Endoplasmic Reticulum stress induces hepatic stellate cell apoptosis and contributes to fibrosis resolution.

BACKGROUND: Survival of hepatic stellate cells (HSCs) is a hallmark of liver fibrosis, while the induction of HSC apoptosis may induce recovery. Activated HSC are resistant to many pro-apoptotic stimuli. To this issue, the role of Endoplasmic Reticulum (ER) stress in promoting apoptosis of HSCs and consequently fibrosis resolution is still debated. AIM: To evaluate the potential ER stress-mediated apoptosis of HSCs and fibrosis resolution METHODS: HSCs were incubated with the ER stress agonists, tunicamycin or thapsigargin. In vivo, HSC were isolated from normal, bile duct-ligated (BDL) and bile duct-diverted (BDD) rats. RESULTS: In activated HSC, the specific inhibitor of ER stress-induced apoptosis, calpastatin, is significantly increased vs. quiescent HSCs. Calpain is conversely reduced in activated HSCs. This pattern of protein expression provides HSCs resistance to the ER stress signals of apoptosis (apoptosis-resistant phenotype). However, both tunicamycin and thapsigargin are able to induce apoptosis in HSCs in vitro, completely reversing the calpain/calpastatin pattern expression. Furthermore, in vivo, the fibrosis resolution observed in rat livers subjected to bile duct ligation (BDL) and subsequent bile duct diversion (BDD), leads to fibrosis resolution through a mechanism of HSCs apoptosis, potentially associated with ER stress: in fact, BDD rat liver shows an increased number of apoptotic HSCs associated with reduced calapstatin and increased calpain protein expression, leading to an apoptosis-sensible phenotype. CONCLUSIONS: ER stress sensitizes HSC to apoptosis both in vitro and in vivo. Thus, ER stress represents a key target to trigger cell death in activated HSC and promotes fibrosis resolution

HCC Development Is Associated to Peripheral Insulin Resistance in a Mouse Model of NASH

NAFLD is the most common liver disease worldwide but it is the potential evolution to NASH and eventually to hepatocellular carcinoma (HCC), even in the absence of cirrhosis, that makes NAFLD of such clinical importance. Aim: we aimed to create a mouse model reproducing the pathological spectrum of NAFLD and to investigate the role of possible co-factors in promoting HCC. Methods: mice were treated with a choline-deficient L-amino-acid-defined-diet (CDAA) or its control (CSAA diet) and subjected to a low-dose i.p. injection of CCl4 or vehicle. Insulin resistance was measured by the euglycemic-hyperinsulinemic clamp method. Steatosis, fibrosis and HCC were evaluated by histological and molecular analysis. Results: CDAA-treated mice showed peripheral insulin resistance at 1 month. At 1-3 months, extensive steatosis and fibrosis were observed in CDAA and CDAA+CCl4 groups. At 6 months, equal increase in steatosis and fibrosis was observed between the two groups, together with the appearance of tumor. At 9 months of treatment, the 100% of CDAA+CCl4 treated mice revealed tumor versus 40% of CDAA mice. Insulin-like Growth Factor-2 (IGF-2) and Osteopontin (SPP-1) were increased in CDAA mice versus CSAA. Furthermore, Immunostaining for p-AKT, p-c-Myc and Glypican-3 revealed increased positivity in the tumors. Conclusions: the CDAA model promotes the development of HCC from NAFLD-NASH in the presence of insulin resistance but in the absence of cirrhosis. Since this condition is increasingly recognized in humans, our study provides a model that may help understanding mechanisms of carcinogenesis in NAFLD

Lack of NLRP3-inflammasome leads to gut-liver axis derangement, gut dysbiosis and a worsened phenotype in a mouse model of NAFLD.

Abstract Non-Alcoholic Fatty Liver Disease (NAFLD) represents the most common form of chronic liver injury and can progress to cirrhosis and hepatocellular carcinoma. A “multi-hit” theory, involving high fat diet and signals from the gut-liver axis, has been hypothesized. The role of the NLRP3-inflammasome, which senses dangerous signals, is controversial. Nlrp3−/− and wild-type mice were fed a Western-lifestyle diet with fructose in drinking water (HFHC) or a chow diet. Nlrp3−/−-HFHC showed higher hepatic expression of PPAR γ2 (that regulates lipid uptake and storage) and triglyceride content, histological score of liver injury and greater adipose tissue inflammation. In Nlrp3−/−-HFHC, dysregulation of gut immune response with impaired antimicrobial peptides expression, increased intestinal permeability and the occurrence of a dysbiotic microbiota led to bacterial translocation, associated with higher hepatic expression of TLR4 (an LPS receptor) and TLR9 (a receptor for double-stranded bacterial DNA). After antibiotic treatment, gram-negative species and bacterial translocation were reduced, and adverse effects restored both in liver and adipose tissue. In conclusion, the combination of a Western-lifestyle diet with innate immune dysfunction leads to NAFLD progression, mediated at least in part by dysbiosis and bacterial translocation, thus identifying new specific targets for NAFLD therapy

Ageing-related expression of Twinfilin-1 regulates cholangiocyte biological response to injury

Disorders of the biliary tree develop and progress differently according to patient age. It is currently not known whether the ageing process affects the response to injury of cholangiocytes

An oestrogen receptor β-selective agonist exerts anti-neoplastic effects in experimental intrahepatic cholangiocarcinoma

Background: Cholangiocarcinoma cells over-express oestrogen receptor-beta, which displays anti-proliferative and pro-apoptotic effects. Aim: To evaluate the effects of a newly developed and highly selective oestrogen receptor-beta agonist (KB9520) on experimental intrahepatic cholangiocarcinoma. Methods: In vitro, the effects of KB9520 on apoptosis and proliferation of HuH-28 cells, HuH-28 cells with selective oestrogen receptor-beta silencing (by small interfering RNA), HepG2 cells (oestrogen receptor-alpha and oestrogen receptor-beta negative) and HepER3 cells (HepG2 cells transformed to stably express oestrogen receptor-alpha) were evaluated. In vivo, the effects of KB9520 on experimental intrahepatic cholangiocarcinoma, induced by thioacetamide administration were tested. Results: In vitro, KB9520 induced apoptosis and inhibited proliferation of HuH-28 cells. KB9520 effects were absent in cells lacking oestrogen receptor-alpha and beta (HepG2) and in cells expressing only oestrogen receptor-alpha (HepER3); its pro-apoptotic effect was impaired in cells where oestrogen receptor-beta expression was decreased by specific small interfering RNA. In vivo, KB9520 inhibited experimental intrahepatic cholangiocarcinoma development in thioacetamide-treated rats and promoted tumour regression in rats where tumour was already established. In treated animals, tumour areas showed reduced proliferation but increased apoptosis. Conclusions: KB9520 induced apoptosis in cholangiocarcinoma by selectively acting on oestrogen receptor-beta, suggesting that oestrogen receptor-beta selective agonists may be a novel and effective therapeutic option for the medical treatment of intrahepatic cholangiocarcinoma. (C) 2011 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved