Hepatic macrophage iron aggravates experimental alcoholic steatohepatitis

One prime feature of alcoholic liver disease (ALD) is iron accumulation in hepatic macrophages/Kupffer cells (KC) associated with enhanced NF-kappaB activation. Our recent work demonstrates a peroxynitrite-mediated transient rise in intracellular labile iron (ILI) as novel signaling for endotoxin-induced IKK and NF-kappaB activation in rodent KC. The present study investigated the mechanism of KC iron accumulation and its effects on ILI response in experimental ALD. We also tested ILI response in human blood monocytes. Chronic alcohol feeding in rats results in increased expression of transferrin (Tf) receptor-1 and hemochromatosis gene (HFE), enhanced iron uptake, an increase in nonheme iron content, and accentuated ILI response for NF-kappaB activation in KC. Ex vivo treatment of these KC with an iron chelator abrogates the increment of iron content, ILI response, and NF-kappaB activation. The ILI response is evident in macrophages derived from human blood monocytes by PMA treatment but not in vehicle-treated monocytes, and this differentiation-associated phenomenon is essential for maximal TNF-alpha release. PMA-induced macrophages load iron dextran and enhance ILI response and TNF-alpha release. These effects are reproduced in KC selectively loaded in vivo with iron dextran in mice and more importantly aggravate experimental ALD. Our results suggest enhanced iron uptake as a mechanism of KC iron loading in ALD and demonstrate the ILI response as a function acquired by differentiated macrophages in humans and as a priming mechanism for ALD.     

Lactobacillus rhamnosus GG reduces hepatic TNFα production and inflammation in chronic alcohol-induced liver injury

The therapeutic effects of probiotic treatment in alcoholic liver disease (ALD) have been studied in both patients and experimental animal models. Although the precise mechanisms of the pathogenesis of ALD are not fully understood, gut-derived endotoxin has been postulated to play a crucial role in hepatic inflammation. Previous studies have demonstrated that probiotic therapy reduces circulating endotoxin derived from intestinal gram-negative bacteria in ALD. In this study, we investigated the effects of probiotics on hepatic tumor necrosis factor-α (TNFα) production and inflammation in response to chronic alcohol ingestion. Mice were fed Lieber DeCarli liquid diet containing 5% alcohol for 8 weeks, and Lactobacillus rhamnosus GG (LGG) was supplemented in the last 2 weeks. Eight-week alcohol feeding caused a significant increase in hepatic inflammation as shown by histological assessment and hepatic tissue myeloperoxidase activity assay. Two weeks of LGG supplementation reduced hepatic inflammation and liver injury and markedly reduced TNFα expression. Alcohol feeding increased hepatic mRNA expression of Toll-like receptors (TLRs) and CYP2E1 and decreased nuclear factor erythroid 2-related factor 2 expression. LGG supplementation attenuated these changes. Using human peripheral blood monocytes-derived macrophages, we also demonstrated that incubation with ethanol primes both lipopolysaccharide- and flagellin-induced TNFα production, and LGG culture supernatant reduced this induction in a dose-dependent manner. In addition, LGG treatment also significantly decreased alcohol-induced phosphorylation of p38 MAP kinase. In conclusion, probiotic LGG treatment reduced alcohol-induced hepatic inflammation by attenuation of TNFα production via inhibition of TLR4- and TLR5-mediated endotoxin activation.     

Nf-kappab activation is associated with free radical generation and endotoxemia and precedes pathological liver injury in experimental alcoholic liver disease.

Endotoxemia and oxidative stress activate nuclear factor kappa B (NF-kappaB) in alcoholic liver injury. In alcohol-fed rats, activation of NF-kappaB is associated with the development of necro-inflammatory changes in the liver. Whether activation of NF-kappaB occurs prior to development of liver injury is unknown. We determined whether activation of NF-kappaB preceded histopathological liver changes. Male Wistar rats were fed a liquid diet containing ethanol by continuous infusion through permanently implanted gastric tubes. Radical intermediates detected by spin trapping were measured in bile prior to killing. After 2 weeks of treatment, samples of liver tissue were obtained for histopathological examination, for evaluation of NF-kappaB, and determination of messenger RNA levels of cytokines, chemokines and cyclo-oxygenase-2. No pathological changes in liver were seen after 2 weeks of intragastric feeding. However, activation of NF-kappaB was seen in the livers from ethanol-fed rats. In addition, elevated mRNA levels of hepatic pro-inflammatory cytokines (TNF-alpha and IL12), chemokines MIPIalpha and MIP-2) and cyclo-oxygenase-2 were seen in association with activation of NF-kappaB and increased levels of free radicals and endotoxin. Thus, activation of NF-kappaB, associated with elevated mRNA levels of pro-inflammatory stimuli, precedes the histopathological liver changes in experimental alcoholic liver disease in rats.     

Curcumin prevents alcohol-induced liver disease in rats by inhibiting the expression of NF-kappa B-dependent genes

Induction of NF-kappaB-mediated gene expression has been implicated in the pathogenesis of alcoholic liver disease (ALD). Curcumin, a phenolic antioxidant, inhibits the activation of NF-kappaB. We determined whether treatment with curcumin would prevent experimental ALD and elucidated the underlying mechanism. Four groups of rats (6 rats/group) were treated by intragastric infusion for 4 wk. One group received fish oil plus ethanol (FE); a second group received fish oil plus dextrose (FD). The third and fourth groups received FE or FD supplemented with 75 mg. kg(-1). day(-1) of curcumin. Liver samples were analyzed for histopathology, lipid peroxidation, NF-kappaB binding, TNF-alpha, IL-12, monocyte chemotactic protein-1, macrophage inflammatory protein-2, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nitrotyrosine. Rats fed FE developed fatty liver, necrosis, and inflammation, which was accompanied by activation of NF-kappaB and the induction of cytokines, chemokines, COX-2, iNOS, and nitrotyrosine formation. Treatment with curcumin prevented both the pathological and biochemical changes induced by alcohol. Because endotoxin and the Kupffer cell are implicated in the pathogenesis of ALD, we investigated whether curcumin suppressed the stimulatory effects of endotoxin in isolated Kupffer cells. Curcumin blocked endotoxin-mediated activation of NF-kappaB and suppressed the expression of cytokines, chemokines, COX-2, and iNOS in Kupffer cells. Thus curcumin prevents experimental ALD, in part by suppressing induction of NF-kappaB-dependent genes.     

Cytochrome P4502E1 primes macrophages to increase TNF-alpha production in response to lipopolysaccharide

Kupffer cells become activated in response to elevated levels of LPS during ethanol feeding, but the role of ethanol in the molecular processes of activation remains unclear. Because cytochrome P4502E1 (CYP2E1) is upregulated in Kupffer cells after ethanol, we hypothesized that this effect primes Kupffer cells, sensitizing them to increase TNF-alpha production in response to LPS. However, cultured Kupffer cells rapidly lose their CYP2E1. This difficulty was overcome by transfecting CYP2E1 to RAW 264.7 macrophages. Macrophages with stable increased CYP2E1 expression (E2) displayed increased levels of CD14/Toll-like receptor 4, NADPH oxidase and H2O2, accompanied by activation of ERK1/2, p38, and NF-kappaB. These increases primed E2 cells, sensitizing them to LPS stimuli, with amplification of LPS signaling, resulting in increased TNF-alpha production. Diphenyleneiodonium, a NADPH oxidase inhibitor, and diallyl sulfide, a CYP2E1 inhibitor, decreased approximately equally H2O2 levels in E2 cells, suggesting that NADPH oxidase and CYP2E1 contribute equally to H2O2 generation. Because CYP2E1 expression also enhanced the levels of the membrane localized NADPH oxidase subunits p47phox and p67phox, thereby contributing to the oxidase activation, it may augment H2O2 generation via this mechanism. H2O2, derived in part from NADPH and CYP2E1, activated ERK1/2 and p38. ERK1/2 stimulated TNF-alpha production via activation of NF-kappaB, whereas p38 promoted TNF-alpha production by stabilizing TNF-alpha mRNA. Oxidant generation after CYP2E1 overexpression appears to be central to macrophage priming and their sensitization to LPS. Accordingly, CYP2E1 priming could explain the sensitization of Kupffer cells to LPS activation by ethanol, a critical early step in alcoholic liver disease.     

Suramin prevents fulminant hepatic failure resulting in reduction of lethality through the suppression of NF-kappaB activity

AIM: Suramin is a symmetrical polysulfonated naphthylamine derivative of urea. There have been few studies on the effect of suramin on cytokines. We examined the effects of suramin on production of inflammatory cytokines. METHODS: We made an acute liver injury model treated with d-galactosamine (GalN) and lipopolysaccharide (LPS). Plasma AST, ALT, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-6 levels were measured. We compared with survival rate, histological found and NF-kappaB activity between with and without treatment of suramin. In macrophage like cell line, TNF-alpha and IL-6 production, TNF-alpha and IL-6 mRNA expression, and NF-kappaB activity was measured. RESULTS: The lethality of mice administered suramin with GalN/LPS was significantly decreased compared with that in mice without suramin. Changes of hepatic necrosis and apoptosis were slight in suramin-treated mice. Serum AST, ALT, TNF-alpha, IL-6 levels and NF-kappaB activity in the liver were significantly lower in mice administered suramin. In an in vitro model, suramin preincubation inhibited TNF-alpha and IL-6 production, TNF-alpha and IL-6 mRNA expression, and NF-kappaB activity. CONCLUSIONS: Suramin inhibits TNF-alpha and IL-6 production through the suppression of NF-kappaB activity from macrophages and shows therapeutic effects on acute liver damage.     

Zinc supplementation inhibits hepatic apoptosis in mice subjected to a long-term ethanol exposure

Hepatocyte apoptosis has been documented in both clinical and experimental alcoholic liver disease. This study was undertaken to examine the effect of dietary zinc supplementation on hepatic apoptosis in mice subjected to a long-term ethanol exposure. Male adult 129S6 mice fed an ethanol-containing liquid diet for 6 months developed hepatitis, as indicated by neutrophil infiltration and elevation of hepatic keratinocyte chemoattractant (KC) and monocyte chemoattractant protein-1 (MCP-1) levels. Apoptotic cell death was detected in ethanol-exposed mice by a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and was confirmed by the increased activities of caspase-3 and -8. Zinc supplementation attenuated alcoholic hepatitis and reduced the number of TUNEL-positive cells in association with inhibition of caspase activities. Ethanol exposure caused oxidative stress, as indicated by reactive oxygen species accumulation, mitochondrial glutathione depletion, and decreased metallothionein levels in the liver, which were suppressed by zinc supplementation. The mRNA levels of tumor necrosis factor (TNF)-alpha, TNF-R1, FasL, Fas, Fas-associated factor-1, and caspase-3 in the liver were upregulated by ethanol exposure, which were attenuated by zinc supplementation. Zinc supplementation also prevented ethanol-elevated serum and hepatic TNF-alpha levels and TNF-R1 and Fas proteins in the liver. In conclusion, zinc supplementation prevented hepatocyte apoptosis in mice subjected to long-term ethanol exposure, and the action of zinc is likely through suppression of oxidative stress and death receptor-mediated pathways.     

Dilinoleoylphosphatidylcholine decreases acetaldehyde-induced TNF-alpha generation in Kupffer cells of ethanol-fed rats

We previously reported that dilinoleoylphosphatidylcholine (DLPC) decreases lipopolysaccharide-induced TNF-alpha generation by Kupffer cells of ethanol-fed rats by blocking p38, ERK1/2, and NF-kappaB activation. Here we show that DLPC also decreases TNF-alpha induction by acetaldehyde, a toxic metabolite released by ethanol oxidation. Acetaldehyde induces TNF-alpha generation with a maximal effect at 200 microM and activates p38 and ERK1/2; the latter in turn activates NF-kappaB. This effect is augmented in Kupffer cells of ethanol-fed rats, with upregulation of cytochrome P4502E1 by ethanol. DLPC decreases TNF-alpha generation by blocking p38, ERK1/2, and NF-kappaB activation. Likewise, SB203580, which abolishes p38 activation, and PD098059, which abrogates ERK1/2 and NF-kappaB activation, diminish TNF-alpha generation. Since increased TNF-alpha generation plays a pathogenic role in alcoholic liver disease, the DLPC action on Kupffer cells may explain, in part, its beneficial effects on liver cell injury after ethanol consumption.     

Oxidative mechanisms in the pathogenesis of alcoholic liver disease

Although the capacity of ethanol to induce oxidative stress in the liver is well established, the mechanisms by which oxidative damage contributes to the pathogenesis of alcoholic liver disease (ALD) is still incompletely understood. Recent reports have implicated oxidative mechanisms in the onset of alcoholic steatosis and in the formation of Mallory's bodies. Moreover, by inducing mitochondrial alterations, oxidative stress promotes hepatocyte necrosis and contributes to alcohol-induced sensitization of hepatocyte to the pro-apoptotic action of TNF-alpha. Oxidative mechanisms play also a role in the progression of liver fibrosis by triggering the release of pro-fibrotic cytokines and activating collagen gene expression in hepatic stellate cells. Finally, immune responses towards antigens originating from the reactions of lipid peroxidation products with hepatic proteins might represent one of the mechanisms that contribute to perpetuate chronic hepatic inflammation in ALD. Altogether these observations give a rationale to the possible clinical application of antioxidants in the therapy of ALD.     

The functional role of microRNAs in alcoholic liver injury

The function of microRNAs (miRNAs) during alcoholic liver disease (ALD) has recently become of great interest in biological research. Studies have shown that ALD associated miRNAs play a crucial role in the regulation of liver‐inflammatory agents such as tumour necrosis factor‐alpha (TNF‐α), one of the key inflammatory agents responsible for liver fibrosis (liver scarring) and the critical contributor of alcoholic liver disease. Lipopolysaccharide (LPS), a component of the cell wall of gram‐negative bacteria, is responsible for TNF‐α release by Kupffer cells. miRNAs are the critical mediators of LPS signalling in Kupffer cells, hepatocytes and hepatic stellate cells. Certain miRNAs, in particular miR‐155 and miR‐21, show a positive correlation in up‐regulation of LPS signalling when they are exposed to ethanol. ALD is related to enhanced gut permeability that allows the levels of LPS to increase, leads to increased secretion of TNF‐α by the Kupffer cells and subsequently promotes alcoholic liver injury through specific miRNAs. Meanwhile, two of the most frequently dysregulated miRNAs in steatohepatitis, miR‐122 and miR‐34a are the critical mediators in ethanol/LPS activated survival signalling during ALD. In this review, we summarize recent findings regarding the experimental and clinical aspects of functions of specific microRNAs, focusing mainly on inflammation and cell survival after ethanol/LPS treatment, and advances on the role of circulating miRNAs in human alcoholic disorders.     

Zinc prevention and treatment of alcoholic liver disease

Alcoholic liver disease (ALD) is associated with decreases in zinc (Zn) and its major binding protein, metallothionein (MT), in the liver. Studies using animal models have shown that Zn supplementation prevents alcohol-induced liver injury under both acute and chronic alcohol exposure conditions. There are hepatic and extrahepatic actions of Zn in the prevention of alcoholic liver injury. Zn supplementation attenuates ethanol-induced hepatic Zn depletion and suppresses ethanol-elevated cytochrome P450 2E1 (CYP2E1) activity, but increases the activity of alcohol dehydrogenase in the liver; an action that is likely responsible for Zn suppression of alcohol-induced oxidative stress. Zn also enhances glutathione-related antioxidant capacity in the liver. At the cellular level, Zn inhibits alcohol-induced hepatic apoptosis partially through suppression of the Fas/FasL-mediated pathway. Zn supplementation preserves intestinal integrity and prevents endotoxemia, leading to inhibition of endotoxin-induced tumor necrosis factor-alpha (TNF-alpha) production in the liver. Zn also directly inhibits the signaling pathway involved in endotoxin-induced TNF-alpha production. These hepatic and extrahepatic effects of Zn are independent of MT. However, low levels of MT in the liver sensitize the organ to alcohol-induced injury, and elevation of MT enhances the endogenous Zn reservoir and makes Zn available when oxidative stress is imposed. Zn has a high potential to be developed as an effective agent in the prevention and treatment of ALD.     

Role of cAMP and phosphodiesterase signaling in liver health and disease

Liver disease is a significant health problem worldwide with mortality reaching around 2 million deaths a year. Non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) are the major causes of chronic liver disease. Pathologically, NAFLD and ALD share similar patterns of hepatic disorders ranging from simple steatosis to steatohepatitis, fibrosis and cirrhosis. It is becoming increasingly important to identify new pharmacological targets, given that there is no FDA-approved therapy yet for either NAFLD or ALD. Since the evolution of liver diseases is a multifactorial process, several mechanisms involving parenchymal and non-parenchymal hepatic cells contribute to the initiation and progression of liver pathologies. Moreover, certain protective molecular pathways become repressed during liver injury including signaling pathways such as the cyclic adenosine monophosphate (cAMP) pathway. cAMP, a key second messenger molecule, regulates various cellular functions including lipid metabolism, inflammation, cell differentiation and injury by affecting gene/protein expression and function. This review addresses the current understanding of the role of cAMP metabolism and consequent cAMP signaling pathway(s) in the context of liver health and disease. The cAMP pathway is extremely sophisticated and complex with specific cellular functions dictated by numerous factors such abundance, localization and degradation by phosphodiesterases (PDEs). Furthermore, because of the distinct yet divergent roles of both of its effector molecules, the cAMP pathway is extensively targeted in liver injury to modify its role from physiological to therapeutic, depending on the hepatic condition. This review also examines the behavior of the cAMP-dependent pathway in NAFLD, ALD and in other liver diseases and focuses on PDE inhibition as an excellent therapeutic target in these conditions.