Onset Of Non-alcoholic Steatohepatitis Research Articles

Pathophysiological characteristics of non-alcoholic steatohepatitis-like changes in cholesterol-loaded type 2 diabetic rats.

Spontaneously Diabetic Torii (SDT) fatty rats, a new obese diabetic model, reportedly presented with features of non-alcoholic steatohepatitis (NASH) after 32 weeks of age. We tried to accelerate the onset of NASH in SDT fatty rats using dietary cholesterol loading and noticed changes in the blood choline level which is expected to be a NASH biomarker. Body weight and biochemical parameters were measured from 8 to 24 weeks of age. At 16, 20, 24 weeks, pathophysiological analysis of the livers were performed. Hepatic lipids, lipid peroxides, and the expression of mRNA related to triglyceride (TG) synthesis, inflammation, and fibrosis were evaluated at 24 weeks. Hepatic fibrosis was observed in SDT fatty rats fed cholesterol-enriched diets (SDT fatty-Cho) from 16 weeks. Furthermore, hepatic lipids and lipid peroxide were significantly higher in SDT fatty-Cho than SDT fatty rats fed normal diets at 24 weeks. Hepatic mRNA expression related to TG secretion decreased in SDT fatty-Cho, and the mRNA expression related to inflammation and fibrosis increased in SDT fatty-Cho at 24 weeks. Furthermore, SDT fatty-Cho presented with increased plasma choline, similar to human NASH. There were no significant changes in the effects of feeding a cholesterol-enriched diet in Sprague-Dawley rats. SDT fatty-Cho has the potential to become a valuable animal model for NASH associated with type 2 diabetes and obesity.

Canagliflozin, an SGLT2 inhibitor, attenuates the development of hepatocellular carcinoma in a mouse model of human NASH

Sodium glucose cotransporter 2 (SGLT2) inhibitors, an antidiabetic drug, promotes urinary excretion of glucose by blocking its reabsorption in the renal proximal tubules. It is unclear whether SGLT2 inhibition could attenuate nonalcoholic steatohepatitis (NASH) and NASH-associated hepatocellular carcinoma. We examined the preventive effects of an SGLT2 inhibitor canagliflozin (CANA) in Western diet (WD)-fed melanocortin 4 receptor-deficient (MC4R-KO) mice, a mouse model of human NASH. An eight-week CANA treatment attenuated hepatic steatosis in WD-fed MC4R-KO mice, with increased epididymal fat mass without inflammatory changes. CANA treatment for 20 weeks inhibited the development of hepatic fibrosis in WD-fed MC4R-KO mice. After one year of CANA treatment, the number of liver tumors was significantly reduced in WD-fed MC4R-KO mice. In adipose tissue, CANA suppressed the ratio of oxidative to reduced forms of glutathiones (GSSG/GSH) in WD-fed MC4R-KO mice. Treatment with GSH significantly attenuated the H2O2-induced upregulation of genes related to NADPH oxidase in 3T3-L1 adipocytes, and that of Il6, Tgfb, and Pdgfb in RAW264.7 cells. This study provides evidence that SGLT2 inhibitors represent the unique class of drugs that can attenuate or delay the onset of NASH and eventually hepatocellular carcinoma, at least partly, through “healthy adipose expansion”.

ASSOCIATION OF POLYMORPHISM OF ACE (I/D) AND PPAR-G2 (PRO12ALA) GENES WITH THE DEVELOPMENT OF NONALCOHOLIC FATTY LIVER DISEASE IN PATIENTS WITH ARTERIAL HYPERTENSION AND OBESITY

Objective – To study the association of polymorphism of ACE (I/D) and PPAR-g2 (Pro12Ala) genes with the onset of non-alcoholic steatohepatitis and steatohepatosis in patients suffering from essential arterial hypertension (EAH) and abdominal obesity (AO). Materials and methods. The prospective study involved 96 patients with non-alcoholic fatty liver disease (NAFLD), stage II EAH of 1-2 degrees of high and very high risk with concomitant AO. There were 41,67 % (40) of men and 58,33 % (56) women, the average age was 53,70±5,34 years. The function of the liver was studied by the activity of organspecific enzymes. The study of polymorphism of PPAR-g2 (Pro12Ala) and АСЕ (I/D) genes was carried out by using the PCR method. The control group consisted of 50 practically healthy individuals. Results. First degree obesity (OB) was diagnosed in 27,08 % (26) individuals, second degree OB was found in 58,33 % (56), 14,58 % (14) of patients had third degree OB; 16,67 % (16) individuals suffered from steatohepatitis with minimal activity of mesenchymal and inflammatory process, the rest 83,33 % (80) of the patients had steatohepatosis. Among the residents of the Northern Bukovyna suffering from NAFLD, OB and EAH the deletion in the 16th intron of the АСЕ (rs 4646994) gene in the homozygous condition occurs in 32,29 % of cases, which is by14,29 % more frequently than in the individuals of the control group (χ2=3.38; р=0,048). The unfavorable D-allele of the ACE gene is associated in patients with NAFLD and EAH with II and III degree obesity (χ2=5,24; р=0,022 and χ2=6,11; р=0.013, respectively) and occurs in general more frequently in patients by12,29 % (χ2=3,99; р=0,046). The DD genotype and D-allele are also associated with a higher incidence of steatohepatosis by 20,57 % (χ2=3,81; р=0,05) and 13,75 % (χ2=4,68; р=0,03), respectively. Frequency of homozygous missense mutation in chromosome 3 of codon 12 of exon B of PPAR-g2 (rs1801282) gene exists in 2,0 % of practically healthy individuals and in 5,21 % of patients with NAFLD, EAH and AO (р>0.05). In general, Pro-allele is prevalent among the examined individuals by 6.14 and 3.85 times (p<0,001), which is more common in the control than in patients with NAFLD, EAH and first degree AO by16.77% (χ2=5,06; р=0,024). Ala-allele, as well as AlaAla- and ProAla-genotypes are associated with a higher incidence of steatohepatitis by 30,25 % (χ2=4,99; р=0,025) and 17,25 % (χ2=4,85; р=0,028) respectively. Limitations of the study / consequences. The limitations are due to the lack of a puncture biopsy of the liver / consequences – the accuracy of diagnosis of NAFLD is based on clinical and laboratory findings and those of ultrasound examination. Novelty / value. This original study provides data allowing to assess the association of ACE (I/D) and PPAR-g2 (Pro12Ala) genes with NAFLD, EAH and AO considering the type of NAFLD and the degrees of obesity. Conclusions. In patients with Non-alcoholic Fatty Liver Disease, Essential Arterial Hypertension the unfavorable D-allele of the ACE (rs4646994) gene is associated with II and III obesity degree and higher incidence of steatohepatosis; Ala-allele of the PPAR-g2 (rs1801282) gene is associated with a higher incidence of steatohepatitis.

Uncovering a Predictive Molecular Signature for the Onset of NASH-Related Fibrosis in a Translational NASH Mouse Model.

Background & AimsThe incidence of nonalcoholic steatohepatitis (NASH) is increasing. The pathophysiological mechanisms of NASH and the sequence of events leading to hepatic fibrosis are incompletely understood. The aim of this study was to gain insight into the dynamics of key molecular processes involved in NASH and to rank early markers for hepatic fibrosis.MethodsA time-course study in low-density lipoprotein–receptor knockout. Leiden mice on a high-fat diet was performed to identify the temporal dynamics of key processes contributing to NASH and fibrosis. An integrative systems biology approach was used to elucidate candidate markers linked to the active fibrosis process by combining transcriptomics, dynamic proteomics, and histopathology. The translational value of these findings were confirmed using human NASH data sets.ResultsHigh-fat-diet feeding resulted in obesity, hyperlipidemia, insulin resistance, and NASH with fibrosis in a time-dependent manner. Temporal dynamics of key molecular processes involved in the development of NASH were identified, including lipid metabolism, inflammation, oxidative stress, and fibrosis. A data-integrative approach enabled identification of the active fibrotic process preceding histopathologic detection using a novel molecular fibrosis signature. Human studies were used to identify overlap of genes and processes and to perform a network biology-based prioritization to rank top candidate markers representing the early manifestation of fibrosis.ConclusionsAn early predictive molecular signature was identified that marked the active profibrotic process before histopathologic fibrosis becomes manifest. Early detection of the onset of NASH and fibrosis enables identification of novel blood-based biomarkers to stratify patients at risk, development of new therapeutics, and help shorten (pre)clinical experimental time frames.

Oral arginine supplementation protects female mice from the onset of non-alcoholic steatohepatitis

Dietary arginine (Arg) supplementation has been proposed to have positive effects on the development of liver diseases. In the present study, we investigate if an oral Arg supplementation in diet protects mice fed a fructose, fat and cholesterol enriched Western-style diet (WSD) from the development of non-alcoholic steatohepatitis (NASH). Female C57BL/6J mice were fed a liquid control diet or a liquid WSD ± Arg (2.49 g/kg body weight/day) for 6 weeks. Indices of liver injury, glucose metabolism and intestinal permeability were determined. While Arg supplementation had no effects on body weight gain, fasting blood glucose levels were significantly lower in WSD+Arg-fed mice than in C+Arg-fed animals. WSD-fed mice developed liver steatosis accompanied with inflammation, both being significantly attenuated in WSD+Arg-fed mice. These effects of Arg supplementation went along with a protection against WSD-induced decreased tight junction protein levels in the upper parts of the small intestine, increased levels of bacterial endotoxin in portal plasma as well as increased hepatic toll-like receptor-4 mRNA and 4-hydroxynonenal protein adduct levels. In conclusion, Arg supplementation may protect mice from the development of NASH.

Rosuvastatin limits the activation of hepatic stellate cells in diet-induced obese mice.

The aim of this study was to investigate the effects of rosuvastatin in a model of diet-induced obesity and non-alcoholic fatty liver disease, with attention to the activation of hepatic stellate cells (HSCs). Male C57BL/6 mice received a control diet (C; 10% energy as lipids) or a high-fat diet (HF; 50% energy as lipids) for 12weeks, followed by 7weeks of treatment. Group CR received control diet + rosuvastatin; group HFR received high-fat diet + rosuvastatin. The HF group showed higher insulin, total cholesterol, triacylglycerol, and leptin levels than the C group, all of which were significantly diminished by rosuvastatin in the HFR group. The HF group had greater steatosis and activated HSCs than the C group, whereas rosuvastatin diminished the steatosis (less 21%, P < 0.001) and significantly inhibited the activation of the HSCs in the HFR group compared to the HF group. The sterol regulatory element-binding protein-1 and the peroxisome proliferator-activated receptor (PPAR)-γ protein expressions were increased in HF animals and reduced after treatment in the HFR group. By contrast, low PPAR-α and carnitine palmitoyltransferase-1 expressions were found in the HF group, and were restored by rosuvastatin treatment in the HFR group. Rosuvastatin mitigated hepatic steatosis by modulating PPAR balance, favoring PPAR-α over PPAR-γ downstream effects. The effects were accompanied by a diminishing of insulin resistance, the anti-inflammatory adipokine profile, and HSC activation, avoiding non-alcoholic fatty liver disease progression and non-alcoholic steatohepatitis onset in this model.

Interleukin-18–deficient mice develop dyslipidemia resulting in nonalcoholic fatty liver disease and steatohepatitis

We investigated potential pathophysiological relationships between interleukin 18 (IL-18) and dyslipidemia, nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH). Compared with Il18(+/+) mice, IL-18 knockout (Il18(-/-)) micedeveloped hypercholesterolemia and hyper-high-density-lipoprotein-cholesterolemia as well as hypertriglyceridemia as they aged, and these disorders occurred before the manifestation of obesity and might cause secondary NASH. The analyses of molecular mechanisms involved in the onset of dyslipidemia, NAFLD, and NASH in Il18(-/-) mice identified a number of genes associated with these metabolic diseases. In addition, molecules related to circadian rhythm might affect these extracted genes. The intravenous administration of recombinant IL-18 significantly improved dyslipidemia, inhibited the body weight gain of Il18(+/+) mice, and prevented the onset of NASH. The expression of genes related to these dysfunctions was also affected by recombinant IL-18 administration. In conclusion, this study demonstrated the critical function of IL-18 in lipid metabolism and these findings might contribute to the progress of novel treatments for NAFLD or NASH.

Lipotoxicity in steatohepatitis occurs despite an increase in tricarboxylic acid cycle activity.

The hepatic tricarboxylic acid (TCA) cycle is central to integrating macronutrient metabolism and is closely coupled to cellular respiration, free radical generation, and inflammation. Oxidative flux through the TCA cycle is induced during hepatic insulin resistance, in mice and humans with simple steatosis, reflecting early compensatory remodeling of mitochondrial energetics. We hypothesized that progressive severity of hepatic insulin resistance and the onset of nonalcoholic steatohepatitis (NASH) would impair oxidative flux through the hepatic TCA cycle. Mice (C57/BL6) were fed a high-trans-fat high-fructose diet (TFD) for 8 wk to induce simple steatosis and NASH by 24 wk. In vivo fasting hepatic mitochondrial fluxes were determined by(13)C-nuclear magnetic resonance (NMR)-based isotopomer analysis. Hepatic metabolic intermediates were quantified using mass spectrometry-based targeted metabolomics. Hepatic triglyceride accumulation and insulin resistance preceded alterations in mitochondrial metabolism, since TCA cycle fluxes remained normal during simple steatosis. However, mice with NASH had a twofold induction (P< 0.05) of mitochondrial fluxes (μmol/min) through the TCA cycle (2.6 ± 0.5 vs. 5.4 ± 0.6), anaplerosis (9.1 ± 1.2 vs. 16.9 ± 2.2), and pyruvate cycling (4.9 ± 1.0 vs. 11.1 ± 1.9) compared with their age-matched controls. Induction of the TCA cycle activity during NASH was concurrent with blunted ketogenesis and accumulation of hepatic diacylglycerols (DAGs), ceramides (Cer), and long-chain acylcarnitines, suggesting inefficient oxidation and disposal of excess free fatty acids (FFA). Sustained induction of mitochondrial TCA cycle failed to prevent accretion of "lipotoxic" metabolites in the liver and could hasten inflammation and the metabolic transition to NASH.

Increased ratio of neutrophil elastase to α1‐antitrypsin is closely associated with liver inflammation in patients with nonalcoholic steatohepatitis

An imbalance between neutrophil elastase (NE) and its inhibitor α1-antitrypsin (A1 AT) is known to contribute to the development of obesity-related inflammation. This study aimed to investigate the role of the NE-A1 AT system in the histological progression of non-alcoholic fatty liver disease (NAFLD), and to evaluate the ability of it to predict nonalcoholic steatohepatitis (NASH). A total of 252 adults (NAFLD group, n=202; healthy group, n=50) were recruited. Clinical biochemical characteristics, NE and A1 AT concentrations were measured in all subjects. Among the NAFLD group, 86 patients had previously undergone liver biopsy and information on histological characteristics was consequently available. The area under the receiver operating characteristic curve (AUC) was used to determine the predictive accuracy of the NE-A1 AT system for NASH. NAFLD patients had an elevated serum NE concentration and a reduced A1 AT level with consequent NE/A1 AT imbalance. NE increased in the early stage of steatosis, preceding the decline in A1 AT, dating from the onset of NASH (NAS 3-4), and subsequently NE/A1 AT increased in the presence of NASH. Nonetheless, this increase began to resolve as the disease state progressed to advanced fibrosis. A1 AT had a sensitivity (SEN) of 83.8% and a specificity (SP) of 83.3% with the optimal cut-off of -1459.43, NE/A1 AT had a SEN of 88.8% and a SP of 83.3% with cut-off of 0.363 to predict NASH. An increased NE: A1 AT ratio is closely associated with liver Inflammation in patients with NASH and could serve as a novel marker to predict NASH in humans.

Cross talk of the immune system in the adipose tissue and the liver in non-alcoholic steatohepatitis: Pathology and beyond.

Non-alcoholic steatohepatitis (NASH) is considered to be the hepatic manifestation of the metabolic syndrome, thus has a tight correlation with systemic metabolic impairment. The complex mechanisms underlying the pathogenesis of NASH involve different organs and systems that cross talk together contributing to the onset of NASH. A crucial role is played by inflammatory mediators, especially those deriving from the adipose tissue and the liver, which are involved in the cascade of inflammation, fibrosis and eventually tumorigenesis. In this setting cytokines and adipokines as well as immunity are emerging drivers of the key features of NASH. The immune system participates in this process with disturbances of the cells constituting both the innate and the adaptive immune systems that have been reported in different organs, such as in the liver and in the adipose tissue, in clinical and preclinical studies. The role of the immune system in NASH is increasingly studied, not only because of its contribution to the pathogenetic mechanisms of NASH but also because of the new potential therapeutic options it offers in this setting. Indeed, novel treatments acting on the immune system could offer new options in the management of NASH and the correlated clinical consequences.

β7-Integrins and MAdCAM-1 play an opposing role during the development of Non-alcoholic steatohepatitis (NASH)

Introduction: Non-alcoholic steatohepatitis (NASH), the hepatic correlate of the metabolic syndrome, is the third most common cause of chronic liver disease in western countries. Its global incidence is on the rise and reflects one of the fastest growing medical problems. Aim: The significance of leukocyte infiltration during the development of NASH remains unclear until now. We therefore investigated the role of β7-Integrin and one of its receptors MAdCAM-1 in a mouse model of NASH development. Methods: Constitutive MAdCAM-1 and β7-Integrin knockout (KO) mice were fed a MCD diet (methionine and choline deficient) for 4 weeks. Results: Interestingly β7-deficient mice displayed an earlier and faster progressing steatohepatitis during MCD treatment, while MAdCAM-1 KO showed less histomorphological changes in contrast to wildtype mice. Oxidative stress analysis in β7 KO mice showed a stronger response as indicated by DHE (dihydroethidium) staining and a down regulation of transcription factors involved in anti-oxidative stress and fatty acid metabolism (e.g. FAS and SREBP). MAdCAM-1 KO mice on the other hand had an upregulation of the anti-oxidative stress response. Moreover, we detected a stronger hepatic infiltration of inflammatory cells (CD4+ and CD11b+) in the β7 KO group, reflecting an earlier onset of NASH. Those changes finally resulted in an earlier and stronger collagen accumulation (Sirius red) in the β7-Integrin deficient group, while MAdCAM-1 KO mice were protected. Conclusion: MAdCAM-1 and β7 Integrins mediate opposing effects in the MCD model, with protection in MAdCAM-1 KO animals and a more severe phenotype and significantly stronger fibrosis progression in β7-Integrin KO mice. Therefore, the interaction of β7 Integrins and their receptor MAdCAM-1 provide a novel interesting target for therapeutic interventions during NASH development.

Dysregulated Bile Acid Synthesis, Metabolism and Excretion in a High Fat-Cholesterol Diet-Induced Fibrotic Steatohepatitis in Rats

Background and AimsCholesterol over-intake is involved in the onset of nonalcoholic steatohepatitis (NASH), and hepatocellular bile acid (BA) accumulation correlates with liver injuries. However, how dietary cholesterol influences cholesterol and BA kinetics in NASH liver remains ambiguous and needs to be clarified.MethodsMolecular markers involved in cholesterol and BA kinetics were investigated at protein and mRNA levels in an already-established stroke-prone spontaneously hypertensive 5/Dmcr rat model with fibrotic steatohepatitis, by feeding a high fat-cholesterol (HFC) diet.ResultsUnlike the control diet, the HFC diet deposited cholesterol greatly in rat livers, where 3-hydroxy-3-methylglutaryl CoA reductase, low-density lipoprotein (LDL) receptor and LDL receptor-related protein-1 were expectedly downregulated, especially at 8 and 14 weeks, suggesting that cholesterol synthesis and uptake in response to cholesterol accumulation may not be disorganized. The HFC diet did not upregulate liver X receptor-α, conversely, it enhanced classic BA synthesis by upregulating cholesterol 7α-hydroxylase but downregulating sterol 12α-hydroxylase, and influenced alternative synthesis by downregulating sterol 27-hydroxylase but upregulating oxysterol 7α-hydroxylase, mainly at 8 and 14 weeks, indicating that there were different productions of primary BA species. Unexpectedly, no feedback inhibition of BA synthesis by farnesoid X receptor occurred. Additionally, the HFC diet impaired BA detoxification by UDP-glucuronosyltransferase and sulfotransferase 2A1, and decreased excretion by bile salt export pump at 8 and 14 weeks, although it induced compensatory export by multidrug resistance-associated protein-3. The disturbed BA detoxification may correlate with suppressed pregnane X receptor and constitutive androstane receptor.ConclusionsThe HFC diet may accumulate BA in rat livers, which influences fibrotic steatohepatitis progression.Electronic supplementary materialThe online version of this article (doi:10.1007/s10620-013-2747-1) contains supplementary material, which is available to authorized users.

Hepatic recruitment of macrophages promotes nonalcoholic steatohepatitis through CCR2

Inflammatory cell infiltration in the liver is a hallmark of nonalcoholic steatohepatitis (NASH). The chemokine-chemokine receptor interaction induces inflammatory cell recruitment. CC-chemokine receptor (CCR)2 is expressed on hepatic macrophages and hepatic stellate cells. This study aims to investigate the therapeutic potential of CCR2 to NASH. Twenty-two weeks on a choline-deficient amino acid-defined (CDAA) diet induced steatosis, inflammatory cell infiltration, and liver fibrosis with increased CCR2 and monocyte chemoattractant protein (MCP)-1 expression in the wild-type livers. The infiltrated macrophages expressed CD68, CCR2, and a marker of bone marrow-derived monocytes, Ly6C. CCR2(-/-) mice had less steatosis, inflammatory cell infiltration, and fibrosis, and hepatic macrophages expressing CD68 and Ly6C were decreased. Toll-like receptor (TLR)4(-/-), TLR9(-/-), and MyD88(-/-) mice had reduced hepatic macrophage infiltration with decreased MCP-1 and CCR2 expression because TLR signaling is a potent inducer of MCP-1. To assess the role of Kupffer cells at the onset of NASH, Kupffer cells were depleted by liposomal clodronate. The Kupffer cell depletion ameliorated steatohepatitis with a decrease in the MCP-1 expression and recruitment of Ly6C-expressing macrophages at the onset of NASH. Finally, to test the therapeutic potential of targeting CCR2, a CCR2 inhibitor was administered to mice on a CDAA diet. The pharmaceutical inhibition of CCR2 prevented infiltration of the Ly6C-positive macrophages, resulting in an inhibition of liver inflammation and fibrosis. We concluded that CCR2 and Kupffer cells contribute to the progression of NASH by recruiting bone marrow-derived monocytes.

Plasma free choline is a novel non‐invasive biomarker for early‐stage non‐alcoholic steatohepatitis: A multi‐center validation study

Choline is a dietary component that is crucial for normal cellular function. Choline is predominantly absorbed from the small intestine and completely metabolized in the liver. We recently demonstrated that free choline (fCh) levels in blood reflect the level of phosphatidylcholine synthesis in the liver and is correlated with the onset of non-alcoholic steatohepatitis (NASH). Our aim here was to validate the utility of this biomarker for NASH diagnosis. Our cohort consisted of 110 patients with biopsy proven non-alcoholic fatty liver disease (NAFLD) from four centers across Japan and 25 age-matched healthy controls. Plasma fCh levels were measured using high-performance liquid chromatography. Patients with diagnosed or borderline NASH had significantly increased plasma fCh levels when compared with control subjects, or patients not diagnosed with NASH. Interestingly, an association between plasma fCh levels and expression of microsomal triglyceride transfer protein, which catalyzes the transfer of triglyceride, was reflected in the markedly negative correlation between these two variables in patients with NAFLD. Moreover, the grade of liver steatosis and fibrosis stage increased with increasing plasma fCh levels (P < 0.05). The area under the receiver-operating characteristic (ROC) curves for NASH, including borderline diagnosis, was 0.811. Additionally, the areas under the ROC for fibrosis stage were 0.816 for >stage 1, 0.805 for >stage 2, 0.809 for >stage 3 and 0.818 for >stage 4. Plasma fCh levels are closely related to the grade of liver steatosis and fibrosis, and predict NASH severity. Plasma fCh levels are therefore a potential diagnostic marker for early-stage NASH in clinical practice.

Hydrogen Rich Water Prevents Progression of NASH and Accompanying Hepatic Tumorigenesis in Mice

(Background/Aims) Non-alcoholic fatty liver disease (NAFLD) is one of the common causes of chronic liver disease and a major sign of metabolic syndrome. Non-alcoholic steatohepatitis(NASH) is a more severe form of NAFLD and is broadly defined by the presence of steatosis with inflammation and progressive fibrosis, and can ultimately lead to cirrhosis and hepatocellular carcinoma (HCC). The prevalence of NAFLD is gradually increasing. It is known that the presence of reactive oxygen stress (ROS) plays important role for the onset of NASH. Molecular hydrogen has recently been shown to have therapeutic value as an antioxidant through its ability to reduce cytotoxic ROS. Therefore, it may be of potential therapeutic in the treatment of ROS-induced pathologies. However, there is no report that demonstrates the effects of hydrogen for chronic liver disease such as NASH. In this study, we evaluated the effects of hydrogen rich water in the NASH model mouse. (Method) A methionine-choline-deficient (MCD) diet mouse NASH model was introduced. Eight-weekold C57BL/6 mice were divided into 3 experimental groups and fed for 8 weeks as follows : (1) MCD diet + normal water (NW group) (2) MCD diet + hydrogen rich water (HW group) (3) MCD diet mixed with 0.01% of the PPAR-agonist pioglitazone + normal water (PGZ group). Hepatic inflammatory grade and fibrosis stage of each group was compared histologically. Hepatic 8-OHdG concentrationwasmeasured by commercially available ELISA kit. Real time PCR was administered for quantifying the expression of hepatic inflammation genes such as TNF-α and IL-6, and lipogenic genes such as fatty acid synthase (FAS), fatty acid translocase (FAT), and fatty acid transport protein (FATP). Next, a STAM mouse NASH hepatocellular carcinoma model (purchased from Charles River Laboratories Japan Inc.) was introduced. This model mice were divided into 2 experimental groups and fed for 8 weeks as follows: (1) High fat ( HF ) diet + normal water (NW-STAM group) (2) HF diet + hydrogen rich water (HW-STAM group). After 8 weeks, mice were sacrificed and counted for hepatic tumor number. (Results) HW group and PGZ group showed low inflammation and fibrosis compared with NW group histologically. The concentration of 8-OHdG was reduced in HW and PGZ group compared with NW group. The hepatic mRNA expression of TNF-α, IL-6, FAS, FAT, and FATP were also downregulated in HW and PGZ groups. Hepatic tumor number was reduced in HW-STAM group compared with NW-STAM group. (Conclusion) Hydrogen rich water reduces hepatic lipogenesis, oxidative stress and results in improvement of NASH and accompanying hepatic tumors.

Intravenous administration of multipotent stromal cells prevents the onset of non-alcoholic steatohepatitis in obese mice with metabolic syndrome

Metabolic syndrome is secondary to obesity and characterized by dyslipidemia, insulin resistance, and hypertension. Non-alcoholic fatty liver disease is its hepatic manifestation, whose progression-limiting step is non-alcoholic steatohepatitis (NASH). The latter is characterized by lipid accumulation, hepatocyte damage, leukocyte infiltration, and fibrosis. NASH is a prodrome to cirrhosis and hepatocellular carcinoma. Multipotent stromal cells (MSCs) have been shown to be immunomodulatory and contribute to liver regeneration in acute failure conditions. Our aim was to evaluate whether MSC administration prevents the onset of NASH in obese mice with metabolic syndrome. C57BL/6 mice were chronically fed with high-fat diet. At week 33, mice received intravenously either the vehicle (obese untreated) or two doses of 0.5×10(6) syngeneic MSCs (obese MSC-treated). Four months later, liver function and structure, and metabolic syndrome markers were assessed. The persistence of donor MSCs(GFP) in obese mice was evaluated 17 weeks after their administration. Obese untreated mice presented high plasma levels of hepatic enzyme, hepatomegaly, liver fibrosis, inflammatory cell infiltration, and hepatic triglyceride accumulation. Furthermore, they showed high expression levels of fibrosis markers and pro-inflammatory cytokines. By contrast, obese MSC-treated mice only presented steatosis. Mice kept obese, hypercholesterolemic, hyperglycemic, and insulin resistant irrespective of whether they received MSCs or not. Donor MSCs(GFP) were found in liver, bone marrow, heart, and kidney of obese mice. MSC administration prevents the onset of NASH in obese mice. Observed hepatoprotection is not related to a reversion of the metabolic syndrome but to the preclusion of the inflammatory process.

Inhibition of JNK along with activation of ERK1/2 MAPK pathways improve steatohepatitis among the rats

Oxidative stress-induced hepatocyte apoptosis is implicated in the onset of non-alcoholic steatohepatitis (NASH). Regarding the pronounced roles of mitogen activated protein kinases (MAPKs) in oxidative stress-induced cellular damages and the development of NASH, we evaluated the effect of Teucrium polium ethyl acetate (EtoAc) extract on rats with NASH and on the modulation of MAPKs activities. To induce NASH, a methionine choline deficient diet (MCD) was given to N-Mary rats. These animals were then compared to the control group receiving normal diet and/or MCD diet plus EtoAc extract (0.5g/kg/day). After 8 weeks, liver histopathology, malondialdehyde (MDA) and immunoblot analyses of caspase-3 cleavage, ERK/pERK and JNK/pJNK were examined. Simultaneous treatments with MCD diet and the EtoAc extract resulted in pronounced improvements in liver steatosis, ballooning degeneration and inflammation. Elevated MDA and caspase-3 levels among MCD-fed rats were also markedly decreased. In addition, the EtoAc extract treatments caused significant reduction in the phosphorylated form of JNK along with an increase in the phosphorylated level of ERK1/2. These results indicate that the anti-apoptotic effect of T. polium extract is mediated through inhibition of ROS generation. Moreover, the inhibitory effect of T. polium on the development and progression of NASH is apparently governed by the attenuation of JNK activation and augmentation of ERK1/2 activity through phosphorylation.

Principal Component Analysis of Direct Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Data Related to Metabolites of Fatty Liver

Non-alcoholic steatohepatitis (NASH) is a common liver disease. NASH is characterized by fatty liver, along with inflammation. Most people with NASH are not aware of their condition, even though NASH can lead to hepatic cirrhosis. Several approaches have been tested to clarify the pathology of NASH. However, the mechanism of onset of NASH was not well-defined. In this study, a supervised multivariate analysis (principal component analysis) approach using direct matrix-assisted laser desorption/ionization mass spectrometry (dMALDI-MS) was applied to the analysis of metabolites in starvation-induced fatty liver tissue sections. This approach does not require complex pretreatments. We investigated the characteristic dynamics of metabolites in fatty liver. This approach can be applied to the analysis of human biopsy specimens of fatty liver in future studies.