The Apparent Asymmetrical Relationship Between Small Bowel Bacterial Overgrowth, Endotoxemia, and Liver Steatosis and Fibrosis in Cirrhotic and Non-Cirrhotic Patients: A Single-Center Pilot Study.

Introduction: Gut microbiota are a complex ecosystem harboring our intestine. They maintain human body equilibrium, while their derangement, namely, "dysbiosis", has been associated with several gastrointestinal diseases, such as liver steatosis (NAFLD) and liver cirrhosis. Small intestinal bacterial overgrowth (SIBO) is an example of dysbiosis of the upper gastrointestinal (GI) tract. Aim: The aim of this study is to evaluate the relationship between SIBO and levels of endotoxemia and grade of liver steatosis (LS) and liver fibrosis (LF) in hepatologic patients. Materials and Methods: Consecutive outpatients referred to our hepatology clinic were tested for SIBO by the lactulose breath test (LBT) and peripheral blood levels of endotoxemia; LS grading and LF were assessed by abdominal ultrasound and transient elastography, respectively. Results: Fifty-two consecutive patients (17 with alcohol abuse (4.5 ± 0.8 alcohol units per day), 4 with HCV and 2 with HBV infection, 24 of metabolic origin, 2 of autoimmune origin, and 3 with cholangiopathies; mean age 54.7 ± 8.3 years, 31 F, BMI 24.1 ± 1.1 Kg/m2) and 14 healthy volunteers (HV) (mean age 50.1 ± 4.3 years, 9 F, BMI 23.3 ± 1.1 Kg/m2) were enrolled. SIBO prevalence was significantly higher in cirrhotic (LC) vs. non-cirrhotic (LNC) patients and vs. HV (all, p < 0.05), with a significant positive trend according to Child-Pugh status (all, p < 0.05). SIBO prevalence was not correlated with LS stages (all, p = NS). Consensually, endotoxin levels were significantly higher in LC vs. LNC and vs. HV (all, p < 0.05) and significantly correlated with LF in patients with LC, according to Child-Pugh status (all, p < 0.05). Conclusion: This study shows that SIBO prevalence and relative endotoxin blood levels seem to be significantly associated with the grade of LF vs. LS in LC. SIBO is also present under pre-cirrhotic conditions, but its prevalence seems to correlate with liver disease irreversible derangement.

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.

Hepatocellular carcinoma recurrence in patients with curative resection or ablation: impact of HCV eradication does not depend on the use of interferon.

BACKGROUND: In HCV-infected cirrhotic patients with successfully treated early hepatocellular carcinoma (HCC), the time to HCC recurrence and the effects of sustained viral eradication (SVR) by interferon (IFN)-based or IFN-free regimens on HCC recurrence remain unclear. AIM: To perform an indirect comparison of time to recurrence (TTR) in patients with successfully treated early HCC and active HCV infection with those of patients with SVR by IFN-based and by IFN-free regimens. METHODS: We evaluated 443 patients with HCV-related cirrhosis and Barcelona Clinic Liver Cancer Stage A/0 HCC who had a complete radiological response after curative resection or ablation. Active HCV infection was present in 328, selected from the Italian Liver Cancer group cohort; 58 patients had SVR achieved by IFN-free regimens after HCC cure, and 57 patients had SVR achieved by IFN-based regimens after HCC cure. Individual data of patients in the last two groups were extracted from available publications. RESULTS: TTR by Kaplan-Meier curve was significantly lower in patients with active HCV infection compared with those with SVR both by IFN-free (P = 0.02) and by IFN-based (P < 0.001) treatments. TTR was similar in patients with SVR by IFN-free or by IFN-based (P = 0.49) strategies. CONCLUSION: In HCV-infected, successfully treated patients with early HCC, SVR obtained by IFN-based or IFN-free regimens significantly reduce tumour recurrence without differences related to the anti-viral strategy used.

Prolonged n-3 polyunsaturated fatty acid supplementation ameliorates hepatic steatosis in patients with non-alcoholic fatty liver disease: a pilot study.

BACKGROUND: Recent studies suggest a role of n-3 long-chain polyunsaturated fatty acids (n-3 PUFA) as peroxisome proliferator-activated receptor-alpha ligands in improving non-alcoholic fatty liver disease (NAFLD) in rodents. However, data in humans are still lacking. AIM: To evaluate the efficacy of prolonged PUFA supplementation in patients with NAFLD. METHODS: Fifty-six patients with NAFLD were enrolled. Among the overall eligible patients, 42 assumed n-3 PUFA 1-g capsule daily for 12 months, whereas 14 refused the treatment and were analysed as controls. All patients underwent haematochemical and ultrasound follow-up. RESULTS: Polyunsaturated fatty acid supplementation significantly decreased serum aspartate transaminase (P = 0.003), alanine transaminase (P = 0.002), gamma-glutamyl transpeptidase (P = 0.03), triglycerides (P = 0.02) and fasting glucose (P = 0.02) in comparison with controls. Circulating arachidonate and n-6/n-3 fatty acid ratio was reduced (P = 0.0002, and P = 0.0001 respectively) in treated patients. Moreover, ultrasonography demonstrated improvement of liver echotexture after PUFA (P = 0.0001), and increase of Doppler perfusion index (P = 0.001), whereas no significant changes occurred in controls. CONCLUSIONS: Supplementation with n-3 PUFA improves biochemical, ultrasonographic and haemodynamic features of liver steatosis. Our study supports the efficacy of n-3 PUFA as a new therapeutic approach in the treatment of NAFLD.

Acetaldehyde-collagen adducts in N-nitrosodimethylamine-induced liver cirrhosis in rats.

Increased acetaldehyde levels have been found in non-alcoholic liver diseases and an acetaldehyde-collagen adduct has been reported in rats with CCl4-induced cirrhosis. In cytosol and microsomes of rats with cirrhosis produced by N-nitrosodimethylamine, a similar acetaldehyde-protein adduct of approximately 200 kD was recognized by rabbit IgG raised against either an in vitro produced hemocyanin-acetaldehyde adduct or an in vivo occurring P4502E1-acetaldehyde adduct isolated from alcohol-fed rats, as well as by anti-rat collagen (I) IgG. Its immune complexes contained 3 proteins that reacted with the anti-collagen IgG and were digested by collagenase: 2 proteins with molecular weights similar to procollagens alpha 1 and alpha 2, and a beta 1,2(I)-like protein which was readily produced by in vitro modification of cytosol with acetaldehyde.

Wine: risk factors for liver disease and antifibrotic compounds.

Wine can be considered an integral part of the Mediterranean diet and moderate alcohol intake can be beneficial for health. This health-promoting effect is presumably due to the presence of antioxidant substances. It is also known that excessive alcohol intake can lead to liver cirrhosis. A main pathogenetic mechanism in liver cirrhosis is the activation of hepatic stellate cells which acquire a myofibroblast-like phenotype. Excessive production of oxidative stress products may initiate the activation process. Phenolic compounds contained in red wine have been shown to have antifibrotic properties on activated hepatic stellate cells. In vitro and in vivo studies are needed for a better evaluation of the clinical relevance of these findings.

[L-sulpiride versus metoclopramide in functional dyspepsia: a randomized double-blind study]. / L-sulpiride contro metoclopramide nella dispepsia funzionale: studio randomizzato in doppio cieco.

Fifty patients suffering from functional dyspepsia have been treated in a double-blind study either with 1-sulpiride (75 mg die per os) or with metoclopramide (30 mg die per os) for 30 days. The frequency and severity of the symptoms in the two patient groups were similar. The administration of either drug was followed by a reduction of the symptoms, but 1-sulpiride was more effective on nausea, headache, pyrosis, epigastric pain, and showed an earlier effect than metoclopramide in inducing total regression of symptoms.

Liver transplantation in neurological Wilson's Disease: is there indication? A case report.

Wilson's disease (WD) is an autosomal recessive disorder characterized by copper overload. In this disease, inadequate hepatic excretion leads to copper accumulation in the liver, brain, kidney, and cornea. Severe neurological symptoms can develop in patients with WD, often in the absence of relevant liver damage: it is unclear whether liver transplantation (LT) could reverse neurological symptoms, and at present LT is not recommended in this setting. We report a case of regression of neurological symptoms in a patient affected by WD with prevalent neurological involvement. A 19-year-old man with disabling neuropsychiatric symptoms from WD that included frontal ataxia, akinesia, dystonia, tremors, and behavioral disorders in the presence of preserved liver function (Model for End-Stage Liver Disease score=7; Child-Turcotte-Pugh score=A5) underwent LT in November 2009. At the time of LT, encephalic magnetic resonance imaging (MRI) indicated diffuse neurodegenerative alterations involving subtentorial and supratentorial structures; bilateral Kayser-Fleischer ring was present. Four years after LT, laboratory tests show normalized copper metabolism and excellent liver function test results. Encephalic MRI shows a substantial improvement of already-known signal alterations at nuclei thalamus and putamen, mesencephalon, and pons. Kayser-Fleischer ring disappeared from the right eye, but a little remnant is still visible in the left eye. At neurological examination, all of the previous symptoms and signs are no longer present and behavioral disorders are no longer present; psychosocial functions are completely restored. The present case provides some evidence that LT may be a valid therapeutic option for WD patients with marked neurological impairment, particularly in those no longer responsive to chelation therapy.

Post-load insulin resistance is an independent predictor of hepatic fibrosis in virus C chronic hepatitis and in non-alcoholic fatty liver disease.

BACKGROUND: Insulin resistance is a significant risk factor for hepatic fibrosis in patients with both non-alcoholic fatty liver disease (NAFLD) and chronic hepatitis C (CHC), either directly or by favouring hepatic steatosis. Several methods are available to assess insulin resistance, but their impact on this issue has never been evaluated. AIMS: To determine the relative contribution of steatosis, metabolic abnormalities and insulin resistance, measured by different basal and post-load parameters, to hepatic fibrosis in CHC and in NAFLD patients. METHODS: In 90 patients with CHC and 90 pair-matched patients with NAFLD, the degree of basal insulin resistance (by the homeostasis model assessment, (HOMA)) and post-load insulin sensitivity (by the oral glucose insulin sensitivity (OGIS) index) was assessed, together with the features of the metabolic syndrome according to Adult Treatment Panel III definition. Data were correlated with hepatic histopathology. RESULTS: The prevalence of basal insulin resistance (HOMA values >75th percentile of normal) was 23.3% in CHC patients and 57.8% in NAFLD, but it increased to 28.8 and 67.8% when measured by post-load insulin resistance (OGIS <25th percentile). In a multivariate model, after adjustment for age, gender and body mass index, OGIS was a predictor of severe fibrosis in CHC and in NAFLD patients, independently of steatosis. An OGIS value below the cut-off of the 25th percentile increased the likelihood ratio of severe fibrosis by a factor of 1.5-2 and proved to be a more sensitive and generally more specific test than HOMA-R for the identification of subjects with severe fibrosis both in NAFLD and in CHC. CONCLUSIONS: Post-load insulin resistance (OGIS <9.8 mg/kg/min) is associated with severe hepatic fibrosis in both NAFLD and CHC patients, and may help identify subjects at risk of progressive disease.

Hepatic portal venous gas in a patient with enterovascular fistula.

Hepatic portal venous gas is an uncommon clinical condition that is often characterized by acute onset of abdominal pain and is associated with a high rate of mortality despite clinical and/or surgical treatment. Radiologic diagnosis is important and usually includes abdominal radiography, ultrasound, and computed tomography. We describe the clinical, computed tomographic, and angiographic data of a patient with sigmoid diverticulitis who developed a massive embolism of the intra- and extrahepatic portal systems due to an enterovascular fistula and was treated with fistula embolization and subsequent sigmoidectomy.

Regulation of intracellular pH in isolated periportal and perivenular rat hepatocytes.

BACKGROUND: Liver acinus shows a well-known metabolic zonation. The aim of this study was to investigate intracellular pH (pHi) regulation in isolated periportal (PP) and perivenular (PV) rat hepatocytes. METHODS: 2,7-bis(carboxyethyl)-5(6)-carboxy-fluorescein was used as pH-sensitive dye. Hepatocyte subconfluent monolayers were acid-loaded by exposure to 20 mmol/L NH4Cl and alkali-loaded by reducing external CO2 and HCO3- at an external pH of 7.4. RESULTS: In the presence of HCO3-/CO2, (1) baseline pHi was higher in PP (7.25 +/- 0.018) than in PV hepatocytes (7.20 +/- 0.013) (P < 0.05); (2) pHi recovery from an acid load was 40% higher in PP than in PV hepatocytes (P < 0.02) and was inhibited by amiloride by 36% in PV and 7% in PP hepatocytes; (3) DIDS inhibited amiloride-independent pHi recovery from an acid load by 65% in PP and 52% in PV cells. In the absence of HCO3-/CO2, baseline pHi and pHi recovery from an acid load were not significantly different in PP and PV hepatocytes. pHi recovery from an alkali load was 30% higher in PV than in PP cells (P < 0.02). CONCLUSIONS: Our data suggest that isolated PP rat hepatocytes show higher activity for Na(+)-HCO3- cotransport, whereas PV cells show greater activity for Cl-/HCO3- exchanger.

Collagen-acetaldehyde adducts in alcoholic and nonalcoholic liver diseases.

Alcoholic and, to a lesser extent, nonalcoholic patients with liver disease have serum antibodies to acetaldehyde-protein adducts produced in vitro. These antibodies presumably reflect the presence of adducts in the liver, but the protein that triggers this immune response has not been identified. To study this, we measured the reactivity of cytosolic proteins to rabbit IgG developed against a P-450 2E1-acetaldehyde adduct, isolated from alcohol-fed rats, that recognizes acetaldehyde-modified epitopes in proteins. Adducts were determined on Western blots by scanning densitometry of antibody-linked alkaline phosphatase activity in 4 normal livers and in needle biopsy specimens from subjects with liver disease, 17 alcoholic and 14 nonalcoholic. In all livers, except for a normal one, we found a reactive protein of at least 200 kD, similar to the collagen-acetaldehyde adduct we reported to be markedly increased in rats with experimentally induced cirrhosis. The immunostaining intensity in the alcoholic patients with liver disease was eightfold (p < 0.01) and that in nonalcoholic patients with liver disease was fourfold, greater (p < 0.02) than the weak staining in normal livers; it correlated with the degree of inflammation and serum AST or gamma-glutamyl transpeptidase activities. The adduct was reproduced on incubation of normal cytosolic proteins with 2.5 mmol/L acetaldehyde, whereas higher concentrations yielded many additional adducts; the adduct also reacted with IgG antibody to rat collagen type I and disappeared after digestion with collagenase, suggesting that the target protein is a form of collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenotypic analysis of inflammatory infiltrate in rats with dimethylnitrosamine-induced cirrhosis.

The present study is concerned with the immunohistochemical characterization in situ of the mononuclear infiltrate accompanying the formation of septa and the development of cirrhosis in the liver of rats treated with dimethylnitrosamine (DMN), i.p. (10 microliters/kg, 3 days a week for 3 weeks). Monoclonal antibodies against macrophages, pan T cells, T cell subsets and B cells have been applied on cryostat sections of animals given DMN for 7, 14 and 21 days. The maximum increase of macrophages and lymphocytes was observed at days 7 and 14 respectively. At all times T lymphocytes appeared as the major component of the inflammatory infiltrate with a largely predominant population of cytotoxic/suppressor T cells. At day 21, with evidence of nodulation of parenchyma, macrophages levelled off while T cells remained numerous without changes in the inducer-helper T cells/cytotoxic-suppressor T cells ratio which remained always less than 1. B cells were always few. These findings illustrate the early influx of lymphocytes in DMN-induced liver injury and help to define the lymphocyte subsets associated with inflammation and fibrosis in a reproducible animal model.

Transforming growth factor beta 1 increases the number of apoptotic bodies and decreases intracellular pH in isolated periportal and perivenular rat hepatocytes.

Transforming growth factor beta 1 (TGF beta 1) is involved in promoting cell death by apoptosis in the liver, whereas the activation of Na+/H+ exchanger has been related to cell proliferation. The aim of this study was to gain information on the effects of TGF beta 1 on intracellular pH and Na+/H+ exchange activity in isolated periportal (PP) and perivenular (PV) rat hepatocytes using the pH-sensitive dye BCECF in a perfused subconfluent hepatocyte monolayer. Steady-state intracellular pH (pHi) in a bicarbonate-free solution (HEPES) were 7.17 +/- 0.031 in PP and 7.15 +/- 0.041 in PV cells. Treatment with TGF beta 1 (120 pmol/L) for 7 hours increased the number of apoptotic bodies by 25% and 38%, and decreased steady-state pHi to 7.11 +/- 0.018 (P = .05) and to 7.07 +/- 0.021 (P < .02), respectively, in PP and PV hepatocytes. In HEPES, cells recovered from an acid load, extruding protons at a rate (JH) of 4.85 +/- 1.01 mmol/L/min in PP cells and of 4.91 +/- 0.99 mmol/L/min in PV hepatocytes. This recovery appeared amiloride inhibitable (1 mmol/L). Culture with TGF beta 1 for 7 hours induced (in HEPES) a decrease of pHi recovery rate from an acid load more in PV (by 46%) than in PP hepatocytes (by 35%, P < .05). Acute administration of epidermal growth factor (EGF) (10 to 100/mL) induced an increase in Na+/H+ exchange activity by 32% and 27%, respectively, in PP and PV cells compared with controls. In contrast, in cells cultured for 7 hours with 120 pmol/L TGF beta 1, the acute administration of EGF slightly increased Na+/H+ exchange activity (by 18%, P < .05) only in PP cells. This study demonstrates that pHi and Na+/H+ exchange activity are decreased by TGF beta 1, which increases the number of apoptotic bodies in periportal and perivenular rat hepatocyte primary cultures.

Collagen synthesis by liver stellate cells is released from its normal feedback regulation by acetaldehyde-induced modification of the carboxyl-terminal propeptide of procollagen.

Acetaldehyde stimulates collagen synthesis in stellate cells and forms adducts with procollagen in the liver of alcoholics. To assess the possibility that modification of the carboxyl-terminal propeptide by acetaldehyde affects its capacity to exert a feedback inhibition of collagen synthesis after splitting from procollagen, the propeptide was prepared by gel filtration of the bacterial collagenase digests of procollagen type I (obtained from 10(9) calvaria fibroblasts of newborn rats) and reacted with either 250 mM acetaldehyde and 100 mM CNBH3 or with 170 microM acetaldehyde without reducing agents, to mimick in vivo conditions. The unmodified propeptide produced a concentration-dependent inhibition of collagen synthesis by Ito cells. By contrast, the acetaldehyde-modified propeptide produced a lesser inhibition of procollagen synthesis in the cells, associated with a greater accumulation of collagen in the media. The incubation with 170 microM acetaldehyde and, to a lesser extent, 50 mM ethanol produced collagenase-digestible adducts in stellate cells. Thus, the formation of acetaldehyde adducts with the carboxyl-terminal propeptide of procollagen may account, at least in part, for the stimulatory effect of acetaldehyde on collagen synthesis by stellate cells and may lead to collagen accumulation through a decrease of the normal feedback regulation of collagen synthesis by the propeptide.

Involvement of reactive oxygen species and nitric oxide radicals in activation and proliferation of rat hepatic stellate cells.

BACKGROUND/AIMS: Reactive oxygen species (ROS) induce HSCs activation, proliferation and collagen gene expression in vitro. Nitric oxide (NO) represents a reactive molecule that reacts with ROS, yielding peroxynitrite. We thus verified the effect of NO on ROS-induced HSCs proliferation in vitro and correlated iNOS expression and ROS formation to HSCs activation in the early phase of liver injury leading to hepatic fibrosis in vivo. METHODS/RESULTS: HSCs were incubated with iron ascorbate (FeAsc) in vitro, which induced ROS production, ERK1/2 phosphorylation and increased cell proliferation. This effect was significantly reduced by the presence of the NO donor S-nitroso-N-acetylpenicillamine. Liver injury was induced in vivo in rats by dimethylnitrosamine administration. HSCs activation started 6 h after DMN administration and peaked at 1 week. ROS generation and neutrophil infiltration were evident for at least 48 h after DMN treatment, showing an identical distribution pattern. Only a few inflammatory cells expressed iNOS 6 h after DMN administration. CONCLUSIONS: we have shown that NO acts as a ROS scavenger in vitro, thus inhibiting HSCs proliferation. ROS production by infiltrating neutrophils occurs in the early phase of liver fibrosis and can represent a stimulus to HSCs activation in vivo. The reduced iNOS expression may account for the low NO levels and the inability to prevent the ROS-induced HSC activation in vivo.

Observer agreement in endoscopic assessment of ulcerative colitis.

BACKGROUND: Colonoscopy is the investigation of choice to evaluate ulcerative colitis, but the reliability of the assessment of endoscopic signs is not clear. AIMS: The aim of this study was to evaluate interobserver agreement for the identification of endoscopic lesions typical of ulcerative colitis, and the influence of training. MATERIAL AND METHODS: Four experienced observers and 11 endoscopists under training assessed 49 still images selected from endoscopic video recordings. RESULTS: The agreement rate between experienced observers was excellent or good (k > 0.39) for recognition of 10 out of 14 signs or patterns (loss of vascular pattern, erythema, oedema, granular mucosa, blood, pseudopolyp, erosion, ulcer, normal pattern, severe activity), and was poor for pus, stricture, mild activity, moderate activity. The rates between endoscopists under training were excellent or good for 6 items (loss of vascular pattern, erythema, oedema, pseudopolyp, normal pattern, severe activity). CONCLUSIONS: Trained observers can reproducibly record most endoscopic signs of ulcerative colitis. A reliable overall scoring of severity should be based on a simple three-grading scale, i.e. normal pattern, moderate activity, severe activity. Acceptable agreement rates can be obtained by endoscopists under training on some well-defined endoscopic appearances.

Intracellular signaling pathways involved in acetaldehyde-induced collagen and fibronectin gene expression in human hepatic stellate cells.

Ethanol induces liver fibrosis by several means that include, among others, the direct fibrogenic action of acetaldehyde on hepatic stellate cells (HSC). However the mechanisms responsible for this effect are not well understood. In this communication we investigated signal transduction pathways triggered by acetaldehyde leading to upregulation of alpha2(I) collagen and fibronectin gene expression in human HSC. Run-on assays showed that acetaldehyde-enhanced transcription of these 2 genes as early as 2 hours, via de novo protein synthesis-independent and -dependent mechanisms. It also stimulated a time-dependent induction in phosphorylation of pp70(S6K) and extracellular-regulated kinase (1/2) (ERK1/2). These effects were completely prevented by calphostin C, a protein kinase C inhibitor. As expected, acetaldehyde-elicited ERK1/2 phosphorylation was inhibited by PD98059, a MEK inhibitor, but not by wortmannin, a PI3K inhibitor. On the other hand, both of these inhibitors partially inhibited phosphorylation of pp70(S6K) induced by acetaldehyde suggesting that its activation is ERK1/2- and PI3K-dependent. Acetaldehyde-elicited fibronectin and alpha2(I) collagen upregulation was inhibited by calphostin C. However, while PD98059, wortmannin and rapamycin (a pp70(S6K) inhibitor) completely abrogated alpha2(I) collagen upregulation, they had no effect on fibronectin expression. Overall, these data suggest that protein kinase C is an upstream component from which acetaldehyde signals are transduced to other pathways such as PI3K and ERK1/2. In addition, differential activation of these pathways is needed for the increase in fibronectin and alpha2(I) collagen gene expression induced by acetaldehyde in human HSC.

Plasma membrane order parameter in periportal and perivenular hepatocytes isolated from ethanol-treated rats.

Hepatic ethanol (EtOH) metabolism has been assumed to involve hepatocytes differently, according to their location in the hepatic acinus. This study's aim was to gain information on plasma membrane (PM) order parameter in periportal (PP) and perivenular (PV) hepatocyte-enriched fractions isolated by a digitonin-collagenase perfusion technique from rats pair-fed for 6-8 wk liquid diets containing either EtOH or isocaloric carbohydrates. Fluorescence polarization (P) studies have been performed to measure PM order parameter of PP and PV hepatocytes cultured for 2-6 h on glass cover slips and labeled with 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), a specific probe for PM of living cells. Fluorescence polarization and microscopy indicated that TMA-DPH is a suitable probe to study PM order parameter in subconfluent rat hepatocyte monolayers where it labeled, after a rapid incorporation, PM of cells. In pair-fed control rats, PM order parameter was lower in PP hepatocytes than in PV cells (P = 0.366 +/- 0.013 vs. 0.381 +/- 0.021, respectively, P < 0.02; n = 7). In EtOH-treated rats, these zonal differences tended to disappear (P = 0.419 +/- 0.012 in PP cells vs. 0.417 +/- 0.007 in PV cells; n = 7). In addition, the order parameter was significantly higher either in PP or PV hepatocytes compared with pair-fed control animals (P < 0.002 and 0.003, respectively). A 30-min culture of cells in the presence of 40-200 mM EtOH significantly decreased the PM order parameter of hepatocytes isolated from pair-fed control rats with respect to EtOH-treated animals both in PP and PV cells (P < 0.01 and 0.02, respectively; n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the NA(+)/H(+) exchanger reduces rat hepatic stellate cell activity and liver fibrosis: an in vitro and in vivo study.

BACKGROUND & AIMS: The Na(+)/H(+) exchanger is the main intracellular pH (pH(i)) regulator in hepatic stellate cells (HSCs) and plays a key role in regulating proliferation and gene expression. We evaluated the effect of specific inhibition of this exchanger on HSC proliferation and collagen synthesis in vivo and in vitro. METHODS: Rat HSCs were incubated in the presence of platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta1, iron ascorbate (FeAsc), and ferric nitrilotriacetate solution (FeNTA) with or without the Na(+)/H(+) exchanger inhibitor 5-N-ethyl-N-isopropyl-amiloride (EIPA). pH(i) and Na(+)/H(+) exchanger activity, cell proliferation, and type I collagen accumulation were measured by using the fluorescent dye 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein, by immunohistochemistry for bromodeoxyuridine, and by enzyme-linked immunosorbent assay, respectively. In vivo liver fibrosis was induced by dimethylnitrosamine administration and bile duct ligation (BDL) in rats treated or not treated with amiloride. RESULTS: PDGF, FeAsc, and FeNTA increased Na(+)/H(+) exchange activity and induced HSC proliferation. TGF-beta1 had no effect on the Na(+)/H(+) exchanger and was able, as for FeAsc and FeNTA, to induce type I collagen accumulation. EIPA inhibited all the effects determined by PDGF, FeAsc, and FeNTA and had no effect on TGF-beta1-induced collagen accumulation. In vivo, amiloride reduced HSC proliferation, activation, collagen deposition, and collagen synthesis. CONCLUSIONS: The Na(+)/H(+) exchanger can play a key role in the development of liver fibrosis and in HSC activation in vivo.