Non-alcoholic Steatohepatitis Pathology Research Articles

Identification cholesterol metabolites altered before the onset of nonalcoholic steatohepatitis by targeted metabolomics

Nonalcoholic steatohepatitis (NASH) is a disease with symptoms similar to those of alcoholic liver inflammation without alcohol intake. As an effective treatment strategy has not been established for this disease, a detailed understanding of the pathological progression mechanism is required. We focused on cholesterol metabolites, which are suspected to regulate NASH pathology, and investigated their relationship with the pathological progression in the early stages of NASH.First, the LC/MS/MS methods for bile acids and sterols were optimized and validated. Next, NASH model mice were established by feeding a choline-deficient, methionine-reduced high-fat diet, and the levels of hepatic cholesterol metabolites were measured. As a result, before the onset of NASH, desmosterol, 4β-hydroxycholesterol, campesterol, sitosterol, secondary bile acids such as taurodeoxycholic acid significantly decreased by up to 1/38 of NASH model group. Autoxidation-generated sterols significantly increased 2- to 5-fold, and various primary bile acids such as conjugated β-muricholic acids and cholic acids significantly increased 2- to 7-fold.In this study, the levels of cholesterol metabolites changed in the before the onset of NASH. These metabolic alterations involved in inflammation induction and detoxification for NASH may help the discovery of early diagnostic biomarkers in the future.

Targeting CCN2 protects against progressive non-alcoholic steatohepatitis in a preclinical model induced by high-fat feeding and type 2 diabetes

Type 2 diabetes is an independent risk factor for non-alcoholic steatohepatitis (NASH) progression and its mediators have not been resolved. In this study, a pathogenic role of cellular communication network factor 2 (CCN2) protein in NASH pathology, was investigated in an established preclinical NASH model. Male wild type C57BL/6 mice received either Chow or high fat diet (HFD) for 26 weeks, with some mice in each group randomly selected to receive low dose streptozotocin (STZ: 3 i.p. injections, 65 mg/kg) at 15 weeks to induce type 2 diabetes. In the final 10 of the 26 weeks mice from each group were administered i.p. either rabbit anti-CCN2 neutralizing antibody (CCN2Ab) or as control normal rabbit IgG, at a dose of 150 µg per mouse twice/week. NASH developed in the HFD plus diabetes (HFD+DM) group. Administration of CCN2Ab significantly downregulated collagen I and collagen III mRNA induction and prevented pro-inflammatory MCP-1 mRNA induction in HFD+DM mice. At the protein level, CCN2Ab significantly attenuated collagen accumulation by PSR stain and collagen I protein induction in HFD+DM. Phosphorylation of the pro-fibrotic ERK signalling pathway in liver in HFD+DM was attenuated by CCN2Ab treatment. Intrahepatic CCN1 mRNA was induced, whereas CCN3 was downregulated at both the mRNA and protein levels in HFD+DM. CCN3 down-regulation was prevented by CCN2Ab treatment. This in vivo study indicates that CCN2 is a molecular target in NASH with high fat diet and diabetes, and that regulation of ERK signalling is implicated in this process.

Hypoxia-Inducible Factors as Key Players in the Pathogenesis of Non-alcoholic Fatty Liver Disease and Non-alcoholic Steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) and its more severe form non-alcoholic steatohepatitis (NASH) are a major public health concern with high and increasing global prevalence, and a significant disease burden owing to its progression to more severe forms of liver disease and the associated risk of cardiovascular disease. Treatment options, however, remain scarce, and a better understanding of the pathological and physiological processes involved could enable the development of new therapeutic strategies. One process implicated in the pathology of NAFLD and NASH is cellular oxygen sensing, coordinated largely by the hypoxia-inducible factor (HIF) family of transcription factors. Activation of HIFs has been demonstrated in patients and mouse models of NAFLD and NASH and studies of activation and inhibition of HIFs using pharmacological and genetic tools point toward important roles for these transcription factors in modulating central aspects of the disease. HIFs appear to act in several cell types in the liver to worsen steatosis, inflammation, and fibrosis, but may nevertheless improve insulin sensitivity. Moreover, in liver and other tissues, HIF activation alters mitochondrial respiratory function and metabolism, having an impact on energetic and redox homeostasis. This article aims to provide an overview of current understanding of the roles of HIFs in NAFLD, highlighting areas where further research is needed.

Accumulation of Toxic Advanced Glycation End-Products Induces Cytotoxicity and Inflammation in Hepatocyte-Like Cells Differentiated from Human Induced Pluripotent Stem Cells.

Nonalcoholic steatohepatitis (NASH), the aggressive form of the most common chronic liver disease nonalcoholic fatty liver disease, is characterized by inflammation and damage in the liver. Although hepatocyte injury and cell death have been identified as cardinal pathological features of NASH, its pathogenesis has not yet been elucidated in detail. Immortalized cell lines and primary cultured cells have been used as in vitro models of NASH. However, these cells have several disadvantages, such as specialized characteristics by immortalization or limited growth potential. To overcome these difficulties and develop a strategy to analyze the pathology of NASH, we employed hepatocyte-like cells differentiated from human induced pluripotent stem cells (hiPSC-HLCs) as an in vitro model of NASH to clarify the intracellular effects of glyceraldehyde-derived advanced glycation end-products (AGEs), also named toxic AGEs (TAGE). The viability of hiPSC-HLCs decreased with the accumulation of TAGE in the cells, which was consistent with previous findings on human hepatocellular carcinoma cells and human primary cultured hepatocytes. In addition, the TAGE accumulation up-regulated the expression of inflammation-related genes (interleukin 6, interleukin 8, and monocyte chemoattractant protein-1) in hiPSC-HLCs. These results indicated that the accumulation of TAGE induced hiPSC-HLC cytotoxicity and inflammation, which are features of the pathology of NASH. Therefore, we suggest the use of hiPSC-HLCs as an important strategy for analyses of the pathology of NASH.

Nonobese mice with nonalcoholic steatohepatitis fed on a choline-deficient, l-amino acid-defined, high-fat diet exhibit alterations in signaling pathways.

Nonalcoholic steatohepatitis (NASH) is often associated with obesity, but some patients develop NASH without obesity. The physiological processes by which nonobese patients develop NASH and cirrhosis have not yet been determined. Here, we analyzed the effects of dietary methionine content on NASH induced in mice fed on a choline‐deficient, methionine‐lowered, l‐amino acid‐defined high‐fat diet (CDAHFD). CDAHFD with insufficient methionine induced insulin sensitivity and enhanced NASH pathology, but without obesity. In contrast, CDAHFD with sufficient methionine induced steatosis, and unlike CDAHFD with insufficient methionine, also induced obesity and insulin resistance. Gene profile analysis revealed that the disease severity in CDAHFD may partially be due to upregulation of the Rho family GTPases pathway and mitochondrial and nuclear receptor signal dysfunction. The signaling factors/pathways detected in this study may assist in future study of NASH regulation, especially its ‘nonobese’ subtype.

Genistein and exercise treatment reduced NASH related HDAC3, IL-13 and MMP-12 expressions in ovariectomized rats fed with high fat high fructose diet

Background and aimGenistein (GEN) and exercise (Ex) may be regarded as an alternative treatment for non-alcoholic steatohepatitis (NASH). However, the mechanisms behind their therapeutic effects in NASH are not well-understood. Experimental procedureThis study investigated the roles of histone deacetylase (HDAC)3 and interleukin-(IL-)13 in the NASH model of ovariectomized (OVX) rats fed with high fat high fructose (HFHF) diet. Results and conclusionNine weeks after being fed with HFHF diet, severe NASH pathology with mild fibrosis were seen along with an increase in HDAC3, IL-13 and matrix metalloelastase (MMP-12) expressions in OVX rats. Five weeks of either GEN or Ex treatments abrogated the increase in both HDAC3 and IL-13 expressions in OVX rats fed with HFHF diet and ameliorated NASH features, liver fibrosis and MMP-12 expression. The combination of Gen and Ex, however, did not provide additional benefits on NASH features in OVX rats fed with HFHF diet. These results suggested that GEN and Ex treatments improved HFHF diet induced NASH in OVX rats through the suppression of HDAC3, IL-13 and MMP-12 expression.

Development of liver organoids from NASH model mice and their application to drug discovery

Nonalcoholic steatohepatitis (NASH) is one of the most common causes of chronic liver disease, with the increased prevalence of obesity, type 2 diabetes, and metabolic disorders in recent years. As the disease progresses, it leads to hepatic fibrosis, which may progress to hepatocellular carcinoma, but there is still no cure for severe hepatic fibrosis. Currently, in order to develop drugs for the treatment of NASH, the effects of candidate drugs are evaluated by a long-term administration to mice and rats that are fed a high-fat or methionine-deficient diet to reproduce the pathology of fatty liver and liver fibrosis. Since drug development using these experimental animals is time-consuming and costly, in vitro models that reproduce the pathology of NASH have recently been developing. In this review, we will outline the current issues in the diagnosis and treatment of NASH, and introduce our research for the discovery of early diagnostic markers and the development of new therapeutic agents using liver organoid cultures derived from mouse models of NASH.

Vesicular ATP release from hepatocytes plays a role in the progression of nonalcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is becoming a growing public health problem along with the increase of metabolic syndrome worldwide. Extracellular nucleotides are known to serve as a danger signal by initiating purinergic signaling in many inflammatory disorders, although the role of purinergic signaling in the progression of NASH remains to be clarified. Vesicular nucleotide transporter (VNUT) is a key molecule responsible for vesicular ATP release to initiate purinergic signaling. Here, we studied the role of VNUT in the progression of nonalcoholic steatohepatitis. VNUT was expressed in mouse hepatocytes and associated, at least in part, with apolipoprotein B (apoB)-containing vesicles. High glucose stimulation evoked release of appreciable amount of ATP from hepatocytes, which disappeared in hepatocytes of Vnut knockout (Vnut−/−) mice. Glucose treatment also stimulated triglyceride secretion from hepatocytes, which was inhibited by PPADS and MRS211, antagonists of P2Y receptors, and clodronate, a VNUT inhibitor, and was significantly reduced in Vnut−/− mice. In vivo, postprandial secretion of triglyceride from hepatocytes was observed, while the serum triglyceride level was significantly reduced in Vnut−/− mice. On a high-fat diet, the liver of wild type mice exhibited severe inflammation, fibrosis, and macrophage infiltration, which is similar to NASH in humans, while this NASH pathology was not observed in Vnut−/− mice. These results suggest that VNUT-mediated vesicular ATP release regulates triglyceride secretion and involves in chronic inflammation in hepatocytes. Since blockade of vesicular ATP release protects against progression of steatohepatitis, VNUT may be a pharmacological target for NASH.

The Nuclear Envelope in Lipid Metabolism and Pathogenesis of NAFLD.

Simple SummaryThe liver is a major organ regulating lipid metabolism and a proper liver function is essential to health. Nonalcoholic fatty liver disease (NAFLD) is a condition with abnormal fat accumulation in the liver without heavy alcohol use. NAFLD is becoming one of the most common liver diseases with the increase in obesity in many parts of the world. There is no approved cure for the disease and a better understanding of disease mechanism is needed for effective prevention and treatment. The nuclear envelope, a membranous structure that surrounds the cell nucleus, is connected to the endoplasmic reticulum where the vast majority of cellular lipids are synthesized. Growing evidence indicates that components in the nuclear envelope are involved in cellular lipid metabolism. We review published studies with various cell and animal models, indicating the essential roles of nuclear envelope proteins in lipid metabolism. We also discuss how defects in these proteins affect cellular lipid metabolism and possibly contribute to the pathogenesis of NAFLD.Nonalcoholic fatty liver disease (NAFLD) is a burgeoning public health problem worldwide. Despite its tremendous significance for public health, we lack a comprehensive understanding of the pathogenic mechanisms of NAFLD and its more advanced stage, nonalcoholic steatohepatitis (NASH). Identification of novel pathways or cellular mechanisms that regulate liver lipid metabolism has profound implications for the understanding of the pathology of NAFLD and NASH. The nuclear envelope is topologically connected to the ER, where protein synthesis and lipid synthesis occurs. Emerging evidence points toward that the nuclear lamins and nuclear membrane-associated proteins are involved in lipid metabolism and homeostasis. We review published reports that link these nuclear envelope proteins to lipid metabolism. In particular, we focus on the recent work demonstrating the essential roles for the nuclear envelope-localized torsinA/lamina-associated polypeptide (LAP1) complex in hepatic steatosis, lipid secretion, and NASH development. We also discuss plausible pathogenic mechanisms by which the loss of either protein in hepatocytes leads to hepatic dyslipidemia and NASH development.

Effects of surface-deacetylated chitin nanofibers on non-alcoholic steatohepatitis model rats and their gut microbiota

Nonalcoholic steatohepatitis (NASH), a more advanced form of nonalcoholic fatty liver disease (NAFLD), is associated with increased cardiovascular and liver-related mortality. Stroke-prone spontaneously hypertensive rats (SHRSP5/Dmcr) that are fed a high-fat and high-cholesterol diet develop hepatic lesions that are similar to those observed in human NASH pathology. We investigated the hepatic protective and antioxidant effects of surface-deacetylated chitin nanofibers (SDACNFs) that were administered to SHRSP5/Dmcr rats for 8 weeks. The administration of SDACNFs (80 mg/kg/day) resulted in a significant decrease in hepatic injury, oxidative stress, compared with the non-treatment. The SDACNFs also caused a reduction in the population of harmful members of the Morganella and Prevotella genus, and increased the abundance of the Blautia genus, a useful bacterium in gut microbiota. We therefore conclude that SDACNF exerts anti-hepatic and antioxidative effects not only by adsorbing lipid substances but also by reforming the community of intestinal microflora in the intestinal tract.

Exogenous Liposomal Ceramide-C6 Ameliorates Lipidomic Profile, Energy Homeostasis, and Anti-Oxidant Systems in NASH.

In non-alcoholic steatohepatitis (NASH), many lines of investigation have reported a dysregulation in lipid homeostasis, leading to intrahepatic lipid accumulation. Recently, the role of dysfunctional sphingolipid metabolism has also been proposed. Human and animal models of NASH have been associated with elevated levels of long chain ceramides and pro-apoptotic sphingolipid metabolites, implicated in regulating fatty acid oxidation and inflammation. Importantly, inhibition of de novo ceramide biosynthesis or knock-down of ceramide synthases reverse some of the pathology of NASH. In contrast, cell permeable, short chain ceramides have shown anti-inflammatory actions in multiple models of inflammatory disease. Here, we investigated non-apoptotic doses of a liposome containing short chain C6-Ceramide (Lip-C6) administered to human hepatic stellate cells (hHSC), a key effector of hepatic fibrogenesis, and an animal model characterized by inflammation and elevated liver fat content. On the basis of the results from unbiased liver transcriptomic studies from non-alcoholic fatty liver disease patients, we chose to focus on adenosine monophosphate activated kinase (AMPK) and nuclear factor-erythroid 2-related factor (Nrf2) signaling pathways, which showed an abnormal profile. Lip-C6 administration inhibited hHSC proliferation while improving anti-oxidant protection and energy homeostasis, as indicated by upregulation of Nrf2, activation of AMPK and an increase in ATP. To confirm these in vitro data, we investigated the effect of a single tail-vein injection of Lip-C6 in the methionine-choline deficient (MCD) diet mouse model. Lip-C6, but not control liposomes, upregulated phospho-AMPK, without inducing liver toxicity, apoptosis, or exacerbating inflammatory signaling pathways. Alluding to mechanism, mass spectrometry lipidomics showed that Lip-C6-treatment reversed the imbalance in hepatic phosphatidylcholines and diacylglycerides species induced by the MCD-fed diet. These results reveal that short-term Lip-C6 administration reverses energy/metabolic depletion and increases protective anti-oxidant signaling pathways, possibly by restoring homeostatic lipid function in a model of liver inflammation with fat accumulation.

Sweroside Prevents Non-Alcoholic Steatohepatitis by Suppressing Activation of the NLRP3 Inflammasome.

Non-alcoholic steatohepatitis (NASH), a type of non-alcoholic fatty liver disease, is characterized as steatosis and inflammation in the liver. NLRP3 inflammasome activation is associated with NASH pathology. We hypothesized that suppressing the NLRP3 inflammasome could be effective in preventing NASH. We searched substances that could inhibit the activation of the NLRP3 inflammasome and identified sweroside as an NLRP3 inhibitor. We investigated whether sweroside can be applied to prevent the pathological symptoms associated with NASH in a methionine–choline-deficient (MCD) diet-induced NASH mouse model. The activation of the NLRP3 inflammasome was determined by detecting the production of caspase-1 and IL-1β from pro-caspase-1 and pro-IL-1β in primary mouse macrophages and mouse liver. In a NASH model, mice were fed an MCD diet for two weeks with daily intraperitoneal injections of sweroside. Sweroside effectively inhibited NLRP3 inflammasome activation in primary macrophages as shown by a decrease in IL-1β and caspase-1 production. In a MCD diet-induced NASH mouse model, intraperitoneal injection of sweroside significantly reduced serum aspartate transaminase and alanine transaminase levels, hepatic immune cell infiltration, hepatic triglyceride accumulation, and liver fibrosis. The improvement of NASH symptoms by sweroside was accompanied with its inhibitory effects on the hepatic NLRP3 inflammasome as hepatic IL-1β and caspase-1 were decreased. Furthermore, sweroside blocked de novo synthesis of mitochondrial DNA in the liver, contributing to suppression of the NLRP3 inflammasome. These results suggest that targeting the NLRP3 inflammasome with sweroside could be beneficially employed to improve NASH symptoms.

Flow cytometric quantification of neutral lipids in a human skin stem cell-derived model of NASH

Non-alcoholic steatohepatitis (NASH) is a severe chronic liver disease that affects 3 to 5 percent of the world population. It is characterized by hepatic lipid accumulation and inflammation and can progress towards fibrosis, cirrhosis and hepatocellular carcinoma. Until today, no drug has been approved for the treatment of NASH. This delay relates to the complex pathogenesis of NASH and also to a lack of appropriate predictive preclinical testing systems. Furthermore, the human specificity of the NASH pathology hampers a fortiori clinical translation of animal studies.Therefore, we recently employed human skin-derived precursors (hSKP) differentiated to hepatocyte-like cells (hSKP-HPC) as a human-relevant cell source for modelling NASH in vitro. Using this in vitro NASH model, it was possible to test novel drugs being developed for anti-NASH therapy, such as elafibranor. Since steatosis is an important aspect of NASH and multiple drugs are being developed to decelerate and reduce lipid accumulation in the liver, we optimized a flow cytometric method for quantifying neutral lipids in ‘NASH’-triggered hSKP-HPC. This methodology enables efficient identification of anti-steatotic properties of new medicines.• NASH-triggered hSKP-HPC robustly accumulate lipids intracellularly.• Flow cytometric quantification of neutral lipids in NASH-triggered hSKP-HPC allows for accurate determination of the steatotic response.• This method enables efficient identification of potential anti-steatotic drugs in a human-specific model

Imaging Fibrogenesis in a Diet-Induced Model of Nonalcoholic Steatohepatitis (NASH).

Purpose Liver fibrosis is the hallmark of chronic nonalcoholic steatohepatitis (NASH) and is characterised by the excessive deposition of extracellular matrix proteins. Early detection and accurate staging of liver fibrosis is critically important for patient management. One of the earliest pathological markers in NASH is the activation of hepatic stellate cells (HSCs) which may be exploited as a marker of fibrogenesis. Activated HSCs secreting factors such as integrin αvβ3 propagate fibrosis. The purpose of the current study was to assess the utility of the integrin αvβ3 imaging agent [18F]FtRGD for the early detection of fibrosis in a diet-induced model of NASH longitudinally using PET imaging. Procedures Mice were fed with either standard chow diet (SD), high-fat diet (HFD), or a choline-deficient, L-amino acid-defined high-fat fibrogenic diet (CDAHFD) to mimic the clinical pathology of liver disease and followed longitudinally for 10 weeks to assess the development of liver fibrosis using [18F]FtRGD positron emission tomography (PET) imaging. Standard blood biochemistry, histological measures, and qPCR were used to quantify integrin αvβ3, smooth muscle actin, and collagen types 1 and 6 to assess the extent of NASH pathology and accurately stage liver fibrosis. Results The CDAHFD fibrogenic diet predictably developed hepatic inflammation and steatosis over the 10 weeks studied with little NASH pathology detected in high fat diet-treated animals. Stage 1 fibrosis was detected early by histology at day 21 and progressed to stage 2 by day 35 and stage 3 by day 56 in mice fed with CDAHFD diet only. Noninvasive imaging with [18F]FtRGD correlated well with integrin αvβ3 and was able to distinguish early mild stage 2 fibrosis in CDAHFD animals compared with standard chow diet-fed animals at day 35. When compared with high fat diet-fed animals, [18F]FtRGD was only able to distinguish later moderate stage 2 fibrosis in CDAHFD animals at day 49. Conclusions The diet-induced progression of liver fibrosis was confirmed using histology and correlated well with the mRNA of integrin αvβ3 and extracellular matrix protein expression. [18F]FtRGD showed very good correlation between liver uptake and integrin αvβ3 expression and similar detection sensitivity to the current clinical gold standard modalities for staging of liver fibrosis.

Role of Angiogenesis and Chronic Inflammation in Fat Hypertrophy in NASH Pathology

Tissue expansion and chronic inflammation in adipose tissue (AT) are closely related to nonalcoholic steatohepatitis (NASH) pathology. Angiogenesis is initiated by the detachment of pericytes (PCs) from vessels in AT. This process is necessary for the development of AT in obesity. The detachment is caused by excessive platelet-derived growth factor B (PDGF-B) derived from M1-macrophages (Mφ) infiltrating obese AT. On the other hand, AT of tamoxifen-induced systemic PDGF receptor-β knockout mice showed decreased detachment of PCs from vessels in obesity, thereby attenuating hypertrophy of AT mediated by neoangiogenesis, resulting in protection from the development of chronic AT inflammation and systemic insulin resistance. The selective mineralocorticoid receptor (MR) inhibitor eplerenone (Ep) suppresses chronic inflammation in fat and the liver, improves glucose and lipid metabolism, and inhibits body weight and fat mass gain in mice fed a high-fat diet. As a novel mechanism, Ep increases energy expenditure and suppresses fat accumulation, thereby controlling the polarity of visceral AT Mφ from inflammatory M1 to anti-inflammatory M2 dominant. In addition, Ep directly inhibits the activation of signals 1 and 2 of NLRP3-inflammasomes in Mφ, which is an inflammatory mechanism closely involved in the development of NASH. Thus, we propose novel therapeutic approaches to NASH. Inhibition of PDGF receptor-β signaling prevents AT hypertrophy by regulating AT angiogenesis, and MR inhibitors directly suppress chronic inflammation in the AT and liver.

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.

Hepatoprotection by Green Tea Extract Along the Gut-liver Axis in Mice with Nonalcoholic Steatohepatitis Is Mediated by Epigallocatechin Gallate but Not Catechin (OR34-03-19)

ObjectivesGreen tea extract (GTE) alleviates nonalcoholic steatohepatitis (NASH) through a mechanism involving the gut-liver axis by limiting endotoxin-TLR4-NFκB inflammation, but the independent bioactivities of specific green tea catechins have not been defined. We hypothesized that epigallocatechin gallate (EGCG) and catechin (CAT) would protect against NASH, but to a lesser extent than GTE. MethodsMale, 6-wk old C57BL6/J mice were provided a diet formulated to be low-fat (LF), high-fat (HF), or HF containing GTE (2%), EGCG (0.3%), or CAT (0.3%) for 8-wk. Then, NASH, endotoxin-TLR4-NFκB inflammation, and gut health were assessed. EGCG was supplemented equivalent to that in GTE, whereas CAT was matched to EGCG to evaluate their relative bioactivities. ResultsHF-feeding induced NASH in association with increased adiposity, serum endotoxin and hepatic TLR4/NFκB inflammation. HF-fed mice also had lower expression of ileal and colonic hypoxia inducible factor-1α (HIF-1α) and tight junction proteins (TJPs) whereas fecal calprotectin and the Firmicutes: Bacteroidetes (F: B) ratio were increased. Relative to HF controls, EGCG and CAT attenuated adiposity, which was further lowered by GTE. NASH (steatosis, ballooning) was attenuated by GTE, EGCG, and CAT, but only GTE and EGCG normalized liver pathology to that of LF controls. Serum endotoxin and hepatic TLR4/NFκB inflammation were similarly attenuated by GTE, EGCG and CAT. Each treatment also similarly maintained mRNA expression of ileal and colonic HIF-1α and TJPs, and attenuated fecal calprotectin and the F: B ratio. ConclusionsEGCG and CAT protected against gut barrier dysfunction and endotoxin-TLR4-NFκB inflammation similar to GTE, whereas EGCG but not CAT lowered NASH pathology similar to GTE. Although EGCG and CAT independently protect against NASH-associated inflammation, GTE bioactivities are likely attributed primarily to EGCG. Funding SourcesUSDA-NIFA and OSU Center for Applied Plant Sciences.

Development of a mouse iron overload-induced liver injury model and evaluation of the beneficial effects of placenta extract on iron metabolism

Hepatic iron deposition is seen in cases of chronic hepatitis and cirrhosis, and is a hallmark of a poorer prognosis. Iron deposition is also found in non-alcoholic steatohepatitis (NASH) patients. We have now developed a mouse model of NASH with hepatic iron deposition by combining a methione- and choline-deficient (MCD) diet with an iron-overload diet. Using this model, we evaluated the effects of human placenta extract (HPE), which has been shown to ameliorate the pathology of NASH. Four-week-old male C57BL/6 mice were fed the MCD diet with 2% iron for 12 weeks. In liver sections, iron deposition was first detected around the portal vein after 1 week. From there it spread throughout the parenchyma. Biliary iron concentrations were continuously elevated throughout the entire 12-week diet. As a compensatory response, the diet caused elevation of serum hepcidin, which accelerates excretion of iron from the body. Accumulation of F4/80-positive macrophages was detected within the sinusoids from the first week onward, and real-time PCR analysis revealed elevated hepatic expression of genes related inflammation and oxidative stress. In the model mice, HPE treatment led to a marked reduction of hepatic iron deposition with a corresponding increase in biliary iron excretion. Macrophage accumulation was much reduced by HPE treatment, as was the serum oxidation-reduction potential, an index of oxidative stress. These data indicate that by suppressing inflammation, oxidative stress and iron deposition, and enhancing iron excretion, HPE effectively ameliorates iron overload-induced liver injury. HPE administration may thus be an effective strategy for treating NASH.

The Relevance of Toxic AGEs (TAGE) Cytotoxicity to NASH Pathogenesis: A Mini-Review.

Non-alcoholic fatty liver disease (NAFLD) is currently the most common feature of chronic liver disease. Non-alcoholic steatohepatitis (NASH) is a severe form of NAFLD, and one of its risk factors is hyperglycemia. The chronic ingestion of excessive amounts of high-fructose corn syrup is associated with an increased prevalence of fatty liver. Under hyperglycemic conditions, advanced glycation end-products (AGEs) are generated through a non-enzymatic glycation reaction between the ketone or aldehyde groups of sugars and amino groups of proteins. Glyceraldehyde (GA) is a metabolic intermediate of sugars, and GA-derived AGEs (known as toxic AGEs (TAGE)) have been implicated in the development of NASH. TAGE accumulates more in serum or liver tissue in NASH patients than in healthy controls or patients with simple steatosis. Furthermore, the TAGE precursor, GA, causes cell damage through protein dysfunctions by TAGE modifications and induces necrotic-type hepatocyte death. Intracellular TAGE may leak outside of necrotic-type cells. Extracellular TAGE then induce inflammatory or fibrotic responses related to the pathology of NASH in surrounding cells, including hepatocytes and hepatic stellate cells. This review focuses on the contribution of TAGE to the pathology of NASH, particularly hepatic cell death related to NASH.

Molecular drivers of non-alcoholic steatohepatitis are sustained in mild-to-late fibrosis progression in a guinea pig model.

Hepatic fibrosis increases mortality in humans with non-alcoholic steatohepatitis (NASH), but it remains unclear how fibrosis stage and progression affect the pathogenic mechanisms of NASH. This study investigates the transcriptional regulation and the impact of fibrosis stage, of pathways relating to hepatic lipid and cholesterol homeostasis, inflammation and fibrosis using RT-qPCR in the guinea pig NASH model. Animals were fed a chow (4% fat), a high-fat (20% fat, 0.35% cholesterol) or high-fat/high-sucrose (20% fat, 15% sucrose, 0.35% cholesterol) diet for 16 or 25 weeks (n = 7/group/time point). High-fat diets induced NASH. In NASH, markers of hepatic de novo lipogenesis were enhanced (e.g. FASN, > twofold, p < 0.05) while markers of mitochondrial, peroxisomal and cytochrome fatty acid oxidation were reduced (e.g. CPT1A > twofold, p < 0.05). Markers of fatty acid uptake were unaltered or decreased. Likewise, expression of cholesterol uptake and synthesis markers were decreased, whereas genes relating to lipid and cholesterol export were unaltered. Inflammatory and chemotactic cytokines were enhanced alongside fibrogenic pathways including increased hepatic stellate cell activation and migration, matrix deposition (e.g. MCP1, TNFα, β-PDGF and Col1a1, > threefold, p < 0.05) and decreased matrix degradation. Fibrosis stage (mild vs. severe) and progression did generally not affect the expression of the investigated pathways. This suggests that liver dysfunction at the transcriptional level is induced early and maintained throughout fibrosis progression, allowing potential treatments to target dysregulated pathways already at early disease stages. As the guinea pig NASH model mimics several aspects of human molecular pathophysiology, these results may be used to increase the current understanding of NASH pathology and explore future treatment targets.