CD4+ T Cells Mediate the Development of Liver Fibrosis in High Fat Diet-Induced NAFLD in Humanized Mice.

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.

Novel oral plasminogen activator inhibitor­1 inhibitor TM5275 attenuates hepatic fibrosis under metabolic syndrome via suppression of activated hepatic stellate cells in rats.

An orally bioavailable small molecule inhibitor of plasminogen activator inhibitor­1 (PAI­1) is currently being clinically assessed as a novel antithrombotic agent. Although PAI­1 is known to serve a key role in the pathogenesis of metabolic syndrome (MetS) including nonalcoholic steatohepatitis (NASH), the pharmacological action of an oral PAI­1 inhibitor against the development of MetS­related liver fibrosis remains unclear. The current study was designed to explicate the effect of TM5275, an oral PAI­1 inhibitor, on MetS­related hepatic fibrogenesis. The in vivo antifibrotic effect of orally administered TM5275 was investigated in two different rat MetS models. Fischer 344 rats received a choline­deficient L­amino­acid­defined diet for 12 weeks to induce steatohepatitis with development of severe hepatic fibrosis. Otsuka Long­Evans Tokushima Fatty rats, used to model congenital diabetes, underwent intraperitoneal injection of porcine serum for 6 weeks to induce hepatic fibrosis under diabetic conditions. In each experimental model, TM5275 markedly ameliorated the development of hepatic fibrosis and suppressed the proliferation of activated hepatic stellate cells (HSCs). Additionally, the hepatic production of tumor growth factor (TGF)­ß1 and total collagen was suppressed. In vitro assays revealed that TGF­ß1 stimulated the upregulation of Serpine1 mRNA expression, which was inhibited by TM5275 treatment in cultured HSC­T6 cells, a rat HSC cell line. Furthermore, TM5275 substantially attenuated the TGF­ß1­stimulated proliferative and fibrogenic activity of HSCs by inhibiting AKT phosphorylation. Collectively, TM5275 demonstrated an antifibrotic effect on liver fibrosis in different rat MetS models, suppressing TGF­ß1­induced HSC proliferation and collagen synthesis. Thus, PAI­1 inhibitors may serve as effective future therapeutic agents against NASH­based hepatic fibrosis.

11ß­hydroxysteroid dehydrogenase­1 is associated with the activation of hepatic stellate cells in the development of hepatic fibrosis.

Hepatic fibrosis (HF) is a common complication of numerous chronic liver diseases, but predominantly results from persistent liver inflammation or injury. If left untreated, HF can progress and develop into liver cirrhosis and even hepatocellular carcinoma. However, the underlying molecular mechanisms of HF remain unknown. The present study aimed to investigate the role of 11ß­hydroxysteroid dehydrogenase­1 (11ß­HSD1) during the development of hepatic fibrosis. An experimental rat model of liver fibrosis was induced using porcine serum. 11ß­HSD1 gene expression levels and enzyme activity during hepatic fibrogenesis were assessed. 11ß­HSD1 gene knockdown using small interfering RNA and overexpression were performed in LX2­human hepatic stellate cells (HSCs). HSCs were stimulated with transforming growth factor­ß1 (TGF­ß1). Cell cycle distribution, proliferation, collagen secretion and 11ß­HSD1 gene activity in HSCs were compared before and after stimulation. As hepatic fibrosis progressed, 11ß­HSD1 gene expression and activity increased, indicating a positive correlation with typical markers of liver fibrosis. 11ß­HSD1 inhibition markedly reduced the degree of fibrosis. The cell proliferation was increased, the number of cells in the G0/G1 phase decreased and the number of cells in the S and G2/M phases increased in the pSuper transfected group compared with the N group. In addition, the overexpression of 11ß­HSD1 enhanced the TGF­ß1­induced activation of LX2­HSCs and enzyme activity of connective tissue growth factor. 11ß­HSD1 knockdown suppressed cell proliferation by blocking the G0/G1 phase of the cell cycle, which was associated with HSC stimulation and inhibition of 11ß­HSD1 enzyme activity. In conclusion, increased 11ß­HSD1 expression in the liver may be partially responsible for hepatic fibrogenesis, which is potentially associated with HSC activation and proliferation.

Protective Effects of Spermidine Against Cirrhotic Cardiomyopathy in Bile Duct-Ligated Rats.

Cirrhotic cardiomyopathy is a critical factor that causes morbidity and mortality in crucial conditions such as liver transplantation. In animal model, the common pathophysiologic mechanisms of cirrhotic cardiomyopathy are similar to those associated with bile duct ligation (BDL). Overproduction of inflammatory and oxidant markers plays a crucial role in cirrhotic cardiomyopathy. Spermidine, a multifunctional polyamine, is known for its antioxidant and anti-inflammatory effects. In this study, we investigated the effects of spermidine on development of cirrhotic cardiomyopathy in BDL rats. Rats were randomly housed in 6 groups. Except the normal and sham groups, BDL was performed for all the control and spermidine groups. Seven days after operation, 3 different doses of spermidine (5, 10 and 50 mg/kg) were administrated until day 28, in spermidine groups. At the end of the fourth week, the electrocardiography (ECG) and papillary muscle isolation were performed. The serum level of tumor necrosis factor-a (TNF-α), interleukin-1ß (IL-1ß), and IL-10 and cardiac level of superoxide dismutase, glutathione (GSH). and malondialdehyde (MDA) were assessed. Furthermore, the nuclear factor-κB (NF-κB) expression was assessed by western blot. Cardiac histopathological changes were monitored. The serum levels of magnesium (Mg) and potassium (K) were investigated. Control group, exhibited exaggerated signs of cirrhotic cardiomyopathy in comparison with the sham group. Co-administration of spermidine at the dose of 10 mg/kg in BDL rats significantly improved the cardiac condition, reduced the inflammatory mediators, and increased antioxidant enzymes. In addition, the histopathologic findings were in accordance with the other results of the study. Besides, there was no significant alteration in serum levels of Mg and K. This study demonstrates that spermidine at the dose of 10 mg/kg significantly improved the cirrhotic cardiomyopathy in BDL model in rats.

A novel model of non-alcoholic steatohepatitis with fibrosis and carcinogenesis in connexin 32 dominant-negative transgenic rats.

Non-alcoholic steatohepatitis (NASH) is a recognized risk factor for liver fibrosis and malignancies, and is associated with features of metabolic syndrome, such as obesity and insulin resistance (IR). We previously demonstrated that the disturbance of connexin 32 (Cx32), a gap junctional protein of hepatocytes, exacerbated NASH in Cx32 dominant-negative transgenic (Cx32ΔTg) rats fed methionine choline-deficient diet (MCDD). MCDD is well-established means of inducing NASH in rodents; however, the Cx32ΔTg-MCDD NASH model does not reproduce obesity and IR. In this study, we aimed to establish an improved NASH model. Eight-week-old male Cx32ΔTg and wild-type (Wt) rats received a high-fat diet (HFD) with dimethylnitrosamine (DMN) for 12 weeks. The HFD with DMN led to gains in body, liver, and visceral fat weights in both genotypes. IR was significantly greater in Cx32ΔTg than in Wt rats. Elevation of serum hepatic enzymes (AST, ALT), inflammatory cytokine expressions (Tnfα, Il-6, Tgf-ß1, Il-1ß, Timp2, and Col1a1), steatohepatitis, and fibrosis were significantly greater in Cx32ΔTg as compared with Wt rats. Regarding carcinogenesis, the number and area of glutathione S-transferase placental form (GST-P)-positive preneoplastic hepatic foci were significantly increased in Cx32ΔTg versus Wt rats. Moreover, activation of NF-κB and JNK contributed to the progression of NASH in Cx32ΔTg rats. These results suggest that Cx32 dysfunction promoted the progression of NASH, metabolic syndrome, and carcinogenesis. Therefore, the novel Cx32ΔTg-HFD-DMN NASH model may be a rapid and useful tool for evaluating the progression of NASH.

HWL-088, a new and highly effective FFA1/PPARδ dual agonist, attenuates nonalcoholic steatohepatitis by regulating lipid metabolism, inflammation and fibrosis.

OBJECTIVES: Nonalcoholic fatty liver (NAFLD), a chronic progressive liver disease, is highly correlated with pathoglycemia, dyslipidemia and oxidative stress. The free fatty acid receptor 1 (FFA1) agonists have been reported to improve liver steatosis and fibrosis, and the peroxisome proliferator-activated receptor δ (PPARδ) plays a synergistic role with FFA1 in energy metabolism and fibrosis. HWL-088, a PPARδ/FFA1 dual agonist, exerts better glucose-lowering effects than the representative FFA1 agonist TAK-875. However, the ability of HWL-088 to protect NAFLD was unknown. This study aimed to discover a new strategy for the treatment of NAFLD. METHODS: The methionine- and choline-deficient diet (MCD)-induced Nonalcoholic steatohepatitis (NASH) model was constructed to evaluate the effects of HWL-088. KEY FINDINGS: Administration of HWL-088 exerted multiple benefits on glucose control, lipid metabolism and fatty liver. Further mechanism research indicated that HWL-088 promotes lipid metabolism by decreasing lipogenesis and increasing lipolysis. Moreover, HWL-088 attenuates NASH by regulating the expression levels of genes related to inflammation, fibrosis and oxidative stress. CONCLUSIONS: These positive results indicated that PPARδ/FFA1 dual agonist HWL-088 might be a potential candidate to improve multiple pathogenesis of NASH.

Costunolide represses hepatic fibrosis through WW domain-containing protein 2-mediated Notch3 degradation.

BACKGROUND AND PURPOSE: This study investigates the antifibrotic activities and potential mechanisms of costunolide (COS), a natural sesquiterpene compound. EXPERIMENTAL APPROACH: Rats subjected to bile duct ligation and mice challenged with CCl4 were used to study the antifibrotic effects of COS in vivo. Mouse primary hepatic stellate cells (pHSCs) and human HSC line LX-2 also served as an in vitro liver fibrosis models. The expression of fibrogenic genes and signaling proteins in the neurogenic locus notch homologue protein 3 (Notch3)-hairy/enhancer of split-1 (HES1) pathway was examined using western blot and/or real-time PCR. Notch3 degradation was analysed using immunofluorescence and coimmunoprecipitation. KEY RESULTS: In animals, COS administration attenuated hepatic histopathological injury and collagen accumulation and reduced the expression of fibrogenic genes. COS time- and dose-dependently suppressed the levels of fibrotic markers in LX-2 cells and mouse pHSCs. Mechanistic studies showed COS destabilized Notch3 and subsequently inhibited the Notch3-HES1 pathway, thus inhibiting HSC activation. Furthermore, COS blocked the WW domain-containing protein 2 (WWP2)/protein phosphatase 1G (PPM1G) interaction and enhanced the effect of WWP2 on Notch3 degradation. CONCLUSIONS AND IMPLICATIONS: COS exerted potent antifibrotic effects in vitro and in vivo by disrupting the WWP2/PPM1G complex, promoting Notch3 degradation and inhibiting the Notch3/HES1 pathway. This indicates that COS may be a potential therapeutic candidate for the treatment of liver fibrosis.

Comparison of murine steatohepatitis models identifies a dietary intervention with robust fibrosis, ductular reaction, and rapid progression to cirrhosis and cancer.

Progressive fibrosis, functional liver failure, and cancer are the central liver-related outcomes of nonalcoholic steatohepatitis (NASH) but notoriously difficult to achieve in mouse models. We performed a direct, quantitative comparison of hepatic fibrosis progression in well-defined methionine- and choline-deficient (MCD) and choline-deficient, amino-acid defined (CDAA) diets with increasing fat content (10-60% by calories) in C57Bl/6J and BALB/cAnNCrl mice. In C57Bl/6J mice, MCD feeding resulted in moderate fibrosis at week 8 (up to twofold increase in total hepatic collagen content) and progressive weight loss irrespective of dietary fat. In contrast, CDAA-fed mice did not lose weight and developed progressive fibrosis starting from week 4. High dietary fat in the CDAA diet model induced the lipid metabolism genes for sterol regulatory element-binding protein and stearoyl-CoA desaturase-2 and increased ductular reaction and fibrosis in a dose-dependent manner. Longitudinal analysis of CDAA with 60% fat (HF-CDAA) feeding revealed pronounced ductular reaction and perisinusoidal bridging fibrosis, with a sevenfold increase of hepatic collagen at week 12, which showed limited spontaneous reversibility. At 24 wk, HF-CDAA mice developed signs of cirrhosis with pan-lobular "chicken wire" fibrosis, 10-fold hydroxyproline increase, regenerative nodules, portal hypertension and elevated serum bilirubin and ammonia levels; 80% of mice (8/10) developed multiple glypican-3- and/or glutamine synthetase-positive hepatocellular carcinomas (HCC). High-fat (60%) supplementation of MCD in C57Bl/6J or feeding the HF-CDAA diet fibrosis-prone BALB/cAnNCrl strain failed to result in increased fibrosis. In conclusion, HF-CDAA feeding in C57Bl/6J mice was identified as an optimal model of steatohepatitis with robust fibrosis and ductular proliferations that progress to cirrhosis and HCC within 24 wk. This robust model will aid the testing of interventions and drugs for severe NASH.NEW & NOTEWORTHY Via quantitative comparison of several dietary models, we report HF-CDAA feeding in C57Bl/6 mice as an excellent model recapitulating several key aspects of fibrotic NASH: 1) robust, poorly reversible liver fibrosis, 2) prominent ductular reaction, and 3) progression to cirrhosis, portal hypertension, and liver cancer within 24 wk. High fat dose-dependently activates SREBP2/SCD2 genes and drives liver fibrosis in e HF-CDAA model. These features qualify the model as a robust and practical tool to study mechanisms and novel treatments addressing severe human NASH.

Short hairpin RNA attenuates liver fibrosis by regulating the PPAR­Î³ and NF­κB pathways in HBV­induced liver fibrosis in mice.

Progressive liver fibrosis, caused by chronic viral infection and metabolic disorders, results in the development of cirrhosis and hepatocellular carcinoma. However, no antifibrotic therapies have been approved to date. In our previous study, adeno­associated virus (AAV) short hairpin RNAs (shRNAs) targeting hepatitis B virus (HBV) and transforming growth factor (TGF)­ß administration could persistently inhibit HBV replication and concomitantly prevent liver fibrosis. However, the differentially expressed proteins and critical regulatory networks of AAV­shRNA treatment remain unclear. Accordingly, in the present study, we aimed to analyze differentially expressed proteins in the liver of AAV­shRNA­treated mice with HBV infection and liver fibrosis using isobaric tags for relative and absolute quantitation (iTRAQ)­based quantitative proteomics and to elucidate the underlying antifibrotic mechanisms. In total 2,743 proteins were recognized by iTRAQ­based quantitative proteomics analysis. Gene Ontology analysis revealed that the differentially expressed proteins mostly participated in peptide metabolism in the biological process category, cytosolic ribosomes in the cell component category, and structural constituents of ribosomes in the molecular function category. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that oxidative stress and the peroxisome proliferator­activated receptor (PPAR) signaling pathway were activated after treatment. Verification studies revealed that AAV­shRNAs inhibited hepatic stellate cell activation and inflammation by suppressing nuclear factor­κB p65 phosphorylation and α­smooth muscle actin expression via upregulation of PPAR­Î³. Hepatocytes steatosis was also decreased by activating the PPAR signaling pathway and improving lipid metabolism. The expression level of TGF­ß was decreased due to upregulation of PPAR­Î³ expression and direct inhibition using AAV­shRNA targeting TGF­ß. TGF­ß­induced oxidative stress was suppressed by increasing glutathione S­transferase Pi 1 and reducing peroxiredoxin 1. Collectively, the present results indicated that AAV­shRNAs were effective in modulating liver fibrosis by reducing oxidative stress, inflammation and activating the PPAR signaling pathway.

Dietary fat, cholesterol, and cholic acid affect the histopathologic severity of nonalcoholic steatohepatitis in Sprague-Dawley rats.

Understanding of the pathogenesis of nonalcoholic steatohepatitis (NASH)-associated fibrosis has been hampered by the lack of a comprehensive and physiological small animal model of NASH with fibrosis. Feeding a high-fat and high-cholesterol (HFC) diet supplemented with cholic acid to rats is known to replicate human NASH pathology, and it induces fibrosis earlier than with an HFC diet alone. In the present study, physiological and histopathological observations from 65 Sprague-Dawley (SD) rats fed an HFC diet with or without cholic acid for 9 or 18 weeks in our laboratory between January 2013 and February 2018 were retrospectively reviewed. The liver weight/body weight ratio at the end of the rearing period was higher in rats fed an HFC diet than in rats fed a normal diet in a cholesterol dose-, cholic acid dose-, or rearing period dependent manner. Dietary fat, cholesterol and/or cholic acid and rearing period affected the histopathologic severity of NASH. Overall, 56 (86.2%) of 65 SD rats fed an HFC diet for 9 or 18 weeks developed histopathologically proven NASH. It is noted that the SD rats fed an HFC diet supplemented with 2% (w/w) cholic acid for 18 weeks frequently developed advanced fibrosis, including cirrhosis. Thus, this diet-induced NASH rat model is likely to be a highly reproducible.

Sub-Chronic Microcystin-LR Liver Toxicity in Preexisting Diet-Induced Nonalcoholic Steatohepatitis in Rats.

Microcystin-LR (MCLR) is a hepatotoxic cyanotoxin reported to cause a phenotype similar to nonalcoholic steatohepatitis (NASH). NASH is a common progressive liver disease that advances in severity due to exogenous stressors such as poor diet and toxicant exposure. Our objective was to determine how sub-chronic MCLR toxicity affects preexisting diet-induced NASH. Sprague-Dawley rats were fed one of three diets for 10 weeks: control, methionine and choline deficient (MCD), or high fat/high cholesterol (HFHC). After six weeks of diet, animals received vehicle, 10 µg/kg, or 30 µg/kg MCLR via intraperitoneal injection every other day for the final 4 weeks. Incidence and severity scoring of histopathology endpoints suggested that MCLR toxicity drove NASH to a less fatty and more fibrotic state. In general, expression of genes involved in de novo lipogenesis and fatty acid esterification were altered in favor of decreased steatosis. The higher MCLR dose increased expression of genes involved in fibrosis and inflammation in the control and HFHC groups. These data suggest MCLR toxicity in the context of preexisting NASH may drive the liver to a more severe phenotype that resembles burnt-out NASH.

Hepatocyte Deletion of Triglyceride-Synthesis Enzyme Acyl CoA: Diacylglycerol Acyltransferase 2 Reduces Steatosis Without Increasing Inflammation or Fibrosis in Mice.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excess lipid accumulation in hepatocytes and represents a huge public health problem owing to its propensity to progress to nonalcoholic steatohepatitis, fibrosis, and liver failure. The lipids stored in hepatic steatosis (HS) are primarily triglycerides (TGs) synthesized by two acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. Either DGAT1 or DGAT2 catalyzes this reaction, and these enzymes have been suggested to differentially utilize exogenous or endogenously synthesized fatty acids, respectively. DGAT2 has been linked to storage of fatty acids from de novo lipogenesis, a process increased in NAFLD. However, whether DGAT2 is more responsible for lipid accumulation in NAFLD and progression to fibrosis is currently unknown. Also, it is unresolved whether DGAT2 can be safely inhibited as a therapy for NAFLD. Here, we induced NAFLD-like disease in mice by feeding a diet rich in fructose, saturated fat, and cholesterol and found that hepatocyte-specific Dgat2 deficiency reduced expression of de novo lipogenesis genes and lowered liver TGs by ~70%. Importantly, the reduction in steatosis was not accompanied by increased inflammation or fibrosis, and insulin and glucose metabolism were unchanged. Conclusion: This study suggests that hepatic DGAT2 deficiency successfully reduces diet-induced HS and supports development of DGAT2 inhibitors as a therapeutic strategy for treating NAFLD and preventing downstream consequences.

Ablation of Hepatocyte Smad1, Smad5, and Smad8 Causes Severe Tissue Iron Loading and Liver Fibrosis in Mice.

A failure of iron to appropriately regulate liver hepcidin production is central to the pathogenesis of hereditary hemochromatosis. SMAD1/5 transcription factors, activated by bone morphogenetic protein (BMP) signaling, are major regulators of hepcidin production in response to iron; however, the role of SMAD8 and the contribution of SMADs to hepcidin production by other systemic cues remain uncertain. Here, we generated hepatocyte Smad8 single (Smad8fl/fl ;Alb-Cre+ ), Smad1/5/8 triple (Smad158;Alb-Cre+ ), and littermate Smad1/5 double (Smad15;Alb-Cre+ ) knockout mice to investigate the role of SMAD8 in hepcidin and iron homeostasis regulation and liver injury. We found that Smad8;Alb-Cre+ mice exhibited no iron phenotype, whereas Smad158;Alb-Cre+ mice had greater iron overload than Smad15;Alb-Cre+ mice. In contrast to the sexual dimorphism reported for wild-type mice and other hemochromatosis models, hepcidin deficiency and extrahepatic iron loading were similarly severe in Smad15;Alb-Cre+ and Smad158;Alb-Cre+ female compared with male mice. Moreover, epidermal growth factor (EGF) failed to suppress hepcidin in Smad15;Alb-Cre+ hepatocytes. Conversely, hepcidin was still increased by lipopolysaccharide in Smad158;Alb-Cre+ mice, although lower basal hepcidin resulted in lower maximal hepcidin. Finally, unlike most mouse hemochromatosis models, Smad158;Alb-Cre+ developed liver injury and fibrosis at 8 weeks. Liver injury and fibrosis were prevented in Smad158;Alb-Cre+ mice by a low-iron diet and were minimal in iron-loaded Cre- mice. Conclusion: Hepatocyte Smad1/5/8 knockout mice are a model of hemochromatosis that encompasses liver injury and fibrosis seen in human disease. These mice reveal the redundant but critical role of SMAD8 in hepcidin and iron homeostasis regulation, establish a requirement for SMAD1/5/8 in hepcidin regulation by testosterone and EGF but not inflammation, and suggest a pathogenic role for both iron loading and SMAD1/5/8 deficiency in liver injury and fibrosis.

Perilipin-2 promotes obesity and progressive fatty liver disease in mice through mechanistically distinct hepatocyte and extra-hepatocyte actions.

KEY POINTS: Wild-type mice and mice with hepatocyte-specific or whole-body deletions of perilipin-2 (Plin2) were used to define hepatocyte and extra-hepatocyte effects of altered cellular lipid storage on obesity and non-alcoholic fatty liver disease (NAFLD) pathophysiology in a Western-diet (WD) model of these disorders. Extra-hepatocyte actions of Plin2 are responsible for obesity, adipose inflammation and glucose clearance abnormalities in WD-fed mice. Hepatocyte and extra-hepatic actions of Plin2 mediate fatty liver formation in WD-fed mice through distinct mechanisms. Hepatocyte-specific actions of Plin2 are primary mediators of immune cell infiltration and fibrotic injury in livers of obese mice. ABSTRACT: Non-alcoholic fatty liver disease (NAFLD) is an obesity- and insulin resistance-related metabolic disorder with progressive pathology. Perilipin-2 (Plin2), a ubiquitously expressed cytoplasmic lipid droplet scaffolding protein, is hypothesized to contribute to NAFLD in humans and rodent models through effects on cellular lipid metabolism. In this study, we delineate hepatocyte-specific and extra-hepatocyte Plin2 mechanisms regulating the effects of obesity and insulin resistance on NAFLD pathophysiology in mice fed an obesogenic Western-style diet (WD). Total Plin2 deletion (Plin2-Null) fully protected WD-fed mice from obesity, insulin resistance, adipose inflammation, steatohepatitis (NASH) and liver fibrosis found in WT animals. Hepatocyte-specific Plin2 deletion (Plin2-HepKO) largely protected against NASH and fibrosis and partially protected against steatosis in WD-fed animals, but it did not protect against obesity, insulin resistance, or adipose inflammation. Significantly, total or hepatocyte-specific Plin2 deletion impaired WD-induced monocyte recruitment and pro-inflammatory macrophage polarization found in livers of WT mice. Analyses of the molecular and cellular processes mediating steatosis, inflammation and fibrosis identified differences in total and hepatocyte-specific actions of Plin2 on the mechanisms promoting NAFLD pathophysiology. Our results demonstrate that hepatocyte-specific actions of Plin2 are central to the initiation and pathological progression of NAFLD in obese and insulin-resistant mice through effects on immune cell recruitment and fibrogenesis. Conversely, extra-hepatocyte Plin2 actions promote NAFLD pathophysiology through effects on obesity, inflammation and insulin resistance. Our findings provide new insight into hepatocyte and extra-hepatocyte mechanisms underlying NAFLD development and progression.

The small molecule drug diminazene aceturate inhibits liver injury and biliary fibrosis in mice.

There is no established medical therapy to treat biliary fibrosis resulting from chronic inflammation in the biliary tree. We have recently shown that liver-specific over-expression of angiotensin converting enzyme 2 (ACE2) of the renin angiotensin system (RAS) ameliorated liver fibrosis in mice. Diminazene aceturate (DIZE), a small molecule drug approved by the US Food and Drug Administration, which is used to treat human trypanosomiasis, has been shown to have antifibrotic properties by enhancing ACE2 activity. In this study we sought to determine the therapeutic potential of DIZE in biliary fibrosis using bile duct ligated and multiple drug resistant gene-2 knockout mice. Additionally, human hepatic stellate (LX-2) and mouse Kupffer (KUP5) cell lines were used to delineate intracellular pathways. DIZE treatment, both in vivo and in vitro, markedly inhibited the activation of fibroblastic stellate cells which was associated with a reduced activation of Kupffer cells. Moreover, DIZE-inhibited NOX enzyme assembly and ROS generation, activation of profibrotic transcription factors including p38, Erk1/2 and Smad2/3 proteins and proinflammatory and profibrotic cytokine release. These changes led to a major reduction in biliary fibrosis in both models without affecting liver ACE2 activity. We conclude that DIZE has a potential to treat biliary fibrosis.

NLRP3 inflammasome driven liver injury and fibrosis: Roles of IL-17 and TNF in mice.

The NLRP3 inflammasome, a caspase-1 activation platform, plays a key role in the modulation of liver inflammation and fibrosis. Here, we tested the hypothesis that interleukin 17 (IL-17) and tumor necrosis factor (TNF) are key cytokines involved in amplifying and perpetuating the liver damage and fibrosis resulting from NLRP3 activation. To address this hypothesis, gain-of-function Nlrp3A350V knock-in mice were bred onto il17a and Tnf knockout backgrounds allowing for constitutive Nlrp3 activation in myeloid derived cells in mice deficient in IL-17 or TNF. Livers of Nlrp3A350V knock-in mice exhibited severe liver inflammatory changes characterized by infiltration with neutrophils, increased expression of chemokine (C-X-C motif) ligand (CXCL) 1 and CXCL2 chemokines, activated inflammatory macrophages, and elevated levels of IL-17 and TNF. Mutants with ablation of il17a signal showed fewer neutrophils when compared to intact Nlrp3A350V mutants, but still significant inflammatory changes when compared to the nonmutant il17a knockout littermates. The severe inflammatory changes associated with mutant Nlrp3 were almost completely rescued by Tnf knockout in association with a marked decrease in circulating IL-1ß levels. Intact Nlrp3A350V mutants showed changes in liver fibrosis, as evidenced by morphometric quantitation of Sirius Red staining and increased mRNA levels of profibrotic genes, including connective tissue growth factor and tissue inhibitor of matrix metalloproteinase 1. Il17a lacking mutants exhibited amelioration of the aforementioned fibrosis, whereas Tnf-deficient mutants showed no signs of fibrosis when compared to littermate controls. Conclusion: Our study uncovers key roles for TNF and, to a lesser extent, IL-17 as mediators of liver inflammation and fibrosis induced by constitutive NLRP3 inflammasome activation in myeloid-derived cells. These findings may lead to therapeutic strategies aimed at halting the progression of liver injury and fibrogenesis in various liver pathogeneses driven by NLRP3 activation. (Hepatology 2018;67:736-749).

Ascorbate lacks significant influence in rats with bile duct ligation-induced liver injury.

BACKGROUND: Liver inflammation may induce fibrogenesis, cirrhosis and portal hypertension. Liver cirrhosis is characterized by increased intrahepatic resistance and enhanced vasoconstrictive response. The splanchnic vasodilatation, angiogenesis and portosystemic collaterals formation further bring about lethal complications. Ascorbate is a potent antioxidant with anti-inflammation, anti-fibrosis, and anti-angiogenesis effects. However, the relevant influences in chronic liver injury have not been sufficiently explored. METHODS: Chronic liver injury was induced in Spraque-Dawley rats with common bile duct ligation (BDL). Ascorbate (250 mg/kg/day, oral gavage) or vehicle was administered starting on the 1st day after operation. On the 8th (hepatitis) and 29th (cirrhosis) day, serum biochemistry parameters, hepatic concentrations of lipid peroxidation-related substances, protein expressions of α-SMA, TGF-ß, iNOS, eNOS, p-eNOS-Ser1177, p-eNOS-Thr496, VEGF, VEGFR2, p-VEGFR2, and liver histology were evaluated. In three series of paralleled groups, rats treated with 28-day ascorbate or vehicle received hemodynamic measurements, hepatic and collateral vasoresponsiveness perfusion experiments, mesenteric CD31 immunofluorescence staining, and Western blot analyses of mesenteric VEGF, VEGFR2, pVEGFR2, PDGF, PDGFß, COX1, COX2, eNOS, p-eNOS-Thr495, p-eNOS-Ser1177 protein expressions. In another series, the severity of portosystemic shunting was evaluated. RESULTS: Ascorbate did not influence hepatitis, oxidative stress, fibrosis, and hemodynamic parameters in BDL rats. The intrahepatic and collateral vasoresponsiveness were not affected, either from direct incubation or acute treatment with ascorbate. Furthermore, the mesenteric angiogenesis and severity of shunting were not influenced. CONCLUSION: The oxidative stress, fibrosis, hemodynamic derangements, angiogenesis and vascular functional changes in BDL-induced chronic liver injury may be too overwhelming to be modulated by ascorbate.

Annexin A2 promotes liver fibrosis by mediating von Willebrand factor secretion.

BACKGROUND: Liver fibrosis can lead to cirrhosis and hepatocellular carcinoma if not treated in the early stages. The molecular mechanisms of the pathogenesis of hepatic fibrosis remain unclear. AIM: To identify the molecules involved in the pathogenesis of liver fibrosis and to investigate the potential effect and mechanism of Annexin A2 up-regulation during liver fibrosis progression. METHODS: Twenty Sprague-Dawley rats were divided into two groups: the carbon tetrachloride (CCl4)-induced liver fibrosis group and the normal control group. Hematoxylin and eosin staining or Masson Trichrome staining and enzyme-linked immunosorbent assay were applied to assess the degree of liver damage and fibrosis in rats with CCl4-induced liver fibrosis. Liver tissue protein profiles were analyzed using iTRAQ and mass spectrometry. RT-PCR and western blotting analyses were employed to validate differentially expressed proteins. Small interfering RNA-based silencing was performed to study the function of Annexin A2. RESULTS: Twelve weeks after CCl4 injection, significant body weight changes and liver injury and liver fibrosis were observed in rats. In addition, 130 proteins were differentially expressed in the liver fibrosis group. Overexpression of Annexin A2 was confirmed by RT-PCR and Western blotting analysis. Silencing of Annexin A2 expression in HepG2 and LX-2 cells significantly reduced the secretion of von Willebrand factor (vWF). CONCLUSION: Annexin A2 promotes liver fibrosis by mediating vWF secretion, which can be used to mitigate the progression of liver fibrosis.

Thymic NF-κB-inducing kinase regulates CD4+ T cell-elicited liver injury and fibrosis in mice.

BACKGROUND & AIMS: The liver is an immunologically-privileged organ. Breakdown of liver immune privilege has been reported in chronic liver disease; however, the role of adaptive immunity in liver injury is poorly defined. Nuclear factor-κB-inducing kinase (NIK) is known to regulate immune tissue development, but its role in maintaining liver homeostasis remains unknown. This study aimed to assess the role of NIK, particularly thymic NIK, in regulating liver adaptive immunity. METHODS: NIK was deleted systemically or conditionally using the Cre/loxp system. Cluster of differentiation [CD]4+ or CD8+ T cells were depleted using anti-CD4 or anti-CD8 antibody. Donor bone marrows or thymi were transferred into recipient mice. Immune cells were assessed by immunohistochemistry and flow cytometry. RESULTS: Global, but not liver-specific or hematopoietic lineage cell-specific, deletion of NIK induced fatal liver injury, inflammation, and fibrosis. Likewise, adoptive transfer of NIK-null, but not wild-type, thymi into immune-deficient mice induced liver inflammation, injury, and fibrosis in recipients. Liver inflammation was characterized by a massive expansion of T cells, particularly the CD4+ T cell subpopulation. Depletion of CD4+, but not CD8+, T cells fully protected against liver injury, inflammation, and fibrosis in NIK-null mice. NIK deficiency also resulted in inflammation in the lung, kidney, and pancreas, but to a lesser degree relative to the liver. CONCLUSIONS: Thymic NIK suppresses development of autoreactive T cells against liver antigens, and NIK deficiency in the thymus results in CD4+ T cell-orchestrated autoimmune hepatitis and liver fibrosis. Thus, thymic NIK is essential for the maintenance of liver immune privilege and liver homeostasis. LAY SUMMARY: We found that global or thymus-specific ablation of the NIK gene results in fatal autoimmune liver disease in mice. NIK-deficient mice develop liver inflammation, injury, and fibrosis. Our findings indicate that thymic NIK is essential for the maintenance of liver integrity and homeostasis.

Quercetin protects liver injury induced by bile duct ligation via attenuation of Rac1 and NADPH oxidase1 expression in rats.

BACKGROUND: Bile duct ligation (BDL) and subsequent cholestasis are correlated with oxidative stress, hepatocellular injury and fibrosis. Quercetin is a flavonoid with antifibrotic, and hepatoprotective properties. However, the molecular mechanism underlying quercetin-mediated hepatoprotection is not fully understood. The current study was to evaluate mechanisms of hepatoprotective effect of quercetin in BDL rat model. METHODS: We divided male Wistar rats into 4 groups (n=8 for each): sham, sham+quercetin (30 mg/kg per day), BDL, and BDL+quercetin (30 mg/kg per day). Four weeks later, the rats were sacrificed, the blood was collected for liver enzyme measurements and liver for the measurement of Rac1, Rac1-GTP and NOX1 mRNA and protein levels by quantitative PCR and Western blotting, respectively. RESULTS: Quercetin significantly alleviated liver injury in BDL rats as evidenced by histology and reduced liver enzymes. Furthermore, the mRNA and protein expression of Rac1, Rac1-GTP and NOX1 were significantly increased in BDL rats compared with those in the sham group (P<0.05); quercetin treatment reversed these variables back toward normal (P<0.05). Another interesting finding was that the antioxidant markers e.g. superoxide dismutase and catalase were elevated in quercetin-treated BDL rats compared to BDL rats (P<0.05). CONCLUSION: Quercetin demonstrated hepatoprotective activity against BDL-induced liver injury through increasing antioxidant capacity of the liver tissue, while preventing the production of Rac1, Rac1-GTP and NOX1 proteins.