N-n-Butyl haloperidol iodide ameliorates liver fibrosis and hepatic stellate cell activation in mice.

N-n-Butyl haloperidol iodide (F2) is a novel compound that has antiproliferative and antifibrogenic activities. In this study we investigated the therapeutic potential of F2 against liver fibrosis in mice and the underlying mechanisms. Two widely used mouse models of fibrosis was established in mice by injection of either carbon tetrachloride (CCl4) or thioacetamide (TAA). The mice received F2 (0.75, 1.5 or 3 mg·kg-1·d-1, ip) for 4 weeks of fibrosis induction. We showed that F2 administration dose-dependently ameliorated CCl4- or TAA-induced liver fibrosis, evidenced by significant decreases in collagen deposition and c-Jun, TGF-ß receptor II (TGFBR2), α-smooth muscle actin (α-SMA), and collagen I expression in the liver. In transforming growth factor beta 1 (TGF-ß1)-stimulated LX-2 cells (a human hepatic stellate cell line) and primary mouse hepatic stellate cells, treatment with F2 (0.1, 1, 10 µM) concentration-dependently inhibited the expression of α-SMA, and collagen I. In LX-2 cells, F2 inhibited TGF-ß/Smad signaling through reducing the levels of TGFBR2; pretreatment with LY2109761 (TGF-ß signaling inhibitor) or SP600125 (c-Jun signaling inhibitor) markedly inhibited TGF-ß1-induced induction of α-SMA and collagen I. Knockdown of c-Jun decreased TGF-ß signaling genes, including TGFBR2 levels. We revealed that c-Jun was bound to the TGFBR2 promoter, whereas F2 suppressed the binding of c-Jun to the TGFBR2 promoter to restrain TGF-ß signaling and inhibit α-SMA and collagen I upregulation. In conclusion, the therapeutic benefit of F2 against liver fibrosis results from inhibition of c-Jun expression to reduce TGFBR2 and concomitant reduction of the responsiveness of hepatic stellate cells to TGF-ß1. F2 may thus be a potentially new effective pharmacotherapy for human liver fibrosis.

Inducible T-Cell Costimulator Mediates Lymphocyte/Macrophage Interactions During Liver Repair.

The liver capacity to recover from acute liver injury is a critical factor in the development of acute liver failure (ALF) caused by viral infections, ischemia/reperfusion or drug toxicity. Liver healing requires the switching of pro-inflammatory monocyte-derived macrophages(MoMFs) to a reparative phenotype. However, the mechanisms involved are still incompletely characterized. In this study we investigated the contribution of T-lymphocyte/macrophage interaction through the co-stimulatory molecule Inducible T-cell co-stimulator (ICOS; CD278) and its ligand (ICOSL; CD275) in modulating liver repair. The role of ICOS/ICOSL dyad was investigated during the recovery from acute liver damage induced by a single dose of carbon tetrachloride (CCl4). Flow cytometry of non-parenchymal liver cells obtained from CCl4-treated wild-type mice revealed that the recovery from acute liver injury associated with a specific up-regulation of ICOS in CD8+ T-lymphocytes and with an increase in ICOSL expression involving CD11bhigh/F4-80+ hepatic MoMFs. Although ICOS deficiency did not influence the severity of liver damage and the evolution of inflammation, CCl4-treated ICOS knockout (ICOS-/- ) mice showed delayed clearance of liver necrosis and increased mortality. These animals were also characterized by a significant reduction of hepatic reparative MoMFs due to an increased rate of cell apoptosis. An impaired liver healing and loss of reparative MoMFs was similarly evident in ICOSL-deficient mice or following CD8+ T-cells ablation in wild-type mice. The loss of reparative MoMFs was prevented by supplementing CCl4-treated ICOS-/- mice with recombinant ICOS (ICOS-Fc) which also stimulated full recovery from liver injury. These data demonstrated that CD8+ T-lymphocytes play a key role in supporting the survival of reparative MoMFs during liver healing trough ICOS/ICOSL-mediated signaling. These observations open the possibility of targeting ICOS/ICOSL dyad as a novel tool for promoting efficient healing following acute liver injury.

Up-regulation of Nrf2/P62/Keap1 involves in the anti-fibrotic effect of combination of monoammonium glycyrrhizinate and cysteine hydrochloride induced by CCl4.

Combination of monoammonium glycyrrhizinate and cysteine hydrochloride (MG-CH) has been used in the treatment of chronic liver disease for decades, however, its mechanism is still unclear. Our previous studies showed that MG-CH confers the optimal therapeutic effect at the ratio of 2:1 to against acute liver damage. In this study, it was used to investigate the anti-fibrotic effect induced by CCl4. The results showed that injection of MG-CH produced anti-fibrotic effect ranged from 30 mg/kg to 60 mg/kg, evidenced by decreased the collagens deposition and inhibited the production of hydroxyproline. Mechanism study found that Nrf2/ARE signaling pathway was activated by MG-CH, whereas loss of hepatocytic Nrf2 abolished its anti-fibrotic effect significantly. Furthermore, it was demonstrated that MG-CH is a non-canonical NRF2 inducer, which promoted the autophagy activity and release the Nrf2 from keap 1 by promoting the phosphorylation of p62 at Ser351. Knockdown of p62 abolished the enhancement of nuclear accumulation of Nrf2 by MG-CH. All of these results suggested that up-regulation of Nrf2/P62/Keap1 involves in the anti-fibrotic effect of MG-CH, which provide a rational explanation for the usage of MG-CH in the treatment of fibrosis.

Synergistic effect of ursolic acid and piperine in CCl4 induced hepatotoxicity.

BACKGROUND: Ursolic acid (UA) is a potent plant-based hepatoprotective agent having poor bioavailability, which hampers its therapeutic efficacy. The present study tries to overcome this limitation by combining it with piperine (PIP), a proven bioenhancer and hepatoprotective agent. METHODS: The type of interaction (synergism, addition, or antagonism) resulting between UA and PIP was analyzed and quantified by isobologram and combination index analysis. The hepatoprotective activity of UA and PIP was evaluated by measuring the level of hepatic marker enzymes. Pharmacokinetic analysis was carried out to ascertain the improvement of bioavailability. RESULTS: The combinations significantly decrease the enzyme levels, which indicate better hepatoprotective activity compared to single drugs. The relative oral bioavailability of UA was increased about tenfold (from AUC0-t =12.78 ± 2.59 µg/h/ml to 125.15 ± 1.84 µg/h/ml) along with the improvement of plasma concentration and elimination half-life. CONCLUSIONS: The findings indicated that the combination of PIP and UA is an effective strategy in enhancing the bioavailability and hepatoprotective potential of UA.KEY MESSAGESUrsolic acid in a combination with piperine provides a synergistic hepatoprotective effect in carbon tetrachloride induced liver damage in rats.Piperine improves the pharmacokinetic properties of ursolic acid when given in combination.Piperine improves the relative oral bioavailability of ursolic acid by tenfold when combined together.

Curc-mPEG454, a PEGylated curcumin derivative, as a multi-target anti-fibrotic prodrug.

Our previous studies demonstrated that Curc-mPEG454, a curcumin derivative modified with short-chain polyethylene glycol (PEG), not only increased the blood concentration of curcumin, but also retained its anti-inflammatory activity. Here, we aimed to evaluate the anti-fibrotic effect of Curc-mPEG454 on a rat liver fibrosis model induced by carbon tetrachloride (CCl4), and to explore the underlying mechanisms by integrating our total liver RNA sequencing (RNA-seq) data with recent liver single-cell sequencing (scRNA-seq) studies. 50 mg/kg and 100 mg/kg Curc-mPEG454 treatment significantly reduced the elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) induced by CCl4, and the incidence of liver cirrhosis decreased from 75% to 37% and 35%, respectively. RNA-seq analysis revealed that Curc-mPEG454 significantly upregulated aldehyde oxidase 1 (AOX1) while downregulated cytochrome p450 26A1 (CYP26A1) and cytochrome p450 26B1 (CYP26B1) resulting in restoring liver retinoic acid (RA) level, increased glutamate-cysteine ligase catalytic subunit (GCLC) and glutamate-cysteine ligase modifier subunit (GCLM) expression to synthesize hepatic glutathione (GSH), and inhibited liver inflammation via down-regulating the Prostaglandin E Synthase 2 (PTGES2)/prostacyclin E2 (PGE2) signaling. Integrating scRNA-seq data revealed that Curc-mPEG454 effectively inhibited the expansion of scar-associated macrophage subpopulation and scar-producing myofibroblasts in the damaged liver, and remodeled the fibrotic niche via regulation of ligand-receptor interactions including platelet-derived growth factor-B (PDGF-B)/platelet-derived growth factor receptor-α (PDGFR-α) signaling. As a multi-target prodrug, PEGylated curcumin deserves further attention and research.

Environmental eustress modulates ß-ARs/CCL2 axis to induce anti-tumor immunity and sensitize immunotherapy against liver cancer in mice.

Although psycho-social stress is a well-known factor that contributes to the development of cancer, it remains largely unclear whether and how environmental eustress influences malignant diseases and regulates cancer-related therapeutic responses. Using an established eustress model, we demonstrate that mice living in an enriched environment (EE) are protected from carcinogen-induced liver neoplasia and transplantable syngeneic liver tumors, owning to a CD8+ T cell-dependent tumor control. We identify a peripheral Neuro-Endocrine-Immune pathway in eustress, including Sympathetic nervous system (SNS)/ß-adrenergic receptors (ß-ARs)/CCL2 that relieves tumor immunosuppression and overcomes PD-L1 resistance to immunotherapy. Notably, EE activates peripheral SNS and ß-ARs signaling in tumor cells and tumor infiltrated myeloid cells, leading to suppression of CCL2 expression and activation of anti-tumor immunity. Either blockade of CCL2/CCR2 or ß-AR signaling in EE mice lose the tumor protection capability. Our study reveales that environmental eustress via EE stimulates anti-tumor immunity, resulting in more efficient tumor control and a better outcome of immunotherapy.

Selective depletion of hepatic stellate cells-specific LOXL1 alleviates liver fibrosis.

The role of LOXL1 in fibrosis via mediating ECM crosslinking and stabilization is well established; however, the role of hepatic stellate cells (HSCs)-specific LOXL1 in the development of fibrosis remains unknown. We generated HSCs-specific Loxl1-depleted mice (Loxl1Gfap-cre mice) to investigate the HSCs-specific contribution of LOXL1 in the pathogenesis of fibrosis. Loxl1fl/fl mice were used as the control. Furthermore, we used RNA sequencing to explore the underlying changes in the transcriptome. Results of the sirius red staining, type I collagen immunolabeling, and hydroxyproline content analysis, coupled with the reduced expression of profibrogenic genes revealed that Loxl1Gfap-cre mice with CCl4 -induced fibrosis exhibited decreased hepatic fibrosis. In addition, Loxl1Gfap-cre mice exhibited reduced macrophage tissue infiltration by CD68-positive cells and decreased expression of inflammatory genes compared with the controls. RNA sequencing identified integrin α8 (ITGA8) as a key modulator of LOXL1-mediated liver fibrosis. Functional analyses showed that siRNA silencing of Itga8 in cultured fibroblasts led to a decline in the LOXL1 expression and inhibition of fibroblast activation. Mechanistic analyses indicated that LOXL1 activated the FAK/PI3K/AKT/HIF1a signaling pathway, and the addition of inhibitors of FAK or PI3K reversed these results via downregulation of LOXL1. Furthermore, HIF1a directly interacted with LOXL1 and upregulated its expression, indicating that LOXL1 can positively self-regulate by forming a positive feedback loop with the FAK/PI3K/AKT/HIF1a pathway. We demonstrated that HSCs-specific Loxl1 deficiency prevented fibrosis, inflammation and that ITGA8/FAK/PI3K/AKT/HIF1a was essential for the function and expression of LOXL1. Knowledge of this approach can provide novel mechanisms and targets to treat fibrosis in the future.

Multiparametric magnetic resonance imaging/magnetic resonance elastography assesses progression and regression of steatosis, inflammation, and fibrosis in alcohol-associated liver disease.

BACKGROUND: Magnetic resonance imaging (MRI) and MRI-based elastography (MRE) are the most promising noninvasive techniques in assessing liver diseases. The purpose of this study was to evaluate an advanced multiparametric imaging method for staging disease and assessing treatment response in realistic preclinical alcohol-associated liver disease (ALD). METHODS: We utilized four different preclinical mouse models in our study: Model 1-mice were fed a fast-food diet and fructose water for 48 weeks to induce nonalcoholic fatty liver disease; Model 2-mice were fed chronic-binge ethanol (EtOH) for 10 days or 8 weeks to induce liver steatosis/inflammation. Two groups of mice were treated with interleukin-22 at different time points to induce disease regression; Model 3-mice were administered CCl4 for 2 to 4 weeks to establish liver fibrosis followed by 2 or 4 weeks of recovery; and Model 4-mice were administered EtOH plus CCl4 for 12 weeks. Mouse liver imaging biomarkers including proton density fat fraction (PDFF), liver stiffness (LS), loss modulus (LM), and damping ratio (DR) were assessed. Liver and serum samples were obtained for histologic and biochemical analyses. Ordinal logistic regression and generalized linear regression analyses were used to model the severity of steatosis, inflammation, and fibrosis, and to assess the regression of these conditions. RESULTS: Multiparametric models with combinations of biomarkers (LS, LM, DR, and PDFF) used noninvasively to predict the histologic severity and regression of steatosis, inflammation, and fibrosis were highly accurate (area under the curve > 0.84 for all). A three-parameter model that incorporates LS, DR, and ALT predicted histologic fibrosis progression (r = 0.84, p < 0.0001) and regression (r = 0.79, p < 0.0001) as measured by collagen content in livers. CONCLUSION: This preclinical study provides evidence that multiparametric MRI/MRE can be used noninvasively to assess disease severity and monitor treatment response in ALD.

Comparison of the Effects of Intraperitoneal Injection with Carbon Tetrachloride on Acute Liver Toxicity in Male and Female Kunming Mice.

BACKGROUND Acute chemical liver injury needs to be further explored. The present study aimed to compare the effects of intraperitoneal injection with carbon tetrachloride on acute liver toxicity after 24 h in male and female Kunming mice. MATERIAL AND METHODS In this study, female and male mice were simultaneously divided into 3 different groups. Each group was treated differently, and after 24 h, blood samples were collected to check for changes in the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which were used to assess liver toxicity. Liver samples were used for hematoxylin-eosin staining, and periodic acid Schiff reagent staining was performed to detect the pathological changes of each group. The expression level of biomarker molecules in liver cells was also systematically analyzed. RESULTS Our results showed that, compared with male mice, female mice showed more serious damage: reduced glycogen and higher degree of necrosis, and the levels of heatshock protein 27 (HSP27), heat-shock protein 70 (HSP70), proliferating cell nuclear antigen (PCNA) and B cell lymphoma/lewkmia-2 (Bcl-2) were significantly lower than in the male group (P<0.05 or P<0.01), while the results of Bcl-2-associated X protein (Bax), cysteinyl aspartate specific proteinase 3 (Caspase3), and cytochrome P450 2E1 (CYP2E1) were the opposite (P<0.05 or P<0.01). CONCLUSIONS The findings from this study showed that, compared with male mice, at 24 h after CCl4 toxicity, female mice showed more severe changes of hepatocyte necrosis and PAS-positivity, with significantly reduced expression of HSP27, HSP70, PCNA, and Bcl-2, and significantly increased expression of Bax, caspase-3, and CYP2E1.

Splicing Factor SLU7 Prevents Oxidative Stress-Mediated Hepatocyte Nuclear Factor 4α Degradation, Preserving Hepatic Differentiation and Protecting From Liver Damage.

BACKGROUND AND AIMS: Hepatocellular dedifferentiation is emerging as an important determinant in liver disease progression. Preservation of mature hepatocyte identity relies on a set of key genes, predominantly the transcription factor hepatocyte nuclear factor 4α (HNF4α) but also splicing factors like SLU7. How these factors interact and become dysregulated and the impact of their impairment in driving liver disease are not fully understood. APPROACH AND RESULTS: Expression of SLU7 and that of the adult and oncofetal isoforms of HNF4α, driven by its promoter 1 (P1) and P2, respectively, was studied in diseased human and mouse livers. Hepatic function and damage response were analyzed in wild-type and Slu7-haploinsufficient/heterozygous (Slu7+/- ) mice undergoing chronic (CCl4 ) and acute (acetaminophen) injury. SLU7 expression was restored in CCl4 -injured mice using SLU7-expressing adeno-associated viruses (AAV-SLU7). The hepatocellular SLU7 interactome was characterized by mass spectrometry. Reduced SLU7 expression in human and mouse diseased livers correlated with a switch in HNF4α P1 to P2 usage. This response was reproduced in Slu7+/- mice, which displayed increased sensitivity to chronic and acute liver injury, enhanced oxidative stress, and marked impairment of hepatic functions. AAV-SLU7 infection prevented liver injury and hepatocellular dedifferentiation. Mechanistically we demonstrate a unique role for SLU7 in the preservation of HNF4α1 protein stability through its capacity to protect the liver against oxidative stress. SLU7 is herein identified as a key component of the stress granule proteome, an essential part of the cell's antioxidant machinery. CONCLUSIONS: Our results place SLU7 at the highest level of hepatocellular identity control, identifying SLU7 as a link between stress-protective mechanisms and liver differentiation. These findings emphasize the importance of the preservation of hepatic functions in the protection from liver injury.

Single-Cell Transcriptomic Analysis Reveals a Hepatic Stellate Cell-Activation Roadmap and Myofibroblast Origin During Liver Fibrosis in Mice.

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 CCL4 -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 CCL4 -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 CCL4 -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.

Ketogenic Diet Enhances the Cholesterol Accumulation in Liver and Augments the Severity of CCl4 and TAA-Induced Liver Fibrosis in Mice.

Persistent chronic liver diseases increase the scar formation and extracellular matrix accumulation that further progress to liver fibrosis and cirrhosis. Nevertheless, there is no antifibrotic therapy to date. The ketogenic diet is composed of high fat, moderate to low-protein, and very low carbohydrate content. It is mainly used in epilepsy and Alzheimer's disease. However, the effects of the ketogenic diet on liver fibrosis remains unknown. Through ketogenic diet consumption, ß-hydroxybutyrate (bHB) and acetoacetate (AcAc) are two ketone bodies that are mainly produced in the liver. It is reported that bHB and AcAc treatment decreases cancer cell proliferation and promotes apoptosis. However, the influence of bHB and AcAc in hepatic stellate cell (HSC) activation and liver fibrosis are still unclear. Therefore, this study aimed to investigate the effect of the ketogenic diet and ketone bodies in affecting liver fibrosis progression. Our study revealed that feeding a high-fat ketogenic diet increased cholesterol accumulation in the liver, which further enhanced the carbon tetrachloride (CCl4)- and thioacetamide (TAA)-induced liver fibrosis. In addition, more severe liver inflammation and the loss of hepatic antioxidant and detoxification ability were also found in ketogenic diet-fed fibrotic mouse groups. However, the treatment with ketone bodies (bHB and AcAc) did not suppress transforming growth factor-ß (TGF-ß)-induced HSC activation, platelet-derived growth factor (PDGF)-BB-triggered proliferation, and the severity of CCl4-induced liver fibrosis in mice. In conclusion, our study demonstrated that feeding a high-fat ketogenic diet may trigger severe steatohepatitis and thereby promote liver fibrosis progression. Since a different ketogenic diet composition may exert different metabolic effects, more evidence is necessary to clarify the effects of a ketogenic diet on disease treatment.

Ginsenoside Rg1 alleviates acute liver injury through the induction of autophagy and suppressing NF-κB/NLRP3 inflammasome signaling pathway.

Background: Severe hepatitis is a common cause of chronic or acute liver disease and autophagy might play an important role in cellular response to inflammation and injury. It has been reported that Ginsenoside-Rg1 (G-Rg1) has strong hepatoprotective effects for acute liver injury, but its protective mechanisms have not yet been elucidated. This study aims to explore the detailed molecular mechanisms of G-Rg1 on acute liver injury via autophagy. Methods: The role of G-Rg1 by autophagic induction was studied in the mouse model of acute liver injury which induced by carbon tetrachloride (CCl4). Liver function, inflammatory reaction and apoptosis were detected when autophagy has been inhibited by 3-MA or stimulated by RPA. MCC950 and ATP were applied to investigate the role of NLRP3 inflammasome in acute liver injury. The differential expression of NF-κB, NLRP3 inflammasome, caspase 1, caspase 3, IL-1ß, IL-18, LC3-I, LC3-II, Beclin-1, PINK1 and Parkin have been detected by the quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Results: G-Rg1 could decrease ALT, AST, TNF-α, IL-1ß and IL-6 in mice with CCl4-induced acute liver injury. The change of autophagy and apoptosis after the treatment of 3-MA or RPA demonstrated that the autophagy played a key role in the protective effect of G-Rg1 in acute liver injury. The enhancement of G-Rg1 promoted-autophagy resulted in the significant decrease in NF-κB, NLRP3 inflammasome, caspase 1, caspase 3, IL-1ß and IL-18, which suggesting that NF-κB/NLRP3 inflammasome signaling pathway was associated with the autophagy induced by G-Rg1 in acute liver injury. Conclusion: G-Rg1 ameliorated acute liver injury via the autophagy, which may be related to NF-κB/NLRP3 inflammasome signaling pathway.

Albumin inhibits the nuclear translocation of Smad3 via interleukin-1beta signaling in hepatic stellate cells.

Activation of quiescent hepatic stellate cells (HSCs) to myofibroblasts plays a key role in liver fibrosis. We had previously shown that albumin and its derivative, R-III (a retinol-binding protein-albumin domain III fusion protein), inhibited HSC activation by sequestering retinoic acid (RA) and that R-III administration reduced carbon tetrachloride (CCl4)-induced liver fibrosis. In this study, we aimed to elucidate the mechanism of action of albumin downstream of RA sequestration. Nuclear factor-κB p65 was evenly distributed in the cytoplasm in activated mouse HSCs, whereas albumin expression or R-III treatment (albumin/R-III) caused the nuclear translocation of p65, probably via RA sequestration, resulting in a dramatic increase in interleukin-1beta (IL-1ß) expression. Albumin/R-III in turn induced the phosphorylation of Smad3 at the linker region, inhibiting its nuclear import in an IL-1ß-dependent manner. Consistent with the in vitro results, the level of IL-1ß mRNA expression was higher in CCl4/R-III-treated livers than in CCl4-treated livers. These findings reveal that albumin/R-III inhibits the transforming growth factor-ß-Smad3 signaling as well as the retinoic acid receptor-mediated pathway, which probably contributes to the inhibition of HSC activation, and suggest that R-III may be an anti-fibrotic drug candidate.

The Genetic Architecture of Carbon Tetrachloride-Induced Liver Fibrosis in Mice.

BACKGROUND & AIMS: Liver fibrosis is a multifactorial trait that develops in response to chronic liver injury. Our aim was to characterize the genetic architecture of carbon tetrachloride (CCl4)-induced liver fibrosis using the Hybrid Mouse Diversity Panel, a panel of more than 100 genetically distinct mouse strains optimized for genome-wide association studies and systems genetics. METHODS: Chronic liver injury was induced by CCl4 injections twice weekly for 6 weeks. Four hundred thirty-seven mice received CCl4 and 256 received vehicle, after which animals were euthanized for liver histology and gene expression. Using automated digital image analysis, we quantified fibrosis as the collagen proportionate area of the whole section, excluding normal collagen. RESULTS: We discovered broad variation in fibrosis among the Hybrid Mouse Diversity Panel strains, demonstrating a significant genetic influence. Genome-wide association analyses revealed significant and suggestive loci underlying susceptibility to fibrosis, some of which overlapped with loci identified in mouse crosses and human population studies. Liver global gene expression was assessed by RNA sequencing across the strains, and candidate genes were identified using differential expression and expression quantitative trait locus analyses. Gene set enrichment analyses identified the underlying pathways, of which stellate cell involvement was prominent, and coexpression network modeling identified modules associated with fibrosis. CONCLUSIONS: Our results provide a rich resource for the design of experiments to understand mechanisms underlying fibrosis and for rational strain selection when testing antifibrotic drugs.

Oxidative stress and Liver X Receptor agonist induce hepatocellular carcinoma in Non-alcoholic steatohepatitis model.

BACKGROUND AND AIM: The incidence of non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) is progressively increasing. However, the pathophysiology and etiology of NASH progression to HCC are unknown. We hypothesized that steatosis was the key factor in NASH-related hepatocarcinogenesis and aimed to evaluate the effects of long-term liver X receptor (LXR) agonist stimulation on hepatic steatosis induced by a high-fat diet and oxidative stress. METHODS: We used an LXR agonist (T0901317) and CCl4 to induce hepatic steatosis and oxidative stress, respectively. C57BL/6 mice fed with a high-fat diet were treated with either T0901317 + CCl4 (T09 + CCl4 group) or CCl4 alone (CCl4 group). T0901317 (2.5 mg/kg) and CCl4 (0.1 mL/kg) were intraperitoneally administered twice weekly for 24 weeks. RESULTS: The liver-to-body weight ratio was significantly higher in the T09 + CCl4 group than in the CCl4 group. Mice in the T09 + CCl4 group exhibited abnormal lipid metabolism and NASH-like histopathological features. Additionally, all mice in the T09 + CCl4 group developed liver tumors diagnosed as well-differentiated HCC. The genes identified via microarray analysis were related to NASH and HCC development. CONCLUSIONS: By combining long-term LXR agonist stimulation with oxidative stress and a high-fat diet, we successfully reproduced liver conditions in mice similar to those in humans with NASH and progression to HCC. Our results provide new insight into NASH-related HCC progression and therapy.

TMEM9-v-ATPase Activates Wnt/ß-Catenin Signaling Via APC Lysosomal Degradation for Liver Regeneration and Tumorigenesis.

BACKGROUND AND AIMS: How Wnt signaling is orchestrated in liver regeneration and tumorigenesis remains elusive. Recently, we identified transmembrane protein 9 (TMEM9) as a Wnt signaling amplifier. APPROACH AND RESULTS: TMEM9 facilitates v-ATPase assembly for vesicular acidification and lysosomal protein degradation. TMEM9 is highly expressed in regenerating liver and hepatocellular carcinoma (HCC) cells. TMEM9 expression is enriched in the hepatocytes around the central vein and acutely induced by injury. In mice, Tmem9 knockout impairs hepatic regeneration with aberrantly increased adenomatosis polyposis coli (Apc) and reduced Wnt signaling. Mechanistically, TMEM9 down-regulates APC through lysosomal protein degradation through v-ATPase. In HCC, TMEM9 is overexpressed and necessary to maintain ß-catenin hyperactivation. TMEM9-up-regulated APC binds to and inhibits nuclear translocation of ß-catenin, independent of HCC-associated ß-catenin mutations. Pharmacological blockade of TMEM9-v-ATPase or lysosomal degradation suppresses Wnt/ß-catenin through APC stabilization and ß-catenin cytosolic retention. CONCLUSIONS: Our results reveal that TMEM9 hyperactivates Wnt signaling for liver regeneration and tumorigenesis through lysosomal degradation of APC.

Regeneration Defects in Yap and Taz Mutant Mouse Livers Are Caused by Bile Duct Disruption and Cholestasis.

BACKGROUND AND AIMS: The Hippo pathway and its downstream effectors YAP and TAZ (YAP/TAZ) are heralded as important regulators of organ growth and regeneration. However, different studies provided contradictory conclusions about their role during regeneration of different organs, ranging from promoting proliferation to inhibiting it. Here we resolve the function of YAP/TAZ during regeneration of the liver, where Hippo's role in growth control has been studied most intensely. METHODS: We evaluated liver regeneration after carbon tetrachloride toxic liver injury in mice with conditional deletion of Yap/Taz in hepatocytes and/or biliary epithelial cells, and measured the behavior of different cell types during regeneration by histology, RNA sequencing, and flow cytometry. RESULTS: We found that YAP/TAZ were activated in hepatocytes in response to carbon tetrachloride toxic injury. However, their targeted deletion in adult hepatocytes did not noticeably impair liver regeneration. In contrast, Yap/Taz deletion in adult bile ducts caused severe defects and delay in liver regeneration. Mechanistically, we showed that Yap/Taz mutant bile ducts degenerated, causing cholestasis, which stalled the recruitment of phagocytic macrophages and the removal of cellular corpses from injury sites. Elevated bile acids activated pregnane X receptor, which was sufficient to recapitulate the phenotype observed in mutant mice. CONCLUSIONS: Our data show that YAP/TAZ are practically dispensable in hepatocytes for liver development and regeneration. Rather, YAP/TAZ play an indirect role in liver regeneration by preserving bile duct integrity and securing immune cell recruitment and function.

Severity assessment in mice subjected to carbon tetrachloride.

The Directive 2010/63 EU requires classifying burden and severity in all procedures using laboratory animals. This study evaluated the severity of liver fibrosis induction by intraperitoneal carbon tetrachloride (CCl4) injections in mice. 29 male C57BL/6N mice were treated three times per week for 4 weeks with an intraperitoneal injection (50 µl) of either 0.6 ml/kg body weight CCl4-vehicle solution, germ oil (vehicle-control) or handling only. Severity assessment was performed using serum analysis, behavioral tests (open field test, rotarod, burrowing and nesting behavior), fecal corticosterone metabolite (FCM) measurement, and survival. The most significant group differences were noticed in the second week of treatment when the highest AST (1463 ± 1404 vs. 123.8 ± 93 U/L, p < 0.0001) and nesting values were measured. In addition, respective animals showed lower moving distances (4622 ± 1577 vs. 6157 ± 2060 cm, p < 0.01) and velocity in the Open field, identified as main factors in principal component analysis (PCA). Overall, a 50% survival rate was observed within the treatment group, in which the open field performance was a good tracer parameter for survival. In summary, this study demonstrates the feasibility of assessing severity in mice using behavioral tests and highlight the open field test as a possible threshold parameter for risk assessment of mortality.

Assessment of a biofluid mechanics-based model for calculating portal pressure in canines.

BACKGROUND: Portal hypertension is a severe complication caused by various chronic liver diseases. The standard methods for detecting portal hypertension (hepatic venous pressure gradient and free portal pressure) are available in only a few hospitals due to their technical difficulty and invasiveness; thus, non-invasive measuring methods are needed. This study aimed to establish and assess a novel model to calculate free portal pressure based on biofluid mechanics. RESULT: Comparison of each dog's virtual and actual free portal pressure showed that a biofluid mechanics-based model could accurately predict free portal pressure (mean difference: -0.220, 95% CI: - 0.738 to 0.298; upper limit of agreement: 2.24, 95% CI: 1.34 to 3.14; lower limit of agreement: -2.68, 95% CI: - 3.58 to - 1.78; intraclass correlation coefficient: 0.98, 95% CI: 0.96 to 0.99; concordance correlation coefficient: 0.97, 95% CI: 0.93 to 0.99) and had a high AUC (0.984, 95% CI: 0.834 to 1.000), sensitivity (92.3, 95% CI: 64.0 to 99.8), specificity (91.7, 95% CI: 61.5 to 99.8), positive likelihood ratio (11.1, 95% CI: 1.7 to 72.8), and low negative likelihood ratio (0.08, 95% CI: 0.01 to 0.6) for detecting portal hypertension. CONCLUSIONS: Our study suggests that the biofluid mechanics-based model was able to accurately predict free portal pressure and detect portal hypertension in canines. With further research and validation, this model might be applicable for calculating human portal pressure, detecting portal hypertensive patients, and evaluating disease progression and treatment efficacy.