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Background & aims Myostatin is mainly expressed in skeletal muscle, where it negatively regulates trophism. This myokine is implicated in the pathophysiology of nonalcoholic steatohepatitis, an emerging cause of liver fibrosis. In this study we explored the effects of myostatin on the biology of hepatic stellate cells. Methods The effects of myostatin were assessed both in LX-2 and in human primary stellate cells. Cell migration was determined in Boyden chambers. Activation of intracellular pathways was evaluated by Western blotting. Procollagen type 1 secretion was measured by enzyme immunoassay. The role of c-Jun N-terminal kinase was assessed by pharmacologic and genetic inhibition. Results Activin receptor-2B was up-regulated in livers of mice with experimental fibrosis, and detectable in human stellate cells. Serum myostatin levels increased in a model of acute liver injury. Myostatin reduced HSC proliferation, induced cell migration, and increased expression of procollagen type1, tissue inhibitor of metalloproteinase-1, and transforming growth factor-β1. Myostatin activated different signaling pathways, including c-Jun N-terminal kinase and Smad3. Genetic and/or pharmacologic inhibition of c-Jun N-terminal kinase activity significantly reduced cell migration and procollagen secretion in response to myostatin. Conclusions Activation of activin receptor-2B by myostatin modulates the fibrogenic phenotype of human stellate cells, indicating that a myokine may be implicated in the pathogenesis of hepatic fibrosis.

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 microg 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 alpha-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.

Hepatic stellate cells (HSC) coordinate the liver wound-healing response through secretion of several cytokines and chemokines, including CCL2 (formerly known as monocyte chemoattractant protein-1). In this study, we evaluated the role of different proteins of the MAPK family (ERK, p38MAPK, and JNK) in the regulation of CCL2 expression by HSC, as an index of their proinflammatory activity. Several mediators activated all three MAPK, including TNF, IL-1, and PDGF. To assess the relative role of the different MAPKs, specific pharmacological inhibitors were used; namely, SB203580 (p38MAPK), SP600125 (JNK), and PD98059 (MEK/ERK). The efficacy and specificity of the different inhibitors in our cellular system were verified analyzing the enzymatic activity of the different MAPKs using in vitro kinase assays and/or testing the inhibition of phosphorylation of downstream substrates. SB203580 and SP600125 dose-dependently inhibited CCL2 secretion and gene expression induced by IL-1 or TNF. In contrast, inhibition of ERK did not affect the upregulation of CCL2 induced by the two cytokines. Finally, activin A was also found to stimulate CCL2 expression and to activate ERK, JNK, p38, and their downstream targets. Unlike in cells exposed to proinflammatory cytokines, all three MAPKs were required to induce CCL2 secretion in response to activin. We conclude that members of the MAPK family differentially regulate cytokine-induced chemokine expression in human HSC.

Background & Aims 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. 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, 25 mg together with 75 mg OO), or OO alone (100 mg), two times a week by intragastric gavage. Results After 8 weeks, 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

Thrombopoietin (TPO), a cytokine that participates in the differentiation and maturation of megakaryocytes, is produced in the liver, but only limited information is available on the biological response of liver-derived cells to TPO. In this study, we investigated whether HepG2 cells express c-Mpl, the receptor for TPO, and whether TPO elicits biological responses and intracellular signaling in this cell type. Specific transcripts for c-Mpl were detected in HepG2 cells by RT-PCR, and expression of the protein was demonstrated by Western blot analysis and immunofluorescence. Exposure of HepG2 cells to TPO was associated with a dose-dependent increase in cell migration and chemoinvasion through Matrigel-coated filters. A checkerboard analysis showed that the effects of TPO on cell migration were dependent on both chemotaxis and chemokinesis. Exposure of HepG2 cells to TPO resulted in the activation of different members of the MAPK family, including ERK and JNK, as assessed using phosphorylation-specific antibodies and immune complex kinase assays. TPO also activated phosphatidylinositol 3-kinase (PI3K) and the downstream kinase Akt in a time-dependent manner. Finally, activation of c-Mpl was associated with increased activation of nuclear factor-κB. With the use of specific inhibitors, tyrosine phosphorylation and activation of PI3K were found to be required for the induction of migration in response to TPO. We conclude that TPO exerts biological actions on cultured hepatoblastoma cells via activation of c-Mpl and its downstream signaling.

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.

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.

as treatments for steatohepatitis. The mechanisms implicated remain poorly understood. Activation of PPARg induces expression of adiponectin, an anti-inflammatory and insulin-sensitising adipocytokine. Upon binding to specific receptors on liver cells, adiponectin activates the AMP-activated protein kinase (AMPK), stimulates b-oxidation and represses hepatic de novo lipogenesis. Also, adiponectin, through PPARa, activates fat oxidation and inhibits inflammatory reaction. Here we tested the hypothesis that the beneficial effect of PGZ on steatohepatitis results from reduced hepatic fat accumulation due to adiponectin-dependent stimulation of AMPK and PPARa. Methods: Mice lacking adiponectin (adipo−/−), the AMPK catalytic subunit alpha1 (AMPKa1−/−) and their respective littermates were fed a methionine and choline (MCD) deficient diet, supplemented or not with pioglitazone 0.01%. Histopathology, hepatic lipid content, serum adiponectin levels were assessed. In liver tissues, we evaluated AMPK (total, a1, a2) activities, proteins and mRNA levels, PPARa mRNA and the expression of downstream targets of AMPK or PPARa. Results: In wt mice, PGZ reduces the severity of MCD diet-induced steatohepatitis, while it increased circulating levels of adiponectin. By contrast, in adiponectin−/− mice, PGZ did not alter the severity of steatohepatitis. AMPKa1−/− mice and their littermates were genetically less sensitive to dietary steatohepatitis than C57 mice. In AMPKa1−/− mice fed the MCD diet, PGZ did not reduce the severity of steatohepatitis, although it significantly increased serum adiponectin levels. In wt mice, PGZ almost completely repressed the expression and activation of SREBP1c, a key transcription factor for de novo lipogenesis and downstream target of AMPK. This effect of PGZ was lacking in adiponectin−/− and in AMPKa1−/− mice. In the strains analysed, PGZ did not alter the expression of PPARa or of its downstream targets implicated in the regulation of lipid oxidation. In conclusion, we showed that the preventive effect of PGZ on MCD-induced steatohepatitis is dependent on the up-regulation of adiponectin. While the adiponectin-PPARa-boxidation axis does not appear to be implicated, our data strongly suggest that adiponectin produces its effect by controlling the expression and activity of SREBP-1c via the activation of the AMPK complex.