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Background and Aims: HSCs and portal fibroblasts (PFs) are the major sources of collagen-producing myofibroblasts during liver fibrosis, depending on different etiologies. However, the mechanisms by which their dynamic gene expression directs the transition from the quiescent to the activated state-as well as their contributions to fibrotic myofibroblasts-remain unclear. Here, we analyze the activation of HSCs and PFs in CCL 4 -induced and bile duct ligation-induced fibrosis mouse models, using single-cell RNA sequencing and lineage tracing., Approach and Results: We demonstrate that HSCs, rather than PFs, undergo dramatic transcriptomic changes, with the sequential activation of inflammatory, migrative, and extracellular matrix-producing programs. The data also reveal that HSCs are the exclusive source of myofibroblasts in CCL 4 -treated liver, while PFs are the major source of myofibroblasts in early cholestatic liver fibrosis. Single-cell and lineage-tracing analysis also uncovers differential gene-expression features between HSCs and PFs; for example, nitric oxide receptor soluble guanylate cyclase is exclusively expressed in HSCs, but not in PFs. The soluble guanylate cyclase stimulator Riociguat potently reduced liver fibrosis in CCL 4 -treated livers but showed no therapeutic efficacy in bile duct ligation livers., Conclusions: This study provides a transcriptional roadmap for the activation of HSCs during liver fibrosis and yields comprehensive evidence that the differential transcriptomic features of HSCs and PFs, along with their relative contributions to liver fibrosis of different etiologies, should be considered in developing effective antifibrotic therapeutic strategies., (© 2021 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Background and Aims: During liver injury, quiescent hepatic stellate cells (qHSCs) transdifferentiate into proliferative and fibrogenic activated myofibroblastic phenotype (activated hepatic stellate cell; aHSCs) expressing smooth muscle α-actin (αSMA) and platelet-derived growth factor beta receptor (PDGFβR). Their interactions with gut-derived bacterial lipopolysaccharide (LPS) are implicated in hepatic fibrogenesis. However, LPS can also attenuate fibrogenic characteristics of aHSCs., Approach and Results: We examined molecular mechanisms of antifibrogenic effects of LPS on aHSCs in vitro and in vivo. Culture-activated rat HSCs were exposed to 0-100 ng/mL of LPS or its active component, diphosphoryl-lipid A (DPLA), and parameters of fibrosis and inflammatory cytokines/chemokines were determined by qRT-PCR, western, and immunohistochemical analyses. In vivo, HSCs were activated by repeated CCl 4 administration to rats every 3 days for 3 or 8 weeks, then challenged with LPS (5 mg/kg; IP). HSCs were isolated 24 hours later, and fibrogenic/inflammatory parameters were analyzed. LPS induced phenotypic changes in aHSCs (rounding, size reduction) and loss of proliferation. LPS down-regulated expression of αSMA, PDGFβR, transforming growth factor beta receptor 1 (TGFβR1), collagen 1α1 (Col1α1), and fibronectin while up-regulating tumor necrosis factor alpha, interleukin-6, and C-X-C motif chemokine ligand 1 expression. LPS did not increase peroxisome proliferation-activated receptor gamma expression or lipid accumulation typical of qHSCs. DPLA elicited the same effects as LPS on aHSCs, indicating specificity, and monophosphoryl lipid A down-regulated fibrogenic markers, but elicited very weak inflammatory response. LPS down-regulated the expression of cMyb, a transcription factor for αSMA, and up-regulated small mother against decapentaplegic (SMAD)7 and CCAAT/enhancer-binding protein (C/EBP)δ, the transcriptional inhibitors of Col1α1 expression. In vivo LPS treatment of aHSCs inhibited their proliferation, down-regulated PDGFβR, αSMA, TGFβR1, Col1α1, and cMyb expression, and increased expression of SMAD7, C/EBPα, and C/EBPδ., Conclusions: In conclusion, LPS induces a unique phenotype in aHSCs associated with down-regulation of key fibrogenic mechanisms and thus may have an important role in limiting fibrosis., (© 2020 by the American Association for the Study of Liver Diseases.)

Non-alcoholic fatty liver disease (NAFLD) has been on a global rise. While animal models have rendered valuable insights to the pathogenesis of NAFLD, discrepancy with patient data still exists. Since non-alcoholic steatohepatitis (NASH) involves chronic inflammation, and CD4 + T cell infiltration of the liver is characteristic of NASH patients, we established and characterized a humanized mouse model to identify human-specific immune response(s) associated with NAFLD progression. Immunodeficient mice engrafted with human immune cells (HIL mice) were fed with high fat and high calorie (HFHC) or chow diet for 20 weeks. Liver histology and immune profile of HIL mice were analyzed and compared with patient data. HIL mice on HFHC diet developed steatosis, inflammation and fibrosis of the liver. Human CD4 + central and effector memory T cells increased within the liver and in the peripheral blood of our HIL mice, accompanied by marked up-regulation of pro-inflammatory cytokines (IL-17A and IFNγ). In vivo depletion of human CD4 + T cells in HIL mice reduced liver inflammation and fibrosis, but not steatosis. Our results highlight CD4 + memory T cell subsets as important drivers of NAFLD progression from steatosis to fibrosis and provides a humanized mouse model for pre-clinical evaluation of potential therapeutics., (Copyright © 2020 Her, Tan, Lim, Tan, Chan, Tan, Liu, Yong, Lai, Ceccarello, Zheng, Fan, Chang, Sun, Chang, Chin, Lee, Dan, Chan, Lim, Chan, Chandy and Chen.)

Background: Liver fibrosis is a common health problem worldwide and there is still a lack of effective medicines. The Chinese herbal medicine, Gan Shen Fu Fang (GSFF) is composed of salvianolic acid B and diammonium glycyrrhizinate. In this study, we observed the effects of GSFF on liver fibrosis in vivo and in vitro in an attempt to provide some hope for the treatment., Aim: To observe the effects of GSFF on liver fibrosis in vivo and in vitro and investigate the mechanism from the perspective of the inflammatory response and extracellular signal-regulated kinase (ERK) phosphorylation., Methods: Common bile duct-ligated rats were used for in vivo experiments. Hepatic stellate cells-T6 (HSC-T6) cells were used for in vitro experiments. Hematoxylin and eosin staining and Masson staining, biochemical assays, hydroxyproline (Hyp) assays, enzyme-linked immunoasorbent assay and western blotting were performed to evaluate the degree of liver fibrosis, liver function, the inflammatory response and ERK phosphorylation. The CCK8 assay, immunofluorescence and western blotting were applied to test the effect of GSFF on HSC-T6 cell activation and determine whether GSFF had an effect on ERK phosphorylation in HSC-T6 cells., Results: GSFF improved liver function and inhibited liver fibrosis in common bile duct-ligated rats after 3 wk of treatment, as demonstrated by histological changes, hydroxyproline assays and collagen I concentrations. GSFF alleviated inflammatory cell infiltration and reduced the synthesis of pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α) and interlukin-1β] and NF-κB. In addition, GSFF decreased ERK phosphorylation. In vitro , GSFF inhibited the viability of HSC-T6 cells with and without transforming growth factor β1 (TGF-β1) stimulation and decreased the synthesis of collagen I. GSFF had the greatest effect at a concentration of 0.5 μmol/L. GSFF inhibited the expression of α-smooth muscle actin (α-SMA), a marker of HSC activation, in HSC-T6 cells. Consistent with the in vivo results, GSFF also inhibited the phosphorylation of ERK and downregulated the expression of NF-κB., Conclusion: GSFF inhibited liver fibrosis progression in vivo and HSC-T6 cell activation in vitro . These effects may be related to an alleviated inflammatory response and downregulated ERK phosphorylation., Competing Interests: Conflict-of-interest statement: All other authors have nothing to disclose., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

Background and Aims: Monocyte chemotactic protein-1 (MCP-1) is a potent chemoattractant for monocytes. It is involved in pathogenesis of several inflammatory diseases. Hepatic MCP-1 is a readout of macrophage activation. While inflammation is a major driver of liver disease progression, the origin and role of circulating MCP-1 as a biomarker remains unclear. Methods: Hepatic CC-chemokine ligand 2 ( CCL2 ) expression and F4/80 staining for Kupffer cells were measured and correlated in a mouse model of chronic liver disease (inhalative CCl 4 for 7 weeks). Next, hepatic RNA levels of CCL2 were measured in explanted livers of 39 patients after transplantation and correlated with severity of disease. Changes in MCP-1 were further evaluated in a rat model of experimental cirrhosis and acute-on-chronic liver failure (ACLF). Finally, we analyzed portal and hepatic vein levels of MCP-1 in patients receiving transjugular intrahepatic portosystemic shunt insertion for complications of portal hypertension. Results: In this mouse model of fibrotic hepatitis, hepatic expression of CCL2 ( P = 0.009) and the amount of F4/80 positive cells in the liver ( P < 0.001) significantly increased after induction of hepatitis by CCl 4 compared to control animals. Moreover, strong correlation of hepatic CCL2 expression and F4/80 positive cells were seen ( P = 0.023). Furthermore, in human liver explants, hepatic transcription levels of CCL2 correlated with the MELD score of the patients, and thus disease severity ( P = 0.007). The experimental model of ACLF in rats revealed significantly higher levels of MCP-1 plasma ( P = 0.028) and correlation of hepatic CCL2 expression ( R = 0.69, P = 0.003). Particularly, plasma MCP-1 levels did not correlate with peripheral blood monocyte CCL2 expression. Finally, higher levels of MCP-1 were observed in the hepatic compared to the portal vein ( P = 0.01) in patients receiving TIPS. Similarly, a positive correlation of MCP-1 with Child-Pugh score was observed ( P = 0.018). Further, in the presence of ACLF, portal and hepatic vein levels of MCP-1 were significantly higher compared to patients without ACLF (both P = 0.039). Conclusion: Circulating levels of MCP-1 mainly derive from the injured liver and are associated with severity of liver disease. Therefore, liver macrophages contribute significantly to disease progression. Circulating MCP-1 may reflect the extent of hepatic macrophage activation., (Copyright © 2020 Queck, Bode, Uschner, Brol, Graf, Schulz, Jansen, Praktiknjo, Schierwagen, Klein, Trautwein, Wasmuth, Berres, Trebicka and Lehmann.)

Background & Aims: Liver inflammation has been recognized as a hallmark of hepatocarcinogenesis. Although Forkhead Box M1 (FoxM1) is a well-defined oncogenic transcription factor that is overexpressed in hepatocellular carcinoma (HCC), its role in liver inflammation has never been explored., Methods: We generated hepatocyte-specific FoxM1 conditional transgenic (TG) mice by using the Cre-loxP and Tetracycline (Tet)-on systems to induce FoxM1 expression in a hepatocyte-specific and time-dependent manner., Results: After treatment of Tet-derivatives doxycycline (DOX) to induce FoxM1, TG mice exhibited spontaneous development of hepatocyte death with elevated serum alanine aminotransferase levels and hepatic infiltration of macrophages. The removal of DOX in TG mice completely removed this effect, suggesting that spontaneous inflammation in TG mice occurs in a hepatocyte FoxM1-dependent manner. In addition, liver inflammation in TG mice was associated with increased levels of hepatic and serum chemokine (C-C motif) ligand 2 (CCL2). In vitro transcriptional analysis confirmed that CCL2 is a direct target of FoxM1 in murine hepatocytes. After receiving FoxM1 induction since birth, all TG mice exhibited spontaneous HCC with liver fibrosis at 12 months of age. Hepatic expression of FoxM1 was significantly increased in liver injury models. Finally, pharmacologic inhibition of FoxM1 reduced liver inflammation in models of liver injury., Conclusions: Hepatocyte FoxM1 acts as a crucial regulator to orchestrate liver inflammation linking to hepatocarcinogenesis. Thus, hepatocyte FoxM1 may be a potential target not only for the treatment of liver injury but also for the prevention toward HCC., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Clinically, liver fibrosis and cholestasis are two major disease entities, ultimately leading to hepatic failure. Although autophagy plays a substantial role in the pathogenesis of these diseases, its precise mechanism has not been determined yet., Materials and Methods: Mouse models of liver fibrosis or cholestasis were obtained after the serial administration of thioacetamide (TAA) or surgical bile duct ligation (BDL), respectively. Then, after obtaining liver specimens at specific time points, we compared the expression of makers related to apoptosis (cleaved caspases), inflammation (CD68), necrosis (high-mobility group box 1), phospho-c-Jun N-terminal kinase (p-JNK), and autophagy (microtubule-associated protein light chain 3B and p62) in the fibrotic or cholestatic mouse livers, by polymerase chain reaction, Western blot analysis, immunohistochemistry, and immunofluorescence., Results: Although cholestatic livers exhibited the tendency of progressively increasing the expression of most apoptosis-related markers (cleaved caspases), it was not prominent when it was compared with the tendency found in the livers of TAA-treated mice. Contrastingly, the necrosis-related factor (high-mobility group box 1) was significantly increased in the livers of BDL mice over time, reaching their peak values on day 7 after BDL. In addition, the inflammation-related factor (CD68) was highly expressed in BDL mice compared with TAA-treated mice over time. Autophagy marker studies indicated that autophagy was upregulated in fibrotic livers, whereas it was downregulated in cholestatic livers. We also observed mild to moderate activation of p-JNK in the livers of TAA-treated mice, whereas significantly higher p-JNK activation was detected in the livers of BDL mice., Conclusions: Unlike TAA-treated mice, BDL mice exhibited higher expression of the markers related with inflammation and necrosis, especially including p-JNK, while maintaining low levels of autophagic process. Therefore, obstructive cholestasis is characterized by higher p-JNK activation, which could be related with marked necrotic cell death resulting from extensive inflammation and little chance of compensatory autophagy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Splenic immune cells, especially macrophages, play a key role in multiple pathological processes. With a proved anti-inflammatory and immunoregulatory function of mesencephalicastrocyte-derived neurotrophic factor (MANF) in inflammatory disorders, how MANF affects splenic immune cells in physiological and pathophysiological situations is still unknown. In this study, we constructed mono-macrophage-specific MANF knockout (Mø MANF -/- ) mice and found the increased splenic M1 macrophages, but no significant change of splenic morphology and size compared with wild type (WT) mice. Also, we established the pathophysiological situation of carbon tetrachloride (CCl 4 )-induced hepatic fibrosis. Under the hepatic fibrosis, splenic M2 macrophages and CD138 + plasma cells were significantly increased in Mø MANF -/- mice. Consistently, we found the increased TGF-β1 level in serum and spleen of Mø MANF -/- mice as well. Mono-macrophage-specific MANF knockout did not affect the number of splenic T and B cells under both the normal and hepatic fibrosis conditions. Our results suggest a distinct regulation of MANF on splenic immune cells and a specific regulation of MANF on the differentiation of splenic macrophages, which may exert a significant impact on physiological and pathophysiological processes of the spleen., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Aim: The pathological features of non-alcoholic steatohepatitis (NASH) have not been determined, so fundamental treatment has not been established. Adipose-tissue-derived stromal/stem cells (ADSCs) are beneficial for repair/regenerative therapy of impaired organs because of their immuno-modulatory capability. In this study, we assessed how liver damage progresses during the early development phase of the murine NASH model and investigated whether ADSCs are preventatively efficacious against the fibrosis progression of NASH., Methods: C57BL/6J mice were fed with atherogenic high fat or high-fat diet 60 developing into NASH or simple steatosis. Their hepatic inflammatory cells (HICs) were analyzed by cDNA microarray. NASH mice were treated with ADSCs injected into spleen when hepatic inflammation was initially observed, and liver samples were analyzed. The effect of ADSCs on the mice hepatic stellate cell (HSC) line stimulated by recombinant IL-17 and HICs from NASH mice was analyzed., Results: The gene expression features of HICs implicated as humoral cytokine mediators of lymphoid cells during NASH development, compared with a simple steatosis model. One of the featured cytokines was IL-17. The development of hepatic fibrosis was alleviated when NASH mice were treated with ADSCs as well as treated with anti-IL-17 antibody, and the frequency of IL-17-secreting HICs decreased. NASH-HICs enhanced proliferation of HSCs, in which proliferation was sensitive to IL-17 stimulation. The stimulatory effect of NASH-HICs on the activation of HSCs was attenuated by co-culture with ADSCs., Conclusion: ADSCs treatment prevented progression of NASH fibrosis by suppressing IL-17-mediated inflammation, which was associated with HSCs activation., (© 2019 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Background: Chronic inflammation is the driver of liver injury and results in progressive fibrosis and eventual cirrhosis with consequences including both liver failure and liver cancer. We have previously described increased expression of the highly multifunctional glycoprotein CD147 in liver injury. This work describes a novel role of CD147 in liver inflammation and the importance of leukocyte aggregates in determining the extent of liver injury., Methods: Non-diseased, progressive injury, and cirrhotic liver from humans and mice were examined using a mAb targeting CD147. Inflammatory cell subsets were assessed by multiparameter flow cytometry., Results: In liver injury, we observe abundant, intrahepatic leukocyte clusters defined as ≥5 adjacent CD45+ cells which we have termed "leukocyte aggregates". We have shown that these leukocyte aggregates have a significant effect in determining the extent of liver injury. If CD147 is blocked in vivo, these leukocyte aggregates diminish in size and number, together with a marked significant reduction in liver injury including fibrosis. This is accompanied by no change in overall intrahepatic leukocyte numbers. Further, blocking of aggregation formation occurs prior to an appreciable increase in inflammatory markers or fibrosis. Additionally, there were no observed, "off-target" or unpredicted effects in targeting CD147., Conclusion: CD147 mediates leukocyte aggregation which is associated with the development of liver injury. This is not a secondary effect, but a cause of injury as aggregate formation proceeds other markers of injury. Leukocyte aggregation has been previously described in inflammation dating back over many decades. Here we demonstrate that leukocyte aggregates determine the extent of liver injury., Competing Interests: The authors have declared that no competing interests exist.

An ingredient was isolated from Acanthus ilicifolius and identified as 4-hydroxy-2(3H)-benzoxazolone (HBOA). Its protective effects and underlying mechanism on liver fibrosis were investigated. Briefly, rats were intragastrically administrated with 50% CCl 4 twice a week for 12 weeks to induce liver fibrosis. Meanwhile, the animals were treated with various medicines from weeks 8 to 12. Then the histological change, serum biochemical index, inflammatory factors and hepatocyte apoptosis were detected. Moreover, the TGF-β1/Smads, NF-κB and ERK signaling pathways were also detected to illustrate the underlying mechanism. The results showed that HBOA significantly ameliorated CCl 4 -induced liver injury and collagen accumulation in rats, as evidenced by the histopathologic improvement. Moreover, HBOA markedly decreased hepatocyte apoptosis by regulating the expression levels of caspase-3, -9 and -12, as well as the Bcl-2 family. The mechanism study showed that HBOA significantly decreased the expressions of α-smooth muscle actin (α-SMA) and collagen and inhibited the generation of excessive extracellular matrix (ECM) components by restoring the balance between matrix metalloproteinases (MMPs) and its inhibitor (TIMPs). HBOA markedly alleviated oxidative stress and inflammatory cytokines through inhibiting the NF-κB pathway. In addition, HBOA significantly down-regulated the levels of TGF-β1, Smad2/3, Smad4 and up-regulated the level of Smad7, inhibiting the TGF-β1/Smads signaling pathway. Moreover, HBOA significantly blocked the ERK signaling pathway, leading to the inactivation of hepatic stellate cells. This study suggests that HBOA exerts a protective effect against liver fibrosis via modulating the TGF-β1/Smads, NF-κB and ERK signaling pathways, which will be developed as a potential agent for the treatment of liver fibrosis., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Objective: Liver injury impacts hepatic inflammation in part via Toll-like receptor (TLR) signalling. Triggering receptor expressed on myeloid cells 2 (TREM-2) modulates TLR4-mediated inflammation in bone marrow (BM)-derived macrophages but its function in liver injury is unknown. Here we hypothesised that the anti-inflammatory effects of TREM-2 on TLR signalling may limit hepatic injury., Design: TREM-2 expression was analysed in livers of humans with various forms of liver injury compared with control individuals. Acute and chronic liver injury models were performed in wild type and Trem-2 -/- mice. Primary liver cells from both genotypes of mice were isolated for in vitro experiments., Results: TREM-2 was expressed on non-parenchymal hepatic cells and induced during liver injury in mice and man. Mice lacking TREM-2 exhibited heightened liver damage and inflammation during acute and repetitive carbon tetrachloride and acetaminophen (APAP) intoxication, the latter of which TREM-2 deficiency was remarkably associated with worsened survival. Liver damage in Trem-2 -/- mice following chronic injury and APAP challenge was associated with elevated hepatic lipid peroxidation and macrophage content. BM transplantation experiments and cellular reactive oxygen species assays revealed effects of TREM-2 in the context of chronic injury depended on both immune and resident TREM-2 expression. Consistent with effects of TREM-2 on inflammation-associated injury, primary hepatic macrophages and hepatic stellate cells lacking TREM-2 exhibited augmented TLR4-driven proinflammatory responses., Conclusion: Our data indicate that by acting as a natural brake on inflammation during hepatocellular injury, TREM-2 is a critical regulator of diverse types of hepatotoxic injury., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY. Published by BMJ.)

Liver fibrosis is a major health problem worldwide due to its serious complications including cirrhosis and liver cancer. 2-Methoxyestradiol (2-ME) is an end metabolite of estradiol with anti-proliferative, antioxidant, and anti-inflammatory activities. However, the protective role of 2-ME in liver fibrosis has not been fully investigated. The aim of this study was to determine the protective effect of 2-ME in carbon tetrachloride (CCl 4 )-induced liver fibrosis in mice. Animals were injected intraperitoneally with CCl 4 twice weekly for 6 weeks. 2-ME 50 mg/kg or 100 mg/kg was administrated intraperitoneally every day over the same period. Our data showed that 2-ME reduced the extent of liver toxicity and fibrosis due to CCl 4 exposure. It restored the elevated serum liver enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) levels and ameliorated oxidative status. In addition, 2-ME significantly reduced collagen deposition and alpha-smooth muscle actin (α-SMA) protein expressions. Furthermore, 2-ME markedly lowered macrophage infiltration and macrophage alternative activation marker chitinase-like molecules (CHI3L3/YM1). The results of this study indicate an important protective activity of 2-ME in liver fibrosis and highlight the role of macrophage recruitment and alternative activation as a possible target.

Objective: Little is known about the exact underlying molecular mechanisms of the hepatoprotective effect of carvacrol against liver fibrosis. In the current study, we aimed to investigate the effect of carvacrol on the suppression of liver fibrosis progression via regulation of yes-associated protein (YAP) and transcriptional coactivators with a PDZ-binding motif (TAZ) and transforming growth factor beta (TGF-β) pathway. Materials and methods: To fulfill our target, rats received carbon tetrachloride (CCl 4 ) and carvacrol intraperitoneally, and orally, respectively for 10 weeks. Body weight, liver weight, serum biochemical parameters, hepatic hydroxyproline content, and histological changes were determined. Furthermore, gene expression of collagen and key elements of Hippo and TGF-β pathways were analyzed and then the protein levels of YAP, TAZ, and TGF-β were detected in liver tissue. Results: Carvacrol administration normalized liver and body weight, serum biochemical parameters and hepatic hydroxyproline in CCl 4 treated rats. Also, carvacrol downregulated TAZ and TGF-β signaling pathway at transcriptional levels. Furthermore, carvacrol decreased hepatic protein levels of TGF-β, TAZ, and YAP. Low expression of TAZ and YAP were accompanied with inhibition of TGF-β signaling pathway. Conclusion: Our data clearly revealed that carvacrol suppresses the progression of liver fibrosis via targeting of TAZ, YAP, and TGF-β signaling pathway.

Objective: To investigate the potential antifibrotic mechanisms of Chinese medicine Ganshuang Granules (, GSG) and to provide clinical therapeutic evidence of its effects., Methods: A cirrhotic mouse model was established by intraperitoneally injecting a mixture of CCl 4 (40%) and oil (60%) at 0.2 mL per 100 g of body weight twice a week for 12 weeks. After 12-week modeling, GSG was intragastric administrated to the mice for 2 weeks, and the mice were divided into low-, medium- and high-dose groups at doses of 1, 2 and 4 g/(kg·day), respectively. Liver morphology changes were observed using Masson's trichrome staining and B-ultrasound. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hyaluronic acid (HA) in serum were detected using an automatic biochemistry analyzer. The expressions of desmin, smooth muscle actin (SMA) and Foxp3 in liver were detected by immunoflfluorescence. The regulatory T cell (Treg) frequency was determined through flflow cytometry analysis. Collagen-I, SMA, IL-6, tumor necrosis factor α (TNF-α), interleukin (IL)-1β and transforming growth factor β1 (TGF-β1) expression levels were measured using quantitative polymerase chain reaction (qPCR)., Results: Masson's staining result showed fewer pseudolobule structures and fibrous connective tissue in the GSG-treatment groups than in the spontaneous recovery group. Ultrasonography showed that GSG treatment reduced the number of punctate hyperechoic lesions in mice cirrhotic livers. The serum ALT, AST, HA levels were significantly ameliorated by GSG treatment (ALT: F=8.104, P=0.000; AST: F=7.078, P=0.002; and HA: F=7.621, P=0.001). The expression levels of collagen-I and SMA in the cirrhotic livers were also attenuated by GSG treatment (collagen-I: F=3.938, P=0.011; SMA: F=4.115, P=0.009). Tregs, which were elevated in the fibrotic livers, were suppressed by GSG treatment (F=8.268, P=0.001). The expressions of IL-6, TNF-α and IL-1β increased, and TGF-β levels decreased in the cirrhotic livers after GSG treatment (IL-6: F=5.457, P=0.004; TNF-α: F=6.023, P=0.002; IL-1β: F=6.658, P=0.001; and TGF-β1: F=11.239, P=0.000)., Conclusions: GSG promoted the resolution/regression of cirrhosis and restored liver functions in part by suppressing Treg cell differentiation, which may be mediated by hepatic stellate cells.

Type 3 innate lymphoid cell (ILC3) has recently emerged as a crucial effector in inflammatory and fibrotic diseases. The present study was designed to determine the roles of ILC3 in liver fibrosis. By flow cytometry, we documented increased frequencies of peripheral ILC3 (Lin - CD127 + CD117 + CD294 - lymphocytes) in patients, especially at the advanced stage of hepatitis B virus (HBV)-related chronic liver diseases, and demonstrated their correlations with disease progression. The in vitro fibrogenic effects by ILC3 were determined by co-culture experiments with LX-2 (a human hepatic stellate cell (HSC) line). The data indicate that pathogenic ILC3 can directly promote LX-2 fibrogenesis in non-contact manners by producing interleukin (IL)-17A and IL-22. Additionally, they had indirect fibrogenic effects by producing IL-22 to suppress interferon (IFN)-γ (a well-known anti-fibrotic cytokine) production by other immune cells. In carbon tetrachloride (CCl 4 )-induced wild-type mouse liver fibrosis models, we also documented significantly increased frequencies of both non-natural killer (NK) ILC (Lin - CD127 + lymphocytes) and ILC3 (Lin - CD127 + RORγt + lymphocytes) in liver and spleen specimens. Furthermore, the ILC3 from fibrotic mice contained more IL-17A + ILC3 and IL-22 + ILC3 subsets than those from normal and less-fibrotic mice. The in vivo effects of ILC3 in liver fibrogenesis were further determined using RAG-1 -/- mice with ILC depletion and further adoptive transfer of ILC3 from wild-type mice. The immunohistochemical staining of liver specimens showed the beneficial effects by ILC depletion and the detrimental effects by ILC3 transfer in CCl 4 -induced mouse liver fibrosis models. Collectively, ILC3 plays a pro-fibrotic role in liver fibrosis progression., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Aim: To investigate whether Yiguanjian decoction (YGJ) has an anti-liver cirrhotic effect and whether it regulates hepatic stem cell differentiation., Methods: A rat model of liver cirrhosis was established via subcutaneous injection of carbon tetrachloride (CCl 4 ) for 8 wk. From the beginning of the ninth week, the rats received 2-acetylaminofluorene (2-AAF) by oral gavage and a DLK-1 + fetal liver stem/progenitor cell (FLSPC) transplant or an FLSPC transplant in combination with YGJ treatment for 4 wk. In vitro , lipopolysaccharide (LPS)-activated macrophages were co-cultured with WB-F344 cells, and the differentiation of WB-F344 cells was observed in the presence and absence of YGJ treatment., Results: FLSPC transplantation improved liver function and histopathology, and inhibited the activation of the non-canonical Wnt signaling pathway, while activating the canonical Wnt signaling pathway. YGJ enhanced the therapeutic effects of FLSPCs and also promoted the liver regeneration differentiation of FLSPCs into hepatocytes. In vitro , LPS-activated macrophages promoted the differentiation of WB-F344 cells into myofibroblasts, and the canonical Wnt signaling was inhibited while the non-canonical Wnt signaling was activated in WB-F344 cells. YGJ suppressed the activation of macrophages and then inhibited non-canonical Wnt signaling and promoted canonical Wnt signaling., Conclusion: YGJ enhances FLSPC-mediated repair of liver cirrhosis through regulation of macrophage activation state, and YGJ in combination with stem cell transplantation may be a suitable treatment for end-stage liver cirrhosis., Competing Interests: Conflict-of-interest statement: The authors declare that they have no conflicts of interest.

Yu Gan Long (YGL) is a Chinese traditional herbal medicine that has been used in the treatment of liver fibrosis for many years in clinical practice. However, its anti-hepatofibrotic mechanism has not been studied yet. In this study, the effect and mechanism of YGL in reducing liver fibrosis was demonstrated in vivo. Our results showed that liver fibrosis biomarkers collagen IV (Col IV), type III precollagen (PCIII), hyaluronuc acid (HA) and laminin (LN), were increased after CCl 4 treatment and decreased by YGL. Among the liver fibrosis indicators, α-smooth muscle actin (α-SMA) was decreased by YGL in the CCl 4 -treated rats, while MMP2 and MMP9 was upregulated followed by TIMP1 downregulation. Proteins involved in liver fibrosis such as p-Smad2, p-Smad3 and Smad4 were down-regulated, while Smad7 protein was up-regulated by YGL after CCl 4 -induced liver damage. YGL also suppressed the increase of TGF-β1, TNF-α, IL-1β, IL-6, IL-4 and IL-17 A induced by CCl 4 treatment, while promoted IFN-γ expression. Finally, the transcription factors ROR-γt and GATA3 were decreased, while T-bet was increased after YGL treatment. These results suggested that YGL attenuated CCl 4 -induced hepatic fibrosis by accelerating the extracellular matrix degradation, blocking the TGF-β1/Smad signaling pathway and modulating the balance among IL-4, IL-17 A and IFN-γ, demonstrating YGL protective effect and its potential mechanisms in treating liver fibrosis., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Aim: To reveal the protective mechanism of the combined use of vitamin D and puerarin in the progression of hepatic fibrosis induced by carbon tetrachloride (CCl 4 )., Methods: Eight-week-old male Wistar rats were randomly divided into a normal control group (C group), a CCl 4 group (CCl 4 group), a vitamin D group (V group), a puerarin group (P group), and a combined group of vitamin D and puerarin (V + P group), each of which contained ten rats. In this way, we built a rat model of CCl 4 -induced hepatic fibrosis with intervention by vitamin D, puerarin, or a combination of the two. After eight weeks, the mice were sacrificed to collect serum and liver specimens. Blood was collected to detect the hyaluronic acid (HA). We also measured hydroxyproline (Hyp) and prepared paraffin sections of liver. After Sirius red staining, the liver specimens were observed under a microscope. RT-PCR and western blot analysis were adopted to detect the mRNA and the protein levels of Collagen I, Collagen III, Wnt1, and β-catenin in the liver tissues, respectively., Results: Hepatic fibrosis was observed in the CCl 4 group. In comparison, hepatic fibrosis was attenuated in the V, P, and V + P groups: the HA level in blood and the Hyp level in liver were reduced, and the mRNA levels of Collagen I, Collagen III, Wnt, and β-catenin in liver were also decreased, as well as the protein levels of Wnt1 and β-catenin. Among these groups, the V + P group demonstrated the greatest amelioration of hepatic fibrosis., Conclusion: The combined application of vitamin D and puerarin is capable of alleviating CCl 4 -induced hepatic fibrosis of rats. As to the mechanism, it is probably because the combined use is able to silence the Wnt1/β-catenin pathway, suppress the activation of hepatic stellate cells, and reduce the secretion of collagen fibers, therefore improving the anti-hepatic fibrosis effect., Competing Interests: Conflict-of-interest statement: All authors have no conflict of interest.

Epalrestat (EPS), an aldose reductase inhibitor, is widely prescribed to manage diabetic neuropathy. It is generally believed that EPS is beneficial to diabetic patients because it can protect endothelial cells, Schwann cells, or other neural cells from oxidative stress. However, several clinical studies revealed that EPS therapy led to liver dysfunction, which limited its clinical applications. Currently, the underlying mechanism by which EPS causes liver dysfunction is unknown. This study aimed to investigate the mechanism responsible for EPS-induced liver injury. In mouse liver, EPS 1) increased oxidative stress, indicated by increased expression of manganese superoxide dismutase, Ho-1, and Nqo1, 2) induced inflammation, indicated by infiltration of inflammatory cells, and induced expression of tumor necrosis factor-alpha, CD11b, and CD11c, as well as 3) predisposed to induce fibrosis, evidenced by increased mRNA and protein expression of early profibrotic biomarker genes procollagen I and alpha-smooth muscle actin, and by increased collagen deposition. In cultured mouse and human hepatoma cells, EPS treatment induced oxidative stress, decreased cell viability, and triggered apoptosis evidenced by increased Caspase-3 cleavage/activation. In addition, EPS increased mRNA and protein expression of cytoglobin in mouse liver, indicating that EPS activated hepatic stellate cells (HSCs). Furthermore, EPS treatment in cultured human HSCs increased cell viability. In summary, EPS administration induced oxidative stress and inflammation in mouse liver, and stimulated liver fibrogenesis. Therefore, cautions should be exercised during EPS therapy.

The ability of quercetin to reverse an established cirrhosis has not yet been investigated. Therefore, the aim of this study was to examine the efficacy of this flavonoid in reversing experimental cirrhosis. Cirrhosis was induced by intraperitoneal administration of TAA (200 mg/kg of body weight) three times per week for 8 weeks or by intraperitoneal petrolatum-CCl 4 (400 mg/kg of body weight) administration three times per week for 8 weeks. To determine the capacity of quercetin to prevent liver fibrosis, the flavonoid (50 mg/kg of body weight, p.o.) was administered daily to rats during the CCl 4 or TAA treatment. To evaluate the ability of quercetin to reverse the previously induced cirrhosis, we first treated rats with CCl 4 for 8 weeks, as previously described and then the flavonoid was administered for four more weeks. We found that the liver anti-inflammatory and antinecrotic effects of quercetin are associated with its antioxidant properties, to the ability of the flavonoid to block NF-κB activation and in consequence to reduce cytokine IL-1. The ability of quercetin to reverse fibrosis may be associated with the capacity of the flavonoid to decrease TGF-β levels, hepatic stellate cell activation, and to promote degradation of the ECM by increasing metalloproteinases. The main conclusion is that quercetin, in addition to its liver protective activity against TAA chronic intoxication, is also capable of reversing a well-stablished cirrhosis by blocking the prooxidant processes and by downregulating the inflammatory and profibrotic responses., (© 2017 Société Française de Pharmacologie et de Thérapeutique.)

Patients with liver cirrhosis present an increased incidence of infections. The main cause has been founded in alterations of the enteric flora and of the intestinal barrier probably due to portal hypertension, in addition to a reticulo-endothelial system dysfunction. Furthermore, those living with cirrhosis can report a high predisposition to sepsis and septic shock, due to the excessive response of pro-inflammatory cytokines and a complessive hemodynamic derangement. By the analysis in the experimental model of the cirrhotic rat, it was demonstrated that radio-labelled Escherichia coli given by the oral route resulted in the location of the bacteria in the gut, the ascitic fluid and mesenteric lymph nodes, a phenomenon known as bacterial translocation. Bacteria encountered with the highest frequency are those colonizing the intestinal tract, such as E. coli, Klebsiella pneumoniae and Enterobacteriaceae, intracellular bacteria and parasites are reported with a lower frequency. Multi-drug resistant bacteria are cultured with the highest frequency in those with frequent hospitalisations and report both high septic shock and mortality rates. Spontaneous bacterial peritonitis (SBP) is the commonest infection in cirrhotic, estimated to occur in 10-30% of the cases with ascites. A practical approach may include administration of a protected penicillin, III generation cephalosporin or quinolones in uncomplicated cases. Instead, in complicated cases and in nosocomial SBP, administration of cephalosporin or quinolones can be burned by the high resistance rate and drugs active against ESBL-producing bacteria and multi-drug resistant Gram positive bacteria have to be considered as empiric therapy, until cultures are available. When cultures are not readily available and patients fail to improve a repeated diagnostic paracentesis should be performed. Current investigations suggest that norfloxacin 400 mg/day orally has been reported to successfully prevent SBP in patients with low-protein ascites and patients with prior SBP.

Objective: Patients with liver cirrhosis suffer from increased susceptibility to life-threatening bacterial infections that cause substantial morbidity., Methods: Experimental liver fibrosis in mice induced by bile duct ligation or CCl 4 application was used to characterise the mechanisms determining failure of innate immunity to control bacterial infections., Results: In murine liver fibrosis, translocation of gut microbiota induced tonic type I interferon (IFN) expression in the liver. Such tonic IFN expression conditioned liver myeloid cells to produce high concentrations of IFN upon intracellular infection with Listeria that activate cytosolic pattern recognition receptors. Such IFN-receptor signalling caused myeloid cell interleukin (IL)-10 production that corrupted antibacterial immunity, leading to loss of infection-control and to infection-associated mortality. In patients with liver cirrhosis, we also found a prominent liver IFN signature and myeloid cells showed increased IL-10 production after bacterial infection. Thus, myeloid cells are both source and target of IFN-induced and IL-10-mediated immune dysfunction. Antibody-mediated blockade of IFN-receptor or IL-10-receptor signalling reconstituted antibacterial immunity and prevented infection-associated mortality in mice with liver fibrosis., Conclusions: In severe liver fibrosis and cirrhosis, failure to control bacterial infection is caused by augmented IFN and IL-10 expression that incapacitates antibacterial immunity of myeloid cells. Targeted interference with the immune regulatory host factors IL-10 and IFN reconstitutes antibacterial immunity and may be used as therapeutic strategy to control bacterial infections in patients with liver cirrhosis., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Interleukin-17A has been identified as a driver of hepatic stellate cell activation and plays a critical role in the pathogenesis of hepatic fibrosis. However, the underlining fibrosis-promoting mechanism of IL-17A is far from understood. Here we aimed to define whether hepatocytes directly respond to IL-17A stimulation and are associated with the development of hepatic fibrosis. The functional significance of IL-17A was evaluated in bile duct ligation (BDL) or thioacetamide (TAA) injection-induced mouse models of hepatic fibrosis. Human cirrhosis and control tissues were obtained from the patients with cirrhosis who received an open surgical repair process. Neutralizing IL-17A promoted the resolution of BDL or TAA-induced acute or chronic inflammation and fibrosis, resulted in a shift of the suppressive immune response in fibrotic liver toward a Th1-type immune response, and restored autophagy activity in both cholestatic and hepatotoxic liver injury induced fibrotic liver tissues, which was accompanied by a significant inhibition of STAT3 phosphorylation. Moreover, we found that IL-17A stimulated the concentration-and time-dependent phosphorylation of STAT3 in AML-12 liver cells. Blocking STAT3 with a specific inhibitor STATTIC or STAT3 siRNA protected from the IL-17A-induced autophagy suppression in AML-12 cells, indicating that STAT3 mediates IL-17A-suppressed autophagy. Administration of IL-10, which activated STAT3 and inhibited autophagy, reversed the therapeutic effect of IL-17A antagonism in vivo. Our study suggests that the IL-17A/STAT3 signaling pathway plays a crucial role in the pathogenesis of hepatic fibrosis through suppressing hepatocellular autophagy and that blocking this pathway may provide therapeutic benefits for the treatment of hepatic fibrosis.

Exposure to the halogenated hydrocarbon carbon tetrachloride (CCl 4 ) leads to hepatic lipid peroxidation, inflammation and fibrosis. Dietary supplementation of ω-3 fatty acids has been increasingly advocated as being generally anti-inflammatory, though its effect in models of liver fibrosis is mixed. This raises the question of whether diets high in ω-3 fatty acids will result in a greater sensitivity or resistance to liver fibrosis as a result of environmental toxicants like CCl 4 . In this study, we fed CCl 4 -treated mice a high ω-3 diet (using a mix of docosahexaenoic acid and eicosapentaenoic acid ethyl esters). We also co-administered an inhibitor of soluble epoxide hydrolase, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), which has been shown to boost anti-inflammatory epoxy fatty acids that are produced from both ω-3 and ω-6 dietary lipids. We showed that soluble epoxide inhibitors reduced CCl 4 -induced liver fibrosis. Three major results were obtained. First, the ω-3-enriched diet did not attenuate CCl 4 -induced liver fibrosis as judged by collagen deposition and collagen mRNA expression. Second, the ω-3-enriched diet raised hepatic tissue levels of several inflammatory lipoxygenase metabolites and prostaglandins, including PGE2. Third, treatment with TPPU in drinking water in conjunction with the ω-3-enriched diet resulted in a reduction in liver fibrosis compared to all other groups. Taken together, these results indicate that dietary ω-3 supplementation alone did not attenuate CCl 4 -induced liver fibrosis. Additionally, oxylipin signaling molecules may play role in the CCl 4 -induced liver fibrosis in the high ω-3 diet groups., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of this study was to investigate the effect of betaine (BET) on alcoholic liver fibrosis in rats. Fibrosis was experimentally generated with ethanol plus carbon tetrachloride (ETH+CCl4) treatment. Rats were treated with ETH (5% v/v in drinking water) for 14 weeks. CCl4 was administered intraperitoneally (i.p.) 0.2mL/kg twice a week to rats in the last 6 weeks with/without commercial food containing BET (2% w/w). Serum hepatic damage markers, tumor necrosis factor-α, hepatic triglyceride (TG) and hydroxyproline (HYP) levels, and oxidative stress parameters were measured together with histopathologic observations. In addition, α-smooth muscle-actin (α-SMA), transforming growth factor-β1 (TGF-β1) and type I collagen (COL1A1) protein expressions were assayed immunohistochemically to evaluate stellate cell (HSC) activation. mRNA expressions of matrix metalloproteinase-2 (MMP-2) and its inhibitors (TIMP-1 and TIMP-2) were also determined. BET treatment diminished TG and HYP levels; prooxidant status and fibrotic changes; α-SMA, COL1A1 and TGF-β protein expressions; MMP-2, TIMP-1 and TIMP-2 mRNA expressions in the liver of fibrotic rats. In conclusion, these results indicate that the antifibrotic effect of BET may be related to its suppressive effects on oxidant and inflammatory processes together with HSC activation in alcoholic liver fibrosis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Aim: To evaluate the effect of artesunate (AS) supplementation on bacterial translocation (BT) and gut microbiota in a rat model of liver cirrhosis., Methods: Fifty-four male Sprague-Dawley rats were randomly divided into a normal control group (N), a liver cirrhosis group (M) and a liver cirrhosis group intervened with AS (MA). Each group was sampled at 4, 6 and 8 wk. Liver cirrhosis was induced by injection of carbon tetrachloride (CCl4), intragastric administration of 10% ethanol, and feeding a high fat diet. Rats in the MA group were intragastrically administered with AS (25 mg/kg body weight, once daily). Injuries of the liver and intestinal mucosa were assessed by hematoxylin-eosin or Masson's trichrome staining. Liver index was calculated as a ratio of the organ weight (g) to body weight (g). The gut microbiota was examined by automated ribosomal intergenic-spacer analysis of fecal DNA. BT was assessed by standard microbiological techniques in the blood, mesenteric lymph nodes (MLNs), liver, spleen, and kidney., Results: Compared to group N, the body weight was reduced significantly in groups M and MA due to the development of liver cirrhosis over the period of 8 wk. The body weight was higher in group MA than in group M. The liver indices were significantly elevated at 4, 6 and 8 wk in groups M and MA compared to group N. AS supplementation partially decreased the liver indices in group MA. Marked histopathologic changes in the liver and small intestinal mucosa in group M were observed, which were alleviated in group MA. Levels of pro-inflammatory interleukin-6 and tumor necrosis factor-α were significantly elevated at 8 wk in ileal homogenates in group M compared to group N, which were decreased after AS supplementation in group MA. The dysbiosis of gut microbiota indicated by the mean diversity (Shannon index) and mean similarity (Sorenson index) was severe as the liver cirrhosis developed, and AS supplementation had an apparent intervention effect on the dysbiosis of gut microbiota at 4 wk. The occurrence of BT was increased in the liver of group M compared to that of group N. AS supplementation reduced BT in group MA at 8 wk. BT also occurred in the MLNs, spleen, and kidney, which was reduced by AS supplementation. BT was not detected in the blood in any group., Conclusion: Dysbiosis of gut microbiota, injury of intestinal mucosal barrier and BT occurred as liver cirrhosis progressed, which might enhance inflammation and aggravate liver injury. AS may have other non-antimalarial effects that modulate gut microbiota, inhibit BT and alleviate inflammation, resulting in a reduction in CCl4, alcohol and high fat-caused damages to the liver and intestine.

The aim of this study was to establish the effective hepatoprotective properties of traditional Chinese medicines (TCMs) in fibrotic rat liver regeneration after partial hepatectomy (PHx). Fibrosis was induced in rats by ethanol (EtOH, 5 ml/kg) administration for 6, 24, 72, and 168 h. The rats were then fed four TCMs (1 g/kg/day, Codonopsis pilosula (CP), Salvia miltorrhiza Bunge (SMB), Bupleurum kasi (BK), and Elephantopus scaber L (ESL)) to Spraque-Dawley rats for 6, 24, 72 and 168 h, respectively. Surgical 70% cirrhotic fibrosis PHx was then conducted at 6, 24, 72, and 168 h. The effects on liver regeneration were examined to estimate and measure hepatocyte growth factor (HGF), focal adhesion kinase (FAK), Cyclin D1, Cyclin E, and retinoblastoma protein (pRb) protein expression using Western blotting analysis. Cyclin D1, matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2 and TIMP-3 mRNA by Reverse transcription polymerase chain reaction (RT-PCR) were analyzed in cirrhotic fibrosis rats. Transforming growth factor-β1 (TGF-β1), Cyclin D1, Cyclin E, pRb and E2F mRNA expression levels were determined in fibrotic rats following PHx using RT-PCR. We found elevated glutamyl oxaloacetic transaminase (GOT), glutamyl pyrubic transaminase (GPT), alkaline phosphatase (ALP), gammaglutamyl transpeptidase (γ-GT), glutathione (GSH), nonprotein sulfhydryl (NPSH) and total bilirubin in serum after 6 h EtOH administration. These levels were progressively decreased over 168 h. Total protein and albumin were reduced in serum after 6 h administration and then progressively increased. In contrast, tissues disorder histology and morphology were determined in liver sections. After rats were fed TCMs we found that SMB extraction not only induced HGF, FAK, Cyclin D1, and pRb protein expression and Cyclin D1 mRNA increases, but also reduced MMP-2 and MMP-9 after 24 and 72 h post injury. In the cell cycle S phase the Cyclin E protein expression was increased by ESL. CP induced TIMP-1, TIMP-2 and TIMP-3 mRNA increases in fibrotic rats. We detected liver regeneration in fibrotic rats. We also found that the liver regeneration index increased from 6 to 168 h post PHx. After 168 h fibrotic liver regeneration rats exhibited reduced TGF-β1 mRNA expression and enhanced Cyclin D1, Cyclin E, pRb and E2F mRNA expression. TCMs play a crucial role in the early mediating process in fibrotic rat liver regeneration after PHx.

Chronic liver diseases are characterized by a sustained inflammatory response in which chemokines and chemokine-receptors orchestrate inflammatory cell recruitment. In this study we investigated the role of the chemokine receptor CCR6 in acute and chronic liver injury. In the absence of liver injury Ccr6-/- mice presented a higher number of hepatic macrophages and increased expression of pro-inflammatory cytokines and M1 markers Tnf-α, Il6 and Mcp1. Inflammation and cell recruitment were increased after carbon tetrachloride-induced acute liver injury in Ccr6-/- mice. Moreover, chronic liver injury by carbon tetrachloride in Ccr6-/- mice was associated with enhanced inflammation and fibrosis, altered macrophage recruitment, enhanced CD4+ cells and a reduction in Th17 (CD4+IL17+) and mature dendritic (MHCII+CD11c+) cells recruitment. Clodronate depletion of macrophages in Ccr6-/- mice resulted in a reduction of hepatic pro-inflammatory and pro-fibrogenic markers in the absence and after liver injury. Finally, increased CCR6 hepatic expression in patients with alcoholic hepatitis was found to correlate with liver expression of CCL20 and severity of liver disease. In conclusion, CCR6 deficiency affects hepatic inflammatory cell recruitment resulting in the promotion of hepatic inflammation and fibrosis.

The current study was performed in order to explore the effect of artesunate (Art) on experimental hepatic fibrosis and the potential mechanism involved. Art, a water-soluble hemisuccinate derivative of artemisinin extracted from the Chinese herb Artemisia Annua, is a safe and effective antimalarial drug. Hepatic fibrosis was induced in SD rats by multiple pathogenic factors. Rats were treated concurrently with Art (28.8 mg/kg) given daily by oral gavage for 6 or 8 weeks to evaluate its protective effects. Our data demonstrated that Art treatment obviously attenuated hepatic fibrosis, characterized by less inflammatory infiltration and accumulation of extracellular matrix (ECM). Art remarkably decreased endotoxin, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) levels as well. Art significantly downregulated protein and mRNA expression of α-smooth muscle actin (α-SMA), toll-like receptors 4 (TLR4), myeloid differentiation factor 88 (MyD88) and transforming growth factor beta 1 (TGF-β1). Art also significantly inhibited the nuclear transcription factor kappa B p65 (NF-κB p65) translocation into the nucleus. In addition, there were no remarkable differences between the N group and the NA group. In conclusion, we found that Art could alleviate hepatic fibrosis induced by multiple pathogenic factors and inflammation through the inhibition of LPS/TLR4/NF-κB signaling pathway in rats, suggesting that Art may be a potential candidate for the therapy of hepatic fibrosis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Glutathione S-transferase placental form (GST-P) expression in hepatocyte foci is regarded as a preneoplastic change in rats. We aimed to reveal the contribution of polarized macrophages in development of GST-P-positive pseudolobules (PLs) in chemically-induced rat cirrhosis. F344 rats were injected with thioacetamide (100mg/kg BW, twice a week, intraperitoneally). Macrophage immunophenotypes and expression of M1-/M2-related factors were analyzed by immunohistochemistry, real-time RT-PCR and laser microdissection. GST-P-positive foci/clusters were clearly observed at post-first injection week 15. GST-P-positive PLs were distinguishable at weeks 20-32. Microarray analysis revealed upregulation of preneoplastic genes in GST-P-positive PLs at week 32. M1 (CD68(+), Iba1(+))-and M2 (CD163(+), CD204(+), Gal-3(+))-macrophages were greater in number in the GST-P-positive PLs, whereas MHC class II-positive (M1) macrophage number was fewer in the GST-P-positive PLs. Expression of both M1 (IFN-γ, IL-1β, TNF-α, Iba1)- and M2 (IL-4, TGF-β1, IL-10)-related factors were higher in GST-P-positive PLs. Our results showed that both M1- and M2-macrophage populations contribute to the development of hepatic preneoplastic lesions. MHC class II-positive macrophages may be related to anti-tumor progression, since their kinetics showed reverse pattern to other macrophage phenotypes., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Acute schistosomiasis is characterized by pro-inflammatory responses against tissue- or organ-trapped parasite eggs along with granuloma formation. Here, we describe studies in Cx3cr1(-/-) mice and demonstrate the role of Cx3cr1 in the pathoetiology of granuloma formation during acute schistosomiasis. Mice deficient in Cx3cr1 were protected from granuloma formation and hepatic injury induced by Schistosoma japonicum eggs, as manifested by reduced body weight loss and attenuated hepatomegaly along with preserved liver function. Notably, S. japonicum infection induced high levels of hepatic Cx3cr1 expression, which was predominantly expressed by infiltrating macrophages. Loss of Cx3cr1 rendered macrophages preferentially towards M2 polarization, which then led to a characteristic switch of the host immune defense from a conventional Th1 to a typical Th2 response during acute schistosomiasis. This immune switch caused by Cx3cr1 deficiency was probably associated with enhanced STAT6/PPAR-γ signaling and increased expression of indoleamine 2,3-dioxygenase (IDO), an enzyme that promotes M2 polarization of macrophages. Taken together, our data provide evidence suggesting that CX3CR1 could be a viable therapeutic target for treatment of acute schistosomiasis., (© 2015. Published by The Company of Biologists Ltd.)

Liver cirrhosis is responsible for hepatic fibrosis resulting in high mortality and is also a risk factor for developing hepatocellular carcinoma (HCC), which is the fifth most common cancer in men and the seventh in women globally. Several studies have found effective anti-cancer activities of theaflavins, the major black tea polyphenols. The objective of this study was to investigate the protective effects of theaflavin-enriched black tea extracts (TF-BTE) on hepatic fibrosis induced by dimethylnitrosamine (DMN) administration in Sprague-Dawley (SD) rats. Treatment of SD rats with DMN (10 mg per kg bw) for 4 weeks produced inflammation and remarkable liver fibrosis assessed by serum biochemistry and histopathological examination. Fibrotic status and the activation of hepatic stellate cells were improved by oral administration of 40% theaflavins in black tea extracts (40% TF-BTE) as evidenced by histopathological examination. Oral administration of 40% TF-BTE at a low dose of 50 mg per kg bw per day and a high dose of 100 mg per kg bw per day attenuated the DMN-induced elevation of serum GOT (glutamate oxaloacetate transaminase) and GPT (glutamic pyruvic transaminase) levels and reduced necrosis, bile duct proliferation, and inflammation. Western blot analyses revealed that TF-BTE inhibited the expression of liver alpha-smooth muscle actin (α-SMA) and transforming growth factor-β1 (TGF-β1) protein. The histochemical examination showed the inhibitory effect of TF-BTE on the p-Smad3 expression. Overall, these data demonstrated that TF-BTE exhibited hepatoprotective effects on experimental fibrosis, potentially by inhibiting the TGF-β1/Smad signaling.

Aim: To investigate the effect of interleukin (IL)-22 on hepatic fibrosis in mice and the possible mechanism involved., Methods: Liver fibrosis was induced in male BALB/c mice by CCl₄. Recombinant IL-22 (rmIL-22) was administered intraperitoneally in CCl₄-treated mice. Fibrosis was assessed by histology and Masson staining. The activation of hepatic stellate cells (HSCs) was investigated by analysis of α-smooth muscle actin expression. The frequencies of T helper (Th) 22 cells, Th17 cells and Th1 cells, the expression of inflammatory cytokines [IL-22, IL-17A, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), IL-6, IL-1β] and transcription factors [aryl hydrocarbon receptor (AHR), RAR-related orphan receptor (RORγt), T-bet] mRNA in the liver were investigated. In addition, the plasma levels of IL-22, IL-17A, IFN-γ, TNF-α, IL-6 and IL-1β were evaluated., Results: Significant elevations in circulating Th22 cells, Th17 cells, Th1 cells, IL-22, IL-17A, and IFN-γ were observed in the hepatic fibrosis group compared with the control group (P < 0.01). Treatment with rmIL-22 in mice with hepatic fibrosis ameliorated the severity of hepatic fibrosis, which was confirmed by lower hepatic fibrosis pathological scores (P < 0.01). RmIL-22 decreased the frequencies of Th22 cells (6.71% ± 0.97% vs 8.09% ± 0.74%, P < 0.01), Th17 cells (4.34% ± 0.37% vs 5.71% ± 0.24%, P < 0.01), Th1 cells (3.09% ± 0.49% vs 4.91% ± 0.73%, P < 0.01), and the levels of IL-22 (56.23 ± 3.08 vs 70.29 ± 3.01, P < 0.01), IL-17A (30.74 ± 2.77 vs 45.68 ± 2.71, P < 0.01), and IFN-γ (74.78 ± 2.61 vs 124.89 ± 2.82, P < 0.01). Down-regulation of IL-22, IL-17A, IFN-γ, TNF-α, IL-6, IL-1β, AHR RORγt, and T-bet gene expression in the liver was observed in the rmIL-22 group (P < 0.01)., Conclusion: The frequencies of Th22, Th17 and Th1 cells are elevated in hepatic fibrosis. RmIL-22 can attenuate HSC activation and down-regulate the levels of inflammatory cytokines, thereby ameliorating liver fibrogenesis.

Background & Aims: Bacterial endotoxin is present in patients with advanced cirrhosis and can induce an immunogenic response without an overt infection. Norfloxacin is a gram-negative bactericidal drug able to maintain low endotoxin levels and stimulate IL-10 production. We aimed at investigating the role of IL-10 in decreasing endotoxin absorption in cirrhotic mice treated with norfloxacin., Methods: Cirrhosis was induced by carbon tetrachloride or bile duct ligation in wild type and IL10-deficient mice with or without norfloxacin prior to an intragastrical administration of E. coli, K. pneumonia or E. faecalis. Spontaneous and induced bacterial translocation, free endotoxin and cytokine levels were evaluated in mesenteric lymph nodes. Intestinal permeability was followed by fluorimetry and barrier integrity markers were measured in disrupted intestinal samples. The inflammatory-modulating mechanism was characterized in purified intestinal mononuclear cells., Results: Norfloxacin reduced spontaneous and induced MLN positive-cultures in wild type and IL-10-deficient animals. However, reduction of free endotoxin levels was associated with norfloxacin in wild type but not in IL-10-deficient mice. Wild type but not IL-10-deficient mice treated with norfloxacin significantly normalized intestinal permeability and improved gut barrier integrity markers. The toll-like receptor 4-mediated pro-inflammatory milieu was modulated by norfloxacin in a concentration-dependent manner in cultured intestinal mononuclear cells of wild type mice but not of IL-10-deficient mice. The restoration of IL-10 levels in IL-10-deficient animals reactivated the norfloxacin effect on inflammatory-modulation, gut barrier permeability, and luminal endotoxin absorption., Conclusion: Norfloxacin not only reduces gram-negative intestinal flora but also participates in an IL-10-driven modulation of gut barrier permeability, thus reducing luminal free endotoxin absorption in experimental cirrhosis., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Microglia and astrocyte related pro-inflammatory responses are thought to underpin cerebral sequelae of acute liver failure. Conversely, despite background pro-inflammatory responses in cirrhosis, overt brain swelling and coma associated with acute-on-chronic liver failure, is infrequent unless precipitated (e.g. sepsis). Moreover in other chronic neurodegenerative disorders and sepsis, the brain is protected from recurrent microbial insults by compensatory microglial-associated immune responses. To characterise longitudinal cerebral immune responses in a bile duct-ligated (BDL) rat model of cirrhosis., Method: Rats underwent BDL or sham operation before sacrifice at either 1-day, 1, 2 and 4 weeks post-surgery. We analysed consciousness, brain water, biochemistry and immunohistochemistry to assess activation of microglia (ED-1, OX6 and Iba-1), astrocytes (Glial fibrillary acidic protein - GFAP), cellular stress (Heat shock protein - Hsp 25) and pro-inflammatory mediator expression (inducible nitric oxide synthase (iNOS), interleukin-1beta (IL-1β) and tumour growth factor-beta (TGF-β))., Results: BDL significantly increased ammonia and bilirubin (P < 0.01 respectively). The classical microglial markers OX6, ED1 and Iba-1 and pro-inflammatory IL-1β and iNOS were not significantly increased. However, the alternative microglial marker and regulatory cytokine TGF-β was elevated from day 1 to 4 weeks post-BDL. GFAP expression was significantly increased in corpus callosum in all groups. In BDL rats, Hsp 25 was also increased in the corpus callosum, peaking at 2 weeks., Conclusion: BDL triggers early alternative, but not classical, microglial activation. There was a correlation between astrocyte activation and cellular stress. These findings indicate early cerebral immune responses, which may be associated with immune tolerance to further challenge., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background & Aims: Development of the portal-hypertensive syndrome is mediated by splanchnic inflammation and neoangiogenesis. Since peroxisome proliferator-activated receptor gamma (PPARγ) agonists like pioglitazone (PIO) regulate inflammatory response and inhibit angiogenesis in endothelial cells, we evaluated PIO as treatment for experimental portal hypertension., Methods: PIO (10 mg/kg) or vehicle (VEH) was administered from day 21-28 after bile duct ligation (BDL), from day 0-7 after partial portal vein ligation (PPVL) or sham-operation (SO), respectively. After treatment, systemic hemodynamics, splanchnic blood flow (SMABF), portal pressure (PP), and portosystemic shunting (PSS) were assessed. Splanchnic and hepatic tissues were analyzed for angiogenic and inflammatory markers., Results: BDL and PPVL showed significantly increased PP, SMABF, and PSS compared to SO-VEH rats. While PIO treatment did not decrease PP or SMABF, PSS was significantly reduced both in cirrhotic (BDL-VEH: 71% to BDL-PIO: 41%; p<0.001) and non-cirrhotic (PPVL-VEH: 62% to PPVL-PIO: 40%; p=0.041) rats. PIO (10 μM, in vitro) inhibited endothelial cell migration and significantly increased PPARγ activity in vivo. In BDL rats, PIO decreased hepatic mRNA levels of PPARγ (p=0.01) and PlGF (p=0.071), and splanchnic mRNA expression of PPARγ (p=0.017), PDGFβ (p=0.053) and TNFα (p=0.075). Accordingly, splanchnic protein expression of PPARγ (p=0.032), VEGFR2 (p=0.035), CD31 (p=0.060) and PDGFβ (p=0.066) were lower in BDL-PIO vs. BDL-VEH animals. In PPVL rats, PIO treatment decreased splanchnic gene expression of Ang2 (-12.4 fold), eNOS (-9.3 fold), PDGF (-7.0 fold), PlGF (-11.9 fold), TGFb (-8.3 fold), VEGF-A (-11.3 fold), VEGFR1 (-5.9 fold), IL1b (-14.4 fold), and IL6 (-9.6 fold)., Conclusions: Pioglitazone treatment decreases portosystemic shunting via modulation of splanchnic inflammation and neoangiogenesis. Pioglitazone should be assessed for potential beneficial effects in patients with portosystemic collaterals due to portal hypertension., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Vascular endothelial growth factor (VEGF)-induced angiogenesis is implicated in fibrogenesis and portal hypertension. However, the function of VEGF in fibrosis resolution has not been explored., Methods: We developed a cholecystojejunostomy procedure to reconstruct biliary flow after bile duct ligation in C57BL/6 mice to generate a model of fibrosis resolution. These mice were then given injections of VEGF-neutralizing (mcr84) or control antibodies, and other mice received an adenovirus that expressed mouse VEGF or a control vector. The procedure was also performed on macrophage fas-induced apoptosis mice, in which macrophages can be selectively depleted. Liver and blood samples were collected and analyzed in immunohistochemical, morphometric, vascular permeability, real-time polymerase chain reaction, and flow cytometry assays., Results: VEGF-neutralizing antibodies prevented development of fibrosis but also disrupted hepatic tissue repair and fibrosis resolution. During fibrosis resolution, VEGF inhibition impaired liver sinusoidal permeability, which was associated with reduced monocyte migration, adhesion, and infiltration of fibrotic liver. Scar-associated macrophages contributed to this process by producing the chemokine (C-X-C motif) ligand 9 (CXCL9) and matrix metalloproteinase 13. Resolution of fibrosis was impaired in macrophage fas-induced apoptosis mice but increased after overexpression of CXCL9., Conclusions: In a mouse model of liver fibrosis resolution, VEGF promoted fibrogenesis, but was also required for hepatic tissue repair and fibrosis resolution. We observed that VEGF regulates vascular permeability, monocyte infiltration, and scar-associated macrophages function., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: The liver's response to injury is fibrosis, and when chronic, cirrhosis. Age is a critical factor impacting many immune-mediated processes, potentially including the liver's wounding response to injury., Methods: The effects of age on acute and chronic liver injury were evaluated using a carbon tetrachloride model in mice. Lymphocyte and macrophage populations were assessed by flow cytometry and immunohistochemical analysis., Results: Acute liver injury was greater in 18-month-old (old) mice than in 9-month-old (middle-aged) mice as judged by changes in aminotransferases. Similarly, livers of 18-month-old mice had a significantly greater fibrogenic response to injury than did livers of 9-month-old mice after chronic injury (assessed by col1α1 mRNA expression, morphometric analysis and hydroxyproline measurement). Interestingly, livers from young mice (6 weeks old) also exhibited an increase in fibrogenesis compared to 9-month-old mice, albeit not to the same degree as in old mice. Consistent with a role for macrophages in fibrogenesis, the number of liver macrophages in young and 9-month-old mice increased, while in chronically injured livers of 18-month-old mice, the number of macrophages was reduced, and was less than in the livers of young and 9-month-old injured livers., Conclusions: Our data indicate that the fibrogenic response to injury varies substantially with age, and moreover that macrophage recruitment and dynamics may be an important component in differential age-associated fibrotic disease., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Aim: To investigate long-term effects of Garcinia Cambogia (GC), weight-loss supplement, on adiposity and non-alcoholic fatty liver disease in obese mice., Methods: Obesity-prone C57BL/6J mice were fed a high-fat diet (HFD, 45 kcal% fat) with or without GC (1%, w/w) for 16 wk. The HFD contained 45 kcal% fat, 20 kcal% protein and 35 kcal% carbohydrate. They were given free access to food and distilled water, and food consumption and body weight were measured daily and weekly, respectively. Data were expressed as the mean ± SE. Statistical analyses were performed using the statistical package for the social science software program. Student's t test was used to assess the differences between the groups. Statistical significance was considered at P < 0.05., Results: There were no significant changes in body weight and food intake between the groups. However, the supplementation of GC significantly lowered visceral fat accumulation and adipocyte size via inhibition of fatty acid synthase activity and its mRNA expression in visceral adipose tissue, along with enhanced enzymatic activity and gene expression involved in adipose fatty acid β-oxidation. Moreover, GC supplementation resulted in significant reductions in glucose intolerance and the plasma resistin level in the HFD-fed mice. However, we first demonstrated that it increased hepatic collagen accumulation, lipid peroxidation and mRNA levels of genes related to oxidative stress (superoxide dismutase and glutathione peroxidase) and inflammatory responses (tumor necrosis factor-α and monocyte chemoattractant protein-1) as well as plasma alanine transaminase and aspartate transaminase levels, although HFD-induced hepatic steatosis was not altered., Conclusion: GC protects against HFD-induced obesity by modulating adipose fatty acid synthesis and β-oxidation but induces hepatic fibrosis, inflammation and oxidative stress.

Aim: To investigate the expression of dipeptidyl peptidase (DPP) 8 and DPP9 in lymphocytes and various models of liver fibrosis., Methods: DPP8 and DPP9 expression were measured in mouse splenic CD4⁺ T-cells, CD8⁺ T-cells and B-cells (B220⁺), human lymphoma cell lines and mouse splenocytes stimulated with pokeweed mitogen (PWM) or lipopolysaccharide (LPS), and in dithiothreitol (DTT) and mitomycin-C treated Raji cells. DPP8 and DPP9 expression were measured in epidermal growth factor (EGF) treated Huh7 hepatoma cells, in fibrotic liver samples from mice treated with carbon tetrachloride (CCl₄) and from multidrug resistance gene 2 (Mdr2/Abcb4) gene knockout (gko) mice with biliary fibrosis, and in human end stage primary biliary cirrhosis (PBC)., Results: All three lymphocyte subsets expressed DPP8 and DPP9 mRNA. DPP8 and DPP9 expression were upregulated in both PWM and LPS stimulated mouse splenocytes and in both Jurkat T- and Raji B-cell lines. DPP8 and DPP9 were downregulated in DTT treated and upregulated in mitomycin-C treated Raji cells. DPP9-transfected Raji cells exhibited more annexin V⁺ cells and associated apoptosis. DPP8 and DPP9 mRNA were upregulated in CCl₄ induced fibrotic livers but not in the lymphocytes isolated from such livers, while DPP9 was upregulated in EGF stimulated Huh7 cells. In contrast, intrahepatic DPP8 and DPP9 mRNA expression levels were low in the Mdr2 gko mouse and in human PBC compared to non-diseased livers., Conclusion: These expression patterns point to biological roles for DPP8 and DPP9 in lymphocyte activation and apoptosis and in hepatocytes during liver disease pathogenesis.

Background & Aims: Interleukin (IL)-17 signaling has been implicated in lung and skin fibrosis. We examined the role of IL-17 signaling in the pathogenesis of liver fibrosis in mice., Methods: Using cholestatic and hepatotoxic models of liver injury, we compared the development of liver fibrosis in wild-type mice with that of IL-17RA(-/-) mice and of bone marrow chimeric mice devoid of IL-17 signaling in immune and Kupffer cells (IL-17RA(-/-) to wild-type and IL-17A(-/-) to wild-type mice) or liver resident cells (wild-type to IL-17RA(-/-) mice)., Results: In response to liver injury, levels of Il-17A and its receptor increased. IL-17A increased appeared to promote fibrosis by activating inflammatory and liver resident cells. IL-17 signaling facilitated production of IL-6, IL-1, and tumor necrosis factor-α by inflammatory cells and increased the expression of transforming growth factor-1, a fibrogenic cytokine. IL-17 directly induced production of collagen type I in hepatic stellate cells by activating the signal transducer and activator of transcription 3 (Stat3) signaling pathway. Mice devoid of Stat3 signaling in hepatic stellate cells (GFAPStat3(-/-) mice) were less susceptible to fibrosis. Furthermore, deletion of IL-23 from immune cells attenuated liver fibrosis, whereas deletion of IL-22 exacerbated fibrosis. Administration of IL-22 and IL-17E (IL-25, a negative regulator of IL-23) protected mice from bile duct ligation-induced liver fibrosis., Conclusions: IL-17 induces liver fibrosis through multiple mechanisms in mice. Reagents that block these pathways might be developed as therapeutics for patients with cirrhosis., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Although bacterial endotoxin, such as lipopolysaccharide (LPS), plays a key role in the pathogenesis of nonalcoholic steatohepatitis (NASH), detailed mechanisms of this pathogenesis remain unclear. Here, we demonstrate that upregulation of CD14 by leptin-mediated signaling is critical to hyperreactivity against endotoxin during NASH progression. Upregulation of CD14 in Kupffer cells and hyperreactivity against low-dose LPS were observed in high-fat diet (HFD)-induced steatosis mice, but not chow-fed-control mice. Hyperresponsivity against low-dose LPS led to accelerated NASH progression, including liver inflammation and fibrosis. Administering leptin in chow-fed mice caused increased hepatic expression of CD14 via STAT3 signaling, resulting in hyperreactivity against low-dose LPS without steatosis. In contrast, a marked decrease in hepatic CD14 expression was observed in leptin-deficient ob/ob mice, despite severe steatosis. Our results indicate that obesity-induced leptin plays a crucial role in NASH progression via enhanced responsivity to endotoxin, and we propose a mechanism of bacteria-mediated progression of NASH., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: To examine imatinib mesylate's effects on stellate cell responses in vivo and in vitro. The hepatic stellate cell (HSC) is a key target of anti-fibrotic therapies. Imatinib mesylate is a small molecule receptor tyrosine kinase inhibitor indicated for treatment of chronic myelogenous leukaemia and GI stromal tumours., Design: Because imatinib inhibits β-PDGFR signalling, which stimulates HSC proliferation, we assessed its activity in culture and in vivo, and examined downstream targets in a human stellate cell line (LX-2) using cDNA microarray., Methods and Results: Imatinib inhibited proliferation of LX-2 cells (0.5-10 mM) but not primary human stellate cells, with no effect on viability, associated with attenuated β-PDGFR phosphorylation. Mitochondrial activity and superoxide anion production were decreased in response to imatinib. cDNA microarray uncovered up-regulation of 29 genes in response to imatinib, including interleukin-6 (IL-6) mRNA, which was correlated with progressive IL-6 secretion. Imatinib also decreased gene expression of collagen α(1) (I), alpha smooth muscle actin, β-PDGFR, transforming growth factor β receptor type 1, matrix metalloproteinase 2 and tissue inhibitor of metalloproteinase 2. In vivo, imatinib administered to rats beginning 4 weeks after starting thioacetamide (TAA) led to reduced collagen content, with significant reductions in portal pressure and down-regulation of fibrogenic genes in whole liver. Importantly, hepatic IL-6 mRNA levels were significantly increased in TAA-treated animals receiving imatinib., Conclusions: These findings reinforce the anti-fibrotic activity of imatinib and uncover an unexpected link between inhibition of HSC activation by imatinib and enhanced secretion of IL-6, a regenerative cytokine., (© 2012 John Wiley & Sons A/S.)

Cannabinoid 2 (CB2) receptors expressed on immune cells are considered to be antifibrogenic. Hepatic stellate cells (HSCs) directly interact with phagocytosis lymphocytes, but the nature of this interaction is obscure. We aimed to study the effects of CB2 receptors on hepatic fibrosis via their role in mediating immunity. Hepatic fibrosis was induced by carbon-tetrachloride (CCl(4)) administration in C57BL/6 wild-type (WT) and CB2 knockout (CB2(-/-)) mice. Irradiated animals were reconstituted with WT or CB2(-/-) lymphocytes. Lymphocytes from naïve/fibrotic WT animals and healthy/cirrhotic hepatitis C virus were preincubated in vitro with or without CB2 antagonist, evaluated for proliferation and apoptosis, and then cocultured with primary mouse HSCs or a human HSC line (LX2), respectively. Lymphocyte phagocytosis was then evaluated. Following CCl(4)-administration, CB2(-/-) mice developed significant hepatic fibrosis but less necroinflammation. WT mice harbored decreased liver CD4(+) and NK(+) cells but increased CD8(+) subsets. Naïve CB2(-/-) mice had significantly decreased T cell subsets. Adoptive transfer of CB2(-/-) lymphocytes led to decreased fibrosis in the irradiated WT recipient compared with animals receiving WT lymphocytes. Moreover, necroinflammation also tended to decrease. In vitro, a CB2-antagonist directly increased human HSC activation and increased apoptosis and decreased proliferation of mice/human T cells (healthy/fibrotic) and their phagocytosis. We concluded that CB2(-/-) lymphocytes exert an antifibrotic activity, whereas lack of CB2 receptor in HSCs promotes fibrosis. These findings broaden our understanding of cannabinoid signaling in hepatic fibrosis beyond their activity solely in HSCs.

No drugs have been approved clinically for the therapy of hepatic fibrosis. Though interferon-γ (IFN-γ) is a highly effective anti-fibrotic agent in vitro and in some animal models in vivo, its anti-fibrotic potential in clinical trials has been disappointing, due to unwanted off-target effects and a short half-life period which results in poor efficacy. The aims of this study are to develop a new targeted drug delivery system to selectively deliver IFN-γ to hepatic stellate cells (HSCs) and to investigate whether it will improve the anti-fibrotic effect of IFN-γ and reduce its side effects in fibrotic livers. Sterically stable liposomes (SSLs) were modified by cyclic peptides (pPB) with a specific affinity for platelet-derived growth factor receptor-β (PDGFR-β), and then IFN-γ was encapsulated in the targeted liposomes (pPB-SSL-IFN-γ). In vitro, pPB-SSL was found to be taken up and internalized by cultured activated HSCs. The binding of FITC-labeled pPB-SSL to activated HSCs was in a time-dependent and concentration-dependent manner, which could be inhibited by excess unlabelled pPB-SSL, PDGF-BB, suramin or monensin. The inhibitory effect of pPB-SSL-IFN-γ on the proliferation of activated HSCs was respectively 7.24-fold and 2.95-fold higher than that of free IFN-γ and IFN-γ encapsulated in untargeted SSLs. In healthy rats, the tissue distribution, living-body tracing image analyses and pharmacokinetics study showed that pPB-SSL-IFN-γ accumulated mainly in the livers and had a longer half-life than free IFN-γ (3.98±0.52h vs. 0.21±0.03h). Furthermore, in rats with hepatic fibrosis induced by thioacetamide injection, FITC-labeled pPB-SSL was found to predominantly localize in activated HSCs by immunofluorescent double staining for FITC and albumin or α-smooth muscle actin (α-SMA). The enhanced anti-fibrotic effect of pPB-SSL-IFN-γ treatnment was indicated by significant decreases in the histologic Ishak stage, collagen I-staining positive areas, and α-SMA expression levels in fibrotic livers. In addition, pPB-SSL-IFN-γ treatment improved the leukopenia caused by low- and high-dosage free IFN-γ treatments. In conclusion, IFN-γ encapsulated in pPB-SSL had an extended circulation half-life and was selectively delivered to activated HSCs, which enhanced the anti-fibrotic effect of IFN-γ and reduced its side-effects in rats with hepatic fibrosis. Thus, pPB-SSL-IFN-γ may be an effective agent for the therapy of hepatic fibrosis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Females with sepsis have a better prognosis than males, while those of both genders with cirrhosis have a high mortality. Impaired immunity accompanies liver cirrhosis. The potential association between sex and immunologic response of cirrhotic rats in sepsis following immunonutrition was investigated. One hundred and forty-three rats were randomly divided into groups. Liver cirrhosis was produced by weekly feeding of CCl(4) for 8 weeks. Among them, 24 male and 19 female underwent castration one month before studying. The rats were fed with either immune enhancing diet or control diet for five days, then sepsis was induced with cecal ligation and two holes puncture. Main outcomes included mortality and serum cytokines (IL-1β, 6, and 10). Comparisons were made both within and between genders. Cirrhotic non-castrated male rats showed a significant decrease in mortality (64.1% vs. 32.1%, p = 0.032) with better survival than control diet following immune enhancing diet. Lower mortality of cirrhotic non-castrated female rats was found after immune enhancing diet (69.6% vs. 52.1%, p = 0.365). Cirrhotic castrated male rats showed a lower mortality (44.4%) following immune enhancing diet, and cirrhotic castrated female rats also showed significantly lower mortality and better survival than control diet after immune enhancing diet (87.5% vs. 33.3%, p = 0.004). Plasma concentrations of IL-1β were higher in non-oophorectomized female rats fed with control diet compared to immune enhancing diet. Non-orchidectomized males and non-oophorectomized females exhibited similar increases in IL-10 after immune enhancing diet. Our results demonstrated that immunonutrition was more beneficial for male than female cirrhotic rats following sepsis. Though orchidectomy was not found to be more advantageous for the normal male rats in sepsis, immunonutrition seemed to be as important as sex hormone for female rats in sepsis.

Background: The present study investigated the alteration of intestinal microbiota, innate immunity-related genes, and bacterial translocation in rats with cirrhosis and liver transplantation., Methods: Specific pathogen-free Sprague-Dawley rats were randomized into 4 groups: (1) normal controls (N); (2) liver cirrhosis (LC); (3) normal control groups with liver transplantation (LTN); and (4) liver cirrhosis with liver transplantation (LTC). We examined plasma endotoxin, bacterial tacslocation, denaturing gradient gel electrophoresis (DGGE) profile of intestinal mucosa-associated bacteria, abundance of key bacterial populations, and expression of innate immunity-related gene., Results: The LTC and LC group, showed higher endotoxin levels (1.08±0.73 EU/mL and 0.74±0.70 EU/mL, respectively) than the N group (0.27±0.13 EU/mL; P<.05). the incidence of bacterial translocation (BT) to liver and mesenteric lymph nodes (MLN), and the number of total bacteria were increased significantly in the LTC and LC groups compared with the N group (P<.05). The counts of Lactobacilli and Bacteroides were lower, whereas Enterobacteria were higher in the LC than the N group (P<.05). Mucins (MUC2, MUC3) and Toll-like receptors (TLR2, TLR4) messenger RNA (mRNA) expression were significantly higher in the LC and LTC groups than the N group (P<.05). The marked difference between the groups in the overall structure of the bacterial community was also generated by DGGE profiles., Conclusion: Liver cirrhosis disturbs intestinal microbiota and innate immunity-related genes, which contributes to endotoxemia and bacterial translocation. These had not completely recovered in cirrhotic rats until 1 month after orthotopic liver transplantation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Introduction: Immunomodulatory drugs have been reported to have anti-inflammatory and anti-fibrotic properties. Thymic humoral factor (THF), a peptide produced in the thymus, causes a potent immunomodulatory effect on different components of the immune system., Objective: To evaluate the effect of THF on different stages of liver damage and fibrosis induced in rats through the administration of porcine serum (PS)., Material and Methods: PS-induced liver fibrosis models serve as a primarily immunological mechanism in the development of liver damage and fibrosis., Results: The intraperitoneal administration of THF in rats with PS-induced liver damage produced a reduction of ALT and AST after 60 days. Histopathological changes in liver sections showed an improved histological appearance and lower % of fibrosis after 60 days in liver damaged rats that received THF treatment. Serum IL-6 levels were visibly reduced by THF administration after 60 days and in comparison with rats that did not receive the treatment. This was due to an increment in serum IL-10 levels caused by the administration of THF, which appears to reduce the inflammatory process by decreasing immune response., Conclusion: THF had beneficial effects in combating liver damage and fibrosis processes in an autoimmune model of PS-induced liver fibrosis in rats.