Jadwiga Daniluk

Serum cytokine levels in alcohol-related liver cirrhosis

Chronic alcoholism complicated by alcoholic liver disease is characterized by activation of the inflammatory response system. To evaluate the role of cytokines in the progress of alcoholic cirrhosis, we assessed serum level of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, and IL-8 and the antiinflammatory cytokines IL-2, IL-10, and transforming growth factor (TGF)-beta in patients with compensated and decompensated alcoholic liver cirrhosis. Compensated alcoholic cirrhosis was characterized by increased IL-6 (6.3+/-2.9 vs. HP 2.2+/-1.4 pg/ml in controls) and decreased IL-10 (HP 4.1+/-3.5 vs. 6.4+/-5.4 pg/ml in controls). TNF-alpha, IL-8, and TGF-beta1 levels were comparable to those found in controls. In sera of patients with decompensated alcoholic liver cirrhosis, besides increased IL-6 (11.2+/-7.7 pg/ml), increased concentrations of TNF-alpha (25.1+/-4.5 vs. 9.1+/-7.0 pg/ml in controls) and IL-8 (171.7+/-294.0 vs. 2.7+/-2.9 pg/ml in controls) were also detected. TGF-beta1 and IL-10 levels were similar to those found in controls. These results strongly indicate that a significant derangement of the balance between proinflammatory and antiinflammatory signals is characteristic of compensated and especially of decompensated alcoholic cirrhosis.

Betulin and betulinic acid attenuate ethanol-induced liver stellate cell activation by inhibiting reactive oxygen species (ROS), cytokine (TNF-α, TGF-β) production and by influencing intracellular signaling.

BACKGROUND/AIMS Liver fibrosis has been reported to be inhibited in vivo by oleanolic and ursolic acids. However, the mechanisms of the action of those triterpenoids are poorly understood. In this study, we aimed to determine the antifibrotic potential of other triterpenes, betulin and betulinic acid, and to characterize their influence on the signal transduction pathways involved in ethanol-activated hepatic stellate cells (HSCs). METHODS Investigated was the influence of preincubation of rat HSCs with betulin and betulinic acid, at non-toxic concentrations, on ethanol-induced toxicity, migration, and several markers of HSC activation such as smooth muscle α-actin (α-SMA) and procollagen I expression, release of reactive oxygen species (ROS) and cytokines: tumor necrosis factor-α (TNF-α) and tumor growth factor-β1 (TGF-β1), and production of metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2). To assess the mechanism of the action of those triterpenes, intracellular signals such as nuclear factor-κB (NFκB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) induced by ethanol were examined. RESULTS In vitro, betulin, but not betulinic acid, protected HSCs against ethanol toxicity. However, both betulin and betulinic acid inhibited the production of ROS by HSCs treated with ethanol and inhibited their migration as well as ethanol-induced TNF-α, and TGF-β1, production. Betulin and betulinic acid down-regulated ethanol-induced production of TIMP-1 and TIMP-2. Betulin and betulinic acid, also decreased ethanol-induced activity of MMP-2. In ethanol-induced HSCs, betulin inhibited the activation of the p38 MAPK and the JNK transduction pathways, while betulinic acid inhibited the JNK transduction pathway only. They also significantly inhibited phosphorylation of IκB and Smad 3 and attenuated the activation of TGF-β1 and NFκB/IκB transduction signaling. CONCLUSION The results indicated that betulin and betulinic acid inhibited ethanol-induced activation of HSCs on different levels, acting as antioxidants, inhibitors of cytokine production, and inhibitors of TGF-β, and NFκB/IκB transduction signaling. Betulin was also inhibitor of both JNK and p38 MAPK signal transduction, while betulinic acid inhibited only JNK. The remarkable inhibition of several markers of HCS activation makes triterpenes, especially betulin, promising agents for anti-fibrotic combination therapies.

Oxidative stress in blood of patients with alcohol-related pancreatitis

To determine the possible role of oxidative stress in alcoholic pancreatitis, the authors measured the ability of blood neutrophils of 22 patients with acute and 20 patients with chronic alcoholic pancreatitis to produce superoxide anion (O2−) and hydrogen peroxide (H2O2), spontaneously and after in vitro stimulation with phorbol ester and compared it with that of neutrophils isolated from the blood of 16 healthy controls. In addition, they measured serum activities of superoxide dismutase, catalase, and the serum concentration of glutathione peroxidase (GPx). Phorbol ester–induced O2− and H2O2 production in neutrophils of patients with acute and chronic pancreatitis was greater than in controls, but these differences, except of superoxide anion production by neutrophils of patients with chronic pancreatitis, were not statistically significant because of large individual differences. Spontaneous resting production of O2− and H2O2 by neutrophils of patients with chronic pancreatitis was significantly greater than in the controls. Superoxide dismutase and catalase activity was greater in sera of both groups of patients with acute and chronic alcoholic pancreatitis than in controls, but GPx concentration was significantly less in the sera of patients with chronic pancreatitis. Impaired GPx production and increased production of O2− and H2O2 by neutrophils may result in increased lipid peroxidation and could play a role in the pathogenesis of chronic alcoholic pancreatitis.

Zinc supplementation attenuates ethanol- and acetaldehyde-induced liver stellate cell activation by inhibiting reactive oxygen species (ROS) production and by influencing intracellular signaling

BACKGROUND/AIMS Zinc has been reported to prevent and reverse liver fibrosis in vivo; however, the mechanisms of its action are poorly understood. We therefore aimed to determine the antifibrotic potential of zinc. METHODS Assessed was the influence of preincubation of rat HSCs with 30 microM ZnCl2 on ethanol- (in the presence of 4-methyl pyrazole (4-MP)) or acetaldehyde-induced toxicity, apoptosis, migration, expression of smooth muscle alpha-actin (alpha-SMA) and procollagen I, release of reactive oxygen species (ROS), tumor necrosis factor-alpha (TNF-alpha), tumor growth factor-beta1 (TGF-beta1), metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases (TIMPs) production. Intracellular signals such as nuclear factor-kappaB (NFkappaB), C-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) induced by ethanol and its metabolite were also assessed. RESULTS 30 microM zinc protected HSCs against ethanol and acetaldehyde toxicity and inhibited their apoptosis. Zinc inhibited the production of ROS by HSCs treated with ethanol and acetaldehyde and inhibited their migration. Zinc also inhibited ethanol- and acetaldehyde-induced TGF-beta1 and TNF-alpha production. Zinc down-regulated ethanol- and acetaldehyde-induced production of TIMP-1 and TIMP-2 and decreased the activity of MMP-2. In ethanol- and acetaldehyde-induced HSCs, zinc inhibited the activation of the p38 MAPK as well as the JNK transduction pathways and phosphorylation of IkappaB and Smad 3. CONCLUSION The results indicated that zinc supplementation inhibited ethanol- and acetaldehyde-induced activation of HSCs on different levels, acting as an antioxidant and inhibitor of MAPK, TGF-beta and NFkappaB/IkappaB transduction signaling. The remarkable inhibition of several markers of HCS activation makes zinc a promising agent for antifibrotic combination therapies.

Zinc inhibits ethanol-induced HepG2 cell apoptosis

Alcohol consumption produces a variety of metabolic alterations in liver cells, associated with ethanol oxidation and with nonoxidative metabolism of ethanol, among others apoptosis of hepatocytes. As zinc is known as a potent antioxidant and an inhibitor of cell apoptosis, the aim of this paper was to investigate whether zinc supplementation could inhibit ethanol-induced HepG2 apoptosis, and whether this inhibition was connected with attenuation of oxidative stress and modulation of FasR/FasL system expression. The results indicated that zinc supplementation significantly inhibited ethanol-induced HepG2 cell apoptosis (measured by cytochrome c release from mitochondria and caspase-3 activation) by attenuation of reactive oxygen species (ROS) production, increase in the cellular level of GSH, inhibition of ethanol-induced sFasR and FasL overexpression and caspase-8 activation. These results indicate that zinc can inhibit ethanol-induced hepatocyte apoptosis by several independent mechanisms, among others by an indirect antioxidative effect and probably by inhibition of caspase-8 and caspase-9 activation.

Biomarkers of alcohol misuse: recent advances and future prospects

Alcohol abuse and dependence are highly prevalent in many cultures and contribute considerably to the global burden of health and social issues. The current inability to accurately characterise long-term drinking behaviours is a major obstacle to alcoholism diagnosis and treatment. Therefore, it is of great importance to develop objective diagnostic tools to discern subjects with excessive alcohol use and alcoholism or to confirm abstinence. Research over past years has revealed several biochemical compounds with considerable potential for accurate reflection of alcohol intake. This review will address the issue of alcohol biomarker definition, the types of molecules used as so-called traditional biomarkers, and the compounds that can serve as novel biomarker candidates or components of biomarker panels.

Hyperreactivity of Blood Leukocytes in Patients with NAFLD to Ex Vivo Lipopolysaccharide Treatment Is Modulated by Metformin and Phosphatidylcholine but Not by Alpha Ketoglutarate

Introduction and Aims Toll-like receptor 4 and proinflammatory cytokines play a central role in the progression of nonalcoholic fatty liver disease. We investigated IL-1, IL-6 and TNFα production and toll-like receptor 4 in both—obese and lean patients with non-alcoholic fatty liver disease who met different sets of metabolic syndrome criteria and linked the results with the disease burden. Materials and Methods 95 subjects were divided into four groups depending on the following criteria: presence or absence of metabolic syndrome and/or non-alcoholic fatty liver disease, glucose tolerance (prediabetes or normoglycemia) and BMI value (obese or lean). We determined the levels of IL-1β, IL-6, TNFα, and monocyte toll-like receptor 4 expression in fresh blood as well as in blood cultures treated with lipopolysaccharide with or without metformin, alphaketoglutarate or phosphatidylcholine supplementation. Results The blood leukocytes of patients with non-alcoholic fatty liver disease are hypersensitive to lipopolysaccharide treatment and produce elevated levels of pro-inflammatory cytokines in response to ex vivo treatment with lipopolysaccharide. Moreover, they overexpress toll-like receptor-4. Hyperreactivity was typical mainly for obese patients with non-alcoholic fatty liver disease together with metabolic syndrome and decreased with the severity of disease. Metformin was the most effective in attenuation of hyperreactivity in all groups of patients with non-alcoholic fatty liver disease, but in obese patients the effectiveness of metformin was weaker than in lean. The reduction of cytokine level by metformin was accompanied by the decrease in toll-like receptor-4 expression. phosphatidylcholine also attenuated hyperreactivity to lipopolysaccharide but mainly in obese patients. Alpha ketoglutarate did not modulate cytokines’ level and toll-like receptor 4 expression in non-alcoholic fatty liver disease patients. Conclusions Metformin and phosphatidylcholine attenuated lipopolysaccharide induced toll-like receptor 4 overexpression and overproduction of pro-inflammatory cytokines; however, their efficacy depended on combined presence of non-alcoholic fatty liver disease, metabolic syndrome and obesity.

Peripheral blood dendritic cells in alcoholic and autoimmune liver disorders

Little is known about effects of alcohol consumption on dendritic cell (DC) function and resultant immune response. However, quantitative and qualitative disturbances of DCs are speculated to be involved in alcohol-related as well as in other liver pathology. The present study aimed to evaluate changes in circulating DC subsets in alcoholic liver disease (N = 43), autoimmune hepatitis (N = 26) and primary biliary cirrhosis (N = 20). DCs isolated from the peripheral blood of recruited participants were stained with monoclonal antibodies against blood dendritic cell antigens (BDCAs) and estimated using the flow cytometry. Myeloid DCs were defined as BDCA-1+/CD19− cells, and lymphoid DCs as BDCA-2+/CD123+ cells. Total numbers of circulating DCs in subjects with some liver diseases were markedly lower than in the healthy participants (p = 0.03). There was a significantly lower percentage of circulating BDCA-2+/CD123+ (p = 0.02), and a tendency for the percentage of circulating BDCA-1+/CD19− cells to decrease in patients with liver diseases compared to the controls (p = 0.09). These results may suggest that decreased numbers of DCs may be responsible for reduced adaptive immune responses and increased susceptibility to infections and cancer development observed in patients exposed to alcohol. Moreover, numerical abnormalities of DCs may contribute to the breakdown of self-tolerance, a feature of autoimmune diseases.

Ivabradine attenuates the anticonvulsant potency of lamotrigine, but not that of lacosamide, pregabalin and topiramate in the tonic-clonic seizure model in mice

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are involved not only in synaptic transmission and neuronal excitability under physiological conditions, but also in seizure activity. To determine the influence of ivabradine (an HCN channel inhibitor) on the anticonvulsant potency of four novel antiepileptic drugs (AEDs: lacosamide, lamotrigine, pregabalin and topiramate) in the mouse maximal electroshock-induced seizure (MES) model. Adult male albino Swiss mice were challenged with maximal electroconvulsions (electric current of 25mA delivered via auricular electrodes). Total brain concentrations of AEDs were measured with high-pressure liquid chromatography. Ivabradine (10mg/kg, i.p.) significantly reduced the anticonvulsant potency of lamotrigine by elevating the ED50 value of the AED from 7.48 (6.15-9.11) to 10.07 (8.85-11.45) mg/kg (P<0.05) in the mouse MES model. In contrast, ivabradine (10mg/kg, i.p.) did not significantly affect the anticonvulsant potency of lacosamide, pregabalin or topiramate in the mouse MES model. Additionally, ivabradine had no impact on total brain concentrations of all the studied AEDs in mice. A special caution is advised when combining ivabradine with lamotrigine in epilepsy patients due to the possible pharmacodynamic reduction of the anticonvulsant action of the later drug. The combinations of ivabradine with lacosamide, pregabalin and topiramate seem to be pharmacodynamic and neutral from a preclinical viewpoint.

Metformin Changes the Relationship between Blood Monocyte Toll-Like Receptor 4 Levels and Nonalcoholic Fatty Liver Disease—Ex Vivo Studies

Background Toll-like receptor 4 (TLR4) contributes to the development of NAFLD (nonalcoholic fatty liver disease) and MetS (metabolic syndrome). It is unclear whether anti-diabetic metformin affects TLR4 expression on blood monocytes, thereby protecting or improving inflammatory parameters. Therefore, we investigated TLR4 in patients with NAFLD meeting different sets of MetS criteria and linked the results with the disease burden. Methods 70 subjects were characterized and divided into three groups: (I) healthy individuals, (II) nonobese with NAFLD and without MetS, and (III) prediabetic, obese with NAFLD and MetS. We determined the concentrations of IL-1β, IL-6, TNFα, and monocyte TLR4 levels in fresh blood as well as in blood cultures with or without metformin supplementation. Results The characteristics of the study groups revealed a significant association between NAFLD and BMI, MetS and inflammatory parameters, and TLR4. In ex vivo studies, 100 μM of metformin decreased the TLR4 level by 19.9% (II group) or by 35% (III group) as well as IL-1β and TNFα production. A stepwise multiple regression analysis highlighted a strong effect of metformin on attenuation of the link between TLR4 and NAFLD, and TNFα. Conclusion We concluded that, by attenuation of the blood monocyte TLR4 level, metformin reduced their inflammatory potential—critical after recruitment these cells into liver. However, this finding should be confirmed after in vivo metformin administration.