Hepatocellular organellar abnormalities following elimination of hepatitis C virus.

BACKGROUND AND AIMS: The future development of hepatocellular carcinoma (HCC) in patients after sustained virologic response (SVR) is an important issue. The purposes of this study were to investigate pathological alterations in organelle of the liver of SVR patients and to characterize organelle abnormalities that may be related to carcinogenesis after SVR. METHODS: The ultrastructure of liver biopsy specimens from patients with chronic hepatitis C (CHC) and SVR were compared to cell and mouse models and assessed semi-quantitatively using transmission electron microscopy. RESULTS: Hepatocytes in patients with CHC showed abnormalities in the nucleus, mitochondria, endoplasmic reticulum, lipid droplet, and pericellular fibrosis, comparable to those seen in hepatitis C virus (HCV)-infected mice and cells. DAA treatment significantly reduced organelle abnormalities such as the nucleus, mitochondria, and lipid droplet in the hepatocytes of patients and mice after SVR, and cured cells, but it did not change dilated/degranulated endoplasmic reticulum and pericellular fibrosis in patients and mice after SVR. Further, samples from patients with a post-SVR period of >1 year had significantly larger numbers of abnormalities in the mitochondria and endoplasmic reticulum than those of <1 year. A possible cause of organelle abnormalities in patients after SVR could be oxidative stress of the endoplasmic reticulum and mitochondria associated with abnormalities of the vascular system due to fibrosis. Interestingly, abnormal endoplasmic reticulum was associated with patients with HCC for >1 year after SVR. CONCLUSIONS: These results indicate that patients with SVR exhibit a persistent disease state and require long-term follow-up to detect early signs of carcinogenesis.

HBV with precore and basal core promoter mutations exhibits a high replication phenotype and causes ER stress-mediated cell death in humanized liver chimeric mice.

BACKGROUND AND AIMS: Mutations within the precore (PC) and basal core promoter (BCP) regions of the HBV genome are associated with fulminant hepatitis and HBV reactivation. These mutations may enhance viral replication, but little is known about whether they directly induce damage to the liver. We investigated mechanisms of direct cytopathic effects induced by the infection with PC/BCP mutants in the absence of immune response in vitro and in vivo . APPROACH AND RESULTS: Mice with humanized livers and hepatocytes derived from humanized mice were infected with either wild-type or mutant-type PC/BCP HBV, and the HBV replication and human hepatocyte damage were evaluated. HBV proliferated vigorously in mice with PC/BCP-mutant infection, and the severe loss of human hepatocytes with a slight human ALT elevation subsequently occurred only in PC/BCP mutant mice. In PC/BCP mutant infection, the accumulation of HBsAg in humanized livers colocalized with the endoplasmic reticulum, leading to apoptosis through unfolded protein response in HBV-infected hepatocytes. RNA-sequencing revealed the molecular characteristics of the phenotype of PC/BCP mutant infection in a humanized mouse model. Reduced ALT elevation and higher HBV DNA levels in this model are consistent with characteristics of HBV reactivation, indicating that the hepatocyte damage in this model might mimic HBV reactivation followed by hepatocyte damage under immunosuppressive conditions. CONCLUSION: PC and BCP mutations were associated with enhanced viral replication and cell death induced by ER stress using HBV infection models. These mutations might be associated with liver damage in patients with fulminant hepatitis or HBV reactivation.

Serial ultrathin sections to identify ultrastructural localization of GLUT1 molecules in vesicles in brain endothelial cells-correlative light and electron microscopy in depth.

Precise immunolocalization of molecules in relation to ultrastructural features is challenging, especially when the target is small and not frequent enough to be included in tiny ultrathin sections randomly selected for electron microscopy (EM). Glucose transporter 1 (GLUT1) is in charge of transporting glucose across brain capillary endothelial cells (BCECs). Paraformaldehyde-fixed floating sections (50 µm thick) of mouse brain were immunolabeled with anti-GLUT1 antibody and visualized with fluoronanogold. Fluorescent images encompassing the entire hemisphere were tiled to enable selection of GLUT1-positive BCECs suitable for subsequent EM and landmark placement with laser microdissection to guide trimming. Sections were then fixed with glutaraldehyde, gold enhanced to intensify the labeling and fixed with osmium tetroxide to facilitate ultrastructural recognition. Even though a region that contained target BCECs was successfully trimmed in the resin block, it was only after observation of serial ultrathin sections that GLUT1 signals in coated vesicles on the same cross section corresponding to the cross section preidentified by confocal laser microscope. This is the first ultrastructural demonstration of GLUT1 molecules in coated vesicles, which may well explain its functional relevance to transport glucose across BCECs. Successful ultrastructural localization of molecules in relation to well-preserved target structure in native tissue samples, as achieved in this study, will pave the way to understand the functional relevance of molecules and their relation to ultrastructural details.

Activation of Hepatic Stellate Cells Requires Dissociation of E-Cadherin-Containing Adherens Junctions with Hepatocytes.

Hepatic stellate cells (HSCs) are resident mesenchymal cells in the space of Disse interposed between liver sinusoidal endothelial cells and hepatocytes. Thorn-like microprojections, or spines, project out from the cell surface of HSCs, crossing the space of Disse, to establish adherens junctions with neighboring hepatocytes. Although HSC activation is initiated largely from stimulation by adjacent cells, isolated HSCs also activate spontaneously in primary culture on plastic. Therefore, other unknown HSC-initiating factors apart from paracrine stimuli may promote activation. The dissociation of adherens junctions between HSCs and hepatocytes as an activating signal for HSCs was explored, establishing epithelial cadherin (E-cadherin) as an adhesion molecule linking hepatocytes and HSCs. In vivo, following carbon tetrachloride-induced liver injury, HSCs lost their spines and dissociated from adherens junctions in the early stages of injury, and were subsequently activated along with an increase in YAP/TAZ expression. After abrogation of liver injury, HSCs reconstructed their spines and adherens junctions. In vitro, reconstitution of E-cadherin-containing adherens junctions by forced E-cadherin expression quiesced HSCs and suppressed TAZ expression. Additionally, increase of TAZ expression leading to the activation of HSCs by autocrine stimulation of transforming growth factor-ß, was revealed as a mechanism of spontaneous activation. Thus, we have uncovered a critical event required for HSC activation through enhanced TAZ-mediated mechanotransduction after the loss of adherens junctions between HSCs and hepatocytes.

Adult hepatocytes direct liver organogenesis through non-parenchymal cell recruitment in the kidney.

BACKGROUND & AIMS: Since the first account of the myth of Prometheus, the amazing regenerative capacity of the liver has fascinated researchers because of its enormous medical potential. Liver regeneration is promoted by multiple types of liver cells, including hepatocytes and liver non-parenchymal cells (NPCs), through complex intercellular signaling. However, the mechanism of liver organogenesis, especially the role of adult hepatocytes at ectopic sites, remains unknown. In this study, we demonstrate that hepatocytes alone spurred liver organogenesis to form an organ-sized complex 3D liver that exhibited native liver architecture and functions in the kidneys of mice. METHODS: Isolated hepatocytes were transplanted under the kidney capsule of monocrotaline (MCT) and partial hepatectomy (PHx)-treated mice. To determine the origin of NPCs in neo-livers, hepatocytes were transplanted into MCT/PHx-treated green fluorescent protein transgenic mice or wild-type mice transplanted with bone marrow cells isolated from green fluorescent protein-mice. RESULTS: Hepatocytes engrafted at the subrenal space of mice underwent continuous growth in response to a chronic hepatic injury in the native liver. More than 1.5 years later, whole organ-sized liver tissues with greater mass than those of the injured native liver had formed. Most remarkably, we revealed that at least three types of NPCs with similar phenotypic features to the liver NPCs were recruited from the host tissues including bone marrow. The neo-livers in the kidney exhibited liver-specific functions and architectures, including sinusoidal vascular systems, zonal heterogeneity, and emergence of bile duct cells. Furthermore, the neo-livers successfully rescued the mice with lethal liver injury. CONCLUSION: Our data clearly show that adult hepatocytes play a leading role as organizer cells in liver organogenesis at ectopic sites via NPC recruitment. LAY SUMMARY: The role of adult hepatocytes at ectopic locations has not been clarified. In this study, we demonstrated that engrafted hepatocytes in the kidney proliferated, recruited non-parenchymal cells from host tissues including bone marrow, and finally created an organ-sized, complex liver system that exhibited liver-specific architectures and functions. Our results revealed previously undescribed functions of hepatocytes to direct liver organogenesis through non-parenchymal cell recruitment and organize multiple cell types into a complex 3D liver at ectopic sites. Transcript profiling: Microarray data are deposited in GEO (GEO accession: GSE99141).

Roles for Cell-Cell Adhesion and Contact in Obesity-Induced Hepatic Myeloid Cell Accumulation and Glucose Intolerance.

Obesity promotes infiltration of inflammatory cells into various tissues, leading to parenchymal and stromal cell interaction and development of cellular and organ dysfunction. Liver sinusoidal endothelial cells (LSECs) are the first cells that contact portal blood cells and substances in the liver, but their functions in the development of obesity-associated glucose metabolism remain unclear. Here, we find that LSECs are involved in obesity-associated accumulation of myeloid cells via VLA-4-dependent cell-cell adhesion. VLA-4 blockade in mice fed a high-fat diet attenuated myeloid cell accumulation in the liver to improve hepatic inflammation and systemic glucose intolerance. Ex vivo studies further show that cell-cell contact between intrahepatic leukocytes and parenchymal hepatocytes induces gluconeogenesis via a Notch-dependent pathway. These findings suggest that cell-cell interaction between parenchymal and stromal cells regulates hepatic glucose metabolism and offers potential strategies for treatment or prevention of obesity-associated glucose intolerance.

Development of the Heart Endocardium at an Early Stage of Chick Embryos Evaluated at Light- and Electron-Microscopic Levels.

Development of the endocardium in the heart of 4 to 4·1/2-day-incubated chick embryos was observed light and electron microscopically, and these results were evaluated by immunohistochemistry for desmin, FLK1 (VEGFR-2) or CD31, and by in situ hybridization assays for flk1-mRNA expression. At this developmental stage, the atrium and the ventricle were already discriminated by formation of the atrio-ventricular junction. The cardiac wall consisted of three layers; the inner endocardium, the middle myocardium, and the outer epicardium. The developing endocardium was seen as a chain of single-layered endocardial cells. Along its inner surface, numerous clusters of blood corpuscles were distributed, which seemed to contain some undifferentiated endocardial cells estimated from their characteristic ultrastructure and histological topography. Several blood corpuscles were in directly contact with the myocardium at the missing portions of the developing endocardial cell-chains. Differentiating endocardial cells individually showed roundish, small and large crescent, or flat in shapes. Such a prominent change of cell shapes appeared to be in parallel with their secretory activity during the transformation from the undifferentiated cells to the endocardial cells. Furthermore, immunohistochemistry for FLK1 or CD31, and in situ hybridization assays for flk1-mRNA labeled the cells composing developing endocardial cell-chains. Though these expressional analyses could not document clearly the transition of precursor cells into endocardial cells, the present study provided for the first time some important information regarding the morphological transition process toward endocardial cells at ultrastructural levels. Anat Rec, 299:1080-1089, 2016. © 2016 Wiley Periodicals, Inc.

"The sinusoid" in the liver: lessons learned from the original definition by Charles Sedgwick Minot (1900).

The hepatic sinusoid with its associated sinusoidal cells is a multifunctional cell-complex in the liver. Despite recent advances in research on the hepatic sinusoid, no investigator has played a more basic role in its characterization than Charles Sedgwick Minot (1852-1914), a pioneer who distinguished the sinusoid from the blood-capillary as early as 1900. According to Minot, sinusoids are typically larger in diameter than capillaries, particularly at the early embryonic stage. They closely approach the parenchymal tissue, are formed passively by the adjacent parenchymal tissue, and are on rare occasion surrounded with connective tissue. Sinusoids (sinus-like) are small blood-channels formed by subdivision of the lumen of large blood vessels (sinuses) by the invasion of developing parenchymal cell-cords. Although some of Minot's definitions may no longer be accepted, he described some fundamental and interesting characteristics of sinusoids, to which we have not paid much attention. Here, we have attempted to illustrate lessons we have learned from Minot's view point of sinusoids at this occasion of centenary of his death.

Apoptotic response through a high mobility box 1 protein-dependent mechanism in LPS/GalN-induced mouse liver failure and glycyrrhizin-mediated inhibition.

HMGB1 is a nuclear component involved in nucleosome stabilization and transcription regulation, but extracellularly it is able to serve as a potential late mediator of lethality. In the present study, we explored inflammation-promoting activity of HMGB1 and blockade of extracellular release of HMGB1 by glycyrrhizin (GL) in LPS/GalN-triggered mouse liver injury. At 1 to 10 h after LPS/GalN-treatment, mice were anesthetized to collect blood from heart puncture, and serum transaminase and HMGB1 were evaluated. Administration of LPS/GalN precipitated tissue injury associated with time-dependent alteration in HMGB1 serum levels. At 8 h nuclear immunoreactive products were remarkably reduced and extracellular HMGB1 expression was found exclusively in the pericentral foci. The treatment with GL significantly down-regulated the serum levels of ALT, AST, and HMGB1 in addition to the strong inhibition of tissue injury and extracellular immunoreactivity to HMGB1 and to acetylated-lysine. Furthermore, GL brought about a significant decrease in the number of apoptotic hepatocytes labeled with TUNEL-method. On the basis of these results, three apoptosis-associated genes were identified with microarray analysis and real-time PCR. The ChIP-assay revealed the binding of HMGB1 protein to Gsto1 promoter sequence in LPS/GalN-treated mice and the remarkable decrease in combined HMGB1 protein by GL. The current findings claim that a single injection of LPS/GalN might stimulate apoptosis of hepatocytes through the binding of HMGB1 protein to Gsto1 promoter region and that GL-treatment might prevent the apoptosis and inflammatory infiltrates caused with LPS/GalN-injection by disturbing the binding of HMGB1 protein to Gsto1 promoter sequence.

Antiviral activity of glycyrrhizin against hepatitis C virus in vitro.

Glycyrrhizin (GL) has been used in Japan to treat patients with chronic viral hepatitis, as an anti-inflammatory drug to reduce serum alanine aminotransferase levels. GL is also known to exhibit various biological activities, including anti-viral effects, but the anti-hepatitis C virus (HCV) effect of GL remains to be clarified. In this study, we demonstrated that GL treatment of HCV-infected Huh7 cells caused a reduction of infectious HCV production using cell culture-produced HCV (HCVcc). To determine the target step in the HCV lifecycle of GL, we used HCV pseudoparticles (HCVpp), replicon, and HCVcc systems. Significant suppressions of viral entry and replication steps were not observed. Interestingly, extracellular infectivity was decreased, and intracellular infectivity was increased. By immunofluorescence and electron microscopic analysis of GL treated cells, HCV core antigens and electron-dense particles had accumulated on endoplasmic reticulum attached to lipid droplet (LD), respectively, which is thought to act as platforms for HCV assembly. Furthermore, the amount of HCV core antigen in LD fraction increased. Taken together, these results suggest that GL inhibits release of infectious HCV particles. GL is known to have an inhibitory effect on phospholipase A2 (PLA2). We found that group 1B PLA2 (PLA2G1B) inhibitor also decreased HCV release, suggesting that suppression of virus release by GL treatment may be due to its inhibitory effect on PLA2G1B. Finally, we demonstrated that combination treatment with GL augmented IFN-induced reduction of virus in the HCVcc system. GL is identified as a novel anti-HCV agent that targets infectious virus particle release.

Morphological and microarray analyses of human hepatocytes from xenogeneic host livers.

We previously produced mice with human hepatocyte (h-hep) chimeric livers by transplanting h-heps into albumin enhancer/promoter-driven urokinase-type plasminogen activator-transgenic severe combined immunodeficient (SCID) mice with liver disease. The chimeric livers were constructed with h-heps, mouse hepatocytes, and mouse hepatic sinusoidal cells (m-HSCs). Here, we investigated the morphological features of the chimeric livers and the h-hep gene expression profiles in the xenogeneic animal body. To do so, we performed immunohistochemistry, morphometric analyses, and electron microscopic observations on chimeric mouse livers, and used microarray analyses to compare gene expression patterns in hepatocytes derived from chimeric mouse hepatocytes (c-heps) and h-heps. Morphometric analysis revealed that the ratio of hepatocytes to m-HSCs in the chimeric mouse livers were twofold higher than those in the SCID mouse livers, corresponding to twin-cell plates in the chimeric mouse liver. The h-heps in the chimeric mouse did not show hypoxia even in the twin-cell plate structure, probably because of low oxygen consumption by the h-heps relative to the mouse hepatocytes (m-heps). Immunohistochemical and electron microscopic examinations revealed that the sinusoids in the chimeric mouse livers were normally constructed with h-heps and m-HSCs. However, a number of microvilli projected into the intercellular clefts on the lateral aspects of the hepatocytes, features typical of a growth phase. Microarray profiles indicated that ∼82% of 16 605 probes were within a twofold range difference between h-heps and c-heps. Cluster and principal component analyses showed that the gene expression patterns of c-heps were extremely similar to those of h-heps. In conclusion, the chimeric mouse livers were normally reconstructed with h-heps and m-HSCs, and expressed most human genes at levels similar to those in human livers, although the chimeric livers showed morphological characteristics typical of growth.

The scavenger endothelial cell: a new player in homeostasis and immunity.

To maintain homeostasis, the animal body is equipped with a powerful system to remove circulating waste. This review presents evidence that the scavenger endothelial cell (SEC) is responsible for the clearance of blood-borne waste macromolecules in vertebrates. SECs express pattern-recognition endocytosis receptors (mannose and scavenger receptors), and in mammals, the endocytic Fc gamma-receptor IIb2. This cell type has an endocytic machinery capable of super-efficient uptake and degradation of physiological and foreign waste material, including all major classes of biological macromolecules. In terrestrial vertebrates, most SECs line the wall of the liver sinusoid. In phylogenetically older vertebrates, SECs reside instead in heart, kidney, or gills. SECs, thus, by virtue of their efficient nonphagocytic elimination of physiological and microbial substances, play a critical role in the innate immunity of vertebrates. In major invertebrate phyla, including insects, the same function is carried out by nephrocytes. The concept of a dual-cell principle of waste clearance is introduced to emphasize that professional phagocytes (macrophages in vertebrates; hemocytes in invertebrates) eliminate larger particles (>0.5 µm) by phagocytosis, whereas soluble macromolecules and smaller particles are eliminated efficiently and preferentially by clathrin-mediated endocytosis in nonphagocytic SECs in vertebrates or nephrocytes in invertebrates. Including these cells as important players in immunology and physiology provides an additional basis for understanding host defense and tissue homeostasis.

[Anatomists in the anatomical theater of Tartu (Dorpat) University].

In 1632, King Gustav II Adolphus of Sweden founded Academia Gustaviana,the predecessor of the present Tartu University in Estonia. After the reopening of the University in 1802, the development of the Faculty of Medicine started. The number of outstanding anatomists; Burdach, von Baer, Reichert, Bidder, Reissner, Kupffer, and Rauber made various discoveries at the Anatomical Theater (Theatrum Anatomicum). The present paper acquaints readers with profiles of these anatomists and their main contributions, and attempts to consider reasons of a quick development of Tartu University during rather a short period in the 19th century.

[Browicz's phagocytic cells and Kupffer's 'so-called stellate cells'].

Kupffer (1876) attempted to demonstrate nerve fibers in the liver, using the gold chloride method and found the star-shaped perisinusoidal cells by chance. He named these cells 'Sternzellen' (stellate cells). The stellate cells have been studied enthusiastically within the past thirty years. It has been clarified that these cells are the same cells reported as Ito's 'fat-storing cells', as well as Suzuki's 'interstitial cells', store vitamin A in the lipid droplets, and produce collagen type I, III, and IV and intercellular matrix, playing an important role in fibrogenesis in the liver. Kupffer (1898) changed his earlier opinion, and concluded that the 'so-called stellate cells' were the special endothelial cells of hepatic sinusoids, which incorporate foreign substance. Though Kupffer's revised opinion contained a serious misunderstanding, his new concept was accepted widely for several decades. Browicz (1898) at Kracow in Poland, reported phagocytic cells within the lumen of hepatic sinusoids. However, his report was given but scant attention. In this paper, the author introduces Browicz's report and considers the reason why his report has been disregarded.

Protection by glycyrrhizin against warm ischemia-reperfusion-induced cellular injury and derangement of the microcirculatory blood flow in the rat liver.

BACKGROUND/AIM: The mechanism by which ischemia-reperfusion (I/R)-induced derangement of the hepatic microcirculation leads to tissue injury is not fully understood. We postulated that alterations to the hepatic microcirculation, including hemodynamic derangement and increased leukocyte-endothelium interaction, play a role, and that glycyrrhizin exerts its hepatoprotective effects, in part, by reducing these microcirculatory changes. MATERIALS AND METHODS: Wistar rats were subjected to 30-60 minutes segmental hepatic ischemia, followed by 120 minutes of reperfusion. Glycyrrhizin was administered prior to ischemia. Using intravital fluorescence microscopy, the administration of fluorescein isothiocyanate-conjugated erythrocytes allowed the measurement of erythrocyte-velocity (RBC(vel)), lobular, and sinusoidal perfusion. Bleb formation was observed by electron microscopy. Blood and tissue were taken for the assessment of liver injury. RESULTS: Glycyrrhizin reduced I/R-induced liver injury (histology, liver enzymes) and reduced hepatocyte apoptosis (TUNEL, caspase-3 activity). Glycyrrhizin inhibited hepatocyte bleb formation and reversed the I/R-induced reductions in lobular perfusion and RBC(vel). Leukocyte rolling and adherence in postsinusoidal venules and neutrophil infiltration were reduced by glycyrrhizin. I/R-induced elevation in HMGB1 was prevented by glycyrrhizin. CONCLUSIONS: Early bleb formation with deranged microcirculatory flow and leukocyte-endothelium interaction would appear to contribute to I/R-induced hepatocellular injury. Glycyrrhizin exerts its hepatoprotective effect by preventing these changes, in addition to a direct cellular effect.

Glycyrrhizin prevents of lipopolysaccharide/D-galactosamine-induced liver injury through down-regulation of matrix metalloproteinase-9 in mice.

Glycyrrhizin, a biological active compound isolated from the liquorice root, has been used as a treatment for chronic hepatitis. We have examined the involvement of matrix metalloproteinase (MMP)9 in the development of lipopolysaccharide (LPS) and D-galactosamine (GalN)-induced liver injury in mice. We also investigated the effect of glycyrrhizin on expression of MMP-9 in this model. Levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) increased after LPS/ GalN treatment. Expression of MMP-9 mRNA and protein was markedly up-regulated in liver tissues 6-8 h after LPS/GalN treatment. Pretreatment with glycyrrhizin (50 mg kg(-1)) and the MMP inhibitor (5 mg kg(-1)) suppressed increases in serum levels of ALT and AST in mice treated with LPS/GalN. Furthermore, glycyrrhizin inhibited levels of both mRNA and protein for MMP-9. Immunohistochemical reaction for MMP-9 was observed in macrophages/monocytes infiltrated in the inflammatory area of liver injury. Glycyrrhizin reduced the infiltration of inflammatory cells and immunoreactive MMP- 9 in liver injury. The results indicated that MMP-9 played a role in the development of LPS/GalN- induced mouse liver injury, and suggested that an inhibition by glycyrrhizin of the acute liver injury may have been due to a down-regulation of MMP-9.

The inhibition of apoptosis by glycyrrhizin in hepatic injury induced by injection of lipopolysaccharide / D-galactosamine in mice.

The inhibition of apoptosis by glycyrrhizin (GL) in hepatic injury induced by injection of lipopolysaccharide (LPS)/D-galactosamine (D-GalN) was examined in the present study. Morphological and biochemical analyses of LPS/D-GalN-induced mouse liver injury revealed that apoptosis occurred exclusively in injured hepatocytes of the centrilobular area. The degree of hepatic injury was associated with a substantial number of hepatocytes undergoing apoptosis. Transaminase levels were significantly increased at 6 to 8 h after the injection of LPS/D-GalN compared with controls. GL inhibited the elevation of serum transaminase levels when it was given to mice at 30 min before the administration of LPS/D-GalN. Morphological analyses using the TUNEL-method showed GL significantly reduced the number of TUNEL-labeled cells in acute hepatitis induced with LPS/D-GalN-treatment. Cells from the pericentral hepatic injury region were dissected out using a microdissection-method, and the DNA-ladder was clearly documented. Furthermore, results obtained through the TUNEL-method were confirmed with an oligonucleosome-bound DNA ELISA. From the current results, it seems reasonable to conclude that the protective role of GL in LPS/D-GalN-induced liver injury is performed through the inhibition of hepatic apoptosis.

Inhibitory effect of glycyrrhizin on lipopolysaccharide and d-galactosamine-induced mouse liver injury.

The effects of glycyrrhizin isolated from licorice root were investigated on acute hepatitis induced by lipopolysaccharide (LPS) and d-galactosamine in mice. Serum alanine aminotransferase (ALT) activity was markedly increased 6 h to 8 h after administration of LPS/d-galactosamine. Levels in serum of cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-10 and IL-12 reached a maximum by 2 h, whereas levels of IL-18, as well as of ALT, were maximal at 8 h. Glycyrrhizin (ED(50): 14.3 mg/kg) inhibited the increase in ALT levels when it was given to mice at 30 min before administration of LPS/d-galactosamine. Inflammatory responses, including infiltration of neutrophils and macrophages in the liver injury, were modulated by glycyrrhizin. Increases in ALT levels were reduced by an administration of glycyrrhizin at 10 min and 60 min but not 3 h, even after LPS/d-galactosamine treatment. However, glycyrrhizin had no effect on the production of TNF-alpha, IL-6, IL-10 and IL-12, whereas it significantly inhibited IL-18 production. Exogenous IL-18 further increased the elevation in ALT levels in mice treated with LPS/d-galactosamine. Glycyrrhizin completely suppressed the effect of IL-18 of increasing ALT levels. IL-18 was detected by immunohistochemistry in inflammatory cells such neutrophils and macrophages in liver injury. Glycyrrhizin reduced the responsiveness of cells to IL-18 in the liver injury. These results suggest that glycyrrhizin inhibits the LPS/d-galactosamine-induced liver injury through preventing inflammatory responses and IL-18 production. Furthermore, it seems that glycyrrhizin prevents IL-18-mediated inflammation in liver injury.

Characterization of vitamin A-storing cells in mouse fibrous kidneys using Cygb/STAP as a marker of activated stellate cells.

The expression of the cytoglobin/stellate cell activation-associated protein (Cygb/STAP) was recently confirmed in all splanchnic vitamin A-storing cells--including hepatic stellate cells (HSCs)--in normal conditions. In the hepatic fibrous lesion, the expression of Cygb/STAP has been shown to be upregulated in activated HSCs and myofibroblasts (MFs), which have synthesized extracellular matrices. Furthermore, splanchnic vitamin A-storing cells have been reported to be distributed in the kidney. In this study, we clarify the contribution of vitamin A-storing cells to renal fibrosis by focusing on Cygb/ STAP. Adult mice were subjected to unilateral ureteral obstruction (UUO) and kidneys were harvested 1, 3, 7, and 10 days after UUO. Numbers of Cygb/STAP-immunopositive cells as well as Cygb/STAP mRNA 3 days after UUO (UUO day 3 kidney) increased. Vitamin A-autofluorescence was observed in intertubular spaces of controls but gradually declined in a time-dependent manner after UUO. Cygb/STAP+ cells were not completely identical with alpha-smooth muscle actin (alphaSMA)-positive cells in the control or UUO day 7 kidneys. Immunohistochemical analysis for Cygb/STAP and fibulin-2 (Fib), a specific marker for distinguishing MFs from activated HSCs, revealed that the number of Fib+STAP+ cells (MFs) and Fib-STAP+ cells (splanchnic vitamin A-storing cells) significantly increased in UUO day 3 and UUO day 7 kidneys compared with the controls. Our present findings support the concept that Cygb/STAP can be a unique marker for splanchnic fibroblast-like cells, namely the vitamin A-storing cell lineage, and suggest that splanchnic vitamin A-storing cells contribute to renal fibrogenesis in the obstructed kidney.

A new mouse model for intraportal islet transplantation with limited hepatic lobe as a graft site.

Intraportal site is the standard for grafting in clinical islet transplantation. In the mouse model, the whole liver has been used as the grafting site to mimic clinical islet transplantation. However, this model lacks the potency to directly assess the contribution of the islet graft to diabetes control. Only demonstrating the immediate recurrence of diabetes in a surviving recipient after the removal of the islet graft can validate this assessment. In this study, we develop a mouse model of intraportal islet transplantation equipped with the potency of this assessment by injecting islets selectively into the right hepatic lobe under temporal clamp of the left portal vein. The mouse of this model survives after the right hepatectomy by which the islet graft is removed. This model can be applied to investigate both the specific graft-recipient interaction in the liver and the islet graft contribution to the control of diabetes.