Gut-derived Endotoxin Research Articles

Effects of gut barrier dysfunction and NF‐κB activation on aggravating mechanism of severe acute pancreatitis

To study the effects of gut-derived endotoxin translocation and NF-kappaB activation on the aggravating mechanism of severe acute pancreatitis (SAP) and of treatment with pyrrolidine dithiocarbamate (PDTC) on rats with SAP. SD rats were randomly divided into sham operation group (SO), SAP group, SAP + lipopolysaccharide(LPS) group, pyrrolidine dithiocarbamate (PDTC) treatment group and LPS group. Biochemical parameters and cytokines were examined in the serum. Multiple organs pathological slices were examined. Expression of NF-kappaB mRNA in the liver tissue was detected by RT-PCR. Activation of NF-kappaB by the method of streptomycin avidin-peroxidase (SP) and expression of NF-kappaB p65 protein and its binding activity were analyzed by Western blot and electrophoretic mobidity shift assay (EMSA). Compared with sham operation group, the concentration of TNF-alpha, alanine aminotransferase (ALT), and diamine oxidase (DAO) in serum significantly increased in SAP + LPS group (P < 0.05). Pathological changes were markedly observed in tissues and the expression of NF-kappaB mRNA in the liver significantly increased (P < 0.05) also, the activation of NF-kappaB and binding activity of NF-kappaB p65 protein in the liver markedly increased (P < 0.01) in SAP + LPS group. Treatment with PDTC markedly reduced concentration of ALT, DAO and TNF-alpha, and the expression of NF-kappaB, and the pathologic scores, as well as significantly decreased the expression of NF-kappaB p65 protein. The activation and overexpression of NF-kappaB may participate in the aggravating mechanism of SAP. Treatment with PDTC has a protective effect on multiple organs damage in SAP.

Increased intestinal permeability and tight junction alterations in nonalcoholic fatty liver disease†

The role played by the gut in nonalcoholic fatty liver disease (NAFLD) is still a matter of debate, although animal and human studies suggest that gut-derived endotoxin may be important. We investigated intestinal permeability in patients with NAFLD and evaluated the correlations between this phenomenon and the stage of the disease, the integrity of tight junctions within the small intestine, and prevalence of small intestinal bacterial overgrowth (SIBO). We examined 35 consecutive patients with biopsy-proven NAFLD, 27 with untreated celiac disease (as a model of intestinal hyperpermeability) and 24 healthy volunteers. We assessed the presence of SIBO by glucose breath testing (GBT), intestinal permeability by means of urinary excretion of (51)Cr-ethylene diamine tetraacetate ((51)Cr-EDTA) test, and the integrity of tight junctions within the gut by immunohistochemical analysis of zona occludens-1 (ZO-1) expression in duodenal biopsy specimens. Patients with NAFLD had significantly increased gut permeability (compared with healthy subjects; P < 0.001) and a higher prevalence of SIBO, although both were lower than in the untreated celiac patients. In patients with NAFLD, both gut permeability and the prevalence of SIBO correlated with the severity of steatosis but not with presence of NASH. Our results provide the first evidence that NAFLD in humans is associated with increased gut permeability and that this abnormality is related to the increased prevalence of SIBO in these patients. The increased permeability appears to be caused by disruption of intercellular tight junctions in the intestine, and it may play an important role in the pathogenesis of hepatic fat deposition.

Gut-derived endotoxin translocation is the main aggravating mechanism of acute severe pancreatitis

Severe acute pancreatitis (SAP) is a serious systemic disease. It exacerbates when complicated with multiple organ dysfunction syndrome or failure (MODS or MOF). However, the aggravating mechanism of SAP is still unknown up to now. Study showed that maintaining integrity of intestinal mucosal barrier function by given effective antibiotics, selective digestive decontamination (SDD) and enteral nutrition therapy to the patients with SAP could significantly reduce infection of pancreatic necrotic tissue and improve the patient's outcome. Combining the findings of gut-derived bacteria in animals' pancreas, liver, spleen, mesenteric lymph nodes with increasing concentration of inflammatory cytokines and endotoxin in plasma with SAP, we hypothesize that gut-derived endotoxin translocation is the main aggravating mechanism of SAP. The hypothesis holds potential as a target for therapeutic intervention.

Oral administration of heat-killed Lactobacillus brevis SBC8803 ameliorates alcoholic liver disease in ethanol-containing diet-fed C57BL/6N mice

We examined the effect of heat-killed Lactobacillus brevis ( L. brevis) SBC8803 on the development of alcoholic liver disease using ethanol-containing diet-fed mice. Heat-killed L. brevis was orally administered at a dose of 100 or 500 mg/kg once a day for 35 days. Alcoholic liver injury was examined by measuring the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in a serum, and the alcoholic fatty liver was assessed from the content of triglyceride (TG) and total cholesterol in the liver. Quantitative RT-PCR was used to examine mRNA expression of tumor necrosis factor (TNF)-α, sterol regulatory element-binding protein (SREBP)-1, SREBP-2, and peroxisome proliferator-activated receptor α (PPARα) in the liver, as well as E-cadherin, Zonula occludens 1 (ZO-1), and heat shock protein (Hsp) 25 in the small intestine. Oral administration of L. brevis significantly inhibited an increase in the level of serum ALT and AST, as well as the content of TG and total cholesterol in the liver caused by ethanol intake. L. brevis supplementation suppressed the overexpression of TNF-α, SREBP-1, and SREBP-2 mRNA in the liver induced by ethanol intake and up-regulated the expression of Hsp25 mRNA in the small intestine. These results suggest that L. brevis ameliorated the ethanol-induced liver injury and the fatty liver by suppressing the up-regulation of TNF-α and SREBPs in the liver. We speculate that the inhibition of TNF-α and SREBPs up-regulation by L. brevis is due to the inhibition of gut-derived endotoxin migration into the liver through the enhancement of intestinal barrier function by the induction of cytoprotective Hsps.

Obesity-Induced Lymphocyte Hyperresponsiveness to Chemokines: A New Mechanism of Fatty Liver Inflammation in Obese Mice

Hepatic lipid retention (steatosis) predisposes hepatitis. We investigated the mechanisms of lymphocyte homing to fatty liver and the role of lipopolysaccharide (LPS) in the onset of inflammation in ob/ob mice. We decreased intestinal bacterial compounds by oral antibiotic treatment to test the role of endogenous LPS in liver inflammation. Adoptive transfer of lymphocytes was used to study the respective contributions of steatosis and lymphocytes to liver inflammation. We tested lymphocyte response to chemokines by in vitro chemotaxis assays in ob/ob, their lean controls, and "non-obese ob/ob" mice, generated by controlling caloric intake to distinguish between the effects of obesity and leptin deficiency. Antibiotic treatment decreased liver infiltration with CD4(+) T, CD8(+) T, natural killer (NK)T, B, and NK cells. Adoptive transfer of lymphocytes from ob/ob or control mice showed that (1) steatosis increased lymphocyte recruitment to the liver; (2) CD4(+) T, CD8(+) T, and B cells from ob/ob mice had a greater propensity to migrate specifically to the liver. This migration was enhanced by LPS. These results were also observed in a model of high-fat diet-induced obesity. CD4(+) T and B cells were hyperresponsive to CXCL12 and CXCL13, respectively. Weight normalization in "non-obese ob/ob" mice decreased liver inflammation, lymphocyte response to chemokines, and homing to the liver. Our study provides the first evidence that liver inflammation in mice with genetic or diet-induced obesity results from both steatosis and lymphocyte hyperresponsiveness to chemokines expressed in the liver. These abnormalities are reversible with weight normalization.

Abrogation of the Antifibrotic Effects of Natural Killer Cells/Interferon-γ Contributes to Alcohol Acceleration of Liver Fibrosis

Chronic alcohol drinking accelerates liver fibrosis in patients with viral hepatitis that cannot be fully explained by ethanol-enhanced liver damage. Here, we identified a novel mechanism by which alcohol accelerates liver fibrosis: inhibition of the antifibrotic effects of natural killer (NK) cells and interferon-gamma (IFN-gamma). Alcohol administration was achieved by feeding mice with a liquid diet containing 5% ethanol for 8 weeks. Liver fibrosis was induced by administration of carbon tetrachloride (CCl(4)) for 2 weeks. Hepatic stellate cells (HSCs) were also isolated and cultured for in vitro studies. CCl(4) treatment induced greater fibrosis and less apoptosis of HSCs in ethanol-fed mice compared with pair-fed mice. Polyinosinic-polycytidylic acid (Poly I:C) or IFN-gamma treatment inhibited liver fibrosis in pair-fed but not in ethanol-fed mice. Poly I:C activation of NK cell cytotoxicity against HSCs was attenuated in ethanol-fed mice compared with pair-fed mice, which was due to reduced natural killer group 2 member D (NKG2D), tumor necrosis factor-related apoptosis-inducing ligand, and IFN-gamma expression on NK cells from ethanol-fed mice. In vitro, HSCs from ethanol-fed mice were resistant to IFN-gamma-induced cell cycle arrest and apoptosis compared with pair-fed mice. Such resistance was due to diminished IFN-gamma activation of signal transducer and activator of transcription 1 (STAT1) in HSCs from ethanol-fed mice caused by the induction of suppressors of cytokine signaling proteins and the production of oxidative stress. Finally, HSCs from ethanol-fed mice were resistant to NK cell killing, which can be reversed by transforming growth factor-beta1 (TGF-beta1) neutralizing antibody. Chronic ethanol consumption attenuates the antifibrotic effects of NK/IFN-gamma/STAT1 in the liver, representing new and different therapeutic targets with which to treat alcoholic liver fibrosis.

Anti‐inflammatory strategies in alcoholic steatohepatitis

The hepatotoxic effects of alcohol have been described in detail, but mechanisms underlying the hepatotoxicity have been only partially characterized. Recently, increasing lines of evidence indicate that Kupffer cells play multiple roles in initiation and progression of alcoholic steatohepatitis. After ethanol exposure, Kupffer cells are activated via a mechanism dependent on gut-derived endotoxin, and release active mediators such as proinflammatory cytokines and eicosanoids. These mediators are responsible for the pathophysiology of alcoholic steatohepatitis. This review discusses the current concept of Kupffer cell-mediated steatohepatitis and how it relates to the hypothesis on the mechanism by which alcoholic steatohepetitis is caused, as well as several key issues that have to be addressed in this field: (i) How do Kupffer cells undergo priming and activation during alcoholic steatohepatitis?; (ii) What kind of mediators are involved?; and (iii) How does the concept translate into a strategy for therapeutics of alcoholic steatohepatitis?

Expression of Toll‐like receptor 4 in rat liver during the course of carbon tetrachloride‐induced liver injury

It has recently been reported that Toll-like receptor 4 (TLR4) is involved in cellular responses to lipopolysaccharides (LPS) and early liver injury induced by LPS. The aim of the present study was to investigate the alterations of TLR4 gene expression in liver tissues and Kupffer cells during the course of carbon tetrachloride (CCl(4))-induced chronic liver injury and fibrosis and its role in liver injury. Rats were induced with liver injury and fibrosis by CCl(4) administered subcutaneously twice weekly for up to 8 weeks. The Kupffer cells were isolated by the combined collagenase-pronase perfusion method and incubated with varying doses of LPS. The mRNA expression of TLR4 in liver tissues and Kupffer cells was measured by reverse transcriptase polymerase chain reaction. The levels of tumor necrosis factor (TNF)-alpha in Kupffer cell culture supernatants were determined by enzyme-linked immunosorbent assay. The plasma levels of the endotoxin were determined by chromogenic substrate limulus amebocyte lysate assay. The association of the endotoxin receptor expression with plasma endotoxin levels was assessed. CCl(4) administration elicited extensive changes in liver morphology, including steatosis, inflammation, necrosis, and fibrosis. Low levels of TLR4 mRNA were detected in normal rat liver tissues, but no expression was detected in the Kupffer cells. The expression of TLR4 mRNA in liver tissues and Kupffer cells was increased 2 weeks after CCl(4) administration, peaked at 4 and 6 weeks, and declined at 8 weeks. Basic TNF-alpha production of Kupffer cells isolated from CCl(4)-treated rats at 4 and 6 weeks was significantly higher than that of normal rats (P < 0.05). Upon LPS stimulation, production of TNF-alpha was markedly increased in Kupffer cells isolated from normal and 2-,4-, and 6-week CCl(4)-treated rats. Moreover, LPS-induced TNF-alpha production was dose-dependent. The plasma levels of the endotoxin were increased during the time of liver injury. There was a correlation between plasma endotoxin levels and TLR4 gene expression in the early and middle stage of liver injury. The gene expression of TLR4 was upregulated during the course of CCl(4)-induced liver injury, which is associated with the degree of liver injury and Kupffer cell activation. The gut-derived endotoxin may be involved in the upregulation of TLR4 expression.

Gut Mucosa in HIV Infection: “Immune Milk” Solution

In recent weeks, three noteworthy papers, published in PLoS Medicine, The Journal of Virology, and Nature Medicine, direct our attention to the gut as a critical target in HIV-1 infection and portal for therapeutic intervention. In PLoS Medicine, Mehandru and colleagues report that over half of the CD4+ T cells in the gut mucosa are lost within the first few weeks after HIV-1 infection and remain consistently low, compared to peripheral blood sources, despite long term antiretroviral therapy; furthermore, of the few CD4+ T cells that persist in the gut, a significant increase in immune activation is observed [1]. Consistent with earlier observation in SIV models, Veazey reminds us that the battle against HIV-1 should focus on the intestinal mucosa with therapeutic strategies to reduce gut immune activation [2]. The longitudinal study in The Journal of Virology by Guadalupe and colleagues showed a similar discordance in CD4+ T cells between restoration in peripheral blood and significant delay in the gut mucosa of chronic infected individuals during antiretroviral therapy. Here, the depletion in CD4+ T cells was associated with an increase in gut immune activation, CD8+ T cells, and associated inflammation with a corresponding decease in epithelial growth and repair-associated genes in gut mucosal tissue [3]. Brenchley and colleagues suggest in Nature Medicine that HIV infection causes this breakdown in the gut mucosa resulting in a “leaky gut,” thus allowing translocation of gut-derived endotoxin and the subsequent triggering of immune activation [4]. Collectively, these observations suggest that an orally active therapeutic, used in conjunction with antiretroviral therapy, be designed to both block gut-derived microbial translocation and stimulate restitution of the gut epithelium. The hope would be to restore immunological integrity of the intestinal mucosal barrier, thereby controlling immune activation, both locally, in the gut mucosa, and systemically by suppressing cellular targets distal to the gut that may directly contribute to the progression of AIDS [5–8]. The design for such an orally active therapeutic may be found in the complex formula of bovine colostrum and “immune milk,” which has long been recognized to offer passive protection from a broad number of enteric bacterial and viral pathogens, primarily via the transfer of immunoglobulins and suppression of gut-associated inflammation with promotion of mucosal repair and regeneration. The gut in chronic HIV-1-infected individuals appears to be reminiscent of newborn calves. Calves are born with a highly immature mucosal immune system and “leaky gut” which, if not immediately corrected, results in death due to infection and associated systemic immune activation. However, the cow's first milk rescues her calves from harmful gut microbes with a uniquely complex cocktail enriched with neutralizing polyclonal antibodies, cytokine tissue repair factors, and immune enhancing probiotics, such as Lactobacillus species. Regular consumption of biologically active bovine colostrum has been known for years to promote the development of infantile gut-associated lymphoid tissue and enhance CD4+ levels, while suppressing CD8+ and inflammatory bowl disease (IBD), including ulcerative colitis and Crohn disease [5]. The severity of IBD is often correlated with gut microbial-endotoxin translocation, which now appears in chronic HIV-1-infected individuals [4]. Similar to bovine colostrum, “immune milk” from properly vaccinated cows affords passive immunity against bacterial, viral, and fungal infections in the human gastrointestinal tract, as well as taming gut inflammation [8]. Hence, there may be lessons learned from Bessie's “immune milk.” If viewed as a unique formula that has evolved to complement gut immunity, “immune milk” may also provide relief in chronic HIV-infected individuals. Initial studies have already shown that ingestion of colostrum alleviates refractory diarrhea in HIV patients with a corresponding increase in both body weight and peripheral blood CD4+ T cells [9,10]. As we learn more about the gut microenvironment in HIV-infected individuals, Bessie may prove to be a worthwhile platform for the consideration of “immune milk” exhibiting both microbial endotoxin and HIV-neutralizing activity along with its innate anti-inflammatory and tissue repair and regenerative properties.

COMMON PATHOGENIC MECHANISM IN DEVELOPMENT PROGRESSION OF LIVER INJURY CAUSED BY NON-ALCOHOLIC OR ALCOHOLIC STEATOHEPATITIS

This review showed the common pathogenic mechanism in the development of non-alcoholic or alcoholic steatohepatitis. In particular, we describe the role of innate immune system and oxidative stress caused by gut-derived endotoxin. Gut-derived endotoxin plays an important role in alcoholic liver injury. It was reported that acute ethanol administration reduced activation of Kupffer cells. It is therefore possible that alcohol-induced hepatocellular damage occurs as a result of bacterial or endotoxin translocation under a reduction of the reticuloendothelial system (RES) function in alcoholic liver disease (ALD). On the other hand, recently, attention has been directed toward the effect of ethanol ingestion on Kupffer cell function, which is stimulated by gut-derived endotoxin via mechanisms dependent on increased gut permeability and the possible relationship between Kupffer cells and alcohol-induced liver injury. It is generally accepted that activation of the innate immune system and increased release of proinflammatory cytokines and other mediators plays an important role in the development of ALD. It was shown that Kupffer cells activation by endotoxin via Toll-like receptor (TLR-4) is involved in alcohol-induced liver injury and that ethanol-induced oxidative stress is important in the regulation of transcription factor NF-kappaB activation and that cytokine production by Kupffer cells. TNF-alpha and free radicals are produced in early alcohol-induced liver injury. In support of this finding, the pathology caused by alcohol was blocked nearly completely in TNF-alpha receptor 1. Many pathways have been suggested to contribute to the ability of ethanol to induce a state of oxidative stress. One central pathway appears to be the induction of the CYP2E1 form of cytochrome P450 enzymes by ethanol. Initial efforts to clarify the mechanisms that promote the progression from steatosis to steatohepatitis somewhat artificially divides disease mechanisms into "first and second" hit. The best candidates for these second hits were considered to be oxidative stress (CYP2E1 induction) and associated lipid peroxidation and cyokines, principally, TNF-alpha. Some of the most definitive data on the importance of the innate immune system or oxidative stress in the pathogenesis of liver disease come from studies of alcoholic and non-alcoholic steatohepatitis in animals.

Role of Kupffer cells in host defense and liver disease

Kupffer cells (KC) constitute 80-90% of the tissue macrophages present in the body. They reside within the lumen of the liver sinusoids, and are therefore constantly exposed to gut-derived bacteria, microbial debris and bacterial endotoxins, known to activate macrophages. Upon activation KC release various products, including cytokines, prostanoides, nitric oxide and reactive oxygen species. These factors regulate the phenotype of KC themselves, and the phenotypes of neighboring cells, such as hepatocytes, stellate cells, endothelial cells and other immune cells that traffic through the liver. Therefore, KC are intimately involved in the liver's response to infection, toxins, ischemia, resection and other stresses. This review summarizes established basic concepts of KC function as well as their role in the pathogenesis of various liver diseases.

Long-term ethanol feeding enhances susceptibility of the liver to orally administered lipopolysaccharides in rats.

Endotoxin has been implicated in the pathogenesis and progression of alcoholic liver disease. However, it is still unclear how long-term ethanol feeding affects absorption of endotoxin from the intestine and susceptibility of the liver to gut-derived endotoxin. The object of this study was to determine the effect of long-term ethanol feeding on hepatic susceptibility to orally administered endotoxin. Male Wistar rats that weighed approximately 150 g were pair-fed with an ethanol-containing liquid diet or a control diet for 35 days. In some experiments, 0, 10, or 20 mg/kg of lipopolysaccharides (LPS) was added to the liquid diet for 7 days beginning on day 29. On day 36, the animals were killed for blood biochemistry and histologic examination of the liver. We also determined plasma endotoxin levels after 20 mg/kg of LPS administration using a gastric tube. In another set of experiments, we determined intestinal permeability using FD4 (fluorescein isothiocyanate-labeled dextran with an average molecular weight of 4000 D). With 10 mg/kg of LPS, serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels were significantly increased in the ethanol-fed rats but not in controls. After 20 mg/kg of LPS administration, more substantial increases in serum ALT and ALP levels were observed in ethanol-fed rats as compared with control diet-fed rats. Plasma endotoxin levels in long-term ethanol-fed rats were higher than those in control rats after intragastric administration of high-dose endotoxin (20 mg/kg). Furthermore, intestinal permeability to FD4 was increased by long-term ethanol administration. Long-term ethanol feeding increases intestinal permeability to and absorption of endotoxin, which can sequentially enhance hepatic susceptibility to orally administered endotoxin. This model has potential as a subclinical experimental model for the study of alcoholic liver disease.

Oxidative stress and septic shock: metabolic aspects of oxygen-derived free radicals generated in the liver during endotoxemia

This review describes the role of oxidative stress caused by endotoxin challenge in sepsis or septic shock symptoms. We observed that endotoxin injection resulted in lipid peroxide formation and membrane damage (near 60-150 kDa) in the livers of experimental animals, causing decreased levels of scavengers or quenchers of free radicals. The administration of alpha-tocopherol completely prevented injury to the liver plasma membrane caused by endotoxin, and suggested that lipid peroxidation by free radicals might occur in a tissue ischemic state, probably by disseminated intravascular coagulation (DIC), in endotoxemia. In mice, depression of Ca(2+)-ATPase activity in the liver plasma membrane may contribute to the membrane damage caused by endotoxin, and the increase of [Ca(2+)](i) in the liver cytoplasm may partially explain the oxidative stress that occurs in endotoxemia. It seems that endotoxin-induced free radical formation is regulated by Ca(2+) mobilization. Moreover, we have suggested that the oxidative stress caused by endotoxin may be due, at least in part, to the changes in endogenous zinc or selenium regulation during endotoxemia. Interestingly, the extent of TNF-alpha-induced oxidative stress may be the result of a synergism between TNF-alpha and gut-derived endotoxin. It is likely that bacterial or endotoxin translocation plays a significant role in TNF-alpha-induced septic shock. On the other hand, although nitric oxide (NO) has been implicated in the pathogenesis of vascular hyporesponsiveness and hypotension in septic shock in our experimental model, it is unlikely that NO plays a significant role in liver injury caused by free radical generation in endotoxemia.

From Fat to Inflammation

From Fat to Inflammation

Ethanol‐Induced Sensitization to Endotoxin in Kupffer Cells Is Dependent upon Oxidative Stress

Activation of Kupffer cells by gut-derived endotoxin plays a pivotal role in alcoholic liver injury. On the other hand, it was reported that acute ethanol administration reduced activation of Kupffer cells. We found that Kupffer cells isolated from rat treated only once with ethanol were sensitized to endotoxin 24 hrs later correlatively with CD14 expression. Moreover, it was shown that Kupffer cell activation by endotoxin via Toll-like receptor (TLR)-4 is involved in alcohol-induced liver injury and ethanol-induced oxidative stress is important in the regulation of transcription factor NFkappaB activation and cytokine production by Kupffer cells. Here, we show that IRAK, one of signaling molecules of TLR-4, regulates tolerance and sensitization to LPS and acute ethanol increases in IRAK expression through a mechanism dependent upon oxidant production. Female C57BL/6 mice were given ethanol (5 g/kg) intragastrically, and LPS was injected 1 or 21 hrs later. Serum transaminase levels were measured. Moreover, some mice were treated with NADPH oxidase inhibitor diphenyleneiodonium sulfate (DPI, 1 mg/kg/day) or infected with adenovirus (1 x 10 plaque-forming units, intravenously) containing IkappaB superrepressor gene, which prevent NFkappaB activation of Kupffer cells, for three days. Kupffer cells were isolated from mice 1 hr and 21 hrs after ethanol treatment. After the addition of LPS, TNF-alpha in the media was measured using ELISA, Electrophoretic mobility shift assay (EMSA) was performed to analyze DNA binding activity of NFkappaB. Further, expression of Interleukin-1 receptor-associated kinase (IRAK) was evaluated by Western blotting. LPS-induced increases in transaminases were blunted in mice treated with ethanol before 1 hr. However, ethanol treatment 21 hrs earlier augmented LPS-increased transaminases three-fold over controls. Pretreatment with nonabsorbable antibiotics blocked these effects of ethanol. LPS-induced TNF-alpha production by Kupffer cells isolated from mice 1 hr after ethanol was reduced to about 60% of values from control Kupffer cells, while LPS-induced TNF-alpha production by Kupffer cells isolated from mice treated with ethanol 21 hrs earlier increased 1.5-fold over control Kupffer cells. In Kupffer cells from mice 1 hr after ethanol treatment, expression of IRAK was decreased, and LPS-induced activation of NFkappaB was decreased correlatively. In contrast, ethanol treatment to mice increased expression of IRAK in Kupffer cells 21hrs later and LPS-induced activation of NFkappaB was elevated significantly. On the other hands, DPI treatment for three days prior to ethanol did not prevent decreases in IRAK expression due to ethanol treatment for 1 hr. However, DPI treatment blunted ethanol-induced increases in IRAK expression. Additionally, inhibition of NKkappaB activation with dominant-negative IkappaBalpha blunted ethanol-induced increase in IRAK expression. Contrary, inhibition of NKkappaB did not affect decrease of IRAK expression due to ethanol treatment for 1 hr. Ethanol causes tolerance in the early phase after ethanol consumption, while sensitization was observed later. Both tolerance and sensitization were induced by gut-derived endotoxin. These findings indicate that ethanol-induced both tolerance and sensitization of Kupffer cells to endotoxin involve IRAK expression. Further, NADPH oxidase plays a pivotal role in the increase in IRAK expression due to ethanol via activation of NFkappaB signaling pathway. In conclusion, these data indicate that acute ethanol causes sensitization to endotoxin through mechanisms dependent upon oxidative stress.

Chronic Systemic Endotoxin Exposure: An Animal Model in Experimental Hepatic Encephalopathy

Plasma levels of gut-derived endotoxins (lipopolysaccharides, LPS) are often elevated in cirrhotics and are thought to contribute to hepatic encephalopathy. Circulating LPS activates macrophages to produce tumor necrosis factor alpha (TNF-alpha) and other potentially cytotoxic proinflammatory mediators. A pathogenic role for endotoxins is supported by studies showing that treatment with Lacto-bacillusor antibiotics, both of which reduce LPS-producing intestinal Gram-negative bacteria, alleviates experimental liver damage. To mimic the "leaky gut" syndrome with endotoxin translocation into the circulation in cirrhotics, a new animal model was developed. Rats were chronically exposed to ethanol and for the four last weeks also infused with endotoxin into the jugular vein from subcutaneously implanted osmotic minipumps. Animals receiving endotoxin had elevated hepatic expression of both pro- and anti-inflammatory cytokines, but compared to ethanol treatment alone hepatic steatosis and inflammatory changes were only marginally increased. This demonstrates marked endotoxin tolerance, probably as a consequence of a counteracting anti-inflammatory cytokine response. The role of gut-derived endotoxin in hepatic encephalopathy has recently received considerable attention. To further delineate the role and actions of endotoxin and its extrahepatic effects, studies applying both acute challenge and chronic infusion seem warranted. The chronic endotoxin model, mimicking the "leaky gut," may best be combined with more robust ways to impair liver function, such as carbon tetrachloride treatment, bile duct ligation, or galactosamine administration.

Glycine as a Therapeutic Immuno‐Nutrient for Alcoholic Liver Disease

Activation of Kupffer cells by gut-derived endotoxin is an important factor in ethanol hepatotoxicity. Further, it was shown that ethanol modulates both the expression and activity of several intracellular signaling molecules and transcription factors in Kupffer cells and chronic ethanol treatment enhances Kupffer cell sensitivity to endotoxin. These findings suggest that inhibition of Kupffer cell activation is effective for clinical application in alcoholic hepatitis. Recently, accumulating lines of evidence suggest a possibility that glycine is useful as an immuno-modulating amino acid. It has been shown that a diet containing glycine improved survival in endotoxin shock by preventing Kupffer cell activation. Glycine most likely prevents the LPS-induced elevation of intracellular Ca concentration in Kupffer cells, thereby minimizing LPS receptor signaling and cytokine production. Indeed, glycine prevents alcohol-induced liver injury in a long-term enteral ethanol feeding rats (Tsukamoto-French) by decreasing production of TNF-alpha in the liver. Moreover, glycine is protective against apoptosis of sinusoidal endothelial cells (SECs) that is one of the initial events in the development of liver injury. On the other hand, epidemiologic data have identified chronic alcohol consumption as a significant risk factor for carcinogenesis. Interestingly, glycine inhibits growth of tumor in vivo most likely because of the inhibition of angiogenesis. It was shown that the inhibitory effect of glycine on growth and migration of endothelial cells is due to activation of a glycine-gated Cl channel. It is hypothesized that the opening of this anion channel hyperpolarizes the cell membrane, blocks influx of Ca through voltage-dependent Ca channel, thereby blunting growth factor-mediated signaling. Therefore, glycine can be used not only for treatment of alcoholic hepatitis, but also for chemoprevention and treatment of hepatocellular carcinoma in alcoholic cirrhosis. Taken together, it is concluded that glycine is a potent therapeutic immuno-nutrient for various kinds of chronic liver diseases including alcoholic liver disease (ALD).

Molecular Pathogenesis of Alcohol‐Induced Hepatic Fibrosis

Alcohol abuse is a main cause of liver fibrosis and cirrhosis in the western world. Although the major mechanisms of fibrogenesis are independent of the origin of liver injury, alcoholic liver fibrosis features distinctive characteristics, including the pronounced inflammatory response of immune cells due to elevated gut-derived endotoxin plasma levels, increased formation of reactive oxygen species (ROS), ethanol-induced pericentral hepatic hypoxia or formation of cell-toxic and pro-fibrogenic ethanol metabolites (e.g., acetaldehyde or lipid oxidation products). These factors are together responsible for increased hepatocellular cell death and activation of hepatic stellate cells (HSCs), the key cell type of liver fibrogenesis. To date, removing the causative agent is the most effective intervention to prevent the manifestation of liver cirrhosis. A novel experimental approach in fibrosis therapy is the selective induction of cell death in HSCs. Substances such as gliotoxin, anandamide or antibody against tissue inhibitor of metalloproteinase (TIMP)-1 can selectively induce cell death in activated HSCs. These new results in basic science are encouraging for the search of new antifibrotic treatment.

Chronic ethanol exposure downregulates hepatic expression of pregnane X receptor and P450 3A11 in female ICR mice

Pregnane X receptor (PXR) is a nuclear receptor that regulates cytochrome P450 3A (CYP3A) gene transcription in a ligand-dependent manner. Ethanol has been reported to be either an inducer or an inhibitor of CYP3A expression. In this study, we investigated the effects of chronic ethanol exposure on PXR and P450 3A11 gene expression in mouse liver. Female ICR mice were administered by gavage with different doses (1000, 2000 and 4000 mg/kg) of ethanol for up to 5 weeks. Hepatic PXR and P450 3A11 mRNA levels were measured using RT-PCR. Erythromycin N-demethylase (ERND) activity was used as an indicator of CYP3A protein expression. Results showed that chronic ethanol exposure markedly decreased hepatic PXR and P450 3A11 mRNA levels. Consistent with downregulation of P450 3A11 mRNA, chronic ethanol exposure significantly decreased ERND activity in a dose-dependent manner. Additional experiment showed that chronic ethanol exposure significantly increased plasma endotoxin level and hepatic CD14 and TLR-4 mRNA expression, all of which were blocked by elimination of Gram-negative bacteria and endotoxin with antibiotics. Correspondingly, pretreatment with antibiotics reversed the downregulation of PXR and P450 3A11 mRNA expression and ERND activity in mouse liver. Furthermore, the downregulation of hepatic PXR and P450 3A11 mRNA expression was significantly attenuated in mice pretreated with GdCl 3, a selective Kupffer cell toxicant. GdCl 3 pretreatment also significantly attenuated chronically ethanol-induced decrease in ERND activity. These results indicated that activation of Kupffer cells by gut-derived endotoxin contributes to downregulation of hepatic PXR and P450 3A11 expression during chronic alcohol intoxication.

311 Primed hepatic stellate cells of leptin-deficient ob/ob mice are more sensitive to gut-derived cell wall bacterial endotoxins

311 Primed hepatic stellate cells of leptin-deficient ob/ob mice are more sensitive to gut-derived cell wall bacterial endotoxins