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C57BL/6J (BL6J) and C57BL/6N (BL6N) inbred substrains are most widely used to understand the pathological roles of target molecules in a variety of diseases, including non-alcoholic steatohepatitis (NASH), based on transgenic mouse technologies. There are notable differences in the metabolic phenotypes, including glucose tolerance, between the BL6J and BL6N substrains, but the phenotypic differences in NASH are still unknown. We performed a comparative analysis of the two mouse substrains to identify the pathological phenotypic differences in NASH models. In the CCl 4 -induced NASH model, the BL6J mice exhibited a more severe degree of oxidative stress and fibrosis in the liver than the BL6N mice. In contrast, in the high-fat diet-induced NASH model, more accumulation of hepatic triglycerides but less weight gain and liver injury were noted in the BL6J mice than in the BL6N mice. Our findings strongly suggest caution be exercised with the use of unmatched mixed genetic background C57BL6 mice for studies related to NASH, especially when generating conditional knockout C57BL6 mice.

We investigated the effect of curcumin on liver injury in diabetic rats induced by streptozotocin (STZ) through modulation of endoplasmic reticulum stress (ERS) and unfolded protein response (UPR). Experimental diabetes was induced by a single intraperitoneal injection of STZ (55 mg/kg), and curcumin was given at 100 mg/kg by gavage for 56 days. We observed that curcumin improved the morphological and histopathological changes, significantly decreased hepatic ERS marker protein: glucose-regulated protein 78, and improved liver function in diabetic rats. Moreover, treatment with curcumin markedly decreased the sub-arm of the UPR signaling protein such as phospho-double-stranded RNA-dependent protein kinase-like ER kinase, CCAAT/enhancer-binding protein homologous protein, tumor necrosis factor receptor-associated factor 2, and inositol-requiring enzyme1α; and inhibited tumor necrosis factor α, interleukin 1β, phospho-p38 mitogen-activated protein kinase, and apoptosis signal-regulating kinase 1 in liver tissues of diabetic rats. Apoptotic and anti-apoptotic signaling proteins, such as cleaved caspase-3 and B-cell lymphoma 2, were significantly increased and decreased, respectively in diabetic rats; curcumin treatment prevented all of these alterations. In summary, our results indicate that curcumin has the potential to protect the diabetic liver by modulating hepatic ERS-mediated apoptosis, and provides a novel therapeutic strategy for the diabetic liver damage.

Lithocholic acid (LCA) feeding causes both liver parenchymal and cholestatic damages in experimental animals. Although pregnenolone-16 alpha-carbonitrile (PCN)-mediated protection against LCA-induced hepatocyte injury may be explained by induction of drug metabolizing enzymes, the protection from the delayed cholestasis remains incompletely understood. Thus, the PCN-mediated protective mechanism has been studied from the point of modification of lipid metabolism. At an early stage of LCA feeding, an imbalance of biliary bile acid and phospholipid excretion was observed. Co-treatment with PCN reversed the increase in serum alanine aminotransferase (ALT) as well as alkaline phosphatase (ALP) activities and hepatic hydrophobic bile acid levels. LCA feeding decreased hepatic mRNA levels of several fatty acid- and phospholipid-related genes before elevation of serum ALT and ALP activities. On the other hand, PCN co-treatment reversed the decrease in the mRNA levels and hepatic levels of phospholipids, triglycerides and free fatty acids. PCN co-treatment also reversed the decrease in biliary phospholipid output in LCA-fed mice. Treatment with PCN alone increased hepatic phospholipid, triglyceride and free fatty acid concentrations. Hepatic fatty acid and phosphatidylcholine synthetic activities increased in mice treated with PCN alone or PCN and LCA, compared to control mice, whereas these activities decreased in LCA-fed mice. These results suggest the possibility that PCN-mediated stimulation of lipogenesis contributes to the protection from lithocholic acid-induced hepatotoxicity., ((c) 2010 Elsevier B.V. All rights reserved.)

Recently the authors proposed a new staging and grading system for primary biliary cirrhosis (PBC) that takes into account necroinflammatory activity and histological heterogeneity. Herein is proposed a convenient version of this system. Scores for fibrosis, bile duct loss, and chronic cholestasis were combined for staging: stage 1, total score of 0; stage 2, score 1-3; stage 3, score 4-6; and stage 4, score 7-9. Cholangitis activity (CA) and hepatitis activity (HA) were graded as CA0-3, and HA0-3, respectively. Analysis of interobserver agreement was then conducted. Digital images of 62 needle liver biopsy specimens of PBC were recorded as virtual slides on DVDs that were sent to 28 pathologists, including five located overseas. All participants were able to apply this version in all 62 cases. For staging, kappa was 0.385 (fair agreement) and the concordance rate was 63.9%. For necroinflammatory activity, the kappa and concordance rate were 0.110 (slight agreement) and 36.9% for CA, and 0.197 (slight agreement) and 47% for HA, respectively. In conclusion, this new staging and grading system for PBC seems to be more convenient and practical than those used at present, but more instruction and guidance are recommended for the grading of necroinflammatory activity in practice.

ME3738 (22beta-methoxyolean-12-ene-3beta, 24(4beta)-diol), a derivative of soyasapogenol, attenuates liver disease in several models of chronic liver inflammation. In the present study, we have investigated a protective effect of ME3738 in a typical bile acid-induced cholestatic liver model, lithocholate (LCA) feeding mouse. Co-administration of ME3738 resulted in decreases in plasma alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities and hepatic bile acid level, and increases in biliary outputs of bile acid and cholesterol, as compared with the results in mice treated with LCA alone. LCA sulfation by hydroxysteroid sulfotransferase 2a and hydroxylation have been reported to be involved in protection against LCA-induced hepatotoxicity. ME3738-treatment, however, had no clear influence on the hydroxysteroid sulfotransferase 2a protein level and LCA 6alpha-, 6beta- and 7alpha-hydroxylase activities, but increased biliary cholesterol output. Cholate (CA)-treatment has been shown to induce hepatotoxicity in farnesoid X receptor-null mice, which is scarcely dependent on bile acid sulfation and hydroxylation but associated with decreased biliary bile acid output. Co-administration of ME3738 decreased the ALT and ALP activities and hepatic bile acid level, and increased biliary outputs of bile acid and cholesterol in farnesoid X receptor-null mice, as compared with the results in the mice treated with CA. Moreover, a clear correlation between biliary outputs of cholesterol and bile acid was observed in these two bile acid-induced hepatotoxicity mouse models. These results suggest that ME3738 protects against bile acid-induced hepatotoxicity through increased biliary bile acid output that is not related to bile acid metabolism but associated with cholesterol output.

Few studies on the long-term culture of postnatal mouse hepatic stem/progenitor cells have been reported. We successfully adapted a serum-free culture system that we employed previously to expand fetal mouse hepatic stem/progenitor cells and maintained them in culture over long periods. The expanded postnatal cells contained immature alpha-fetoprotein-positive cells along with hepatocytic and cholangiocytic lineage-committed cells. These cells expressed CD49f but not CD45, CD34, Thy-1, c-kit, CD31, or flk-1, and oncostatin M induced their differentiation. This heterogeneous population contained side population (SP) cells, which express the ATP-binding cassette transporter ABCG2, and sca-1+ cells. As mice aged, the frequency of SP and sca-1+ cells decreased along with the ability of cultured cells to expand. Approximately 20%-40% of the SP cells expressed sca-1, but only a few sca-1+ cells were also SP cells. Analysis of colonies derived from single SP or sca-1+ cells revealed that, although both cells had dual differentiation potential and self-renewal ability, SP cells formed colonies more efficiently and gave rise to SP and sca-1+ cells, whereas sca-1+ cells generated only sca-1+ progeny. Thus, SP cells are more characteristic of stem cells than are sca-1+ cells. In regenerating livers, ABCG2+ cells and sca-1+ cells were detected around or in the portal area (the putative hepatic stem cell niche). The expanded cells share many features of fetal hepatic stem/progenitor cells or oval cells and may be useful in determining the mechanisms whereby hepatic stem cells self-renew and differentiate.

Background: OK-432 is known to increase the host antitumor response. We previously reported that systemic administration of OK-432 (OK-Lipo) specifically induced hepatic lymphocytes in mice. Here we aimed to investigate the antitumor effect of OK-Lipo on hepatocellular carcinomas (HCC) in experimental rats., Methods: Diethylnitrosamine was administered for 12 weeks to all rats (n = 36). Rats were divided into three groups of 12 rats each. One group was injected with OK-Lipo from week 5 (OK-5w group) and another from week 9 (OK-9w group). A control group was injected with saline from week 5 (Non-OK group). At week 13, five rats from each group were used for histological analysis and immunofluorescence assays (surface phenotypic and intracellular cytokine analysis of the mononuclear cells in the liver, spleen and peripheral blood). The remaining rats were observed for the remainder of their survival period., Results: The mean survival times of Non-OK, OK-5w, and OK-9w groups differed significantly (98.0 +/- 5.3 days, 116.0 +/- 5.8 days, and 106.0 +/- 5.4 days, respectively, P < 0.01). Histological examination revealed many apoptotic tumor cells, infiltration of lymphocytes and macrophages in the OK-5w group. The two-color immunofluorescence assay showed that the proportion of natural killer (NK) cells and IFN-gamma-producing cells in the liver were significantly higher in the OK-5w group., Conclusions: These findings showed that systemic administration of OK-Lipo contributed to prolonging the survival of rats with HCC. OK-Lipo induced NK cells and IFN-gamma-producing cells specifically in the liver and these cells seemed to reduce hepatocarcinogenesis and tumor growth.

Primary graft nonfunction of steatotic liver allograft is one of the factors causing shortage of donor livers. Ischemia/reperfusion (I/R) injury is an important contributory factor to primary graft nonfunction. In this study, we investigated the complex chain of events from transcription factor activation to necrosis through cytokine induction and apoptosis in steatotic rat liver after warm I/R. Rats with alcoholic or nonalcoholic fatty liver were subjected to hepatic warm I/R and compared with control rats. Rats fed an ethanol diet for 6 to 8 weeks developed severe hepatic necrosis accompanied by increased neutrophil recruitment after I/R, compared with rats with nonalcoholic fatty liver or control. Hepatic apoptosis as assessed by DNA fragmentation at 4 hours after I/R, however, increased to a similar degree in each of the 2 fatty liver models compared with the control. Alcoholic fatty liver exposed to I/R showed a rapid increase in nuclear factor-kappaB (NF-kappaB) binding activity at 1 hour after I/R, which preceded an increased expression of tumor necrosis factor alpha (TNF-alpha) and cytokine-induced neutrophil chemoattractant-1 (CINC-1). In contrast, nonalcoholic fatty liver did not show such potentiation of either NF-kappaB activation or cytokine induction after I/R. Our results have indicated that alcoholic fatty liver may differentially induce CINC-1 production and hepatic necrosis after I/R. Furthermore, our results suggest that apoptosis per se does not always lead to necrosis in the liver following I/R.

The isolation and characterization of human liver cell lines are rather difficult due to limited material and poor growth in cell culture. In this report, we present the isolation, culture and characterization of a new epithelial-like liver cell line (AKN-1) with a heterogeneous cell population and many characteristics of the biliary epithelium. The AKN-1 cell line stained positively with antibodies to epithelial cytokeratin polypetides CK 8, 18, and 19. In addition, the cell line expressed the anti-human epithelial-related antigen (MOC-31), the human epithelial antigen (HEA), and the gamma-glutamyl transpeptidase, the hematopoietic growth factor, stem cell factor, and also its receptor, c-kit. The cell line failed to express albumin and factor 8 by immunohistochemistry. It did show, however, a twofold increase in 7-ethoxyresorufin-O-deethylase activity. Cytogenetic characterization revealed rare breakpoints in chromosome 2, which to our knowledge, have not yet been reported in liver cells.

Aims/background: Thick hepatic plates have been considered one of the morphological characteristics of hepatocyte regeneration in cirrhotic nodules. They can be recognized by the sinusoidal surface predominance of their nuclei. We have investigated the prevalence of this in HBV and HCV infections., Methods and Results: This feature was more frequently present in type C chronic hepatitis with low activity of inflammation and low grade of fibrosis, than with type B chronic hepatitis. Additionally, this area of sinusoidal surface hepatocyte nuclear predominance (ASSHNP) was seen in zone II, rather than in periportal zones, in type C chronic hepatitis. Clinical data were analyzed statistically. Immunohistochemical reactivity of type IV collagen, laminin, Ulex europaeus agglutinin 1 lectin (UEA-1), and factor VIII-related antigen were increased in ASSHNP. Immunohistochemical staining of Ki-67 antigen was performed in order to assess the regenerative capacity of this area and showed a low level of regeneration. Ultrastructure of this area in type C chronic hepatitis showed a decrease in the number of mitochondria and an increase of nuclear pleomorphism together with basement membrane formation in the space of Disse., Conclusion: Although the cause of these abnormalities was not clarified in this study, it is suggested that they are related to chronic hepatitis C virus (HCV) infection per se, rather than regeneration or inflammatory activity. These changes may be significant in HCV-associated hepatocarcinogenesis.

We investigated the regulatory expression of the HSP70-1 gene in rat liver after whole body hyperthermia (rectal temperature of 42 degrees C for 30 min). After heat treatment, HSP70-1 mRNA showed a peak at 1 h, and its protein product began to increase at 3 h. Under these conditions, we identified cis-acting elements involved in the regulatory expression of the HSP70-1 gene by in vivo genomic footprinting. We observed that the second heat shock element (HSE-2; -201 to -178) was clearly protected from methylation by dimethyl sulfate in parallel with the increase in HSP70-1 mRNA. On the contrary, the most proximal HSE-1 (-120 to -101) was protected only slightly. Furthermore, the flanking regions of HSE-2, that is, an NF-kappa B-like element on the upstream side and a GC box on the down-stream side were also protected simultaneously in accordance with the induced expression of the HSP70-1 gene. Gel mobility shift assays with nuclear extracts from rat liver at 30 min after heat treatment showed maximal DNA binding activity equally with oligonucleotides containing HSE-1 and HSE-2, respectively, and also demonstrated the participation of heat shock factor-1 (HSF1) in both cases. These results suggest that, in addition to HSF1, other regulatory factors might be involved in the dominant protection of HSE-2 in the promoter of the HSP70-1 gene in rat liver after whole body hyperthermia.

Our previous work demonstrated a simultaneous induction of Hsp70 and nucleolin mRNA during the prereplicative stage of hepatocytes after partial hepatectomy [Ohmori, H. et al. (1990) Exp. Cell Res. 189, 227-232]. In the present study, changes of intracellular localization of these proteins were examined. Nucleolin, which mainly localized in the nucleus, increased with time and accumulated in the nucleolus around 12-18 h after partial hepatectomy. Hsp70 protein also increased slightly around 6-12 h after the operation. In accordance with this increase, immunohistochemical staining revealed that almost all nuclei of hepatocytes became Hsp70-positive, although Hsp70 was seen to be dispersed throughout the nucleoplasm and nucleolus at all times examined. Next, we isolated a cDNA clone of ribosomal protein (S-17) and examined its behavior. Induction of S-17 mRNA was observed to be essentially similar to that of nucleolin mRNA in regenerating rat liver, although inductions of histone H2A and H4 occurred at a later time, that is, in parallel with DNA synthesis. Furthermore, we observed a simultaneous induction of Hsp70 and nucleolin mRNA by serum-stimulation after serum-depletion in HeLa and IAR-20 (rat) culture cells. These results suggest that the induction of Hsp70, in addition to nucleolin, was not fortuitous but may be involved in the early events of liver regeneration.

Among 1,098 liver biopsy specimens obtained from patients with various liver diseases characterized by liver injury, 58 epithelial cells whose cytoplasms stained positively by the periodic acid-Schiff stain (digested with diastase) were recognized in the interlobular bile ducts of 37 specimens from 36 patients. Light microscopic study revealed that the cytoplasms of these cells were clear or stained weakly eosinophilic on hematoxylin and eosin staining and that the cell limits were distinct. From their reaction with periodic acid-Schiff stain and from electron microscopic observation it was clear that these cells contained an abundance of glycogen and were located among the normal bile duct cells surrounded by basement membrane. On electron microscopy, these cells had microvilli of equal sizes on their luminal surfaces and many irregularly sized microvilluslike cell membrane projections on their basal surfaces. They rested on basement membrane with basal spaces. These cells varied in size from 25.0 to 452.2 microns 2 (mean = 212.2 microns 2). In contrast, the sizes of normal bile duct cells and hepatocytes ranged from 20.0 to 69.3 microns 2 (mean = 34.2 microns 2) and from 113.0 to 860.3 microns 2 (mean = 447.0 microns 2), respectively. Immunohistochemical study with antiserum to cytokeratin 19, albumin and alpha 1-antitrypsin on serially cut frozen sections showed that some of these cells expressed markers of bile duct cells and hepatocytes. Some cells expressed only the markers of hepatocytes. Computer graphic three-dimensional reconstruction clearly demonstrated that these cells were located sparsely (but sometimes in groups) among normal interlobular bile duct cells, without any connection to the surrounding parenchymal hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue-associated fibronectin was extracted with 4 M urea from human normal and cirrhotic livers. The results of rocket immunoelectrophoresis indicated that the amount of fibronectin in cirrhotic liver was twice as much as that in normal liver. The collagen content was much higher in cirrhotic than in normal livers. These data suggested an important role of fibronectin in tissue fibrosis.

By light and electron microscopic examinations, histologic changes in the liver of mice with graft-versus-host reaction (GVHR) were analyzed. To induce GVHR, C57BL/6 (B6) spleen cells were injected into (B6Xbm1)F1, (B6Xbm12)F1, and (bm1Xbm12)F1 mice. In (B6Xbm12)F1 recipient mice, bile duct changes resembling chronic non-suppurative destructive cholangitis (CNSDC) and a formation of epithelioid granulomas were observed during the course of GVHR. An epithelioid granuloma in the liver of (B6Xbm1)F1 or (bm1.Xbm12)F1 recipients was not detected. By electron microscopy, the bile duct epithelia were seen to be in close contact with infiltrating cells, and marked alterations of their cytoplasm and microvilli were demonstrated; ie, vacuolation of the cytoplasm, deterioration of microvilli, and bleb formation were frequently observed in the liver of class II-disparate hosts. Concerning the basement membrane, no marked changes characteristic of primary biliary cirrhosis (PBC), such as many-layered basement membranes containing osmium positive substance, were detected. Because the major histocompatibility complex (MHC) class II-disparate system was used in our experimental system in the GVHR, the antigen expressed on the bile duct might be a target and be associated with the formation of the initial hepatic lesions in PBC such as CNSDC and epithelioid granuloma formation. Thus, GVHR across the MHC class II antigen is believed to play an important role in the development of PBC.