Experimental Steatohepatitis Research Articles

Micro-RNA 21 inhibition of SMAD7 enhances fibrogenesis via leptin-mediated NADPH oxidase in experimental and human nonalcoholic steatohepatitis.

Hepatic fibrosis in nonalcoholic steatohepatitis (NASH) is the common pathophysiological process resulting from chronic liver inflammation and oxidative stress. Although significant research has been carried out on the role of leptin-induced NADPH oxidase in fibrogenesis, the molecular mechanisms that connect the leptin-NADPH oxidase axis in upregulation of transforming growth factor (TGF)-β signaling have been unclear. We aimed to investigate the role of leptin-mediated upregulation of NADPH oxidase and its subsequent induction of micro-RNA 21 (miR21) in fibrogenesis. Human NASH livers and a high-fat (60% kcal) diet-fed chronic mouse model, where hepatotoxin bromodichloromethane was used to induce NASH, were used for this study. To prove the role of the leptin-NADPH oxidase-miR21 axis, mice deficient in genes for leptin, p47phox, and miR21 were used. Results showed that wild-type mice and human livers with NASH had increased oxidative stress, increased p47phox expression, augmented NF-κB activation, and increased miR21 levels. These mice and human livers showed increased TGF-β, SMAD2/3-SMAD4 colocalizations in the nucleus, increased immunoreactivity against Col1α, and α-SMA with a concomitant decrease in protein levels of SMAD7. Mice that were deficient in leptin or p47phox had decreased activated NF-κB and miR21 levels, suggesting the role of leptin and NADPH oxidase in inducing NF-κB-mediated miR21 expression. Further miR21 knockout mice had decreased colocalization events of SMAD2/3-SMAD4 in the nucleus, increased SMAD7 levels, and decreased fibrogenesis. Taken together, the studies show the novel role of leptin-NADPH oxidase induction of miR21 as a key regulator of TGF-β signaling and fibrogenesis in experimental and human NASH.

CXCL10 plays a key role as an inflammatory mediator and a non-invasive biomarker of non-alcoholic steatohepatitis

Perpetuate liver inflammation is crucial in the pathogenesis of non-alcoholic steatohepatitis (NASH). Expression of CXCL10, a pro-inflammatory cytokine, correlates positively with obesity and type 2 diabetes. Whether CXCL10 plays a role in NASH was unknown. We aimed to investigate the functional and clinical impact of CXCL10 in NASH. Cxcl10 gene-deleted (Cxcl10(-/-)) and C57BL/6 wild type (WT) mice were fed a methionine- and choline-deficient (MCD) diet for 4 or 8 weeks. In other experiments, we injected neutralizing anti-CXCL10 mAb into MCD-fed WT mice. Human serum was obtained from 147 patients with biopsy-proven non-alcoholic fatty liver disease and 73 control subjects. WT mice, fed the MCD diet, developed steatohepatitis with higher hepatic CXCL10 expression. Cxcl10(-/-) mice were refractory to MCD-induced steatohepatitis. We further revealed that CXCL10 was associated with the induction of important pro-inflammatory cytokines (TNF-α, IL-1β, and MCP-1) and activation of the NF-κB pathway. CXCL10 was linked to steatosis through upregulation of the lipogenic factors SREBP-1c and LXR, and also to oxidative stress (upregulation of CYP2E1 and C/EBPβ). Blockade of CXCL10 protected against hepatocyte injury in vitro and against steatohepatitis development in mice. We further investigated the clinical impact of CXCL10 and found circulating and hepatic CXCL10 levels were significantly higher in human NASH. Importantly, the circulating CXCL10 level was correlated with the degree of lobular inflammation and was an independent risk factor for NASH patients. We demonstrate for the first time that CXCL10 plays a pivotal role in the pathogenesis of experimental steatohepatitis. CXCL10 maybe a potential non-invasive biomarker for NASH patients.

N‐3 polyunsaturated fatty acids worsen inflammation and fibrosis in experimental nonalcoholic steatohepatitis

n-3 polyunsaturated fatty acids (PUFA) ameliorate fatty liver in experimental models, but their effects on inflammation and fibrosis during steatohepatitis are either controversial or lacking. We compared the effects of supplementation with olive oil (OO) alone or OO and n-3 PUFA on the development and progression of experimental steatohepatitis. Balb/C mice (≥5 mice/group) were fed a methionine- and choline-deficient (MCD) diet or a control diet for 4 or 8weeks. At the same time, mice were supplemented with n-3 PUFA (eicosapentaenoic and docosahexahenoic acid, 25mg together with 75mg OO), or OO alone (100mg), two times a week by intragastric gavage. After 8weeks, mice on MCD/n-3 had higher ALT levels compared to MCD/OO and more severe scores of inflammation, including a significant increase in the number of lipogranulomas (26.4±8.4 vs. 5.1±5 per field, P<0.001). Intrahepatic expression of TNF-α and CCL2 was higher in MCD/n-3 mice at both time points. In addition, increased expression of the profibrogenic genes TIMP-1 and TGF-β, and more severe histological scores of fibrosis were evident in MCD/n-3 mice. After 8week of MCD diet, portal pressure was higher in mice receiving n-3 than in those on OO alone (5.1±1.4 vs. 7.0±0.9mmHg, P<0.05). Analysis of hepatic fatty acid profile showed that supplementation resulted in effective incorporation of n-3 PUFA. In a murine model of steatohepatitis, supplementation with n-3 PUFA and OO is associated with more severe necro-inflammation and fibrosis than in mice treated with OO only.

The inflammasome pathway as a target of berberine in experimental steatohepatitis and acetaminophen toxicity

The inflammasome pathway as a target of berberine in experimental steatohepatitis and acetaminophen toxicity

Polyphenol-rich extract of Nelumbo nucifera leaves inhibits alcohol-induced steatohepatitis via reducing hepatic lipid accumulation and anti-inflammation in C57BL/6J mice

The present study was undertaken to evaluate the hepatoprotective effect mechanisms of Nelumbo nucifera leaves extract (NLE) in experimental alcoholic steatohepatitis animal models. We found that the NLE contained polyphenols (phenolic acids and flavonoids), and more than 70% of the main functional components in NLE could potentially provide benefits for alcoholic liver disease. The parameters of histopathology, immunohistochemistry, antioxidant defense, proinflammatory mediator and lipid synthesis-related proteins demonstrated the inhibitory effect of NLE on alcoholic steatohepatitis. Plasma and hepatic content analysis showed that NLE inhibited lipid accumulation by altering the levels of triglycerides (TG) and cholesterol (TC). Treatment with NLE increased the expression of the p-AMPK/AMPK ratio and PPAR-α. Furthermore, fatty acid oxidation and transport via carnitine palmitoyltransferase-1 (CPT1) and microsomal triglyceride transfer protein (MTP) were through the activation of the AMPK and PPAR-α signal. These results revealed that the polyphenol-rich component of NLE prevents alcoholic steatohepatitis by multiple pathways, including reduced lipid synthesis, enhanced fatty acid oxidation and transport responses, inhibited oxidative stress and facilitated anti-inflammation. Suggesting that NLE might be regarded as a beneficial food that has the potential to be developed as a natural agent for preventing alcoholic steatohepatitis.

Hepatic-specific lipin-1 deficiency exacerbates experimental alcohol-induced steatohepatitis in mice

Lipin-1 regulates lipid metabolism by way of its function as an enzyme in the triglyceride synthesis pathway and as a transcriptional coregulatory protein and is highly up-regulated in alcoholic fatty liver disease. In the present study, using a liver-specific lipin-1-deficient (lipin-1LKO) mouse model, we aimed to investigate the functional role of lipin-1 in the development of alcoholic steatohepatitis and explore the underlying mechanisms. Alcoholic liver injury was achieved by pair feeding wild-type and lipin-1LKO mice with modified Lieber-DeCarli ethanol-containing low-fat diets for 4 weeks. Surprisingly, chronically ethanol-fed lipin-1LKO mice showed markedly greater hepatic triglyceride and cholesterol accumulation, and augmented elevation of serum liver enzymes accompanied by increased hepatic proinflammatory cytokine expression. Our studies further revealed that hepatic removal of lipin-1 in mice augmented ethanol-induced impairment of hepatic fatty acid oxidation and lipoprotein production, likely by way of deactivation of peroxisome proliferator-activated receptor γ coactivator-1 alpha, a prominent transcriptional regulator of lipid metabolism. Liver-specific lipin-1 deficiency in mice exacerbates the development and progression of experimental alcohol-induced steatohepatitis. Pharmacological or nutritional modulation of hepatic lipin-1 may be beneficial for the prevention or treatment of human alcoholic fatty liver disease.

Chemokine Receptor CXCR6-Dependent Hepatic NK T Cell Accumulation Promotes Inflammation and Liver Fibrosis

Chronic liver injury characteristically results in hepatic inflammation, which represents a prerequisite for organ fibrosis. Although NKT cells are abundantly present in liver and involved in hepatic inflammation, molecular mechanisms of their recruitment in liver fibrosis remained elusive. We hypothesized that chemokine receptor CXCR6 and its ligand CXCL16 control NKT cell migration and functionality in liver fibrosis. In patients with chronic liver diseases (n = 58), CXCR6 and CXCL16 expression was intrahepatically upregulated compared with controls. In murine liver, Cxcl16 was strongly expressed by endothelium and macrophages, whereas lymphocyte populations (NKT, NK, CD4 T, CD8 T cells) expressed CXCR6. Intravital two-photon microscopy imaging of Cxcr6(+/gfp) and Cxcr6(gfp/gfp) mice and chemotaxis studies in vitro revealed that CXCR6 specifically controls hepatic NKT cell accumulation during the early response upon experimental liver damage. Hepatic invariant NKT cells expressed distinct proinflammatory cytokines including IFN-γ and IL-4 upon injury. CXCR6-deficient mice were protected from liver fibrosis progression in two independent experimental models. Macrophage infiltration and protein levels of inflammatory cytokines IFN-γ, TNF-α, and IL-4 were also reduced in fibrotic livers of Cxcr6(-/-) mice, corroborating that hepatic NKT cells provide essential cytokine signals perpetuating hepatic inflammation and fibrogenesis. Adoptive transfer of NKT cells, but not CD4 T cells, isolated from wild type livers restored hepatic fibrosis in Cxcr6(-/-) mice upon experimental steatohepatitis. Our results demonstrate that hepatic NKT cells accumulate CXCR6-dependent early upon injury, thereby accentuating the inflammatory response in the liver and promoting hepatic fibrogenesis. Interfering with CXCR6/CXCL16 might therefore bear therapeutic potential in liver fibrosis.

Synergistic interaction of dietary cholesterol and dietary fat in inducing experimental steatohepatitis

The majority of patients with nonalcoholic fatty liver disease (NAFLD) have "simple steatosis," which is defined by hepatic steatosis in the absence of substantial inflammation or fibrosis and is considered to be benign. However, 10%-30% of patients with NAFLD progress to fibrosing nonalcoholic steatohepatitis (NASH), which is characterized by varying degrees of hepatic inflammation and fibrosis, in addition to hepatic steatosis, and can lead to cirrhosis. The cause(s) of progression to fibrosing steatohepatitis are unclear. We aimed to test the relative contributions of dietary fat and dietary cholesterol and their interaction on the development of NASH. We assigned C57BL/6J mice to four diets for 30 weeks: control (4% fat and 0% cholesterol); high cholesterol (HC; 4% fat and 1% cholesterol); high fat (HF; 15% fat and 0% cholesterol); and high fat, high cholesterol (HFHC; 15% fat and 1% cholesterol). The HF and HC diets led to increased hepatic fat deposition with little inflammation and no fibrosis (i.e., simple hepatic steatosis). However, the HFHC diet led to significantly more profound hepatic steatosis, substantial inflammation, and perisinusoidal fibrosis (i.e., steatohepatitis), associated with adipose tissue inflammation and a reduction in plasma adiponectin levels. In addition, the HFHC diet led to other features of human NASH, including hypercholesterolemia and obesity. Hepatic and metabolic effects induced by dietary fat and cholesterol together were more than twice as great as the sum of the separate effects of each dietary component alone, demonstrating significant positive interaction. Dietary fat and dietary cholesterol interact synergistically to induce the metabolic and hepatic features of NASH, whereas neither factor alone is sufficient to cause NASH in mice.

Insulin Resistance, Ceramide Accumulation and Endoplasmic Reticulum Stress in Experimental Chronic Alcohol-Induced Steatohepatitis

Chronic alcohol abuse causes steatohepatitis with insulin resistance, which impairs hepatocellular growth, survival and metabolism. However, growing evidence supports the concept that progressive alcohol-related liver injury may be mediated by concurrent mal-signaling through other networks that promote insulin resistance, e.g. pro-inflammatory, pro-ceramide and endoplasmic reticulum (ER) stress cascades. Using the Long Evans rat model of chronic ethanol feeding, we characterized the histopathologic and ultrastructural features of steatohepatitis in relation to biochemical and molecular indices of tissue injury, inflammation, insulin resistance, dysregulated lipid metabolism and ER stress. Chronic steatohepatitis with early chicken-wire fibrosis was associated with enlargement of mitochondria and disruption of ER structure by electron microscopy, elevated indices of lipid storage, lipid peroxidation and DNA damage, increased activation of pro-inflammatory cytokines, impaired signaling through the insulin receptor (InR), InR substrate-1, Akt, ribosomal protein S6 kinase and proline-rich Akt substrate 40kDa, glycogen synthase kinase 3β activation and constitutive up-regulation of ceramide and ER stress-related genes. Liquid chromatography coupled with tandem mass spectrometry demonstrated altered ceramide profiles with higher levels of C14 and C18, and reduced C16 species in ethanol-exposed livers. The histopathologic and ultrastructural abnormalities in chronic alcohol-related steatohepatitis are associated with persistent hepatic insulin resistance and pro-inflammatory cytokine activation, dysregulated lipid metabolism with altered ceramide profiles and both ER and oxidative stress. Corresponding increases in lipid peroxidation, DNA damage and protein carbonylation may have contributed to the chronicity and progression of disease. The findings herein suggest that multi-pronged therapeutic strategies may be needed for effective treatment of chronic alcoholic liver disease in humans.

Lack of CC chemokine ligand 2 differentially affects inflammation and fibrosis according to the genetic background in a murine model of steatohepatitis

Expression of CCL2 (CC chemokine ligand 2) (or monocyte chemoattractant protein-1) regulates inflammatory cell infiltration in the liver and adipose tissue, favouring steatosis. However, its role in the pathogenesis of steatohepatitis is still uncertain. In the present study, we investigated the development of non-alcoholic steatohepatitis induced by an MCD diet (methionine/choline-deficient diet) in mice lacking the CCL2 gene on two different genetic backgrounds, namely Balb/C and C57/Bl6J. WT (wild-type) and CCL2-KO (knockout) mice were fed on a lipid-enriched MCD diet or a control diet for 8 weeks. In Balb/C mice fed on the MCD diet, a lack of CCL2 was associated with lower ALT (alanine transaminase) levels and reduced infiltration of inflammatory cells, together with a lower generation of oxidative-stress-related products. Sirius Red staining demonstrated pericellular fibrosis in zone 3, and image analysis showed a significantly lower matrix accumulation in CCL2-KO mice. This was associated with reduced hepatic expression of TGF-β (transforming growth factor-β), type I procollagen, TIMP-1 (tissue inhibitor of metalloproteinases-1) and α-smooth muscle actin. In contrast, in mice on a C57Bl/6 background, neither ALT levels nor inflammation or fibrosis were significantly different comparing WT and CCL2-KO animals fed on an MCD diet. In agreement, genes related to fibrogenesis were expressed to comparable levels in the two groups of animals. Comparison of the expression of several genes involved in inflammation and repair demonstrated that IL (interleukin)-4 and the M2 marker MGL-1 (macrophage galactose-type C-type lectin 1) were differentially expressed in Balb/C and C57Bl/6 mice. No significant differences in the degree of steatosis were observed in all groups of mice fed on the MCD diet. We conclude that, in experimental murine steatohepatitis, the effects of CCL2 deficiency are markedly dependent on the genetic background.

1256 DIETARY SUPPLEMENTATION WITH OLIVE OIL WITH OR WITHOUT N-3 PUFA DIFFERENTIALLY AFFECTS INFLAMMATION AND PROGRESSION OF EXPERIMENTAL NONALCOHOLIC STEATOHEPATITIS

Background/aims: While it is established that excessive calorie intake plays a role in the pathogenesis of nonalcoholic steatohepatitis, the significance of individual dietary factors is still elusive. Supplementation with n-3 polyunsaturated fatty acids (PUFA) has been shown to ameliorate fatty liver in experimental models, and clinical studies are underway. In this study we compared the effects of supplementation with olive oil (mostly composed of oleic acid, 18:1 n-9) or olive oil and n-3 PUFA on the progression of experimental steatohepatitis. Methods: Balb/C mice (≥5 mice/group) were fed a methionine and choline deficient (MCD) diet or a control diet for 4 or 8 weeks. At the same time, mice were supplemented with n-3 PUFA (eicosapentaenoic and docosahexahenoic acid, 25mg together with 75mg olive oil), or olive oil alone (OO, 100mg), two times a week by intragastric gavage Results: Aminotransferase levels were not different comparing mice supplemented with n-3 or OO after 4w of MCD diet, while after 8w mice on MCD/n-3 had more severe necroinflammation compared to MCD/OO (ALT: 373±170 vs. 87±29 UI/L, P < 0.05). Liver/body weight ratio was significantly lower in MCD/n-3 mice after 8w of treatment (5.1±0.8 vs. 6.8±1.0%, P< 0.05). At 8w, the score of inflammation and fibrosis was significantly higher in mice receiving MCD/n-3 than in MCD/OO animals, with a marked increase in the number of lipogranulomas (26.4±8.4 vs. 5.1±5. per field, P < 0.001). A significant increase in intrahepatic expression of TNF-alpha and CCL2 was observed at both 4w and 8w in MCD/n-3 mice, which also had increased expression of CD11b at 4 weeks. In addition, increased transcript levels of the profibrogenic genes TIMP-1 and TGF-beta, and lower expression of PPAR-alpha was observed in MCD/n-3 mice. After 8 week of MCD diet, portal pressure was higher in mice receiving n-3 than in those on OO (5.1±1.4 vs. 7.0±0.9mmHg, P < 0.05). No major differences were observed comparing n-3 and OO supplementation together with a control diet. Conclusions: In a model of steatohepatiits, supplementation with n-3 PUFA and OO is associated with more severe necroinflammation and fibrosis than in mice treated with OO only.

F-31 Berberine ameliorates necroinflammation in experimental steatohepatitis, but is associated with extrahepatic toxicity

F-31 Berberine ameliorates necroinflammation in experimental steatohepatitis, but is associated with extrahepatic toxicity

Structural Correlates of PPAR Agonist Rescue of Experimental Chronic Alcohol-Induced Steatohepatitis.

Chronic alcoholic liver disease is associated with hepatic insulin resistance, inflammation, oxidative and ER stress, mitochondrial dysfunction, and DNA damage. Peroxisome-proliferator activated receptor (PPAR) agonists are insulin sensitizers that have anti-inflammatory/anti-oxidant effects. We previously showed that PPAR agonists can restore hepatic insulin responsiveness in chronic ethanol-fed rats with steatohepatitis. Herein, we furthered our investigations by characterizing the histological and ultrastructural changes mediated by PPAR agonist rescue of alcohol-induced steatohepatitis. Adult male Long Evans rats were pair fed with isocaloric liquid diets containing 0% or 37% ethanol (caloric) for 8 weeks. After 3 weeks on the diets, rats were treated with vehicle, or a PPAR-α, PPAR-δ, or PPAR-γ agonist twice weekly by i.p. injection. Ethanol-fed rats developed steatohepatitis with disordered hepatic chord architecture, mega-mitochondria, disruption of the RER, increased apoptosis, and increased 4-hydroxynonenal (HNE) and 3-nitrotyrosine (NTyr) immunoreactivity. PPAR-δ and PPAR-γ agonists reduced the severity of steatohepatitis, and restored the hepatic chord-like architectural, mitochondrial morphology, and RER organization, and the PPAR-δ agonist significantly reduced hepatic HNE. On the other hand, prominent RER tubule dilation, which could reflect ER stress, persisted in ethanol-exposed, PPAR-γ treated but not PPAR-δ treated livers. The PPAR-α agonist exacerbated both steatohepatitis and formation of mega-mitochondria, and it failed to restore RER architecture or lower biochemical indices of oxidative stress. In conclusion, improved hepatic insulin responsiveness and decreased inflammation resulting from PPAR-δ or PPAR-γ agonist treatments of alcohol-induced steatohepatitis are likely mediated by enhanced signaling through metabolic pathways with attendant reductions in ER stress, oxidative stress, and mitochondrial dysfunction.

P87 Vascular Adhesion Protein-1 promotes increases in liver infiltrating CD4+ T cells and NKT cells and induction of fibrogenesis in steatohepatitis

IntroductionVascular Adhesion Protein-1 (VAP-1) is an adhesion molecule and membrane-bound amine oxidase. Our group has previously demonstrated that VAP-1 is able to support lymphocyte recruitment across human hepatic sinusoidal endothelium...

The role of heme oxygenase-1 in non-alcoholic steatohepatitis

To investigate the potential role of heme oxygenase-1 on preventing non-alcoholic steatohepatitis (NASH) in mice. Experimental models of NASH were established by feeding male C57BL/6J mice with choline-methionine deficient diet (MCD) for four weeks. Control animals were fed with choline-methionine supplemented diet. The treatment groups were fed with MCD diet combined with HO-1 inducer hemin or inhibitor zinc protoporphyrin IX (ZnPP-IX). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were tested by enzymic method with automatic biochemistry analyzer. The degree of hepatic steatosis, inflammation and fibrosis were examined under HE staining. The hepatic mRNA and protein expressions of HO-1, TNFalpha and IL-6 were analyzed by RT-PCR and Western blot respectively. MCD fed mice showed increased serum ALT and AST levels and moderate to severe hepatic steatosis with inflammatory infiltration, hepatic spot or focal necrosis, light portal and sinus hepaticus fibrosis in the liver sections, which associated with enhanced expression of HO-1, TNFalpha and IL-6 mRNA and protein (1.13+/-0.11, 1.74+/-0.05; 0.20+/-0.01, 1.92+/-0.10; 0.58+/-0.02, 2.06+/-0.05 vs 0.43+/-0.02, 0.75+/-0.05; 0.08+/-0.00, 0.59+/-0.02; 0.22+/-0.01, 0.91+/-0.02). Administration of hemin significantly decreased serum ALT and AST levels and attenuated hepatic steatosis and necroinflammation which associated with up-regulation of antioxidative gene HO-1 and down-regulation of pro-inflammatory cytokines TNFalpha and IL-6 (P < 0.01). A contrary effect on serum aminotransferase levels and liver histopathology was observed in mice injected with ZnPP-IX (P < 0.01). The effect was associated with suppressed HO-1 expression and increased TNFaLPHA and IL-6 expression. The data provided a biochemical, morphological and molecular biological evidence for the protective role of HO-1 in ameliorating hepatic steatosis, necroinflammation in experimental nutritional steatohepatitis.

Anti-apoptotic and anti-inflammatory effects of Silybum marianum in treatment of experimental steatohepatitis

In this study, we were aimed to evaluate the probable effect of the crud extract of Silybum marianum, with high polyphenolic content, on experimental nonalcoholic steatohepatitis (NASH). To induce NASH, a methionine and choline deficient (MCD) diet was given to N-Mary rats for 8 weeks. After NASH development, MCD-fed rats were divided into two groups: MCD groups received MCD diet and MCD + S group was fed MCD diet plus crude extract of S. marianum orally for 3 weeks. Control group was fed a normal diet for 11 weeks. Finally, all rats were sacrificed. Plasma alanine amino transferase (ALT) and aspartate amino transferase (AST) levels were evaluated. In addition, the following hepatic factors were also evaluated: liver histology, malondialdehyde (MDA) and reduced glutathione (GSH) contents, gene expressions of TNF-α and TGF-β and immunoblot evaluations of caspase-3, ERK/p-ERK, JNK/pJNK and p38/pp38. Histopathological evaluations of the liver samples revealed that treatment with the S. marianum extract has abated the severity of NASH among the MCD-fed rats. Also, a significant reduction was observed in the sera ALT and AST activities. In addition, the extract caused dramatic reduction in the elevated hepatic TNF-α and TGF-β mRNA and MDA levels along with an increase in the GSH content. Moreover, the plant extract treatments significantly lowered activation of procaspase-3 to active caspase-3 and also lowered the phosphorylated form of JNK among the same group of rats. These results suggest that the S. marianum crude extract beneficial effects on NASH are mainly due to its antioxidant and anti-inflammatory activities.

Curcumin limits the fibrogenic evolution of experimental steatohepatitis

Nonalcoholic steatohepatitis is characterized by the association of steatosis with hepatic cell injury, lobular inflammation and fibrosis. Curcumin is known for its antioxidant, anti-inflammatory and antifibrotic properties. The aim of this study was to test whether the administration of curcumin limits fibrogenic evolution in a murine model of nonalcoholic steatohepatitis. Male C57BL/6 mice were divided into four groups and fed a diet deficient in methionine and choline (MCD) or the same diet supplemented with methionine and choline for as long as 10 weeks. Curcumin (25 μg per mouse) or its vehicle (DMSO) was administered intraperitoneally every other day. Fibrosis was assessed by Sirius red staining and histomorphometry. Intrahepatic gene expression was measured by quantitative PCR. Hepatic oxidative stress was evaluated by staining for 8-OH deoxyguanosine. Myofibroblastic hepatic stellate cells (HSCs) were isolated from normal human liver tissue. The increase in serum ALT caused by the MCD diet was significantly reduced by curcumin after 4 weeks. Administration of the MCD diet was associated with histological steatosis and necro-inflammation, and this latter was significantly reduced in mice receiving curcumin. Curcumin also inhibited the generation of hepatic oxidative stress. Fibrosis was evident after 8 or 10 weeks of MCD diet and was also significantly reduced by curcumin. Curcumin decreased the intrahepatic gene expression of monocyte chemoattractant protein-1, CD11b, procollagen type I and tissue inhibitor of metalloprotease (TIMP)-1, together with protein levels of α-smooth muscle-actin, a marker of fibrogenic cells. In addition, curcumin reduced the generation of reactive oxygen species in cultured HSCs and inhibited the secretion of TIMP-1 both in basal conditions and after the induction of oxidative stress. In conclusion, curcumin administration effectively limits the development and progression of fibrosis in mice with experimental steatohepatitis, and reduces TIMP-1 secretion and oxidative stress in cultured stellate cells.

Antioxidants vitamin E and 1-aminobenzotriazole prevent experimental non-alcoholic steatohepatitis in mice

Objective. Hepatic oxidative stress plays a key role in the development of non-alcoholic steatohepatitis (NASH). However, the protective effects of antioxidants on NASH are largely unknown. The aim of this study was to elucidate the effect and mechanism of antioxidants on NASH in mice. Material and methods. C57BL6/J mice were fed a methionine-choline-deficient (MCD) diet for 10 days or 3 weeks to induce steatohepatitis. Antioxidants (vitamin E, ABT, or vitamin E plus ABT) were supplemented in mice fed a MCD diet, respectively. The effect of antioxidants on oxidative stress and apoptosis was assessed, and activation of adiponectin and expressions of inflammatory factors, apoptosis-related genes, and fibrosis-related genes were assayed. Results. MCD feeding in mice showed increasing serum alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) levels, and progressive hepatic injury including hepatic steatosis and inflammatory infiltration. Administration of antioxidants vitamin E and/or ABT significantly lowered serum ALAT and ASAT levels (p<0.001) and ameliorated hepatic steatosis and necroinflammation. These effects were associated with repressed hepatic lipid peroxides through reducing hepatic MDA content and enhancing hepatic superoxide dismutase (SOD) activity; down-regulated inflammatory factor COX-2, lowered activity of NF-κB, up-regulated anti-apoptotic gene Bcl-2, and down-regulated pro-apoptotic gene Bax suppressed expression of the fibrotic genes TGF-β1 and MMP2. Moreover, expression of the anti-inflammatory factor adiponectin was also induced by vitamin E or ABT. A combination of vitamin E and ABT showed an additive effect on preventing liver injury. Conclusions. The present study provides morphological and molecular biological evidence for the protective role of the antioxidant vitamins E and ABT in ameliorating oxidative stress, hepatic apoptosis, and necroinflammation in experimental nutritional steatohepatitis.

The Treatment with Antibody of TNF-α Reduces the Inflammation, Necrosis and Fibrosis in the Non-alcoholic Steatohepatitis Induced by Methionine- and Choline-deficient Diet

To assess the effects of anti-TNF-alpha antibody (infliximab) in experimental steatohepatitis induced by methionine- and choline-deficient (MCD) diet. The study included thirty rats. One group received normal rat food, and two groups received MCD diet. The treatment group received a single dose intra-peritoneal infliximab (4 mg/kg), at week 8. MCD diet increased levels of AST, ALT, TNF-alpha, TGF-beta(1), tissue and plasma MDA (p < 0.05 for each). Moreover, it led to steatosis, ballooning degeneration, inflammation, fibrosis and increased actin expression, histopathologically (p < 0.05 for each). In this experimental steatohepatitis anti-TNF-alpha antibody decreased the levels of AST, ALT, TGF-beta(1) and plasma and tissue MDA (p < 0.05 for each). Moreover, inflammation, necrosis, actin expression and fibrosis decreased in anti-TNF-alpha group compared to placebo group (p < 0.05 for each). This study indicates that anti-TNF-alpha antibody is effective on necrosis, inflammation and fibrosis in the experimental model of non-alcoholic steatohepatitis, induced by MCD diet.

Nuclear transcription factors and lipid homeostasis in liver

Nuclear transcription factors and lipid homeostasis in liver