A. Provenzano

Mononuclear cells in liver fibrosis

Fibrosis is a multicellular wound healing process, where myofibroblasts that express extracellular matrix components extensively cross-talk with other cells resident in the liver or recruited from the bloodstream. Macrophages and infiltrating monocytes participate in the development of fibrosis via several mechanisms, including secretion of cytokines and generation of oxidative stress-related products. However, macrophages are also pivotal in the process of fibrosis resolution, where they contribute to matrix degradation. T lymphocytes modulate the fibrogenic process by direct interaction with myofibroblasts and secreting cytokines. In general, Th2 polarized responses promote fibrosis, while Th1 cytokines may be antifibrogenic. NK cells limit the development of fibrosis and favor its resolution, at least in part via killing of fibrogenic cells. The possible role of NKT cells and B cells is emerging in recent studies. Thus, mononuclear cells represent a critical regulatory system during fibrogenesis and may become an appealing target for therapy.

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.

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.

Modulation of Liver Fibrosis by Adipokines

Hepatic fibrosis is an integrated process triggered by chronic liver damage, leading to the accumulation of extracellular matrix. In patients with chronic liver disease, this process is favored by the presence of obesity or overweight, which are also relevant risk factors for the progression of nonalcoholic steatohepatitis. In this paper, we review the available evidence indicating the modulation of the fibrogenic process by adipokines, a group of cytokines secreted primarily by adipose tissue. In particular, we discuss in detail the role of leptin and adiponectin, which favor and limit the fibrogenic process, respectively. The possible involvement of other recently identified adipokines is also briefly outlined.

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.

Hepatic Notch1 deletion predisposes to diabetes and steatosis via glucose-6-phosphatase and perilipin-5 upregulation

Notch signaling pathways have recently been implicated in the pathogenesis of metabolic diseases. However, the role of hepatic Notch signaling in glucose and lipid metabolism remains unclear and needs further investigation as it might be a candidate therapeutic target in metabolic diseases such as nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD). We used hepatocyte-specific Notch1 knockout (KO) mice and liver biopsies from NASH and NAFLD patients to analyze the role of Notch1 in glucose and lipid metabolism. Hepatocyte-specific Notch1 KO mice were fed with a high fat diet (HFD) or a regular diet (RD). We assessed the metabolic phenotype, glucose and insulin tolerance tests, and liver histology. Hepatic mRNA expression was profiled by Affymetrix Mouse Gene arrays and validated by quantitative reverse transcription PCR (qPCR). Akt phosphorylation was visualized by immunoblotting. Gene expression was analyzed in liver biopsies from NASH, NAFLD, and control patients by qPCR. We found that Notch1 KO mice had elevated fasting glucose. Gene expression analysis showed an upregulation of glucose-6-phosphatase, involved in the final step of gluconeogenesis and glucose release from glycogenolysis, and perilipin-5, a regulator of hepatic lipid accumulation. When fed with an HFD KO mice developed overt diabetes and hepatic steatosis. Akt was highly phosphorylated in KO animals and the Foxo1 target gene expression was altered. Accordingly, a reduction in Notch1 and increase in glucose-6-phosphatase and perilipin-5 expression was observed in liver biopsies from NAFLD/NASH compared with controls. Notch1 is a regulator of hepatic glucose and lipid homeostasis. Hepatic impairment of Notch1 expression may be involved in the pathogenesis of human NAFLD/NASH.

Novel Aspects in the Pathogenesis of Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease, characterized by inflammation, hepatocyte injury and fibrogenesis. Overall mortality, and liver-related mortality, are both increased in NASH patients. Considering that nonalcoholic fatty liver disease is the most prevalent hepatic abnormality in the Western world, understanding the mechanisms leading to NASH and its progression to cirrhosis is critical for a better management of these patients. Moreover, a more detailed knowledge of this condition may be helpful to identify those subjects which are more susceptible to develop progressive liver disease. Emerging data indicate that NASH progression results from parallel events originating from the liver as well as from the adipose tissue, and the gastrointestinal tract. In this review we highlight some of the most recent findings reported on the pathogenesis of NASH and its fibrogenic progression to cirrhosis, in an effort to identify possible targets for treatment or biomarkers of disease progression.

Mechanisms of Fibrosis in Steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a frequent cause of cirrhosis and may lead to liver-related mortality. In Western countries, NASH is the most common liver disease and may progress to advanced fibrosis or cirrhosis in a significant portion of cases. Moreover, NASH, even in the absence of cirrhosis, is associated with the development of hepatocellular carcinoma. An increased risk of cardiovascular events and/or diabetes represents another major problem in these patients. In this review, we discuss recent data on the basic mechanisms leading to the development of fibrosis in nonalcoholic steatohepatitis, in particular those which may identify novel approaches to treatment.

1131 THE MYOSTATIN SYSTEM IS EXPRESSED IN THE LIVER AND ITS ACTIVATION MEDIATES PROFIBROGENIC ACTIONS IN HEPATIC STELLATE CELLS (HSC) VIA c-Jun N-TERMINAL KINASE (JNK)

deactivation of HSCs in the absence or presence of donor cells were examined in vivo and by co-culture experiments. Results: Mice received donor cells showed significant regression of fibrosis. The extent of steotosis was also low in the experimental groups of mice. Mice showed decreasing trend of alpha-smooth muscle actin expression in HSCs and collagen I synthesis, confirming decline of HSCs activation and fibrosis. The engrafted cells persisted in the liver till the end of this investigation. A major fraction of them expressed albumin, some of them expressed von Willebrand factor and a few expressed F4/80 antigen. In vitro experiments confirmed that CD45 cells can suppress proliferation and activation of HSCs. Conclusions: Hematopoietic cell therapy improves clinically relevant parameters in experimental mouse model of chronic liver injury. Donor cells appear to extend paracrine effects for suppressing HSCs activity in turn assist regression of scar tissue. Engrafted cells also involve in the regeneration of liver. These results suggest a clinical potential of this therapy to treat chronic liver injury. Acknowledgement: This work was funded by Department of Biotechnology, India under CMM programme.

1305 BERBERINE AMELIORATES HEPATIC INJURY IN MICE ACTING ON THE NALP3 INFLAMMASOME PATHWAY

4. Western blotting using anti-NFuB antibody for inflammatory signaling, anti-Acyl-CoA antibody to estimate the lipid metabolism, and anti-elF2, -Ire1 and -ATF6 antibodies to analyse the endoplasmic reticulum (ER) stress. Results: 1. Ratio of liver weight to body weight and blood ALT level were extremely decreased in MCDD with 0.1% WBPs treated group (Gr.3). 2. Blood albumin, total cholesterol and triglyceride levels increased gradually in proportion from MCDD with 0.2% WBPs treated group (Gr.2) to MCDD with 0.05% WBPs treated group (Gr.4). 3. Degree of steatosis, inflammation, ballooning of hepatocytes, Mallory-Denk hyaline bodies, and fibrosis improved in a WBPsdose dependent manner, and were significantly decreased in Gr.2 compared with MCDD with normal feed treated group (Gr.1). 4. In Western blotting, the expression of NFuB in liver tissues decreased, but that of Acyl-CoA, PERK, elF2 and ATF6 increased in proportion from Gr.1 to Gr.4. Conclusions: WBPs suppress ER stress, inflammation and enhance the suppressive effects of other pathogenetic factors in NASH. These peptides, which can be obtained easily, may be a useful therapy in patients with NASH.