Nikolina Kutinová Canová

Inhibition of endotoxemia-induced nitric oxide synthase expression by cyclosporin A enhances hepatocyte injury in rats: amelioration by NO donors

Abstract The goals of the present study were to provide information into the controversy about nitric oxide (NO) status of the liver during endotoxemia and to assess the role of the phosphatase inhibitor cyclosporin A (CsA) during the insult. Rats were injected with saline, lipopolysaccharide (LPS, 10 mg/kg i.p.) or cyclosporin A (CsA, 5 mg/kg. i.p.)+LPS, S -nitroso- N -acetyl penicillamine (SNAP, 0.1 mM/kg)+CsA+LPS or molsidomine (molsid, 0.2 mg/kg)+CsA+LPS. Rat hepatocytes were isolated and tested for metabolic competence by the rate of urea synthesis and for lipid peroxidation. Hepatocytes were cultured under various treatments as LPS or cytokine mixture (CM, TNF-α 500 U/ml, INF-γ 100 U/ml, IL-1β 200 U/ml) with or without CsA and iNOS expression was evaluated by NO productivity and by RT-PCR. Twenty-four hours after LPS dosing in vivo, the mortality rate was 15%, while CsA pretreatment increased mortality rate to 30% and reduced hepatocyte viability, increased ALT leakage and reduced urea synthesis. SNAP and Molsid resulted in complete survival of rats, increased urea synthesis, increased cell viability and reduced alanine aminotransferase leakage. LPS or CM increased iNOS expression while CsA pretreatment reduced iNOS expression. There was no correlation between lipid peroxide levels in hepatocytes and functional status of hepatocytes under various treatments. This study demonstrates that NO produced during endotoxemia and under the present conditions is protective to the liver and may function as an adaptive mechanism and that the inhibition of iNOS by compounds like CsA produce unfavorable effects.

EFFECTS OF RESVERATROL PRETREATMENT ON TERT-BUTYLHYDROPEROXIDE INDUCED HEPATOCYTE TOXICITY IN IMMOBILIZED PERIFUSED HEPATOCYTES: INVOLVEMENT OF INDUCIBLE NITRIC OXIDE SYNTHASE AND HEMOXYGENASE-1

The aim of this work was to study the effects of resveratrol (RES) as compared to silymarin (SM) pretreatments on tert-butylhydroperoxide (tBH) induced apoptotic/necrotic markers in hepatocytes. Hepatocyte in cultures (48 h) and in perifused immobilized agarose threads (5h) were used as cellular systems. Hepatocyte apoptosis was estimated morphologically using Annexin-V combined with propidium iodide, or toluidine blue staining. Hepatocyte viability and functionality were evaluated by ALT and urea synthesis. Nitric oxide (NO) and carbon monoxide involvements were also examined. Resveratrol and silymarin reduced tBH-induced hepatocyte toxic effects in short term experiments (5h) as measured by a significant reduction in ALT and NO increase produced by tBH. Both inducible nitric oxide synthase (NOS-2) and hemoxygenase-1 (HO-1) gene expression were increased by tBH and reduced by both RES and SM pretreatments. Morphologically, there were ameliorations in both apoptotic and necrotic markers under RES treatment and were similar to biochemical findings. In addition, RES improved hepatocyte stability in both cellular systems. It may be concluded that resveratrol and sylimarin ameliorative effects on tBH hepatocyte toxicity are comparable; involve NOS-2 and HO-1 expression and should be re-evaluated in various in vitro and in vivo experimental conditions.

Nitric oxide synthase inhibitors modulate lipopolysaccharide-induced hepatocyte injury: dissociation between in vivo and in vitro effects

Effects of endotoxemia-induced NO production on rat liver and hepatocytes in culture were investigated. Rats were treated intraperitoneally with saline, lipopolysaccharide (LPS, 10 mg/kg), L-nitroarginine methyl ester (L-NAME)+LPS, aminoguanidine (AG)+LPS, FK 506+LPS, S-nitroso-N-acetyl penicillamine (SNAP)+L-NAME+LPS and SNAP+FK 506+LPS. Mortality, hepatocyte viability and liver function test were estimated. Liver morphology was observed by light and electron microscopy. Hepatocyte cultures were treated with LPS, cytokine mixture (CM) with or without FK 506, L-NAME or AG. Hepatocyte function and inducible form of NOS (iNOS) expression were evaluated. Twenty-four hours after treatments with saline, LPS, L-NAME+LPS, AG+LPS, FK 506+LPS, SNAP+L-NAME+LPS and SNAP+FK 506+LPS, rat mortalities were 0%, 10%, 48%, 8%, 20%, 38% and 0%, and hepatocyte viabilities were 93+/-3%, 80+/-3%, 52+/-8%, 88+/-1%, 70+/-3%, 80+/-4% and 82+/-3%, respectively. AG+LPS or L-NAME+LPS administration was followed by excessive vacuolization of hepatocytes with lesions in the intermediary lobule zone characterized by features of secondary necrosis as a continuation of apoptotic processes. SNAP+L-NAME+LPS resulted in a well-preserved structure of central vein lobules with sparse signs of apoptosis. Treatment with LPS or CM increased iNOS expression in hepatocyte culture, which was inhibited by L-NAME, FK 506 or AG. AG reduced LPS-induced rise in alanine aminotransferase leakage. LPS-induced NO exerts cytoprotective effects in vivo, while LPS-induced NO in vitro appears to be toxic. Based on the data of this report, one cannot use in vitro results to predict in vivo responses to LPS-induced NO production. The pharmacological modulation of iNOS expression or NO production in vivo or in vitro, therefore, by the development of specific NO donors or inhibitors is promising for improvement of hepatocyte functions under the two experimental conditions, respectively.

The involvement of heme oxygenase 1 but not nitric oxide synthase 2 in a hepatoprotective action of quercetin in lipopolysaccharide-induced hepatotoxicity of D-galactosamine sensitized rats.

The objective of this study was to evaluate potential hepatoprotective capabilities of quercetin in relation to its modulation of the HO-1 and NOS-2 activities in an experimental model of fulminant liver failure. Liver insult was induced by in vivo administration of D-galactosamine (d-GalN, 400 mg/kg, i.p.) and lipopolysaccharide (LPS, 10 μg/kg, i.p.). The effects of quercetin (50 mg/kg, i.p) on D-GalN toxicity was evaluated by standard biochemical, RT-PCR and Western blot methods. Administration of d-GalN/LPS combination resulted in significantly higher plasma levels of aminotransferases, as well as increased mRNA and protein expressions of both HO-1 and NOS-2 enzymes. Quercetin exhibited cytoprotective effects on the liver, as evidenced by decreased aminotransferase plasma levels. Additionally, quercetin treatment in D-GalN/LPS treated rats significantly increased HO-1 mRNA and its protein expressions. On the contrary, quercetin did not exhibit any significant effects on the levels of nitrites, and NOS-2 mRNA and protein expressions in D-GalN/LPS treated rats. Quercetin when given alone did not have any significant changes on liver enzymes nor HO-1 and NOS-2 mRNA and protein expressions. It can be concluded that the quercetins induction of HO-1 and its byproducts, without concomitant NOS-2 activity reduction, is among mechanisms contributing to the hepatoprotective effect in D-GalN/LPS hepatotoxicity.

Cryopreservation of precision cut tissue slices (PCTS): Investigation of morphology and reactivity

Precision cut tissue slices (PCTS) represent a suitable and convenient tool for pharmacological, toxicological and morphological studies. Cryopreservation would enable to overcome the shortage of liver tissue, in particular in settings using human liver tissue. We investigated the potential of cryopreservation of porcine PCTS as a morphological tool by rapid freezing with 10% and 30% dimethyl sulfoxide as cryopreservation agents and with or without medium using a Brendel/Vitron tissue slicer. Incubation after thawing was done in a static incubation system. Slices were cultured for 3 h, 6 h, 24 h and 48 h and assessed histologically and immunohistologically for proliferation (Ki67) and spontaneous as well as induced apoptotic activity (M30Cytodeath). Vitality was tested using the Tox-8 test. After cryopreservation, morphology of PCTS was well preserved up to 24 h. A reduction of vitality rate took place. Compared to non-frozen PCTS, the rate of spontaneous proliferation of Kupffer cells and apoptosis of hepatocytes were significantly reduced independent of the freezing conditions. The reactivity of PCTS to apoptotic stimuli was significantly reduced in tissue slices after cryopreservation. Apoptotic stimuli could not induce the same amount of cell deaths compared to non-frozen sections. Thus, cryopreservation of PCTS does interfere with pathomechanisms of apoptosis in PCTS.

Hepatoprotective properties of extensively studied medicinal plant active constituents: possible common mechanisms.

Abstract Context: We focused on certain plant active constituents considered to be the most promising/studied for liver disease and that were critically investigated from the basic science point of view and, to some extent, the clinical one. Due to insufficient pharmacological data, most of the herbal formulations containing these molecules cannot be recommended for the treatment of liver disease. Objective: To present the most promising compounds tested experimentally and/or clinically and describe in brief popular models in experimental testing of potential hepatoprotective compounds. Methods: A literature search using Web of Science (WOS), PubMed, and Google search was performed. Results: Focusing on a few herbal hepatoprotective active constituents is useful to health professionals working in the field of therapeutics to develop evidence-based hepatoprotective agents by conducting research on pure chemical structures or on molecular modifications using computational chemistry. This review demonstrates that multi-pathways in the liver pathobiology can be interrupted at one or more levels by natural hepatoprotective studied, such as interference with the oxidative stress at multiple levels to reduce reactive oxygen/nitrogen species, resulting in ameliorating hepatotoxicity. Conclusion: Hepatoprotective constituents of herbal medications are poorly absorbed after oral administration; methods that can improve their bioavailability are being developed. It is recommended that controlled prospective double-blind multicenter studies on isolated active plant constituents, or on related newly designed molecules after structural modifications, should be performed. This effort will lead to expanding the existing, limited drugs for the vast majority of liver diseases.

Modulation of spontaneous and lipopolysaccharide-induced nitric oxide production and apoptosis by d-galactosamine in rat hepatocyte culture: the significance of combinations of different methods.

ABSTRACT Apoptotic markers and signals produced by xenobiotics as hepatotoxic D-galactosamine (D-GalN) and lipopolysaccharide (LPS) are extensively investigated in vivo. The contribution of various cells and factors as nitric oxide (NO) in mediating hepatocyte apoptosis in a rat model of systemic endotoxemia was reported. Therefore, the aim of the present work was to study the in vitro effect of D-GalN on nonstimulated or LPS-treated rat hepatocytes in culture and the potential involvement of NO in this process. Our results showed that the spontaneous and LPS-induced NO production was completely blocked by D-GalN during 0 to 24 hours. However, D-GalN slightly enhanced NO production during 24 to 48 hours. D-GalN was more potent to induce hepatocyte apoptosis and necrosis during 24 to 48 than 0 to 24 hours as evidenced morphologically (Annexin V/propidium iodide staining) and biochemically (caspase-3-like activity, alanine-aminotransferase leakage, MTT test). Interestingly, D-GalN treatment suppressed mitochondrial cytochrome C release throughout the study. LPS addition to D-GalN considerably aggravated apoptotic/necrotic markers only during 0 to 24 hours. Surprisingly, a share of apoptotic cells was distinctly lower after LPS + GalN treatment than after LPS alone during 0 to 24 hours, while 24- to 48-hour incubation produced massive apoptotic/necrotic hepatocytes. It may be concluded that there is a significant modulation of NO production by D-GalN. Because the role of NO is only partly decisive in the apoptotic/necrotic events, and considering the fraction of the cells completing apoptosis while others that turn toward necrosis (aponecrosis), caution should be exercised in apoptosis data interpretation and combinations of different test methods should be applied.

Inhibition of endotoxemia-induced nitric oxide synthase expression by cyclosporin a enhances hepatocyte injury in rat

The goals of the present study were to provide information into the controversy about nitric oxide (NO) status of the liver during endotoxemia and to assess the role of the phosphatase inhibitor cyclosporin A (CsA) during the insult. Rats were injected with saline, lipopolysaccharide (LPS, 10 mg/kg i.p.) or cyclosporin A (CsA, 5 mg/kg. i.p.) + LPS, S-nitroso-N-acetyl penicillamine (SNAP, 0.1 mMikg) + CsA + LPS or molsidomine (molsid, 0.2 mg/kg) + CsA + LPS. Rat hepatocytes were isolated and tested for metabolic competence by the rate of urea synthesis and for lipid peroxidation. Hepatocytes were cultured under various treatments as LPS or cytokine mixture (CM, TNF-alpha 500 U/ml, INF-gamma 100 U/ml, IL-1beta 200 U/ ml) with or without CsA and iNOS expression was evaluated by NO productivity and by RT-PCR. Twenty-four hours after LPS dosing in vivo, the mortality rate was 15%, while CsA pretreatment increased mortality rate to 30% and reduced hepatocyte viability, increased ALT leakage and reduced urea synthesis. SNAP and Molsid resulted in complete survival of rats, increased urea synthesis, increased cell viability and reduced alanine aminotransferase leakage. LPS or CM increased iNOS expression while CsA pretreatment reduced iNOS expression. There was no correlation between lipid peroxide levels in hepatocytes and functional status of hepatocytes under various treatments. This study demonstrates that NO produced during endotoxemia and under the present conditions is protective to the liver and may function as an adaptive mechanism and that the inhibition of iNOS by compounds like CsA produce unfavorable effects.