Role of endogenous opioids in modulating HSC activity in vitro and liver fibrosis in vivo.

BACKGROUND: Endogenous opioids modulate the growth of nervous and non-nervous cells. Hepatic stellate cells (HSCs) are the main cell phenotype involved in liver fibrogenesis, display molecular markers of neuronal cells and respond to neurotransmitters. AIM: To evaluate the role of endogenous opioids on liver fibrogenesis. METHODS: Activated rat HSCs (passage 1-3) were used to evaluate cell proliferation and intracellular signalling pathway activation. Liver fibrosis was induced in rats by dimethylnitrosamine (DMN) administration. RESULTS: Opioid receptors showed a different pattern of expression when measured in quiescent and activated (in vitro and in vivo) HSCs. The activation of opioid receptors increased HSC proliferation and collagen accumulation. Opioid receptor stimulation induced a calcium-dependent protein kinase C alpha (PKC alpha)/extracellular regulated kinase (ERK)/phosphatidylinositol 3-kinase (PI3K) pathway activation that mediated the effect of endogenous opioids on HSC proliferation and collagen synthesis. In DMN-treated rats, the opioid antagonist naloxone reduced alpha-smooth muscle actin expression (as a marker of HSC activation) and collagen deposition, both measured by morphometry after 5 weeks of treatment. In both DMN-treated rats and human liver biopsies from chronic liver diseases, opioid receptors were observed in HSCs in area of active fibrogenesis. The endogenous opioid met-enkephalin increased its expression in zone 3 hepatocytes close to the area of necrosis after DMN administration and in the cellular target of chronic liver injury in human biopsies, and stimulated HSC proliferation and collagen synthesis. CONCLUSIONS: Endogenous opioids released during chronic liver injury participate in the process of liver fibrogenesis by stimulating HSC proliferation and collagen production in a paracrine manner.

Selective inhibition of ion transport mechanisms regulating intracellular pH reduces proliferation and induces apoptosis in cholangiocarcinoma cells.

BACKGROUND: Cells within the acidic extracellular environment of solid tumours maintain their intracellular pH through the activity of the Na(+)/H(+) exchanger and the Na(+) dependent Cl(-)/HCO(3)(-) exchanger. The inhibition of these mechanisms could therefore inhibit cancer cell growth. AIM: We evaluated the effect of two selective inhibitors of these transporters (cariporide and S3705) on proliferation and apoptosis of human cholangiocarcinoma cells (HUH-28 and Mz-ChA-1 cells) as a function of external pH (7.4 and 6.8). METHODS/RESULTS: HUH-28 cells incubated for 24h at external pH 7.4 or 6.8 without inhibitors maintained intracellular pH at physiological level, whereas incubation with cariporide and/or S3705 caused the intracellular pH of cells to drop. Incubation of HUH-28 cells with cariporide and/or S3705 was able to reduce proliferation, evaluated by a colorimetric ELISA method, and to induce apoptosis, evaluated by measuring caspase-3 activity and Annexin-V staining, and these effects were more evident at external pH 6.8. S3705 but not cariporide was able to inhibit serum-induced phosphorylation of ERK1/2, AKT and BAD, intracellular molecules involved in cancer cell proliferation and survival. Similar results were obtained in Mz-ChA-1 cells. CONCLUSIONS: (1) Inhibition of intracellular pH regulatory mechanisms by cariporide and S3705 reduces proliferation and induces apoptosis in cholangiocarcinoma cells; and (2) these drugs might have potential therapeutic value against cholangiocarcinoma.

Hepatoprotective and antifibrotic effect of a new silybin-phosphatidylcholine-Vitamin E complex in rats.

BACKGROUND: Silybin, the main active component of silymarin, has been reported to reduce hepatic fibrosis by 30% in bile duct ligated rats, whereas Vitamin E alone does not significantly modify liver damage and collagen deposition in chronic liver injury. AIM: The aim of the present study was to evaluate the hepatoprotective and the antifibrotic properties of a new silybin-phosphatidylcholine-Vitamin E complex, characterised by elevated oral bioavailability and lipophilicity, on rat hepatic fibrosis induced by dimethylnitrosamine administration and by bile duct ligation. METHODS/RESULTS: The complex was administered by gastric gavage at a dose of 250 and 75 mg/kg (as silybin and Vitamin E, respectively). Treatment with the complex was able to prevent the dimethylnitrosamine-induced loss in body and liver weight, as well as to reduce the degree of liver injury, as determined by alanine aminotransferase values and necroinflammatory score. This was associated with reduced hepatic stellate cells proliferation both after 1 and 5 weeks of treatment. Treatment with the complex reduced also hepatic stellate cells activation and collagen deposition. Treatment with dimethylnitrosamine induced an increase in alpha1(I) procollagen, TGF(beta1), tissue inhibitor of metalloproteinase 1 and metalloproteinase 2 mRNA expression, which were significantly reduced by administration of the complex. In the bile duct ligation model, the administration of the complex was able to reduce hepatic stellate cells proliferation and activation, as well as collagen deposition and alpha1(I) procollagen mRNA expression. CONCLUSIONS: These results suggest that this new silybin-phosphatidylcholine-Vitamin E complex could be an interesting drug to be tested in patients with chronic liver disease.

The anti-fibrotic effect of pirfenidone in rat liver fibrosis is mediated by downregulation of procollagen alpha1(I), TIMP-1 and MMP-2.

BACKGROUND: Pirfenidone (5 methyl-1-phenyl-2(1H)-pyridone) is a novel anti-fibrotic agent, which has been shown to decrease collagen deposition in a variety of animal models in vivo, and recently in hepatic fibrosis also. At cellular level, we have recently demonstrated that pirfenidone is able to inhibit proliferation of hepatic stellate cells induced by platelet-derived growth factor, as well as collagen type I accumulation and alpha1(I) procollagen mRNA expression. AIMS: To evaluate if pirfenidone maintains its anti-fibrotic properties also when administered after the induction of hepatic damage and to further investigate the molecular mechanisms leading to the anti-fibrotic effect of pirfenidone. METHODS AND RESULTS: Rats treated with dimethylnitrosamine (10 mg/kg) for 5 weeks received a liquid diet containing 0.5% pirfenidone starting from the third week. Pirfenidone treatment reduced the degree of liver injury, as determined by alanine aminotransferase values and necro-inflammatory score, which was associated with reduced hepatic stellate cells proliferation and collagen deposition. Treatment with dimethylnitrosamine increased transcripts levels for transforming growth factorbeta1, procollagen alpha1(I), tissue inhibitors of metalloproteinase-1 and matrix metalloproteinase-2 by 7-, 7-, 4- and 15-fold, respectively. Pirfenidone administration downregulated elevated levels of those transcripts by 50-60%, and this was associated with a 70% reduction in collagen deposition. CONCLUSIONS: (1) Pirfenidone is effective also if administered after the induction of the hepatic damage; (2) the anti-fibrotic effect of pirfenidone is mainly due to the reduced expression of profibrogenic procollagen alpha1(I) and TIMP-1, most likely through the downregulation of transforming growth factorbeta1 mRNA, and of matrix metalloproteinase-2, which is mainly implicated in the degradation of the normal extracellular matrix.