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-1ß, 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.

Butein inhibits ethanol-induced activation of liver stellate cells through TGF-ß, NFκB, p38, and JNK signaling pathways and inhibition of oxidative stress.

BACKGROUND: Butein has been reported to prevent and partly reverse liver fibrosis in vivo; however, the mechanisms of its action are poorly understood. We, therefore, aimed to determine the antifibrotic potential of butein. METHODS: We assessed the influence of the incubation of hepatic stellate cells (HSCs) and hepatoma cells (HepG2) with butein on sensitivity to ethanol- or acetaldehyde-induced toxicity; the production of reactive oxygen species (ROS); the expression of markers of HSC activation, including smooth muscle α-actin (α-SMA) and procollagen I; and the production of transforming growth factor-ß1 (TGF-ß1), metalloproteinases-2 and -13 (MMP-2and MMP-13), and tissue inhibitors of metalloproteinases (TIMPs). The influence of butein on intracellular signals in HSCs; i.e., nuclear factor-κB (NFκB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) induced by ethanol was estimated. RESULTS: Butein protected HSCs and HepG2 cells against ethanol toxicity by the inhibition of ethanol- or acetaldehyde-induced production of ROS when cells were incubated separately or in co-cultures; butein also inhibited HSC activation measured as the production of α-SMA and procollagen I. As well, butein downregulated ethanol- or acetaldehyde-induced HSC migration and the production of TGF-ß, TIMP-1, and TIMP-2; decreased the activity of MMP-2; and increased the activity of MMP-13. In ethanol-induced HSCs, butein inhibited the activation of the p38 MAPK and JNK transduction pathways as well as significantly inhibiting the phosphorylation of NF κB inhibitor (IκB) and Smad3. CONCLUSIONS: The results indicated that butein inhibited ethanol- and acetaldehyde-induced activation of HSCs at different levels, acting as an antioxidant and inhibitor of ethanol-induced MAPK, TGF-ß, and NFκB/IκB transduction signaling; this result makes butein a promising agent for antifibrotic therapies.

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.

Apoptosis of blood mononuclear cells in alcoholic liver cirrhosis. The influence of in vitro ethanol treatment and zinc supplementation.

Ethanol consumption induces apoptosis in a variety of tissues, among others in liver and lymphoid tissue. Zinc has been shown to influence apoptosis of blood mononuclear cells by inhibiting the mitochondrial pathway of cell death. The aim of this study was to examine the influence of zinc on spontaneous and in vitro alcohol-induced apoptosis of peripheral blood mononuclear cells (PBMCs) of patients with alcoholic cirrhosis. PBMCs were isolated from the blood of 26 patients with cirrhosis and 20 healthy controls. PBMCs and among them CD4+ T helper cells of cirrhotic patients exhibited accelerated spontaneous (without treatment) apoptosis in vitro. When apoptosis was induced in vitro by treating cells with 80 mM ethanol, CD8+ T lymphocytes of a healthy control were more sensitive to ethanol treatment than those of cirrhotic patients. Thirty micromolar zinc supplementation inhibited both spontaneous and ethanol-induced apoptosis of immune cells derived from the blood of the healthy control and cirrhotic patients. In sera of patients with cirrhosis, an elevated level of IL-12, but also sFas (CD95) and sFas ligand (sFasL) was detected. Moreover, in vitro, PBMCs of cirrhotic patients spontaneously released more sFas and sFasL than control PBMCs. Ethanol treatment significantly increased sFas, but decreased sFasL release from PBMCs of cirrhotic patients, while it only slightly affected control cells. As zinc supplementation did not significantly influence sFas or sFasL release, it seems likely that it is rather the mitochondrial pathway of ethanol-related immune cell death that may be inhibited by zinc supplementation.