at the dose of 50 mg/kg 3 times a week for 2 months. Treatment with ovothiol A caused a significant reduction of collagen fibers as observed by histopathological changes and serum parameters compared to mice treated with control solution. This antifibrotic effect was associated to the decrease of fibrogenic markers involved in liver fibrosis progression, such as the transforming growth factor (TGF-β), the α-smooth muscle actin (α-SMA), and the tissue metalloproteinases inhibitor (TIMP-1). Finally, we provided evidence that the attenuation of liver fibrosis by ovothiol A treatment can be regulated by the expression and activity of the membrane-bound γ-glutamyl-transpeptidase (GGT), which is a key player in maintaining intracellular redox homoeostasis. Overall, these findings indicate that ovothiol A has significant antifibrotic properties and can be considered as a new marine drug or dietary supplement in potential therapeutic strategies for the treatment of liver fibrosis.

Nootkatone confers hepatoprotective and anti-fibrotic actions in a murine model of liver fibrosis by suppressing oxidative stress, inflammation, and apoptosis.

In this study, the hepatoprotective and anti-fibrotic actions of nootkatone (NTK) were investigated using carbon tetrachloride (CCl4 )-induced liver fibrosis in mice. CCl4 administration elevated serum aspartate and alanine transaminases levels, respectively. In addition, CCl4 produced hepatic oxidative and nitrative stress, characterized by diminished hemeoxygenase-1 expression, antioxidant defenses, and accumulation of 4-hydroxynonenal and 3-nitrotyrosine. Furthermore, CCl4 administration evoked profound expression of pro-inflammatory cytokine expressions such as tumor necrosis factor-α, monocyte chemoattractant protein-1, and interleukin-1β in hepatic tissues, which corroborated with nuclear factor κB activation. Additionally, CCl4 -treated animals exhibited higher apoptosis, characterized by increased caspase 3 activity, DNA fragmentation, and poly (ADP-ribose) polymerase activation. Moreover, histological and biochemical investigations revealed marked fibrosis in the livers of CCl4 -administered animals. However, NTK treatment mitigated CCl4 -induced phenotypic changes. In conclusion, our findings suggest that NTK exerts hepatoprotective and anti-fibrotic actions by suppressing oxidative stress, inflammation, and apoptosis.

Endoglin in human liver disease and murine models of liver fibrosis-A protective factor against liver fibrosis.

Background & AimsLiver fibrosis is the outcome of chronic liver injury. Transforming growth factor‐β (TGF‐β) is a major profibrogenic cytokine modulating hepatic stellate cell (HSC) activation and extracellular matrix homeostasis. This study analyses the effect of Endoglin (Eng), a TGF‐β type III auxiliary receptor, on fibrogenesis in two models of liver injury by HSC‐specific endoglin deletion.MethodsEng expression was measured in human and murine samples of liver injury. After generating GFAPCre(+)EngΔ HSC mice, the impact of Endoglin deletion on chronic liver fibrosis was analysed. For in vitro analysis, Engflox/flox HSCs were infected with Cre‐expressing virus to deplete Endoglin and fibrogenic responses were analysed.ResultsEndoglin is upregulated in human liver injury. The receptor is expressed in liver tissues and mesenchymal liver cells with much higher abundance of the L‐Eng splice variant. Comparing GFAPC re(−)Engf/f to GFAPC re(+)EngΔ HSC mice in toxic liver injury, livers of GFAPC re(+)EngΔ HSC mice showed 39.9% (P < .01) higher Hydroxyproline content compared to GFAPC re(−)Engf/f littermates. Sirius Red staining underlined these findings, showing 58.8% (P < .05) more Collagen deposition in livers of GFAPC re(+)EngΔ HSC mice. Similar results were obtained in mice subjected to cholestatic injury.ConclusionEndoglin isoforms are differentially upregulated in liver samples of patients with chronic and acute liver injury. Endoglin deficiency in HSC significantly aggravates fibrosis in response to injury in two different murine models of liver fibrosis and increases α‐SMA and fibronectin expression in vitro. This suggests that Endoglin protects against fibrotic injury, likely through modulation of TGF‐β signalling.

Oroxylin A prevents angiogenesis of LSECs in liver fibrosis via inhibition of YAP/HIF-1α signaling.

Angiogenesis of liver sinusoidal endothelial cells (LSECs) accompanies with hypoxia in liver fibrosis and they are of mutual promotion, which has raised wide concern. Here we established murine model of liver fibrosis and found that oroxylin A (40 mg/kg) could ameliorate angiogenesis in liver fibrosis may related to hypoxia inducible factor 1α (HIF-1α). The underlying mechanism was further investigated by isolating and culturing murine primary LSECs. Hypoxia induced vascular endothelial growth factor A (VEGF-A), angiopoietin 2 (Ang-2), and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) elevated in LSECs were reduced by oroxylin A or acriflavine (ACF, an HIF-1α inhibitor), indicating HIF-1α involved the angiogenesis of LSECs. Additionally, interference with Yes-associated protein (YAP) significant downregulated the protein expression of HIF-1α and VEGF-A, while YAP plasmid exhibited an opposite effect. We next found that oroxylin A inhibited hypoxia-induced nuclear translocation of YAP, which may influence the accumulation of HIF-1α and subsequently decrease transcription of downstream target gene including VEGF-A and Ang-2, thereby exerting an anti-angiogenic activity.

SerpinB3 Promotes Pro-fibrogenic Responses in Activated Hepatic Stellate Cells

SerpinB3 is a hypoxia- and hypoxia-inducible factor-2α-dependent cystein protease inhibitor that is up-regulated in hepatocellular carcinoma and in parenchymal cells during chronic liver diseases (CLD). SerpinB3 up-regulation in CLD patients has been reported to correlate with the extent of liver fibrosis and the production of transforming growth factor-β1, but the actual role of SerpinB3 in hepatic fibrogenesis is still poorly characterized. In the present study we analyzed the pro-fibrogenic action of SerpinB3 in cell cultures and in two different murine models of liver fibrosis. “In vitro” experiments revealed that SerpinB3 addition to either primary cultures of human activated myofibroblast-like hepatic stellate cells (HSC/MFs) or human stellate cell line (LX2 cells) strongly up-regulated the expression of genes involved in fibrogenesis and promoted oriented migration, but not cell proliferation. Chronic liver injury by CCl4 administration or by feeding a methionine/choline deficient diet to transgenic mice over-expressing human SerpinB3 in hepatocytes confirmed that SerpinB3 over-expression significantly increased the mRNA levels of pro-fibrogenic genes, collagen deposition and αSMA-positive HSC/MFs as compared to wild-type mice, without affecting parenchymal damage. The present study provides for the first time evidence that hepatocyte release of SerpinB3 during CLD can contribute to liver fibrogenesis by acting on HSC/MFs.

SiRNA-mediated in vivo gene knockdown by acid-degradable cationic nanohydrogel particles

Cationic nanohydrogel particles have become an attractive tool for systemic siRNA delivery, but improvement of their in vivo tolerance is desirable, especially to prevent potential long term side effects by tissue and cellular accumulation. Here, we designed novel ketal cross-linked cationic nanohydrogel particles that were assessed for reduced tissue accumulation and robust siRNA delivery in vitro and in vivo. An oligo-amine cross-linker equipped with a ketal moiety in its core was synthesized and applied to nanohydrogel cross-linking of self-assembled reactive ester block copolymers in DMSO. The resulting acid-sensitive cationic nanoparticles spontaneously disassembled over time in acidic milieu, as investigated by dynamic light scattering. Fluorescent correlation spectroscopy showed effective complexation with siRNA as well as its release upon particle degradation at endosomal pH. These properties resulted in an enhanced in vitro gene knockdown for the acid-degradable cationic nanoparticles compared to their non-degradable spermine analogues. In a murine liver fibrosis model enhanced carrier and payload accumulation in the fibrotic tissue facilitated sequence-specific gene knockdown and prevented fibrosis progression. Long-term monitoring of the carrier in the body showed an enhanced clearance for the acid-degradable carrier, even after multiple dosing. Therefore, these acid-degradable cationic nanohydrogel particles can be considered as promising siRNA carriers for in vivo purposes towards therapeutic applications.

Rapamycin ameliorates CCl4-induced liver fibrosis in mice through reciprocal regulation of the Th17/Treg cell balance.

Previous investigations have suggested that the activation of Th17 cells and/or deficiency of regulatory T cells (Tregs) are involved in the pathogenesis of liver fibrosis. The aim of the present study was to investigate the effect of rapamycin on immune responses in a carbon tetrachloride (CCl4)-induced murine liver fibrosis model. Liver fibrosis was induced by intraperitoneal administration with CCl4. Following injection of CCl4, the mice were treated intraperitoneally with rapamycin (1.25 mg/kg/day) for 8 weeks. Hematoxylin and eosin staining and Masson's trichrome staining were used for histological examination. The protein levels of forkhead/winged helix transcription factor P3, retinoic-acid-related orphan receptor (ROR)-γt in liver tissue were determined by western blotting, the frequency of Th17 and Treg cells in the liver was evaluated by flow cytometry, and a suppression assay was measured by incorporating [3H]-thymidine. In addition, to explore the effect of Tregs expanded with rapamycin on hepatic stellate cells (HSC), HSCs were co-cultured with Tregs from rapamycin or phosphate-buffered saline-treated mice. It was found that rapamycin treatment led to a significant reduction in the number of Th17 cells and in the expression levels of ROR-γt in the liver tissues. Simultaneously, the results of the present study showed a significant increase in the frequency of Tregs and a marked enhancement in the expression of forkhead/winged helix transcription factor P3 in the rapamycin-treated mice. Furthermore, the Tregs in rapamycin-treated mice had significantly higher suppressive effects, compared with the cells from mice treated with phospphate-buffered saline. Consequently, rapamycin treatment prevented the development of CCl4-induced hepatic fibrosis, which was shown by its histological appearances. These results suggested that the immunosuppressive effect of rapamycin on liver fibrosis was associated with the suppression of hepatic fibrogenesis and regulation of the Th17/Treg cell balance.

Interaction of TWEAK with Fn14 leads to the progression of fibrotic liver disease by directly modulating hepatic stellate cell proliferation.

Tumour necrosis factor‐like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor‐inducible 14 (Fn14) have been associated with liver regeneration in vivo. To further investigate the role of this pathway we examined their expression in human fibrotic liver disease and the effect of pathway deficiency in a murine model of liver fibrosis. The expression of Fn14 and TWEAK in normal and diseased human and mouse liver tissue and primary human hepatic stellate cells (HSCs) were investigated by qPCR, western blotting and immunohistochemistry. In addition, the levels of Fn14 in HSCs following pro‐fibrogenic and pro‐inflammatory stimuli were assessed and the effects of exogenous TWEAK on HSCs proliferation and activation were studied in vitro. Carbon tetrachloride (CCl4) was used to induce acute and chronic liver injury in TWEAK KO mice. Elevated expression of both Fn14 and TWEAK were detected in acute and chronic human liver injury, and co‐localized with markers of activated HSCs. Fn14 levels were low in quiescent HSCs but were significantly induced in activated HSCs, which could be further enhanced with the profibrogenic cytokine TGFβ in vitro. Stimulation with recombinant TWEAK induced proliferation but not further HSCs activation. Fn14 gene expression was also significantly up‐regulated in CCl4 models of hepatic injury whereas TWEAK KO mice showed reduced levels of liver fibrosis following chronic CCl4 injury. In conclusion, TWEAK/Fn14 interaction leads to the progression of fibrotic liver disease via direct modulation of HSCs proliferation, making it a potential therapeutic target for liver fibrosis. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Ghrelin Attenuates Liver Fibrosis through Regulation of TGF-β1 Expression and Autophagy.

Ghrelin is a stomach-derived growth hormone secretagogue that promotes various physiological effects, including energy metabolism and amelioration of inflammation. The purpose of this study was to investigate the protective mechanism of ghrelin against liver fibrosis. Liver fibrosis was induced in C57BL/6 mice by intraperitoneal injection of CCl4 (2.0 mL/kg of 10% CCl4 v/v solution in peanut oil) two times per week for eight weeks. Ghrelin (10 μg/kg) was intraperitoneally injected two times per week for eight weeks. A second murine liver fibrosis model was induced by bile duct ligation (BDL) and concurrent ghrelin administration for four weeks. Hematoxylin eosin (H&E), and Masson’s trichrome were used to detect pathological changes to liver tissue. Western blotting was used to detect protein levels of transforming growth factor (TGF)-β1, phosphorylated Smad3 (p-Smad3), I-collage, α-smooth muscle actin (α-SMA), matrix metalloproteinases (MMPs) 2, tissue inhibitor of matrix metalloproteinases (TIMPs) 1, phosphorylated NF-κB (p-NF-κB), and microtubule-associated protein light chain 3 (LC3). In addition, qRT-PCR was used to detect mRNA levels of TGF-β1, I-collage, α-SMA, MMP2, TIMP1 and LC3, while levels of TGF-β1, p-Smad3, I-collage, α-SMA, and LC3 were detected immunohistochemically. Levels of aspartate aminotransferase and alanine aminotransferase were significantly decreased by ghrelin treatment. Ghrelin administration also significantly reduced the extent of pathological changes in both murine liver fibrosis models. Expression levels of I-collage and α-SMA in both models were clearly reduced by ghrelin administration. Furthermore, ghrelin treatment decreased protein expression of TGF-β1 and p-Smad3. The protein levels of NF-κB and LC3 were increased in the CCl4- and BDL-treatment groups but were significantly reduced following ghrelin treatment. In addition, ghrelin inhibited extracellular matrix formation by decreasing NF-κB expression and maintaining the balance between MMP2 and TIMP1. Our results demonstrated that ghrelin attenuates liver fibrosis via inhibition of the TGF-β1/Smad3 and NF-κB signaling pathways, as well as autophagy suppression.

Phenotypic and functional characterization of macrophages with therapeutic potential generated from human cirrhotic monocytes in a cohort study

Background aimsMacrophages have complex roles in the liver. The aim of this study was to compare profiles of human monocyte-derived macrophages between controls and cirrhotic patients, to determine whether chronic inflammation affects precursor number or the phenotype, with the eventual aim to develop a cell therapy for cirrhosis. MethodsInfusion of human macrophages in a murine liver fibrosis model demonstrated a decrease in markers of liver injury (alanine transaminase, bilirubin, aspartate transaminase) and fibrosis (transforming growth factor-β, α-smooth muscle actin, phosphatidylserine receptor) and an increase in markers of liver regeneration (matrix metalloproteinases [MMP]-9, MMP-12 and TNF-related weak inducer of apoptosis). CD14+ monocytes were then isolated from controls. Monocytes were matured into macrophages for 7 days using a Good Manufacturing Practice–compatible technique. ResultsThere was no significant difference between the mean number of CD14+ monocytes isolated from cirrhotic patients (n = 9) and controls (n = 10); 2.8 ± SEM 0.54 × 108 and 2.5 ± 0.56 × 108, respectively. The mean yield of mature macrophages cultured was also not significantly different between cirrhotic patients and controls (0.9 × 108 ± 0.38 × 108, with more than 90% viability and 0.65 × 108 ± 0.16 × 108, respectively. Maturation to macrophages resulted in up-regulation of a number of genes (MMP-9, CCL2, interleukin [IL]-10 and TNF-related weak inducer of apoptosis). A cytokine and chemokine polymerase chain reaction array, comparing the control and cirrhotic macrophages, revealed no statistically significant differences. ConclusionsMacrophages can be differentiated from cirrhotic patients' apheresis-derived CD14 monocytes and develop the same pro-resolution phenotype as control macrophages, indicating their suitability for clinical therapy.

Human recombinant endostatin Endostar attenuates hepatic sinusoidal endothelial cell capillarization in CCl4‑induced fibrosis in mice.

The aim of the present study was to detect the effect of the recombinant human endostatin Endostar on hepatic sinusoidal capillarization in CCl4‑induced murine models of liver fibrosis. The liver fibrosis model was induced in BALB/c mice using intraperitoneal injection of CCl4 for 6 weeks. Animals were divided into the following six treatment groups: Group 1, normal animals; group 2, CCl4‑induced liver fibrosis; group 3, CCl4+Endostar 20 mg/kg/day for 6 weeks; group 4, CCl4+Endostar 10 mg/kg/day for 6 weeks; group 5, CCl4+Endostar 20 mg/kg/day for 4 weeks; and group 6, CCl4+Endostar 10 mg/kg/day for 4 weeks. The average number of fenestrae per hepatic sinusoid was determined using transmission electron microscopy. Vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) 1 and 2 expression was detected by western blot analysis. There were significant differences in the number of fenestrae per sinusoid between the normal control and untreated model fibrotic mice (P<0.01), and between the untreated model and Endostar‑treated mice (P<0.05). Endostar treatment was associated with reduced levels of VEGFR1 and VEGFR2 in liver tissues (P<0.01), as well as with decreased hepatic sinusoidal endothelial cell capillarization in CCl4‑induced mouse models of liver fibrosis, and this effect may involve the VEGF pathway. However, further studies are required to confirm its involvement in other causes of liver fibrosis.

Utility of texture analysis for quantifying hepatic fibrosis on proton density MRI

To evaluate the potential utility of texture analysis of proton density maps for quantifying hepatic fibrosis in a murine model of hepatic fibrosis. Following Institutional Animal Care and Use Committee (IACUC) approval, a dietary model of hepatic fibrosis was used and 15 ex vivo murine liver tissues were examined. All images were acquired using a 30 mm bore 11.7T magnetic resonance imaging (MRI) scanner with a multiecho spin-echo sequence. A texture analysis was employed extracting multiple texture features including histogram-based, gray-level co-occurrence matrix-based (GLCM), gray-level run-length-based features (GLRL), gray level gradient matrix (GLGM), and Laws' features. Texture features were correlated with histopathologic and digital image analysis of hepatic fibrosis. Histogram features demonstrated very weak to moderate correlations (r = -0.29 to 0.51) with hepatic fibrosis. GLCM features correlation and contrast demonstrated moderate-to-strong correlations (r = -0.71 and 0.59, respectively) with hepatic fibrosis. Moderate correlations were seen between hepatic fibrosis and the GLRL feature short run low gray-level emphasis (SRLGE) (r = -0. 51). GLGM features demonstrate very weak to weak correlations with hepatic fibrosis (r = -0.27 to 0.09). Moderate correlations were seen between hepatic fibrosis and Laws' features L6 and L7 (r = 0.58). This study demonstrates the utility of texture analysis applied to proton density MRI in a murine liver fibrosis model and validates the potential utility of texture-based features for the noninvasive, quantitative assessment of hepatic fibrosis.

IL-30 (IL27p28) attenuates liver fibrosis through inducing NKG2D-rae1 interaction between NKT and activated hepatic stellate cells in mice.

Chronic hepatic diseases, such as cirrhosis, hepatocellular carcinoma, and virus-mediated immunopathogenic infections, affect billions of people worldwide. These diseases commonly initiate with fibrosis. Owing to the various side effects of antifibrotic therapy and the difficulty of diagnosing asymptomatic patients, suitable medication remains a major concern. To overcome this drawback, the use of cytokine-based sustained therapy might be a suitable alternative with minimal side effects. Here, we studied the therapeutic efficacy and potential mechanisms of interleukin (IL)-30 as antifibrosis therapy in murine liver fibrosis models. CCl4 or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) 0.1% (wt/wt) Purina 5015 Chow (LabDiet, St. Louis, MO) was fed for 3 weeks to induce liver fibrosis. Either control vector (pCtr) or pIL30 was injected hydrodynamically once per week. A significant decrease in collagen deposition and reduced expression of alpha-smooth muscle actin (α-SMA) protein indicated that IL-30-based gene therapy dramatically reduced bridging fibrosis that was induced by CCl4 or DDC. Immunophenotyping and knockout studies showed that IL-30 recruits natural-killer-like T (NKT) cells to the liver to remove activated hepatic stellate cells (HSCs) significantly and ameliorate liver fibrosis. Both flow cytometric and antibody-mediated neutralization studies showed that liver NKT cells up-regulate the natural killer group 2, member D (NKG2D) ligand and bind with the NKG2D ligand, retinoic acid early inducible 1 (Rae1), and positively activated HSCs to ameliorate liver fibrosis. Furthermore, adoptive transfer of liver NKT cells in T-cell-deficient mice showed reduction of fibrosis upon IL-30 administration. Highly target-specific liver NKT cells selectively remove activated HSCs through an NKG2D-Rae1 interaction to ameliorate liver fibrosis after IL-30 treatment.

Novel Fate-Tracing Strategies Show that Hepatic Stellate Cells Mediate Fibrosis In Vivo

Mederacke I, Hsu CC, Troeger JS, et al. Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology. Nat Commun 2013;4:2823. Liver fibrosis often arises as a result of chronic liver damage and is characterized by the deposition of extracellular matrix (ECM) proteins, including collagen at injury sites, which can impair normal liver function and may lead to portal hypertension and hepatic failure (N Engl J Med 2004;350:1646–1654). Clinically, liver fibrosis leads to severe morbidity and mortality in patients (J Clin Invest 2005;115:209–218). Myofibroblast, a cell population that produce ECM proteins, has been shown as the mediator for liver fibrogenesis. However, the identity of cellular sources that give rise to myofibroblast remains elusive. In a recent article published in Nature Communications, Mederacke et al discovered lecithin-retinol acyltrasferase (Lrat) as a novel marker for hepatic stellate cells (HSCs). The expression of Lrat was distinctively high in HSCs, whereas other cells, such as hepatocytes, Kupffer cells, cholangiocytes, or endothelial cells, had undetectable Lrat expression. Consistent with its unique expression pattern, Lrat has been shown to play a pivotal role in regulating the formation of retinyl ester-containing lipid droplet, the signature for quiescent HSCs. With the help of this newly discovered marker for HSCs, Mederacke et al studied the precise contribution of HSCs in promoting liver fibrosis in vivo. Briefly, they designed a novel fate-tracing strategy by generating transgenic mice that have Cre recombinase expression cassette under the control of murine Lrat promoter. These animals were then crossed with floxed Zs-Green fluorescent mice; therefore, HSCs in the progeny mice would be labeled by Zs-Green fluorescence. Mederacke et al first confirmed that Lrat-Cre-labeled cells are mostly (>99%) HSCs, because Lrat-Cre expression and known markers of HSCs (ie, desmin and Pdgfrβ) almost completely overlapped. To investigate the precise role of HSCs in promoting liver fibrogenesis in vivo, they performed fate-tracing experiments in several different fibrotic animal models, including toxin-induced liver fibrosis models (ie, carbon tetrachloride-induced and thioacetamide-induced liver fibrosis models) and biliary liver fibrosis models (ie, bile duct ligation model, 3,5-diethoxycarbonyl-1,4-dihydrocollidine-containing diet model and Mdr2KO mouse model). Strikingly, Mederacke et al found that HSCs are the dominant and universal cell population that gives rise to collagen-secreting myofibroblasts in various murine liver fibrosis models. This was also confirmed by the reduction of liver fibrosis in mice with depletion of HSCs using Lrat-induced diphthelia toxin receptor, which further strengthens the concept that HSCs are the responsible cell types in hepatic fibrosis in general. Another important finding by Mederacke et al is that HSCs do not give rise to epithelial cells after liver damage. They found no increase in the numbers of Lrat-Cre and HNF4a (a hepatocyte marker) double-positive cells during liver regeneration after liver damage caused by 7 different hepatic injury models, including 70% partial hepatectomy model. In addition, by employing series of experiments using bone marrow transplantation, Mederacke et al further concluded that HSCs are endogenous to liver, rather than a bone marrow-derived cell population.

Resolvin E1 reduces hepatic fibrosis in mice with Schistosoma japonicum infection

The aim of this study was to investigate whether resolvin E1 (RvE1) protects against hepatic fibrosis in a murine model of liver fibrosis induced by Schistosoma japonicum infection. A total of 30 pathogen-free Kunming mice were randomly and equally divided into three groups: Control (uninfected, untreated), model (infected, untreated) and RvE1 intervention (infected, RvE1-treated; 100 ng daily). The mice were infected with Schistosoma japonicum by inoculating the abdominal skin with 20±2 cercariae to induce models of liver fibrosis. The area and numbers of the granulomas in the livers were assessed through histopathology after 70 days of treatment. The levels of tumor necrosis factor (TNF)-α and interferon (IFN)-γ were evaluated in the serum by enzyme-linked immunosorbent assay (ELISA). The expression levels of TNF-α were detected in the hepatic tissue by reverse transcription-polymerase chain reaction and western blot analysis. The activity levels of alanine aminotransferase and aspartate aminotransferase were determined in the serum by ELISA. The expression levels of laminin (LN), hyaluronic acid (HA), procollagen type III (PC-III) and type IV collagen (IV-C) were detected in the serum by radioimmunoassays. The results revealed that the mean area of the granulomas was smaller in the RvE1 intervention group compared with that in the model group. Following RvE1 treatment, the serum levels of TNF-α were lower than those in the model group, while the serum levels of IFN-γ were higher compared with those in the model group. The expression levels of TNF-α were lower in the hepatic tissue following RvE1 treatment compared with those in the model group. The indicators of liver fibrosis, the levels of LN, HA, PC-III and IV-C in the serum, were lower following RvE1 treatment than those in the model group. In conclusion, RvE1 treatment may reduce the growth of granulomas, thereby slowing the process of hepatic fibrosis, and this effect may be the result of anti-inflammatory and immune system adjustment.

A Blood-to-Liver CXCL12 Concentration Gradient Parallels Increasing Fibrocytes and Liver Fibrosis in a Murine Model

Purpose: Fibrocytes are circulating hematopoietic cells that home to sites of tissue injury via a chemokine gradient and promote fibrosis in several models of injury. Whether fibrocytes contribute to liver fibrosis has not been examined to date. CXCL12 binds to the chemokine receptor CXCR4 expressed on fibrocytes, potentially enabling their fibrogenic action in the liver. To better understand this association, we performed a pilot study in an established preclinical murine model of liver fibrosis. The use of this model allowed us to assess the kinetics and magnitude of fibrocyte presence in the bone marrow, blood, and liver of mice with developing hepatic fibrosis. We further postulated that a liver-blood CXCL12 concentration gradient contributed to fibrocyte trafficking to injured liver. Methods: We used an established 0.1% DDC model of liver fibrosis. A cohort of 6-8 week old female C57BL/6 mice received a diet of either DDC or control chow for 6 weeks. At weekly intervals, blood and liver were assessed for fibrocyte number and phenotype and concentration of CXCL12, and extent of liver fibrosis. Statistical differences between the DDC chow fed and control chow fed groups were assessed by linear regression. Results: Liver collagen deposition increased progressively 9-fold through week 6 in the DDC chow-fed group as compared to the control group (p<.01). There was a progressive, parallel increase in the number of fibrocytes and the liver-blood concentration gradient of CXCL12 between the DDC chow-fed as compared to control mice through (p <.001). There was no statistically significant increase in fibrocytes or the CXCL12 concentration gradient in the control chow fed mice. Conclusion: These data demonstrate that in a murine model of liver fibrosis, the number of blood and liver fibrocytes increase in parallel with progressive fibrosis, and correlate with a blood-to-liver CXCL12 concentration gradient.

Rosiglitazone prevents murine hepatic fibrosis induced by Schistosoma japonicum

To evaluate the effect of rosiglitazone in a murine model of liver fibrosis induced by Schistosoma japonicum infection. A total of 50 mice were randomly and averagely divided into groups A, B, C, D and E. The mice in group A served as normal controls, while those in the other four groups were infected with Schistosoma japonicum to induce the model of liver fibrosis. Besides, the mice in groups C, D and E were treated with praziquantel, rosiglitazone and praziquantel plus rosiglitazone, respectively. NF-kappaB binding activity and expression of PPAR gamma-mRNA were determined by Western blot assay and real-time quantitative PCR. Radioimmunonassay technique was used to detect the serum content changes of TNF-alpha and IL-6. Histological specimens were stained with HE. Expression of TGF-beta1, a-smooth muscle actin and type I and type III collagen was detected by immunohistochemistry and multimedia color pathographic analysis system. Inflammation and fibrosis in the rosiglitazone plus praziquantel treatment group (group E) were lightest among the mice infected with Schistosoma (P < 0.05). To further explore the mechanism of rosiglitazone action, we found that rosiglitazone can significantly increase the expression of PPAR gamma [E: -18.212 +/- (-3.909) vs B: -27.315 +/- (-6.348) and C: -25.647 +/- (-5.694), P < 0.05], reduce the NF-kappaB binding activity (E: 88.89 +/- 19.34 vs B: 141.11 +/- 15.37, C: 112.89 +/- 20.17 and D: 108.89 +/- 20.47, P < 0.05), and lower the serum level of TNF-alpha (E: 1.613 +/- 0.420 ng/mL vs B: 2.892 +/- 0.587 ng/mL, C: 2.346 +/- 0.371 ng/mL and D: 2.160 +/- 0.395 ng/mL, P < 0.05) and IL-6 (E: 0.106 +/- 0.021 ng/mL vs B: 0.140 +/- 0.031 ng/mL and C: 0.137 +/- 0.027 ng/mL, P < 0.05) in mice with liver fibrosis. Rosiglitazone can also substantially reduce the hepatic expression of TGF-beta1, alpha-SMA type I and type III collagen in mice with liver fibrosis. The activation of PPAR gamma by its ligand can retard liver fibrosis and suggest the use of rosiglitazone for the treatment of liver fibrosis due to Schistosoma japonicum infection.