Activation of hepatic stem cells compartment during hepatocarcinogenesis in a HBsAg HBV-transgenic mouse model

Abstract Chronic infection of Hepatitis B Virus (HBV) is one of the highest risk factors of hepatocellular carcinoma (HCC). The accumulation of HBV surface antigen (HBsAg) into hepatocytes induces inflammation and oxidative stress, impairing their replicative ability and allowing the activation of the hepatic stem cells (SC) compartment. This study aimed to understand the involvement of SC during hepatocarcinogenesis in HBsAg-related liver damage, from early injury until HCC. HBsAg-transgenic (TG) and wild type (WT) mouse were followed at several stages of the liver damage: inflammation, early hepatocytes damage, dysplasia, and HCC. Serum transaminases, liver histology, and diagnostic data were collected. The expressions of SC and cancer stem cells (CSC) markers was analyzed by RT-qPCR, immunohistochemistry and Western blot. Starting from 3 months, TG animals showed a progressive liver damage characterized by transaminases increase. The up-regulations of SCs markers Cd34 and Sca-1 started from the beginning of the inflammatory stage while progressive increase of Krt19 and Sox9 and CSCs markers Epcam and Cd133 from early hepatic injury. The expressions of Cd133, Cd34, and Afp were significantly higher in HCC compared to paired non-HCC tissue, in contrast to Epcam and Krt19. Western blot and IHC confirmed the positivity of Cd34 and Cd133 in small cells subpopulation

An animal model for the juvenile non-alcoholic fatty liver disease and non-alcoholic steatohepatitis

11Non Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH) are the hepatic manifestations of the metabolic syndrome; worrisome is the booming increase in pediatric age. To recreate the full spectrum of juvenile liver pathology and investigate the gender impact, male and female C57Bl/6 mice were fed with high fat diet plus fructose in the drinking water (HFHC) immediately after weaning (equal to 3-years old human), and disease progression followed for 16 weeks, until adults (equal to 30-years old human). 100% of subjects of both genders on HFHC diet developed steatosis in 4weeks, and some degree of fibrosis in 8weeks, with the 86% of males and 15% of females presenting a stage 2 fibrosis at 16weeks. Despite a similar final liver damage both groups, a sex difference in the pathology progression was observed. Alterations in glucose homeostasis, dyslipidemia, hepatomegaly and obese phenotype were evident from the very beginning in males with an increased hepatic inflammatory activity. Conversely, such alterations were present in females only at the end of the HFHC diet (with the exception of insulin resistance and the hepatic inflammatory state). Interestingly, only females showed an altered hepatic redox state. This juvenile model appears a good platform to unravel the underlying gender dependent mechanisms in the progression from NAFLD to NASH, and to characterize novel therapeutic approaches.openopenMarin, Veronica; Rosso, Natalia; Dal Ben, Matteo; Raseni, Alan; Boschelle, Manuela; Degrassi, Cristina; Nemeckova, Ivana; Nachtigal, Petr; Avellini, Claudio; Tiribelli, Claudio; Gazzin, SilviaMarin, Veronica; Rosso, NATALIA CAROLINA; DAL BEN, Matteo; Raseni, Alan; Boschelle, Manuela; Degrassi, Cristina; Nemeckova, Ivana; Nachtigal, Petr; Avellini, Claudio; Tiribelli, Claudio; Gazzin, Silvi

Effects of oral administration of silymarin in a juvenile murine model of non-alcoholic steatohepatitis

The increasing prevalence of non-alcoholic fatty liver disease (NAFLD) in adolescents is challenging the global care system. No therapeutic strategies have been de\ufb01ned so far, and changes in the lifestyle remain the only alternative. In this study, we assessed the protective effects of silymarin in a juvenile non-alcoholic steatohepatitis (NASH) model and the in vitro effects on fat-laden human hepatocytes. C57Bl/6 mice were exposed to HFHC diet immediately after weaning. After eight weeks, animals showed histological signs of NASH. Silymarin was added to the HFHC diet, the treatment continued for additional 12 weeks and the effects on BMI, hepatomegaly, visceral fat, lipid pro\ufb01le, transaminases, HOMA-IR, steatosis, in\ufb02ammation, \ufb01brosis, oxidative stress, and apoptosis were determined. The switch from HFHC to control diet was used to mimic lifestyle changes. In vitro experiments were performed in parallel in human hepatocytes. HFHC diet supplemented with silymarin showed a signi\ufb01cant improvement in glycemia, visceral fat, lipid pro\ufb01le, and liver \ufb01brosis. Moreover, it reduced (both in vitro and in vivo) ALT, hepatic in\ufb02ammation, oxidative stress, and apoptosis. Lifestyle changes restored the control group parameters. The data presented show the bene\ufb01cial effects of the oral administration of silymarin in the absence of changes in the dietary habits in a juvenile model of NASH

Effects of oral administration of silymarin in a juvenile murine model of non-alcoholic steatohepatitis

The increasing prevalence of non-alcoholic fatty liver disease (NAFLD) in adolescents is challenging the global care system. No therapeutic strategies have been defined so far, and changes in the lifestyle remain the only alternative. In this study, we assessed the protective effects of silymarin in a juvenile non-alcoholic steatohepatitis (NASH) model and the in vitro effects on fat-laden human hepatocytes. C57Bl/6 mice were exposed to HFHC diet immediately after weaning. After eight weeks, animals showed histological signs of NASH. Silymarin was added to the HFHC diet, the treatment continued for additional 12 weeks and the effects on BMI, hepatomegaly, visceral fat, lipid profile, transaminases, HOMA-IR, steatosis, inflammation, fibrosis, oxidative stress, and apoptosis were determined. The switch from HFHC to control diet was used to mimic lifestyle changes. In vitro experiments were performed in parallel in human hepatocytes. HFHC diet supplemented with silymarin showed a significant improvement in glycemia, visceral fat, lipid profile, and liver fibrosis. Moreover, it reduced (both in vitro and in vivo) ALT, hepatic inflammation, oxidative stress, and apoptosis. Lifestyle changes restored the control group parameters. The data presented show the beneficial effects of the oral administration of silymarin in the absence of changes in the dietary habits in a juvenile model of NASH

Modulation of Mrp1 (ABCc1) and Pgp (ABCb1) by Bilirubin at the Blood-CSF and Blood-Brain Barriers in the Gunn Rat

Accumulation of unconjugated bilirubin (UCB) in the brain causes bilirubin encephalopathy. Pgp (ABCb1) and Mrp1 (ABCc1), highly expressed in the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) respectively, may modulate the accumulation of UCB in brain. We examined the effect of prolonged exposure to elevated concentrations of UCB on expression of the two transporters in homozygous, jaundiced (jj) Gunn rats compared to heterozygous, not jaundiced (Jj) littermates at different developmental stages (2, 9, 17 and 60 days after birth). BBB Pgp protein expression was low in both jj and Jj pups at 9 days (about 16–27% of adult values), despite the up-regulation in jj animals (2 and 1.3 fold higher than age matched Jj animals at P9 and P17–P60, respectively); Mrp1 protein expression was barely detectable. Conversely, at the BCSFB Mrp1 protein expression was rather high (60–70% of the adult values) in both jj and Jj at P2, but was markedly (50%) down-regulated in jj pups starting at P9, particularly in the 4th ventricle choroid plexuses: Pgp was almost undetectable. The Mrp1 protein down regulation was accompanied by a modest up-regulation of mRNA, suggesting a translational rather than a transcriptional inhibition. In vitro exposure of choroid plexus epithelial cells obtained from normal rats to UCB, also resulted in a down-regulation of Mrp1 protein. These data suggest that down-regulation of Mrp1 protein at the BSCFB, resulting from a direct effect of UCB on epithelial cells, may impact the Mrp1-mediated neuroprotective functions of the blood-cerebrospinal fluid barrier and actually potentiate UCB neurotoxicity

Hyaluronic acid inhibition by 4-methylumbelliferone reduces the expression of cancer stem cells markers during hepatocarcinogenesis

Abstract Hyaluronic acid (HA) is a glycosaminoglycan of extracellular matrix related to cell surface which interacts with various cell types. To understand the role of HA during hepatocarcinogenesis, we assessed the effect of the inhibition of HA deposition and its association with heterogeneous hepatocellular carcinoma (HCC) cells. In this study, we used transgenic mice C57BL/6J-Tg(Alb1HBV)44Bri/J (HBV-TG) and normal C57BL/6 J (WT) for in vivo study, while HCC cells Huh7 and JHH6 as in vitro models. Both models were treated with an HA inhibitor 4-methylumbelliferone (4MU). We observed that 4MU treatments in animal model down-regulated the mRNA expressions of HA-related genes Has3 and Hyal2 only in HBV-TG but not in normal WT. As observed in vivo, in HCC cell lines, the HAS2 mRNA expression was down-regulated in Huh7 while HAS3 in JHH6, both with or without the presence of extrinsic HA. Interestingly, in both models, the expressions of various cancer stem cells (CD44, CD90, CD133, and EpCAM) were also decreased. Further, histological analysis showed that 4MU treatment with dose 25 mg/kg/day reduced fibrosis, inflammation, and steatosis in vivo, in addition to be pro-apoptotic. We concluded that the inhibition of HA reduced the expressions of HA-related genes and stem cells markers in both models, indicating a possible modulation of cells-to-cells and cells-to-matrix interaction

Blood brain barrier localization in rat brain, and microvessels isolated from rats.

<p>Cartoon representation of the lateral, 3^rd, 4^th ventricle choroid plexuses (LV CP, 3^rd V CP and 4^thV CP, respectively) and microvessels (MVs) localization in rat brain (A). Representative microvessels freshly isolated from rat brains. Freshly isolate MVs in 0.1% albumin in Krebbs-Ringer buffer were placed on a microscope slide and directly observed by phase contrast microscopy: In B scale bar 100 µm; in C, D, E, F and G scale bar 25 µm. P: post-natal age in days. jj: hyperbilirubinemic rats; Jj: controls.</p

Primers used for transcript analysis, amplicon length and amplification efficiency.

<p>Primers used for transcript analysis, amplicon length and amplification efficiency.</p

Representative Western blot for Pgp, Mrp1 and Actin on Gunn preparations.

<p>Pgp expression was analyzed on microvessels (MVs) preparations (A, upper panel). Mrp1 was quantified in lateral and 4^th ventricle (LV and 4^thV) choroid plexuses (B, upper panel). Actin staining (both A and B, lower panel) was used to normalize the amount of samples loaded. For the quantification procedure, see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016165#s2" target="_blank">material and methods</a> section.</p