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 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 ... -  《-》

A protective effect of pyrrolidine dithiocarbamate in a rat model of liver cirrhosis.

Nuclear factor kappa B (NF-kappaB) activation, proinflammatory cytokines, and reactive oxygen species have been implicated as mediators of liver injury and fibrogenesis. We have shown recently that pyrrolidine dithiocarbamate (PDTC), an antioxidant and inhibitor of NF-kappaB activation, was protective in a rat model of acute liver failure. The aim of the present study was to examine the efficacy of PDTC in a chronic rat model of thioacetamide (TAA)-induced hepatic fibrosis. Liver cirrhosis was induced by intraperitoneal injections of TAA (200 mg/kg) twice weekly for 12 weeks. Two groups of rats also received PDTC (either 20 or 60 mg/kg, i.p. for 12 weeks). TAA administration induced liver cirrhosis, which was inhibited by PDTC in a dose-dependent manner. The histopathologic score of fibrosis, the spleen weight, and hepatic hydroxyproline were significantly lower in the rats treated with TAA+PDTC compared with TAA only (P<0.001). The hepatic levels of thiobarbituric acid reactive substances and protein carbonyls after 12 weeks of treatment were also lower in the rats treated with TAA+PDTC (P=0.02 and 0.01, respectively), indicating reduced oxidative stress. Immunohistochemical studies and in situ hybridization demonstrated inhibition of stellate cell (alpha smooth muscle actin positive) activation, tissue inhibitor of metalloproteinase-2, and collagen alpha1(I) gene expression in the livers of the PDTC-treated rats. As determined by Northern blot analysis, PDTC had no inhibitory effect on collagen alpha1(I) gene expression in the rat hepatic stellate cells-T6 cells in vitro. PDTC inhibits the development of liver cirrhosis in TAA-treated rats. The mechanism of action is associated with decreased oxidative stress and hepatic necroinflammation.

Bruck R ，Schey R ，Aeed H ，Hochman A ，Genina O ，Pines M ... -  《-》