Adenovirus?mediated overexpression of bone morphogenetic protein?9 promotes methionine choline deficiency?induced non?alcoholic steatohepatitis in non?obese mice.
ABSTRACT: Liver inflammation and macrophage infiltration are critical steps in the progression of non?alcoholic fatty liver to the development of non?alcoholic steatohepatitis. Bone morphogenetic protein?9 is a cytokine involved in the regulation of chemokines and lipogenesis. However, the function of bone morphogenetic protein?9 in non?alcoholic steatohepatitis is still unknown. The present study hypothesized that bone morphogenetic protein?9 may contribute to steatohepatitis in mice fed a methionine choline deficiency diet (MCD). C57BL/6 mice overexpressing bone morphogenetic protein?9 and control mice were fed the MCD diet for 4 weeks. Liver tissue and serum samples were obtained for subsequent measurements. Bone morphogenetic protein?9 overexpression exacerbated steatohepatitis in mice on the MCD diet, as indicated by liver histopathology, increased serum alanine aminotransferase activity, aspartate transaminase activity, hepatic inflammatory gene expression and M1 macrophage recruitment. Although bone morphogenetic protein?9 overexpression did not affect the expression of pro?fibrogenic genes, including Collagen I (?)1 or matrix metalloproteinase (MMP) 9, it did upregulate the expression of transforming growth factor?? and plasminogen activator inhibitor 1, and downregulated the expression of MMP2. The above results indicate that bone morphogenetic protein?9 exerts a pro?inflammatory role in MCD diet?induced non?alcoholic steatohepatitis.
Project description:miR-122 is the most abundant miRNA in the liver particularly in hepatocytes where it targets cholesterol metabolism. Steatosis, a key component of non-alcoholic fatty liver disease, is regulated by hypoxia-inducible factor-1? (HIF-1?). Here, we hypothesized that reduced miR-122 has a pathogenic role in steatohepatitis.miR-122 and its target genes were evaluated in mouse livers and/or isolated hepatocytes after methionine-choline-deficient (MCD) or methionine-choline-supplemented (MCS) diet.Liver and hepatocyte miR-122 expression was significantly decreased in steatohepatitis. A maximum reduction in miR-122 occurred at the fibrosis stage (8 weeks of MCD diet). MAP3K3, a miR-122 target gene, was induced at all stages of non-alcoholic steatohepatitis (NASH; 3-8 weeks) only at the mRNA level. Increased NF-?B activation was found in MCD diet-fed mice and MAP3K3 regulated the NF-?B DNA binding in naive hepatocytes. HIF-1? mRNA and DNA binding and expression of the HIF-1? target gene, profibrotic lysyl oxidase, was increased in advanced steatohepatitis (8 weeks). In addition, increase in vimentin and Sirius red staining (liver fibrosis) was found at 8 weeks of MCD diet. Using miR-122 overexpression and inhibition approaches, we confirmed that HIF-1?, vimentin and MAP3K3 are novel miR-122 targets in hepatocytes. We report transcriptional repression of miR-122 in NASH. Decreased liver miR-122 was associated with elevated circulating miR-122 in both exosome-rich and protein-rich serum fractions.Our novel data suggest that decreased liver miR-122 contributes to upregulation of modulators of tissue remodelling (HIF-1?, vimentin and MAP3K3) and might play a role in NASH-induced liver fibrosis.
Project description:Non-alcoholic fatty liver disease (NAFLD) is associated with obesity and type 2 diabetes and is characterized by the accumulation of fat in the liver (steatosis). NAFLD can transition into non-alcoholic steatohepatitis (NASH), with liver cell injury, inflammation, and an increased risk of fibrosis. We previously found that injections of either 1866, a synthetic ligand for the lectin receptor CD209, or DANA, a sialidase inhibitor, can inhibit inflammation and fibrosis in multiple animal models. The methionine and choline-deficient (MCD) diet is a model of NASH which results in the rapid induction of liver steatosis and inflammation. In this report, we show that for C57BL/6 mice on a MCD diet, injections of both 1866 and DANA reversed MCD diet-induced decreases in white fat, decreases in adipocyte size, and white fat inflammation. However, these effects were not observed in type 2 diabetic db/db mice on a MCD diet. In db/db mice on a MCD diet, 1866 decreased liver steatosis, but these effects were not observed in C57BL/6 mice. There was no correlation between the ability of 1866 or DANA to affect steatosis and the effects of these compounds on the density of liver macrophage cells expressing CLEC4F, CD64, F4/80, or Mac2. Together these results indicate that 1866 and DANA modulate adipocyte size and adipose tissue macrophage populations, that 1866 could be useful for modulating steatosis, and that changes in the local density of 4 different liver macrophages cell types do not correlate with effects on liver steatosis.
Project description:The aim of this study was to examine the effect of standardized extract of Salvia miltiorrhiza (SME) on gene and protein expression of non-alcoholic steatohepatitis (NASH)-related factors in activated human hepatic stellate cells (HSC), and in mice with steatohepatitis induced by a methionine-choline deficient (MCD) diet. Male C57BL/6J mice were placed on an MCD or control diet for 8 weeks and SME (0, 0.1, 0.5 and 1 mg/kg body weight) was administered orally every other day for 4 or 6 weeks. HSCs from the LX-2 cell line were treated with transforming growth factor ?-1 (TGF-?1) or TGF-?1 plus SME (0.1-10 ?g/mL). To investigate the effect of SME on reactive oxygen species (ROS)-induced condition, LX-2 cells were treated with hydrogen peroxide (H2O2) or H2O2 plus SME (0.1-100 ?g/mL). MCD administration for 12 weeks increased mRNA expression of tumor necrosis factor (TNF-?), TGF-?1, interleukin-1? (IL-1?), C-reactive protein (CRP), ?-smooth muscle actin (?-SMA), type I collagen, matrix metalloproteinase-2 (MMP-2) and MMP-9. TGF-?1-induced LX-2 cells exhibited similar gene expression patterns. SME treatment significantly reduced the mRNA and protein expression of NASH-related factors in the mouse model and HSCs. Histopathological liver analysis showed improved non-alcoholic fatty liver disease (NAFLD) activity and fibrosis score in SME-treated mice. The in vivo studies showed that SME had a significant effect at low doses. These results suggest that SME might be a potential therapeutic candidate for NAFLD treatment.
Project description:NOD-like receptor (NLR) NLRP3 inflammasome activation has been implicated in the progression of non-alcoholic fatty liver disease (NAFLD) from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH). It has been also shown that palmitic acid (PA) activates NLRP3 inflammasome and promotes interleukin-1? (IL-1?) secretion in Kupffer cells (KCs). However, the specific mechanism of the NLRP3 inflammasome activation is unclear. We studies the molecular mechanisms by investigating the roles of Thioredoxin-interacting protein (TXNIP) and NLRP3 on NAFLD development in patients, high-fat diet (HFD)-induced NAFL and methionine choline deficient (MCD) diet-induced NASH in wild type (WT), TXNIP-/- (thioredoxin-interacting protein) and NLRP3-/- mice, and isolated KCs. We found that the expressions of NLRP3 and TXNIP in human liver tissues were higher in NASH group than in NAFL group. Furthermore, co-immunoprecipitation analyses show that activation of the TXNIP-NLRP3 inflammasome protein complex occurred in KCs of NASH WT mice rather than NAFL WT mice, thus suggesting that the formation and activation of this protein complex is mainly involved in the development of NASH. NLRP3-/- mice exhibited less severe NASH than WT mice in MCD diet model, whereas TXNIP deficiency enhanced NLRP3 inflammasome activation and exacerbated liver injury. PA triggered the activation and co-localization of the NLRP3 inflammasome protein complex in KCs isolated from WT and TXNIP-/- but not NLRP3-/- mice, and most of the complex co-localized with mitochondria of KCs following PA stimulation. Taken together, our novel findings indicate that TXNIP plays a protective and anti-inflammatory role in the development of NAFLD through binding and suppressing NLRP3.
Project description:The mechanisms underlying the progression of simple steatosis to steatohepatitis are yet to be elucidated. To identify the proteins involved in the development of liver tissue inflammation, we performed comparative proteomic analysis of non-alcoholic steatohepatitis (NASH). Mice fed a methionine and choline deficient diet (MCD) developed hepatic steatosis characterized by increased free fatty acid (FFA) and triglyceride levels as well as alpha-SMA. Two-dimensional proteomic analysis revealed that the change from the normal diet to the MCD diet affected the expressions of 50 proteins. The most-pronounced changes were observed in the expression of proteins involved in Met metabolism and oxidative stress, most of which were significantly downregulated in NASH model animals. Peroxiredoxin (Prx) is the most interesting among the modulated proteins identified in this study. In particular, cross-regulated Prx1 and Prx6 are likely to participate in cellular defense against the development of hepatitis. Thus, these Prx isoforms may be a useful new marker for early stage steatohepatitis. Moreover, curcumin treatment results in alleviation of the severity of hepatic inflammation in steatohepatitis. Notably, curcumin administration in MCD-fed mice dramatically reduced CYP2E1 as well as Prx1 expression, while upregulating Prx6 expression. These findings suggest that curcumin may have a protective role against MCD fed-induced oxidative stress.
Project description:Ginsenosides have offered a wide array of beneficial roles in the pharmacological regulation of hepatic metabolic syndromes, including non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), and obesity. Of the numerous ginsenosides, Rg3 has been widely investigated, but there have been few studies of gypenosides (Gyp). Particularly, no study on Gyp LXXV has been reported to date. Here, to firstly explore the pharmacological effects of Gyp LXXV against NASH and the related mechanism, methionine- and choline-deficient (MCD) diet-induced NASH mice and hepatic cells (stellate cells, hepatic macrophages, and hepatocytes) were selected. Gyp LXXV exhibited markedly alleviated MCD diet-induced hepatic injury, inflammation, and fibrosis by down-regulating hepatic fibrosis markers such as ?-smooth muscle actin(?-SMA), collagen1, transforming growth factors-? (TGF-?1), tumor necrosis factor-? (TNF-?), MCP-1, interleukin (IL)-1?, nuclear factor ?B (NF?B), and GRP78. Remarkably, histopathological studies confirmed that 15 mg/kg of Gyp LXXV administration to MCD diet-induced mice led to effective prevention of liver injury, lipid accumulation, and activation of hepatic macrophages, indicating that Gyp LXXV might be a potential anti-NASH drug.
Project description:BACKGROUND & AIMS:NOD-like receptor protein 3 (NLRP3) inflammasome activation occurs in Non-alcoholic fatty liver disease (NAFLD). We used the first small molecule NLRP3 inhibitor, MCC950, to test whether inflammasome blockade alters inflammatory recruitment and liver fibrosis in two murine models of steatohepatitis. METHODS:We fed foz/foz and wild-type mice an atherogenic diet for 16weeks, gavaged MCC950 or vehicle until 24weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6weeks and determined the effects on liver fibrosis. RESULTS:In vehicle-treated foz/foz mice, hepatic expression of NLRP3, pro-IL-1?, active caspase-1 and IL-1? increased at 24weeks, in association with cholesterol crystal formation and NASH pathology; plasma IL-1?, IL-6, MCP-1, ALT/AST all increased. MCC950 treatment normalized hepatic caspase 1 and IL-1? expression, plasma IL-1?, MCP-1 and IL-6, lowered ALT/AST, and reduced the severity of liver inflammation including designation as NASH pathology, and liver fibrosis. In vitro, cholesterol crystals activated Kupffer cells and macrophages to release IL-1?; MCC950 abolished this, and the associated neutrophil migration. MCD diet-fed mice developed fibrotic steatohepatitis; MCC950 suppressed the increase in hepatic caspase 1 and IL-1?, lowered numbers of macrophages and neutrophils in the liver, and improved liver fibrosis. CONCLUSION:MCC950, an NLRP3 selective inhibitor, improved NAFLD pathology and fibrosis in obese diabetic mice. This is potentially attributable to the blockade of cholesterol crystal-mediated NLRP3 activation in myeloid cells. MCC950 reduced liver fibrosis in MCD-fed mice. Targeting NLRP3 is a logical direction in pharmacotherapy of NASH. LAY SUMMARY:Fatty liver disease caused by being overweight with diabetes and a high risk of heart attack, termed non-alcoholic steatohepatitis (NASH), is the most common serious liver disease with no current treatment. There could be several causes of inflammation in NASH, but activation of a protein scaffold within cells termed the inflammasome (NLRP3) has been suggested to play a role. Here we show that cholesterol crystals could be one pathway to activate the inflammasome in NASH. We used a drug called MCC950, which has already been shown to block NLRP3 activation, in an attempt to reduce liver injury in NASH. This drug partly reversed liver inflammation, particularly in obese diabetic mice that most closely resembles the human context of NASH. In addition, such dampening of liver inflammation in NASH achieved with MCC950 partly reversed liver scarring, the process that links NASH to the development of cirrhosis.
Project description:The liver is a major organ that controls hepatic and systemic homeostasis. Dysregulation of liver metabolism may cause liver injury. Previous studies have demonstrated that carboxylesterase 1 (CES1) regulates hepatic triglyceride metabolism and protects against liver steatosis. In the present study, we investigated whether CES1 played a role in the development of alcoholic liver disease (ALD) and methionine and choline-deficient (MCD) diet-induced liver injury. Both hepatocyte nuclear factor 4? (HNF4?) and CES1 were markedly reduced in patients with alcoholic steatohepatitis. Alcohol repressed both HNF4? and CES1 expression in primary hepatocytes. HNF4? regulated CES1 expression by directly binding to the proximal promoter of CES1. Global inactivation of CES1 aggravated alcohol- or MCD diet-induced liver inflammation and liver injury, likely as a result of increased production of acetaldehyde and reactive oxygen species and mitochondrial dysfunctions. Knockdown of hepatic CES1 exacerbated ethanol-induced steatohepatitis. These data indicate that CES1 plays a crucial role in protection against alcohol- or MCD diet-induced liver injury.
Project description:<h4>Background and aims</h4>Non-alcoholic steatohepatitis (NASH), the potentially progressive form of nonalcoholic fatty liver disease (NAFLD), is the pandemic liver disease of our time. Although there are several animal models of NASH, consensus regarding the optimal model is lacking. We aimed to compare features of NASH in the two most widely-used mouse models: methionine-choline deficient (MCD) diet and Western diet.<h4>Methods</h4>Mice were fed standard chow, MCD diet for 8 weeks, or Western diet (45% energy from fat, predominantly saturated fat, with 0.2% cholesterol, plus drinking water supplemented with fructose and glucose) for 16 weeks. Liver pathology and metabolic profile were compared.<h4>Results</h4>The metabolic profile associated with human NASH was better mimicked by Western diet. Although hepatic steatosis (i.e., triglyceride accumulation) was also more severe, liver non-esterified fatty acid content was lower than in the MCD diet group. NASH was also less severe and less reproducible in the Western diet model, as evidenced by less liver cell death/apoptosis, inflammation, ductular reaction, and fibrosis. Various mechanisms implicated in human NASH pathogenesis/progression were also less robust in the Western diet model, including oxidative stress, ER stress, autophagy deregulation, and hedgehog pathway activation.<h4>Conclusion</h4>Feeding mice a Western diet models metabolic perturbations that are common in humans with mild NASH, whereas administration of a MCD diet better models the pathobiological mechanisms that cause human NAFLD to progress to advanced NASH.
Project description:The cause of progression to non-alcoholic fatty liver disease (NAFLD) is not fully understood. In the present study, we aimed to investigate how curcumin, a natural phytopolyphenol pigment, ameliorates NAFLD. Initially, we demonstrated that curcumin dramatically suppresses fat accumulation and hepatic injury induced in methionine and choline-deficient (MCD) diet mice. The severity of hepatic inflammation was alleviated by curcumin treatment. To identify the proteins involved in the pathogenesis of NAFLD, we also characterized the hepatic proteome in MCD diet mice. As a result of two-dimensional proteomic analysis, it was confirmed that thirteen proteins including antioxidant protein were differentially expressed in hepatic steatosis. However, the difference in expression was markedly improved by curcumin treatment. Interestingly, eight of the identified proteins are known to undergo O-GlcNAcylation modification. Thus, we further focused on elucidating how the regulation of O-linked ?-N-acetylglucosamine (O-GlcNAc) modification is associated with the progression of hepatic steatosis leading to hepatitis in MCD diet mice. In parallel with lipid accumulation and inflammation, the MCD diet significantly up-regulated hexosamine biosynthetic pathway (HBP) and O-GlcNAc transferase (OGT) via ER stress. Curcumin treatment alleviates the severity of hepatic steatosis by relieving the dependence of O-GlcNAcylation on nuclear factor-?B (NF-?B) in inflammation signaling. Conversely, the expressions of superoxide dismutase 1 (SOD1) and SIRT1 were significantly upregulated by curcumin treatment. In conclusion, curcumin inhibits O-GlcNAcylation pathway, leading to antioxidant responses in non-alcoholic steatohepatitis (NASH) mice. Therefore, curcumin will be a promising therapeutic agent for diseases involving hyper-O-GlcNAcylation, including cancer.