Experimental Model Of Non-alcoholic Steatohepatitis Research Articles

Therapeutic targeting of adipose tissue macrophages ameliorates liver fibrosis in non-alcoholic fatty liver disease

Background & Aims: The accumulation of adipose tissue macrophages (ATMs) in obesity has been associated with hepatic injury. However, the contribution of ATMs to hepatic fibrosis in non-alcoholic fatty liver disease (NAFLD) remains to be elucidated. Herein, we investigate the relationship between ATMs and liver fibrosis in patients with patients with NAFLD and evaluate the impact of modulation of ATMs over hepatic fibrosis in an experimental non-alcoholic steatohepatitis (NASH) model. MethodsAdipose tissue and liver biopsies from 42 patients with NAFLD with different fibrosis stages were collected. ATMs were characterised by immunohistochemistry and flow cytometry and the correlation between ATMs and liver fibrosis stages was assessed. Selective modulation of the ATM phenotype was achieved by i.p. administration of dextran coupled with dexamethasone in diet-induced obesity and NASH murine models. Chronic administration effects were evaluated by histology and gene expression analysis in adipose tissue and liver samples. In vitro crosstalk between human ATMs and hepatic stellate cells (HSCs) and liver spheroids was performed. ResultsPatients with NAFLD presented an increased accumulation of pro-inflammatory ATMs that correlated with hepatic fibrosis. Long-term modulation of ATMs significantly reduced pro-inflammatory phenotype and ameliorated adipose tissue inflammation. Moreover, ATMs modulation was associated with an improvement in steatosis and hepatic inflammation and significantly reduced fibrosis progression in an experimental NASH model. In vitro, the reduction of the pro-inflammatory phenotype of human ATMs with dextran–dexamethasone treatment reduced the secretion of inflammatory chemokines and directly attenuated the pro-fibrogenic response in HSCs and liver spheroids. ConclusionsPro-inflammatory ATMs increase in parallel with fibrosis degree in patients with NAFLD and their modulation in an experimental NASH model improves liver fibrosis, uncovering the potential of ATMs as a therapeutic target to mitigate liver fibrosis in NAFLD. Impact and implicationsWe report that human adipose tissue pro-inflammatory macrophages correlate with hepatic fibrosis in non-alcoholic fatty liver disease (NAFLD). Furthermore, the modulation of adipose tissue macrophages (ATMs) by dextran-nanocarrier conjugated with dexamethasone shifts the pro-inflammatory phenotype of ATMs to an anti-inflammatory phenotype in an experimental murine model of non-alcoholic steatohepatitis. This shift ameliorates adipose tissue inflammation, hepatic inflammation, and fibrosis. Our results highlight the relevance of adipose tissue in NAFLD pathophysiology and unveil ATMs as a potential target for NAFLD.

1602-P: Galectin-3 Inhibition Protects ApoE Knockout Mice against Western Diet–Induced Nonalcoholic Steatohepatitis and Glucose Intolerance

Nonalcoholic Fatty Liver Disease represents a broad spectrum of histological abnormalities ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which may progress to cirrhosis or hepatocellular carcinoma. NASH is characterized by steatosis, hepatic inflammation, hepatocellular ballooning, and fibrosis. Therefore, the aim of this work was to evaluate the effect of modified citrus pectin (MCP), inhibitor of galectin-3, in ApoE KO mice fed a western diet (WD). ApoE KO mice were divided into 3 groups: standard diet-fed (SD) group, WD-fed group, and WD-fed + MCP-treated group (WD+MCP). WD-fed mice displayed increased hepatic triacylglycerides (TAG-290%), and plasma ALT (115%) and AST (130%) compared with SD group. MCP treatment decreased hepatic TAG (30%) in WD-fed mice, plasma ALT and AST were reverted. WD-fed mice also displayed increased hepatic collagen deposition (18%), and MCP treatment inhibited this increase. These results were similar with hydroxyproline concentration in liver. WD-fed mice showed an impaired glucose tolerance compared with SD-fed mice, while MCP treatment improved glucose tolerance in WD-fed mice. Also, WD-fed mice presented increased fasting insulin (140%) compared with SD-fed mice, and MCP treatment reverted this increase by 100%. Gene expression of F4/80 was increased by 76% in the WD group compared with SD group, however had not significant difference between WD+MCP and SD group. Gene expression of matrix metalloproteinase 2 was increased by 5-fold in WD group compared with SD group, while this increase was inhibited with MCP treatment. Also, it was observed an increase in JNK phosphorylation by 3-fold in WD group, and MCP treatment inhibited this increase. Taken together, we confirm WD-fed ApoE KO mice as a suitable experimental model of NASH, and the inhibition of galectin-3 by the treatment with MCP protects ApoE KO mice against the development of NASH and glucose intolerance. Disclosure L. Araujo: None. F. Sucupira: None. C.C.B. Dias: None. A. Amaral: None. J. Camporez: None. Funding São Paulo Research Foundation (FAPESP)

Moringa oleifera Improves MAFLD by Inducing Epigenetic Modifications.

Background and aims. Metabolic Associated Fatty Liver Disease (MAFLD) encompasses a spectrum of diseases from simple steatosis to nonalcoholic steatohepatitis (NASH). Here, we investigated the hepatoprotective role of Moringa oleifera aqueous extract on hepatic miRNAs, genes and protein expression, as well as histological and biochemical parameters in an experimental model of NASH. Methods. Male C57BL/6J mice were fed with a high fat diet (HFD, 60% lipids, 42 gr/L sugar in water) for 16 weeks. Moringa extract was administered via gavage during the final 8 weeks. Insulin Tolerance Test (ITT) and HOMA-IR were calculated. Serum levels of insulin, resistin, leptin and PAI-1 and hepatic expression of miR-21a-5p, miR-103-3p, miR-122-5p, miR-34a-5p and SIRT1, AMPKα and SREBP1c protein were evaluated. Alpha-SMA immunohistochemistry and hematoxylin-eosin, Masson’s trichrome and sirius red staining were made. Hepatic transcriptome was analyzed using microarrays. Results. Animals treated with Moringa extract improved ITT and decreased SREBP1c hepatic protein, while SIRT1 increased. Hepatic expression of miR-21a-5p, miR-103-3p and miR-122-5p, miR34a-5p was downregulated. Hepatic histologic analysis showed in Moringa group (HF + MO) a significant decrease in inflammatory nodules, macro steatosis, fibrosis, collagen and αSMA reactivity. Analysis of hepatic transcriptome showed down expression of mRNAs implicated in DNA response to damage, endoplasmic reticulum stress, lipid biosynthesis and insulin resistance. Moringa reduced insulin resistance, de novo lipogenesis, hepatic inflammation and ER stress. Conclusions. Moringa prevented progression of liver damage in a model of NASH and improved biochemical, histological and hepatic expression of genes and miRNAs implicated in MAFLD/NASH development.

Western Diet-Fed ApoE Knockout Male Mice as an Experimental Model of Non-Alcoholic Steatohepatitis.

One of the consequences of the Western lifestyle and high-fat diet is non-alcoholic fatty liver disease (NAFLD) and its aggressive form, non-alcoholic steatohepatitis (NASH), which can progress to cirrhosis and hepatocellular carcinoma (HCC) and is rapidly becoming the leading cause of end-stage liver disease or liver transplantation. Currently, rodent NASH models lack significant aspects of the full NASH spectrum, representing a major problem for NASH research. Therefore, this work aimed to characterize a fast rodent model with all characteristic features of NASH. Eight-week-old male ApoE KO mice were fed with Western diet (WD), high fatty diet (HFD) or normal chow (Chow) for 7 weeks. Whole-body fat was increased by ~2 times in WD mice and HFD mice and was associated with increased glucose intolerance, hepatic triglycerides, and plasma ALT and plasma AST compared with Chow mice. WD mice also showed increased galectin-3 expression compared with Chow or HFD mice and increased plasma cholesterol compared with Chow mice. WD and HFD displayed increased hepatic fibrosis and increased F4/80 expression. WD mice also displayed increased levels of plasma MCP-1. Hepatic inflammatory markers were evaluated, and WD mice showed increased levels of TNF-α, MCP-1, IL-6 and IFN-γ. Taken together, these data demonstrated that the ApoE KO mouse fed with WD is a great model for NASH research, once it presents the fundamental parameters of the disease, including hepatic steatosis, fibrosis, inflammation, and metabolic syndrome.

The Metabolic Sensor Adenosine Monophosphate-Activated Protein Kinase Regulates Apoptosis in Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH), occurring in a subset of patients with nonalcoholic fatty liver disease (NAFLD), is characterized by cellular inflammation and hepatic fibrosis. NASH may lead to the development of end-stage liver disease and its sequelae. Hepatocyte cell death contributes to inflammation and fibrosis in NASH. Although multiple modes of cell death occur in experimental models of NASH (i.e., apoptosis, necroptosis, and pyroptosis), apoptosis is the best characterized. Apoptosis is mediated by activation of a family of cysteine proteases termed caspases, which ultimately cleave a plethora of cellular substrates resulting in cellular demise. Caspases are classified as "initiators" (i.e., caspase-8, caspase-9) and "executioners" (i.e., caspase-3, caspase-7, caspase-6), the former being activated in molecular platforms assembled after engagement of death receptors or mitochondrial permeabilization, and the latter being cleaved and activated by initiator caspases, with the exception of caspase-6, which is often activated by caspase-3 rather than by initiator caspases. Although caspase-3 has been implicated in hepatocyte apoptosis and liver fibrosis in a mouse model of NASH (1), the involvement of caspase-6 was never reported until now.

Diosmin ameliorates inflammation, insulin resistance, and fibrosis in an experimental model of non-alcoholic steatohepatitis in rats

Non-alcoholic steatohepatitis (NASH) is becoming of increasing significance due to its growing global prevalence and risk of progression to end-stage liver disease. This study was carried out to investigate the potential anti-inflammatory, insulin sensitizing, and antifibrotic effects of diosmin in an experimental model of NASH induced in rats using high-fat diet (HFD) and 30 mg/kg streptozotocin (STZ). Diosmin was administered orally at dose of 100 mg/kg for 8 weeks. Stained tissue sections were examined for histopathological signs of NASH, collagen deposition, and alpha smooth muscle actin (α-SMA) expression. In addition, insulin resistance, dyslipidemia, inflammation, and fibrosis markers were assessed. HFD/STZ successfully induced different NASH features such as insulin resistance seen by elevated fasting blood glucose levels and homeostasis model assessment for insulin resistance. Moreover, induced rats demonstrated dyslipidemia, a significant elevation in tumor necrosis factor alpha (TNF-α) and interleukin-6 levels, and an imbalance in the oxidative status of the liver. Those events altogether precipitated initiation of liver fibrosis as confirmed by elevated transforming growth factor beta (TGF-β) levels. Treatment with diosmin demonstrated multiple beneficial effects as it significantly ameliorated histopathological NASH findings, lowered TNF-α, interleukin-6, and malondialdehyde levels, improved lipid and glucose metabolism, and lowered hepatic TGF-β, α-SMA, and collagen content compared to untreated rats. The present study represents a drug repositioning scenario as diosmin is widely used for management of blood vessel disorders and is known to be well tolerated. This encourages the extension of our study to the clinical setting to explore diosmin effects in NASH patients.

Evaluation of the therapeutic potential effect of Fas receptor gene knockdown in experimental model of non-alcoholic steatohepatitis

Aim: Stimulation of Fas death receptor is introduced as a major cause of non-alcoholic steatohepatitis (NASH) progression through suppression of cell viability. Therefore, the blocking of death pathways is hypothesised to be express new approaches to NASH therapy. For this purpose, current experiment applied synthetic small interference RNA (SiRNA) to trigger Fas death receptor and to show its potential therapeutic role in designed NASH model.Methods: Male mice were placed on a western diet (WD) for 8 weeks and exposed to cigarette smoke during the last 4 weeks of feeding to induce NASH model. In the next step, Fas SiRNA was injected to mice aiming to examine specific Fas gene silencing, after 8 weeks. As a control, mice received scrambled SiRNA. Reversible possibility of disease was examined by 3 weeks of recovery.Results: Analysis of data is accompanied with the significant histopathological changes (steatosis, ballooning and inflammation), increased lipid profile and hepatic enzyme activities (AST, ALT, ALP) plus TBARS as well as decreased antioxidants levels in NASH model. Upon Fas-SiRNA injection, almost all measured parameters of NASH such as overexpression of Fas receptor, caspase3, NF-kB genes and marked increase of hepatic TNF-α were significantly restored and were remained nearly unchanged following recovery liking as scrambled groups.Conclusions: The suppression of Fas receptor signalling subsequent RNAi therapy may represent an applicable strategy to decline hepatocyte damages and so NASH progression in mice.

Protection against nonalcoholic steatohepatitis through targeting IL-18 and IL-1alpha by luteolin.

The management of nonalcoholic steatohepatitis (NASH) is still a crosstalk so the current study was designed to evaluate the effect of different luteolin doses on an experimental model of NASH and to elucidate novel anti-inflammatory pathways underlying its effect. Adult male Wistar rats (200-220 g; n = 60) were used. Rats were fed a high carbohydrate/high fat diet (˜ 30% carbohydrate and 42% fat) daily for 12 weeks to induce NASH. Luteolin (10, 25, 50 or 100 mg/kg/day) was administered as a suspension (10% w/v in 0.9% NaCl) using an oral gavage. Histopathological changes (necrosis, inflammation and steatosis) were evaluated. Biomarkers for liver function, lipid peroxidation, extracellular matrix deposition and anti-oxidant activity were measured. Levels of IFN-γ, TNF-α and IL-1α and IL-18 were measured. Obtained results showed ability of luteolin to reduce activity of ALT and AST and to decrease levels of bilirubin, hyaluronic acid and malondialdehyde significantly (p < 0.05). Also, luteolin showed an anti-oxidant activity as indicated by the significant (p < 0.05) increase in reduced glutathione. Finally, a significant (p < 0.05) decrease in IFN-γ, TNF-α, IL-1α and IL-18 levels was observed most notably in groups that received high doses of luteolin (50 and 100 mg/kg). Luteolin can protect against non-alcoholic steatohepatitis through targeting the pro-inflammatory IL-1 and Il-18 pathways in addition to an antioxidant effect.

Nitro-fatty acids protect against steatosis and fibrosis during development of nonalcoholic fatty liver disease in mice.

BackgroundNonalcoholic fatty liver disease (NAFLD) and resulting nonalcoholic steatohepatitis (NASH) are reaching global epidemic proportions. Lack of non-invasive diagnostic tools and effective therapies constitute two of the major hurdles for a bona fide treatment and a reversal of NASH progression and/or regression of the disease. Nitro-oleic acid (OA-NO2) has been proven effective in multiple experimental models of inflammation and fibrosis. Thus, the potential benefit of in vivo administration of OA-NO2 to treat advanced NAFLD was tested herein in a model of long-term NASH diet-induced liver damage.MethodsNon-invasive imaging (e.g. photoacustic-ultrasound (PA-US)) was pursued to establish advanced experimental model of NASH in mice in which both steatosis and fibrosis were diagnosed prior experimental therapy with OA-NO2. Experimental controls included equimolar amounts of the non-nitrated oleic acid (OA). CLAMS and NMR-based analysis was used for energy metabolism.FindingsCLAMS and NMR-based analysis demonstrates that OA-NO2 improves body composition and energy metabolism and inhibits hepatic triglyceride (TG) accumulation. Photoacoustic-ultrasound imaging revealed a robust inhibition of liver steatosis and fibrosis by OA-NO2. RNA-sequencing analysis uncovered inflammation and fibrosis as major pathways suppressed by OA-NO2 administration, as well as regulation of lipogenesis and lipolysis pathways, with a robust inhibition of SREBP1 proteolytic activation and subsequent lipogenesis gene expression by OA-NO2. These results were further supported by histological analysis and quantification of lipid accumulation, lobular inflammation (F4/80 staining) and fibrosis (collagen deposition, αSMA staining) as well as established parameters of liver damage (ALT). In vitro studies indicate that OA-NO2 inhibits TG biosynthesis and accumulation in hepatocytes and inhibits fibrogenesis in human stellate cells.InterpretationOA-NO2 improve steatohepatitis and fibrosis and may constitute an effective therapeutic approach against advanced NAFLD that warrants further clinical evaluation.

Novel non-alcoholic steatohepatitis model with histopathological and insulin-resistant features.

Although several non-alcoholic steatohepatitis (NASH) models have been reported to date, few of these models fully reflect the histopathology and pathophysiology of human NASH. The aim of this study was to establish a novel NASH model by feeding a high-fat (HF) diet and administering both carbon tetrachloride (CCl4 ) and the Liver X receptor agonist T0901317. Male C57BL/6J mice were divided into four groups (each n = 5): HF, HF + CCl4 , HF + T0901317, and the novel NASH model (HF + CCl4 + T0901317). CCl4 (0.1 mL/kg) and T0901317 (2.5 mg/kg) were intraperitoneally administered four times and five times, respectively. The livers of the novel NASH model group presented a whitish colour. The serum levels of TNF-α and IL-6 were significantly increased in the novel NASH model group, and mice in this group exhibited histopathological features and insulin resistance reflective of NASH, i.e., macrovesicular hepatic steatosis, ballooning hepatocytes, Mallory-Denk bodies, lobular inflammation and fibrosis. The novel NASH model group presented significantly upregulated expression levels of mRNAs related to lipogenesis, oxidative stress, fibrosis and steatosis and significantly downregulated expression levels of mRNAs related to triglyceride export. We successfully established a novel experimental NASH model that exhibits similar histopathology and pathophysiology to human NASH.

Non-alcoholic steatohepatitis-associated hepatic fibrosis and hepatocellular carcinoma in a combined mouse model of genetic modification and dietary challenge.

Experimental models of non-alcoholic steatohepatitis (NASH) are still required for understanding the pathophysiology of this disease. This study aimed to examine whether disease progression is accelerated by combining dyslipidemic genetic modification and dietary challenges and develop NASH-associated hepatic fibrosis, cirrhosis, and carcinoma in a short period. Low-density lipoprotein receptor knockout mice were fed a modified choline-deficient amino acid-defined diet, including 1 w/w% cholesterol and 41kcal% fat, and was comprehensively profiled over 1year. Microvesicular and macrovesicular steatosis in the liver was observed from the first week after starting the modified choline-deficient amino acid-defined diet. Macrovesicular steatosis was exacerbated with time and was observed in almost all hepatocytes at week 8, but slightly decreased at week 16. Infiltration of macrophages and neutrophils, and upregulation of hepatic inflammatory cytokines such as tumor necrosis factor-α and interleukin-1β were also observed from week 1. Plasma hepatic transaminase activities were increased at week 1, reached a peak at week 4, and gradually decreased thereafter. In parallel with increases in hepatic gene expression of collagen-I, the hepatic fibrosis area expanded after week 4 and massively spread all over the liver by week 8. Hepatocellular hyperplasia was observed from week 24. Hepatocellular adenoma and carcinoma were observed from week 31 and 39, respectively. These results suggest that, in a rodent NASH model with the combination of genetic modification and dietary challenges, hepatic steatosis, inflammatory cell infiltration and hepatic injury, hepatic fibrosis, hepatocellular hyperplasia, adenoma, and carcinoma can be developed in a relatively short period.

Necroptosis is a key pathogenic event in human and experimental murine models of non-alcoholic steatohepatitis.

Hepatocyte cell death, inflammation and oxidative stress constitute key pathogenic mechanisms underlying non-alcoholic fatty liver disease (NAFLD). We aimed to investigate the role of necroptosis in human and experimental NAFLD and its association with tumour necrosis factor α (TNF-α) and oxidative stress. Serum markers of necrosis, liver receptor-interacting protein 3 (RIP3) and phosphorylated mixed lineage kinase domain-like (MLKL) were evaluated in control individuals and patients with NAFLD. C57BL/6 wild-type (WT) or RIP3-deficient (RIP3(-/-)) mice were fed a high-fat choline-deficient (HFCD) or methionine and choline-deficient (MCD) diet, with subsequent histological and biochemical analysis of hepatic damage. In primary murine hepatocytes, necroptosis and oxidative stress were also assessed after necrostatin-1 (Nec-1) treatment or RIP3 silencing. We show that circulating markers of necrosis and TNF-α, as well as liver RIP3 and MLKL phosphorylation were increased in NAFLD. Likewise, RIP3 and MLKL protein levels and TNF-α expression were increased in the liver of HFCD and MCD diet-fed mice. Moreover, RIP3 and MLKL sequestration in the insoluble protein fraction of NASH (non-alcoholic steatohepatitis) mice liver lysates represented an early event during stetatohepatitis progression. Functional studies in primary murine hepatocytes established the association between TNF-α-induced RIP3 expression, activation of necroptosis and oxidative stress. Strikingly, RIP3 deficiency attenuated MCD diet-induced liver injury, steatosis, inflammation, fibrosis and oxidative stress. In conclusion, necroptosis is increased in the liver of NAFLD patients and in experimental models of NASH. Further, TNF-α triggers RIP3-dependent oxidative stress during hepatocyte necroptosis. As such, targeting necroptosis appears to arrest or at least impair NAFLD progression.

6]-Gingerol dampens hepatic steatosis and inflammation in experimental nonalcoholic steatohepatitis

The aim of the study was to investigate the effects of [6]-gingerol ((S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-decanone) in experimental models of non-alcoholic steatohepatitis. HepG2 cells were exposed to 500 µmol/l oleic acid (OA) for 24 h and preincubated for an additional 24 h with [6]-gingerol (25, 50 or 100 µmol/l). [6]-Gingerol (100 µmol/l) inhibited OA-induced triglyceride and inflammatory marker accumulation in HepG2 cells. After being fed a high-fat diet (HFD) for 2 weeks, male golden hamsters were dosed orally with [6]-gingerol (25, 50 or 100 mg/kg/day) once daily for 8 weeks while maintained on HFD. [6]-Gingerol (100 mg/kg/day) alleviated liver steatosis, inflammation, and reversed plasma markers of metabolic syndrome in HFD-fed hamsters. The expression of inflammatory cytokine genes and nuclear transcription factor-κB (NF-κB) were increased in the HFD group; these effects were attenuated by [6]-gingerol. The hepatic mRNA expression of lipogenic genes such as liver X receptor-α, sterol regulating element binding protein-1c and its target genes including acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase 1, and acyl-CoA:diacylglycerol acyltransferase 2 in HFD-fed hamsters was also blocked by [6]-gingerol. [6]-Gingerol may attenuate HFD-induced steatohepatitis by downregulating NF-κB-mediated inflammatory responses and reducing hepatic lipogenic gene expression.

6-gingerol protects against nutritional steatohepatitis by regulating key genes related to inflammation and lipid metabolism.

Non-alcoholic fatty liver disease, including non-alcoholic steatohepatitis (NASH), appears to be increasingly common worldwide. The aim of the study was to investigate the effects of 6-gingerol ((S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-decanone), a bioactive ingredient of plants belonging to the Zingiberaceae family, on experimental models of NASH. In HepG2 cells, 6-gingerol (100 μmol/L) treatment inhibited free fatty acids mixture (0.33 mmol/L palmitate and 0.66 mmol/L oleate)-induced triglyceride and inflammatory marker accumulations. Male C57BL/6 mice were fed with a methionine and choline-deficient (MCD) diet to induce steatohepatitis. After four weeks of MCD diet feeding, the mice were dosed orally with 6-gingerol (25, 50 or 100 mg/kg/day) once daily for another four weeks. 6-Gingerol (100 mg/kg/day) attenuated liver steatosis and necro-inflammation in MCD diet-fed mice. The expressions of inflammatory cytokine genes, including those for monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin-6, and nuclear transcription factor (NF-κB), which were increased in the livers of MCD diet-fed mice, were attenuated by 6-gingerol. 6-Gingerol possesses a repressive property on hepatic steatosis, which is associated with induction of peroxisome proliferator-activated receptor α. Our study demonstrated the protective role of 6-gingerol in ameliorating nutritional steatohepatitis. The effect was mediated through regulating key genes related to lipid metabolism and inflammation.

Regular exercise decreases liver tumors development in hepatocyte-specific PTEN-deficient mice independently of steatosis

Unhealthy lifestyles predispose people to non-alcoholic steatohepatitis (NASH), which may further result in the development of hepatocellular carcinoma (HCC). Although NASH patients benefit from physical activity, it is unknown whether regular exercise reduces the risk of developing HCC. Therefore, we studied the effect of regular exercise on the development of HCC in male hepatocyte-specific PTEN-deficient mice (AlbCrePten(flox/flox)), which develop steatohepatitis and HCC spontaneously. Mice were fed a standardized 10% fat diet and were randomly divided into exercise or sedentary groups. The exercise group ran on a motorized treadmill for 60 min/day, 5 days/week during 32 weeks. After 32 weeks of regular exercise, 71% of exercised mice developed nodules larger than 15 mm(3)vs. 100% of mice in the sedentary group. The mean number of tumors per liver was reduced by exercise, as well as the total tumoral volume per liver. Exercise did not affect steatosis and had no effect on the non-alcoholic fatty liver disease (NAFLD) Activity Score (NAS). Exercise decreased tumor cell proliferation. Mechanistically, exercise stimulated the phosphorylation of AMPK and its substrate raptor, which decreased the kinase activity of mTOR. These data show a beneficial effect of regular exercise on the development of HCC in an experimental model of NASH and offer a rationale for encouraging predisposed patients to increase their physical activity for the prevention of HCC.

6-Gingerol mitigates nutritional steatohepatitis through regulating key genes related to oxidative stress, inflammation and fibrogenesis

The aim of the study was to investigate the effects of 6-gingerol, a bioactive ingredient of plants belonging to the Zingiberaceae family, on experimental models of non-alcoholic steatohepatitis (NASH). Male C57BL/6 mice were fed with a methionine and choline-deficient diet (MCDD) to induce steatohepatitis. After 8 weeks of feeding, the mice were treated orally with 6-gingerol (100 mg per kg per day). All mice were sacrificed after 4 weeks of treatment, and biochemical, pathological, and molecular analyses were performed. MCDD-fed mice showed severe hepatic injury including hepatic steatosis, necro-inflammation and fibrosis. 6-Gingerol possesses a repressive property on hepatic steatosis, which is associated with inhibition of acyl-CoA:diacylglycerol acyltransferase 2 and induction of peroxisome proliferator-activated receptor α. Administration of 6-gingerol significantly lowered plasma levels of alanine aminotransferase, aspartate aminotransferase, reduced hepatic oxidative stress and ameliorated hepatic inflammation and fibrosis. These effects were associated with down-regulation of cytochrome P450 2E1; reducing Jun N-terminal kinase-signaling; suppression of pro-inflammation genes and chemokines; and inhibition of pro-fibrotic genes. Our study demonstrated the protective role of 6-gingerol in ameliorating nutritional steatohepatitis. The effect was mediated through regulating key genes related to oxidative stress, inflammation and fibrogenesis.

Translational approaches: from fatty liver to non-alcoholic steatohepatitis.

Over the past few decades, non-alcoholic fatty liver disease (NAFLD) has become one, if not the most common, cause of chronic liver disease affecting both adults and children. The increasing number of cases at an early age is the most worrying aspect of this pathology, since it provides more time for its evolution. The spectrum of this disease ranges from liver steatosis to steatohepatitis, fibrosis and in some cases, hepatocellular carcinoma. NAFLD may not always be considered a benign disease and hepatologists must be cautious in the presence of fatty liver. This should prompt the use of the available experimental models to understand better the pathogenesis and to develop a rational treatment of a disease that is dangerously increasing. In spite of the growing efforts, the pathogenesis of NAFLD is still poorly understood. In the present article we review the most relevant hypotheses and evidence that account for the progression of NAFLD to non-alcoholic steatohepatitis (NASH) and fibrosis. The available in vitro and in vivo experimental models of NASH are discussed and revised in terms of their validity in translational studies. These studies must be aimed at the discovery of the still unknown triggers or mediators that induce the progression of hepatic inflammation, apoptosis and fibrosis.

Modeling Human Nonalcoholic Steatohepatitis-Associated Changes in Drug Transporter Expression Using Experimental Rodent Models

Nonalcoholic fatty liver disease is a prevalent form of chronic liver disease that can progress to the more advanced stage of nonalcoholic steatohepatitis (NASH). NASH has been shown to alter drug transporter regulation and may have implications in the development of adverse drug reactions. Several experimental rodent models have been proposed for the study of NASH, but no single model fully recapitulates all aspects of the human disease. The purpose of the current study was to determine which experimental NASH model best reflects the known alterations in human drug transporter expression to enable more accurate drug disposition predictions in NASH. Both rat and mouse NASH models were used in this investigation and include the methionine and choline deficient (MCD) diet model, atherogenic diet model, ob/ob and db/db mice, and fa/fa rats. Pathologic scoring evaluations demonstrated that MCD and atherogenic rats, as well as ob/ob and db/db mice, developed NASH. Liver mRNA and protein expression analyses of drug transporters showed that in general, efflux transporters were induced and uptake transporters were repressed in the rat MCD and the mouse ob/ob and db/db models. Lastly, concordance analyses suggest that both the mouse and rat MCD models as well as mouse ob/ob and db/db NASH models show the most similarity to human transporter mRNA and protein expression. These results suggest that the MCD rat and mouse model, as well as the ob/ob and db/db mouse models, may be useful for predicting altered disposition of drugs with similar kinetics across humans and rodents.

Immunohistochemistry pattern of hepatic inflammatory and insulin resistance markers in experimental model of nonalcoholic steatohepatitis

Introduction:The pathophysiology of nonalcoholic steatohepatitis (NAS) includes, basically, insulin resistance, inflammation and oxidative stress. Thus, a study of immunostaining for liver insulin, adiponectin, tumor necrosis factor alpha (TNF-α), and inducible nitric oxide synthase (iNOS) receptors was conducted.Objective:To expand the knowledge about the pathophysiological and molecular mechanisms underlying the experimental model of steatohepatitis in rats fed a high-fat diet.Method:Twenty Wistar rats were divided into two groups: G1 (control, fed a standard diet), and G2 (fed a high-fat diet containing 58% of energy derived from fat, 18% from protein and 24% from carbohydrate). After eight weeks the animals were sacrificed. Blood glucose, insulin, total cholesterol, high-density lipoprotein (HDL), the very low-density lipoproteins (VLDL), triglycerides, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) were determined. The liver tissue was submitted to histopathological analysis, using a NAS score. In immunohistochemistry, we studied the expression of the insulin receptor, adiponectin, TNF-α and iNOS by tissue microarray method.Results and conclusion:There was marked cytoplasmic immunostaining for TNF-α and iNOS mediators in the group on a fat diet. Regarding insulin and adiponectin molecular markers, a reduction of cytoplasmic immunoreactivity of these antigens was observed in the group on a fat diet, reflecting, respectively, the state of hepatocellular inflammation (steatohepatitis) and insulin resistance in this experimental model of fat liver disease.

Ruscogenin ameliorates experimental nonalcoholic steatohepatitis via suppressing lipogenesis and inflammatory pathway.

The aim of the study was to investigate the protective effects of ruscogenin, a major steroid sapogenin in Ophiopogon japonicus, on experimental models of nonalcoholic steatohepatitis. HepG2 cells were exposed to 300 μmol/l palmitic acid (PA) for 24 h with the preincubation of ruscogenin for another 24 h. Ruscogenin (10.0 μmol/l) had inhibitory effects on PA-induced triglyceride accumulation and inflammatory markers in HepG2 cells. Male golden hamsters were randomly divided into five groups fed a normal diet, a high-fat diet (HFD), or a HFD supplemented with ruscogenin (0.3, 1.0, or 3.0 mg/kg/day) by gavage once daily for 8 weeks. Ruscogenin alleviated dyslipidemia, liver steatosis, and necroinflammation and reversed plasma markers of metabolic syndrome in HFD-fed hamsters. Hepatic mRNA levels involved in fatty acid oxidation were increased in ruscogenin-treated HFD-fed hamsters. Conversely, ruscogenin decreased expression of genes involved in hepatic lipogenesis. Gene expression of inflammatory cytokines, chemoattractive mediator, nuclear transcription factor-(NF-) κB, and α-smooth muscle actin were increased in the HFD group, which were attenuated by ruscogenin. Ruscogenin may attenuate HFD-induced steatohepatitis through downregulation of NF-κB-mediated inflammatory responses, reducing hepatic lipogenic gene expression, and upregulating proteins in β-oxidation pathway.