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Background/aims: Endogenous gut-derived bacterial endotoxins have been implicated as an important cofactor in the pathogenesis of liver injury, although their contribution to the progression of non-alcoholic steatohepatitis (NASH) remains unclear., Methods: Male C57BL/6 mice were fed a methionine-choline-deficient (MCD) diet or a standard diet for 17 days, following which they were injected with lipopolysaccharide (LPS) intraperitoneally and sacrificed after 6h. In an in vitro experiment, RAW264.7 cells, a mouse macrophage cell line, and primary mouse hepatocytes were co-treated with hydrogen peroxide (H(2)O(2)) and LPS or tumour necrosis factor (TNF)-alpha., Results: Compared to the control mice, LPS treatment significantly increased hepatic TNF-alpha production in MCD mice. LPS also significantly increased TUNEL-positive cells, which were especially observed in the perivenular area. The apoptotic change was inhibited by co-treatment with a neutralizing anti-mouse TNF receptor antibody or pentoxifylline. In an in vitro experiment, treatment with H(2)O(2) synergistically enhanced LPS-induced TNF-alpha production in RAW264.7 cells, accompanied by an up-regulation of CD14 mRNA. Moreover, co-treatment with TNF-alpha- and H(2)O(2)-induced apoptosis in primary hepatocytes, although neither TNF-alpha nor H(2)O(2) could do so independently., Conclusions: LPS up-regulated TNF-alpha production, which induced hepatocyte apoptosis in a murine NASH model. LPS may play a key role in the pathogenesis of NASH.

Background/aims: Plasminogen activators and plasminogen activator inhibitors are important regulators of the balance between the proteolytic and antiproteolytic activities that determine extracellular matrix turnover. We examined the expression of plasminogen activator-plasmin system components in experimental liver fibrosis of rats., Methods: Liver fibrosis was produced in rats by injecting carbon tetrachloride for 6 to 12 weeks. Gene expression for plasminogen activator inhibitor-1 (PAI-1), urokinase and tissue plasminogen activators (uPA and tPA), urokinase plasminogen activator receptor (uPAR), and transforming growth factor-beta1 (TGF-beta1) was examined by Northern analysis. Western analysis was performed to detect protein expression of PAI-1, uPA and uPAR. An immunohistochemical study was performed to detect the localization of PAI-1. Additionally, primary cultured liver cells were examined by Northern and Western analyses for this protein with or without prior incubation with TGF-beta1., Results: At 6 weeks, when fibrosis had occurred, uPA and uPAR mRNAs had increased 2.8-fold and 1.8-fold, respectively; PAI-1 and tPA mRNA levels were unchanged. At the cirrhotic stage (9 to 12 weeks), mRNA levels for PAI-1, uPA, uPAR and tPA were all increased. Western analysis also showed increased uPA and uPAR expressions in fibrotic liver, and increased PAI-1, uPA and uPAR expressions in cirrhotic liver. PAI-1 protein was also demonstrated immunohistochemically along sinusoids, vessels, and bile duct cells of normal and fibrotic liver. In liver cell cultures, Kupffer cells, hepatocytes, and especially stellate cells, expressed PAI-1. Expression was enhanced in stellate cells cultured from fibrotic or cirrhotic liver or stimulated in vitro with TGF-beta1., Conclusion: Though increased uPA and uPAR may act on matrix degradation in fibrotic liver, increased PAI-1 together with uPA, uPAR and tPA are associated with overall inhibition of matrix degradation in cirrhotic liver. Hepatic stellate cells are an important source of PAI-1 during liver fibrosis.

Background/aims: Matrix metalloproteinase-13, one of the principal neutral proteinases capable of cleaving native fibrillar collagens, is important in the degradation and remodeling of extracellular matrix. However, its precise expression in liver injury has not been characterized. We examined the kinetics of the expression of matrix metalloproteinase-13 and one of its specific inhibitors, tissue inhibitor of metalloproteinase-1, in acute liver injury in rats., Methods: Acute liver injury was induced by administration of carbon tetrachloride or two different doses of D-galactosamine hydrochloride in Wistar rats. Hepatic matrix metalloproteinase-13 and tissue inhibitor of metalloproteinase-1 mRNA levels were then examined by Northern blotting., Results: All rats survived after liver injury induced by carbon tetrachloride or low doses of D-galactosamine hydrochloride. However, rats died 5 days after induction of liver injury by high doses of D-galactosamine hydrochloride. In carbon tetrachloride-induced liver injury, matrix metalloproteinase-13 mRNA was transiently increased between 6 h and 1 day after injury. Tissue inhibitor of metalloproteinase-1 mRNA expression was increased between 6 h and 3 days after the peak of matrix metalloproteinase-13 expression. Similar patterns of matrix metalloproteinase-13 and tissue inhibitor of metalloproteinase-1 expression were observed in low-dose D-galactosamine hydrochloride-induced liver injury. In contrast, in high-dose D-galactosamine hydrochloride-induced liver injury, tissue inhibitor of metalloproteinase-1 expression peaked before matrix metalloproteinase-13 expression, which was increased 2 days after injury. Both mRNA levels continued to increase until death., Conclusions: Transient expression of matrix metalloproteinase-13, followed by that of tissue inhibitor of metalloproteinase-1, was observed during recovery from acute liver injury induced by carbon tetrachloride and low-dose D-galactosamine hydrochloride. In contrast, disordered expression of matrix metalloproteinase-13 was observed in fatal liver injury caused by high-dose D-galactosamine hydrochloride. These results indicate that matrix metalloproteinase13 plays an important role in the early phase of recovery from liver injury.