n-3 polyunsaturated fatty acids worsen inflammation and fibrosis in experimental nonalcoholic steatohepatitis.

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 < 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-ß, and more severe histological scores of fibrosis were evident 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 alone (5.1 ± 1.4 vs. 7.0 ± 0.9 mmHg, P < 0.05). Analysis of hepatic fatty acid profile showed that supplementation resulted in effective incorporation of n-3 PUFA. CONCLUSIONS: 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.

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-ß (transforming growth factor-ß), 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.

Mammalian target of rapamycin mediates the angiogenic effects of leptin in human hepatic stellate cells.

Leptin modulates the angiogenic properties of hepatic stellate cells (HSC), but the molecular mechanisms involved are poorly understood. We investigated the pathways regulating hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in leptin-stimulated myofibroblastic HSC. Exposure to leptin enhanced the phosphorylation of TSC2 on T1462 residues and of p70 S6 kinase and the translational inhibitor 4E-binding protein-1, indicating the ability of leptin to activate the mammalian target of rapamycin (mTOR) pathway. Similar findings were observed when HSC were exposed to PDGF. Both leptin and PDGF increased the expression of HIF-1α and VEGF in HSC. In the presence of rapamycin, a specific mTOR inhibitor, leptin and PDGF were no longer able to activate mTOR, and expression of VEGF was reduced, whereas HIF-1α abundance was not affected. Moreover, knockdown of Raptor, a component of the mTORC1 complex, reduced the ability of leptin to increase VEGF. mTOR was also necessary for leptin- and PDGF-dependent increase in HSC migration. Leptin increased the generation of reactive oxygen species in HSC, which was reduced by NADP(H) oxidase inhibitors. Both N-acetyl cysteine and diphenylene iodonium, a NADP(H) inhibitor, inhibited the expression of HIF-1α and VEGF stimulated by leptin or PDGF. Finally, conditioned media from HSC treated with leptin or PDGF induced tube formation in cultured human umbilical vein endothelial cells. In conclusion, in HSC exposed to leptin or PDGF, increased expression of VEGF requires both activation of mTOR and generation of reactive oxygen species via NADPH-oxidase. Induction of HIF-1α requires NADP(H) oxidase but not mTOR activation.

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 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.

Myostatin regulates the fibrogenic phenotype of hepatic stellate cells via c-jun N-terminal kinase activation.

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-ß1. 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.

Thrombopoietin stimulates migration and activates multiple signaling pathways in hepatoblastoma cells.

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-kappaB. 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.

Differential requirement of members of the MAPK family for CCL2 expression by hepatic 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, p38(MAPK), 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 (p38(MAPK)), 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.