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

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. To evaluate the potential ER stress-mediated apoptosis of HSCs and fibrosis resolution 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. 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. 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.

De Minicis S ，Candelaresi C ，Agostinelli L ，Taffetani S ，Saccomanno S ，Rychlicki C ，Trozzi L ，Marzioni M ，Benedetti A ，Svegliati-Baroni G ... -  《-》

Ursolic acid ameliorates hepatic fibrosis in the rat by specific induction of apoptosis in hepatic stellate cells.

Specific induction of cell death in activated hepatic stellate cells (HSCs) is a promising therapeutic strategy for hepatic fibrosis. In this study, we evaluated the cell-killing effect of ursolic acid (UA), a pentacyclic triterpenoid, in activated HSCs both in vitro and in vivo. Culture-activated rat HSCs were treated with UA (0-40μM), and the mechanisms of cell death were evaluated. The cell killing effect of UA on activated HSCs in rats chronically treated with thioacetamide (TAA) was detected by dual staining of TdT-mediated dUTP nick-end labeling (TUNEL) and smooth muscle α-actin (αSMA) immunohistochemistry, and resolution of hepatic fibrosis was evaluated. Further, the protective effects of UA on progression of hepatic fibrosis caused by TAA and bile duct ligation (BDL) were evaluated. UA induced apoptotic cell death in culture-activated HSCs, but not in isolated hepatocytes and quiescent HSCs. Mitochodrial permeability transition (MPT) preceded the cleavage of caspase-3 and -9 following UA treatment. UA also decreased phosphorylation levels of Akt, and diminished nuclear localization of NFκB in these cells. In rats pretreated with TAA for 6weeks, a single injection of UA induced remarkable increases in TUNEL- and αSMA-dual-positive cells in 24h, and significant regression of hepatic fibrosis within 48h. Moreover, UA ameliorated hepatic fibrogenesis caused by both chronic TAA administration and BDL. UA ameliorated experimental hepatic fibrosis most likely through specific induction of apoptosis in activated HSCs. It is therefore postulated that UA is a potential therapeutic reagent for resolution of hepatic fibrosis.

Wang X ，Ikejima K ，Kon K ，Arai K ，Aoyama T ，Okumura K ，Abe W ，Sato N ，Watanabe S ... -  《-》

Icaritin induces cell death in activated hepatic stellate cells through mitochondrial activated apoptosis and ameliorates the development of liver fibrosis in rats.

Icaritin is an active ingredient extracted from the plant Herba Epimedium Sagittatum (Sieb. et Zucc.) Maxim. The purpose of this study is to investigate the effects and mechanisms of icaritin-induced cell death in activated hepatic stellate cells (HSCs) and ameliorating the development of liver fibrosis in rats. : In vitro, icaritin-induced cell death rates in HSC-T6 (rat) and LX-2 (human) HSC lines as well as normal hepatocyte cell lines HL-7702 (L02) and WRL-68 were assayed by MTT method, and the apoptotic ratios were detected by both flow cytometry and the Annexin-V-FITC Apoptosis Detection Kit. A Whole Rat Genome Microarray Kit was used to identify expression of interest genes through fold-change screening. In vivo study, experimental liver fibrosis models were built by carbon tetrachloride (CCl(4)) or common bile duct ligation (CBDL) in Wistar rats. Icaritin (1mg/kg/day, three days a week) was administered by gastric gavage for four weeks (n=6 per group). At the end of the study, serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) as well as the contents of hydroxyproline and collagen I in liver tissues were measured. Histopathological changes and the distribution of activated HSCs were observed in the liver tissues using hematoxyline-eosin (HE) staining and immunohistochemical staining for α-smooth muscle actin (α-SMA). Icaritin induced apoptosis in HSC-T6 and LX-2 in a concentration- and time-dependent manner with little toxicity to normal hepatocyte cell lines. The IC(50) of icaritin in HSC-T6 was 12.83 μM at 48 h. Apoptotic ratio of HSC-T6 treated with 24 μM icaritin was 20.19%, and the G2 phase of the cell cycle did not occur (P<0.05). Gene analysis showed that icaritin up-regulated Bak-1, Bmf and Bax expression while significantly down-regulated Bcl-2 expression (vs. control group, P<0.01). These results suggested that mitochondrial pathway played an important role in icaritin-induced apoptosis in activated HSCs. In vivo results showed that icaritin reduced the number of activated HSCs, and brought the elevated levels of AST, ALT, hydroxyproline and collagen I to normal or near normal values (vs. model group, P<0.05). Icaritin can induce cell death in activated HSCs through mitochondria-mediated apoptosis, ameliorate the progression of hepatic fibrosis in rats, and could be a promising drug for treating liver fibrosis.

Li J ，Liu P ，Zhang R ，Cao L ，Qian H ，Liao J ，Xu W ，Wu M ，Yin Z ... -  《-》

Celecoxib induces hepatic stellate cell apoptosis through inhibition of Akt activation and suppresses hepatic fibrosis in rats.

Paik YH ，Kim JK ，Lee JI ，Kang SH ，Kim DY ，An SH ，Lee SJ ，Lee DK ，Han KH ，Chon CY ，Lee SI ，Lee KS ，Brenner DA ... -  《-》

Endoplasmic Reticulum Stress in Hepatic Stellate Cells Promotes Liver Fibrosis via PERK-Mediated Degradation of HNRNPA1 and Up-regulation of SMAD2.

Endoplasmic reticulum (ER) stress has been implicated in a variety of diseases. Hepatic stellate cells (HSCs) contribute to the development of liver fibrosis. Information on the link between ER stress and HSC activation is scarce. We investigated the effects of ER stress in HSCs on the progression of liver fibrosis and the regulation of this process in cells and mice. Proteins and messenger RNAs were measured in 2 sets of liver samples (n = 25 and n = 44) collected from patients with chronic hepatitis C virus infection and/or fibrosis. ER stress was induced in cells and mice using chemical agents. Lentiviral vectors were constructed to express glucose-regulated protein 78 (GRP78; also known as HSPA5) or heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) from the α-smooth muscle actin promoter and injected into C57BL/6 mice for HSC-specific gene expression. Liver tissues and HSCs were collected from mice or rats and analyzed using immunoblottings and quantitative reverse-transcription polymerase chain reaction. LX-2 cells were transfected with small interfering RNAs, microRNA mimics, or overexpression vectors. Hepatic ER stress was much higher in liver tissues from patients with severe vs mild fibrosis. ER stress induced fibrogenic genes in HSCs. Targeted lentiviral delivery of glucose-regulated protein 78 to HSCs in mice reduced fiber accumulation in liver. Levels of SMAD2, but not SMAD3, were increased in fibrotic liver tissues from patients or mice exposed to ER stress; small interfering RNA-mediated knockdown of SMAD2 reduced ER stress-mediated activation of HSCs. In rat HSCs, ER stress increased levels of SMAD2 messenger RNA by decreasing levels of microRNA 18a (MIR18A), an inhibitor of SMAD2 expression, rather than transactivating the SMAD2 gene. ER stress-activated PKR-like endoplasmic reticulum kinase, also known as EIF2AK3 (PERK) phosphorylated HNRNPA1, a protein required for the maturational processing of primary MIR18A, at Thr51, accelerating its degradation. Overexpression of HNRNPA1 (or its T51A mutant) in HSCs of mice inhibited liver fibrosis. Severe fibrotic liver tissues from patients had increased levels of phosphorylated PERK and reduced levels of HNRNPA1 in HSCs, compared with mild fibrotic liver tissues. ER stress in HSCs promotes liver fibrosis by inducing overexpression of SMAD2, via dysregulation of MIR18A; this dysregulation is mediated by PERK phosphorylation and destabilization of HNRNPA1.

Koo JH ，Lee HJ ，Kim W ，Kim SG ... -  《-》