Isoliquiritigenin inhibits cell proliferation by a heme oxygenase-dependent pathway in rat hepatic stellate cells.

We evaluated whether the antiproliferative effects of isoliquiritigenin (ISL) on rat hepatic stellate cells (HSCs) are related to the induction of heme oxygenase 1 (HO-1) expression. ISL significantly inhibited serum- or growth factor-induced HSC proliferation. The inhibition of platelet-derived growth factor (PDGF)-induced proliferation by ISL was associated with the mitogen-activated protein kinase and phosphatidylinositol 3-kinase-Akt-p70 (S6K) pathways. ISL induced the expression of HO-1 in HSCs. Using the chemical inhibitor tin protoporphyrin, we also found that the inhibitory action of ISL on PDGF-induced proliferation is mediated by HO-1. These data suggest that HO-1 expression is responsible for the antiproliferative effect of ISL on HSCs.

Gene expression profiles during the activation of rat hepatic stellate cells evaluated by cDNA microarray.

Hepatic stellate cells (HSCs) are activated by producing potentially injurious connective tissue components during hepatic fibrosis, thereby exerting a pivotal action in the development of liver fibrogenesis. The aim of this study was to investigate differences in gene expression patterns during the activation of HSCs using complementary cDNA microarrays. HSCs were isolated from normal rat livers and cultured for 0 (3 h), 3, 5 and 7 d. RNA was extracted from cultured cells at each point. The target RNA was hybridized to gene-specific sequence probes immobilized on chips. The hybridization signal was assessed using a confocal laser scanner. Comparison of hybridization signals and patterns allows the identification of mRNAs that are expressed differentially. Statistical analysis was used to classify and cluster the genes according to their up- or downregulation. As a result, 33 upregulated early-stage and 36 upregulated late-stage gene candidates were identified. This time-based study revealed a number of newly discovered genes involved in fibrogenesis during the activation of HSCs.

2',4',6'-Tris(methoxymethoxy) chalcone attenuates hepatic stellate cell proliferation by a heme oxygenase-dependent pathway.

Proliferation of hepatic stellate cells (HSCs) is central for the development of fibrosis during liver injury. We have shown previously that butein (3,4,2',4'-tetrahydroxychalcone) suppresses myofibroblastic differentiation of rat HSCs. Our aim in this study was to determine whether a new synthetic chalcone derivative, 2',4',6'-tris(methoxymethoxy) chalcone (TMMC) inhibits HSC proliferation induced by serum- or platelet-derived growth factor (PDGF). TMMC significantly inhibited growth factor-induced HSC proliferation. The inhibition of PDGF-induced proliferation by TMMC was associated with the phosphatidylinositol 3-kinase-Akt-p70(S6K) pathways. TMMC induced the expression of heme oxygenase 1 (HO-1) in HSCs. Using the chemical inhibitor tin protoporphyrin, we also found that the inhibitory action of TMMC on PDGF-induced proliferation is mediated by HO-1. Glutathione (GSH) depletion produced by TMMC activated extracellular signal-regulated kinase (ERK), which led to c-Fos expression and transactivation of activator protein 1 (AP-1) and HO-1 gene expression in the HSCs. These results demonstrate that TMMC preferentially activates ERK and that this activation leads to the transcriptional activation of AP-1 and consequently to HO-1 expression. HO-1 expression might be responsible for the antiproliferative effect of TMMC on HSCs.

Tetrandrine induces apoptosis in hepatic stellate cells.

We have previously reported that tetrandrine reduced hepatic stellate cell activation and collagen accumulation in liver fibrosis induced by biliary obstruction. In the present study, we examined the apoptosis-inducing effect of tetrandrine on activated hepatic stellate cells, as the therapeutic goal in hepatic fibrosis is to eliminate the activated hepatic stellate cells by apoptosis. We used rat hepatic stellate cells transformed by Simian virus 40 (T-HSC/Cl-6) to overcome the limitations inherent in studying primary cultures of hepatic stellate cells. Tetrandrine treatment at doses of 25 and 50 microg/ml for 12 h induced apoptosis as confirmed by DNA fragmentation and increased sub-G1 DNA content as detected by flow cytometric analysis. Tetrandrine also induced the activation of caspase-3 protease and subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. In conclusion, our results demonstrate that tetrandrine induces apoptosis of T-HSC/Cl-6 cells, and these results should contribute to the development of new agents for the treatment of hepatic fibrosis.

Butein suppresses myofibroblastic differentiation of rat hepatic stellate cells in primary culture.

Hepatic stellate cells play a key role in the pathogenesis of hepatic fibrosis. In this study, we investigate the inhibitory effect of butein on the activation and proliferation of rat primary cultured hepatic stellate cells. Possible cytotoxic effects were measured on stellate cells and hepatocytes using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of butein on the production of collagen and smooth muscle alpha-actin proteins were examined at the same concentration, by western blot. The effects of butein on alpha1(I) collagen, tissue inhibitor of metalloproteinase-1, and metalloproteinase-13 gene expression in activated stellate cells were investigated by measuring mRNA levels using reverse transcription polymerase chain reaction. The effect of butein on DNA synthesis was also determined. Butein, at a concentration of 1 microg mL(-1), reduced DNA synthesis without affecting cell viability, and downregulated smooth muscle alpha-actin and type-I collagen expression, and alpha1(I) collagen and tissue inhibitor of metalloproteinase-1 mRNA expression, while treatment with butein induced metalloproteinase-13 mRNA expression. These findings suggest that butein is a potent inhibitor of stellate cell transformation.

The chalcone butein from Rhus verniciflua shows antifibrogenic activity.

Butein is known to be the major component of the bark of Rhus verniciflua Stokes (Anacardiaceae). The aim of this work was to investigate the effects of butein on liver fibrosis induced by carbon tetrachloride (CCl4) in rats, and to explore its antifibrogenic mechanism. Butein (10 mg/kg/day or 25 mg/kg/day) showed a significant reduction of hydroxyproline and malondialdehyde levels in rats. The expression of alpha1(I) collagen and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNAs in liver was clearly reduced in a dose-dependent manner in rats given butein compared with control CCl4-treated rats. These data suggest the potential of butein to serve as an antifibrogenic agent by inhibition of collagen accumulation and lipid peroxidation, and by down-regulation of the expression of both alpha1(I) collagen and TIMP-1 mRNA.

Aloe emodin suppresses myofibroblastic differentiation of rat hepatic stellate cells in primary culture.

We have studied the inhibitory effect of aloe emodin on hepatic stellate cells activation and proliferation, as these cells play a key role in the pathogenesis of hepatic fibrosis. Rat hepatic stellate cells were activated by contact with plastic dishes, resulting in their transformation into myofibroblast-like cells. Primary hepatic stellate cells were exposed to aloe emodin (1-10 microg/ml). Possible cytotoxic effects were measured on stellate cells and hepatocytes using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of aloe emodin on production of type I collagen and smooth muscle cell alpha-actin were examined at the same concentration, by quantitative immunoprecipitation. Antiproliferative effects were examined by bromodeoxyuridine incorporation. Aloe emodin at 10 microg/ml restored the morphological changes characteristic of activated primary stellate cells, reduced DNA synthesis to 95% of control hepatic stellate cells at 10 microg/ml without affecting cell viability, and inhibited type I collagen production and smooth muscle alpha-actin expression by 86.77% and 99%, respectively, which suggest that aloe emodin is a potent inhibitor of stellate cell transformation.

Curcumin inhibits collagen synthesis and hepatic stellate cell activation in-vivo and in-vitro.

We previously demonstrated that curcumin, a well-known antioxidant, inhibits collagen deposition in carbon tetrachloride-induced liver injury in rats. The major effector cells responsibleforcollagensynthesis in the liver are activated hepatic stellate cells. In this study,we investigated the inhibitory effects of curcumin on the collagen synthesis and activation of rat hepatic stellate cells in-vitro, and on hepatic stellate cell activation in-vivo. The effects of curcumin on the production of collagen and smooth muscle alpha-actin proteins and of alpha1(I) collagen mRNA were studied in-vivo and in-vitro. The effect of curcumin on DNA synthesis was also determined in-vitro. In-vivo, treatment with curcumin reduced collagen deposition and smooth muscle alpha-actin-positive areas and lowered mRNA levels of type I collagen in the liver. In-vitro, curcumin at a concentration of 5 microg mL(-1) reduced DNA synthesis, and downregulated smooth muscle alpha-actin and type I collagen expression, and alpha1(I) collagen mRNA expression. We concluded that curcumin inhibits collagen synthesis and hepatic stellate cell activation in-vivo and in-vitro, and thus may prove a valuable anti-fibrogenic agent.