Reduced early alcohol-induced liver injury in CD14-deficient mice

Activation of Kupffer cells by gut-derived endotoxin is associated with alcohol-induced liver injury. Recently, it was shown that CD14-deficient mice are more resistant to endotoxin-induced shock than wild-type controls. Therefore, this study was designed to investigate the role of CD14 receptors in early alcohol-induced liver injury using CD14 knockout and wild-type BALB/c mice in a model of enteral ethanol delivery. Animals were given a high-fat liquid diet continuously with ethanol or isocaloric maltose-dextrin as control for 4 wk. The liver to body weight ratio in wild-type mice (5.8 +/- 0.3%) was increased significantly by ethanol (7.3 +/- 0.2%) but was not altered by ethanol in CD14-deficient mice. Ethanol elevated serum alanine aminotransferase levels nearly 3-fold in wild-type mice, but not in CD14-deficient mice. Wild-type and knockout mice given the control high-fat diet had normal liver histology, whereas ethanol caused severe liver injury (steatosis, inflammation, and necrosis; pathology score = 3.8 +/- 0.4). In contrast, CD14-deficient mice given ethanol showed minimal hepatic changes (score = 1.6 +/- 0.3, p < 0.05). Additionally, NF-kappa B, TGF-beta, and TNF-alpha were increased significantly in wild-type mice fed ethanol but not in the CD14 knockout. Thus, chronic ethanol feeding caused more severe liver injury in wild-type than CD14 knockouts, supporting the hypothesis that endotoxin acting via CD14 plays a major role in the development of early alcohol-induced liver injury.     

Ebselen prevents early alcohol-induced liver injury in rats

Oxidants have been shown to be involved in alcohol-induced liver injury. Moreover, 2-phenyl-1,2-benzisoselenazole-3(2H)-one (ebselen), an organoselenium compound and glutathione peroxidase mimic, decreases oxidative stress and protects against stroke clinically. This study was designed to test the hypothesis that ebselen protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-16 g/kg/d) continuously for up to 4 weeks using the intragastric enteral feeding protocol developed by Tsukamoto and French. Ebselen (50 mg/kg twice daily, intragastrically) or vehicle (1% tylose) was administered throughout the experiment. Mean urine ethanol concentrations were not significantly different between treatment groups, and ebselen did not affect body weight gains or cyclic patterns of ethanol concentrations in urine. After 4 weeks, serum ALT levels were increased significantly about 4-fold over control values (37 +/- 5 IU/l) by enteral ethanol (112 +/- 7 IU/l); ebselen blunted this increase significantly (61 +/- 8 IU/l). Enteral ethanol also caused severe fatty accumulation, mild inflammation, and necrosis in the liver (pathology score: 4.3 +/- 0.3). In contrast, these pathological changes were blunted significantly by ebselen (pathology score: 2.5 +/- 0.4). While there were no significant effects of either ethanol or ebselen on glutathione peroxidase activity in serum or liver tissue, ebselen blocked the increase in serum nitrate/nitrite caused by ethanol. Furthermore, ethanol increased the activity of NF-kappaB over 5-fold, the number of infiltrating neutrophils 4-fold, and the accumulation of 4-hydroxynonenal over 5-fold. Ebselen blunted all of these effects significantly. These results indicate that ebselen prevents early alcohol-induced liver injury, most likely by preventing oxidative stress, which decreases inflammation.     

Role of lipopolysaccharide-binding protein in early alcohol-induced liver injury in mice

Cellular responses to endotoxins are enhanced markedly by LPS-binding protein (LBP). Furthermore, it has been demonstrated that endotoxins and proinflammatory cytokines such as TNF-alpha participate in early alcohol-induced liver injury. Therefore, in this study, a long-term intragastric ethanol feeding model was used to test the hypothesis that LBP is involved in alcoholic hepatitis by comparing LBP knockout and wild-type mice. Two-month-old female mice were fed a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin as control continuously for 4 wk. There was no difference in mean urine alcohol concentrations between the groups fed ethanol. Dietary alcohol significantly increased liver to body weight ratios and serum alanine aminotransferase levels in wild-type mice (189 +/- 31 U/L) over high-fat controls (24 +/- 7 U/L), effects which were blunted significantly in LBP knockout mice (60 +/- 17 U/L). Although no significant pathological changes were observed in high-fat controls, 4 wk of dietary ethanol caused steatosis, mild inflammation, and focal necrosis in wild-type animals as expected (pathology score, 5.9 +/- 0.5). These pathological changes were reduced significantly in LBP knockout mice fed ethanol (score, 2.6 +/- 0.5). Endotoxin levels in the portal vein were increased significantly after 4 wk in both groups fed ethanol. Moreover, ethanol increased TNF-alpha mRNA expression in wild-type, but not in LBP knockout mice. These data are consistent with the hypothesis that LBP plays an important role in early alcohol-induced liver injury by enhancing LPS-induced signal transduction, most likely in Kupffer cells.     

Cocoa extract protects against early alcohol-induced liver injury in the rat

Oxidants have been shown to be involved in alcohol-induced liver injury. This study was designed to determine whether cocoa flavonoid extract, composed mostly of epicatechin and epicatechin oligomers, protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-14 g/kg per day) and cocoa extract (400 mg/kg per day) continuously for 4 weeks using an enteral feeding protocol. Mean body weight gains ( approximately 4 g/day) were not significantly different between treatment groups. Cocoa extract did not affect average daily urine ethanol concentrations ( approximately 200mg/dL). After 4 weeks, serum alanine amino transferase levels of the ethanol group were increased nearly fourfold (110+/-16 IU/L) compared to control values (35+/-3 IU/L); this effect of ethanol was blocked by cocoa extract (60+/-6 IU/L). Additionally, enteral ethanol caused severe fat accumulation, mild inflammation, and necrosis in the liver; cocoa extract significantly blunted these changes. Increases in liver TNFalpha protein levels caused by ethanol were completely blocked by cocoa extract. Further, ethanol significantly increased the accumulation of protein adducts of 4-hydroxynonenal, a product of lipid peroxidation serving as an index of oxidative stress; again this was counteracted by the addition of cocoa extract. These results indicate that dietary flavanols such as those found in cocoa can prevent early alcohol-induced liver injury.     

Green tea extract protects against early alcohol-induced liver injury in rats

Oxidants have been shown to be involved in alcohol-induced liver injury. This study was designed to test the hypothesis that the antioxidant polyphenolic extract of green tea, comprised predominantly of epigallocatechin gallate, protects against early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-14 g kg(-1) day(-1)) and green tea (300 mg kg(-1) day(-1)) continuously for 4 weeks using an intragastric enteral feeding protocol. Mean body weight gains (approximately 4 g/day) were not significantly different between treatment groups, and green tea extract did not the affect average concentration or the cycling of urine ethanol concentrations (0-550 mg dl(-1) day(-1)). After 4 weeks, serum ALT levels were increased significantly about 4-fold over control values (35+/-3 IU/l) by enteral ethanol (114+/-18); inclusion of green tea extract in the diet significantly blunted this increase (65+/-10). Enteral ethanol also caused severe fatty accumulation, mild inflammation, and necrosis in the liver. While not affecting fat accumulation or inflammation, green tea extract significantly blunted increases in necrosis caused by ethanol. Furthermore, ethanol significantly increased the accumulation of protein adducts of 4-hydroxynonenal, a product of lipid peroxidation and an index of oxidative stress; green tea extract blocked this effect almost completely. TNFalpha protein levels were increased in liver by alcohol; this phenomenon was also blunted by green tea extract. These results indicate that simple dietary antioxidants, such as those found in green tea, prevent early alcohol-induced liver injury, most likely by preventing oxidative stress.     

Diphenyleneiodonium sulfate, an NADPH oxidase inhibitor, prevents early alcohol-induced liver injury in the rat

The oxidant source in alcohol-induced liver disease remains unclear. NADPH oxidase (mainly in liver Kupffer cells and infiltrating neutrophils) could be a potential free radical source. We aimed to determine if NADPH oxidase inhibitor diphenyleneiodonium sulfate (DPI) affects nuclear factor-kappaB (NF-kappaB) activation, liver tumor necrosis factor-alpha (TNF-alpha) mRNA expression, and early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10-16 g. kg(-1). day(-1)) continuously for up to 4 wk, using the Tsukamoto-French intragastric enteral feeding protocol. DPI or saline vehicle was administered by subcutaneous injection for 4 wk. Mean urine ethanol concentrations were similar between the ethanol- and ethanol plus DPI-treated groups. Enteral ethanol feeding caused severe fat accumulation, mild inflammation, and necrosis in the liver (pathology score, 4.3 +/- 0.3). In contrast, DPI significantly blunted these changes (pathology score, 0.8 +/- 0.4). Enteral ethanol administration for 4 wk also significantly increased free radical adduct formation, NF-kappaB activity, and TNF-alpha expression in the liver. DPI almost completely blunted these parameters. These results indicate that DPI prevents early alcohol-induced liver injury, most likely by inhibiting free radical formation via NADPH oxidase, thereby preventing NF-kappaB activation and TNF-alpha mRNA expression in the liver.     

Induction of TNF-alpha and MnSOD by endotoxin: role of membrane CD14 and Toll-like receptor-4

Endotoxin (LPS) is a potent inducer oftumor necrosis factor- (TNF-) and manganese superoxide dismutase(MnSOD). Recent evidence suggests that LPS induction of TNF- andMnSOD mRNAs is mediated through distinct intracellular signaltransduction pathways. Membrane CD14 (mCD14) and Toll-like receptor-4(TLR4) mediate LPS induction of TNF- in macrophages. In the current study, we evaluated the role of mCD14 and TLR4 in LPS induction ofMnSOD using peritoneal macrophages from CD14 knockout (CD14-KO) miceand mice with the Tlr4 gene point mutation (C3H/HeJ) ordeletion (C57BL/10ScCr). We studied mCD14-dependent (1 and 10 ng/ml)and mCD14-independent (1,000 ng/ml) concentrations of LPS. Compared with control (BALB/c) macrophages, LPS at 1 and 10 ng/ml failed toinduce TNF- or MnSOD mRNA in CD14-KO macrophages. However, LPS at1,000 ng/ml induced TNF- and MnSOD mRNAs equally in macrophages fromCD14-KO and control mice. LPS (1, 10, or 1,000 ng/ml) failed to induceTNF- or MnSOD mRNA and failed to activate nuclear factor-B inC3H/HeJ or C57BL/10ScCr macrophages. Measurements of TNF- and MnSODenzyme activity paralleled TNF- and MnSOD mRNA levels. These datademonstrate that, like TNF-, induction of MnSOD by LPS is mediatedby mCD14 and TLR4 in murine macrophages.

     

牛磺酸对肢体缺血／再灌注后肝损伤大鼠TNF-α、NF-κB表达的影响

目的：探讨给予牛磺酸预处理对肢体缺血／再灌注（limb ischemia-reperfusion,H／R）后大鼠肝脏损伤及,INn、NF—KB表达的影响及意义。方法：采用Wistar大鼠建立LI/R损伤模型,随机分为4组（n=10）：对照（C）组,缺血／再灌注（I／R）组,牛磺酸（T）组和牛磺酸＋缺血／再灌注（TR）组。比色法测定动物血浆ALT、AST、MDA,肝组织MDA、MPO、DNA裂解率和钙含量,放免法检测血浆及肝组织TNF-α水平;HE染色观察肝脏组织形态改变;免疫组化法观察NF-κB蛋白表达。结果：与C组比较,I／R和TR组各损伤性指标、TNF-α水平均升高,NF-κB蛋白表达增高（P〈0．01）;但TR组上述各项指标较I／R组显著降低。结论：牛磺酸预处理可减轻大鼠LI／R所致肝脏损伤,降低TNF-α、NF-κB表达。     

Sake yeast suppresses acute alcohol-induced liver injury in mice

Brewer's and baker's yeasts appear to have components that protect from liver injury. Whether sake yeast, Saccharomyces cerevisiae Kyokai no. 9, also has a hepatoprotective effect has not been examined. Here we show that sake yeast suppresses acute alcoholic liver injury in mice. Male C57BL/6 mice that had been fed a diet containing 1% sake yeast for two weeks received three doses of ethanol (5 g/kg BW). In the mice fed sake yeast, ethanol-induced increases in triglyceride (TG) and glutamate pyruvate transaminase (GPT) were significantly attenuated and hepatic steatosis was improved. In addition, sake yeast-fed mice showed a smaller decrease in hepatic S-adenosylmethionine (SAM) level and a smaller increase in plasma homocysteine (Hcy) level after ethanol treatment than the control mice, suggesting that a disorder of methionine metabolism in the liver caused by ethanol was relieved by sake yeast. These results indicate that sake yeast protects against alcoholic liver injury through maintenance of methionine metabolism in the liver.     

Hepatoprotective effect of gastrodin against alcohol-induced liver injury in mice

Journal of Physiology and Biochemistry - Alcoholic liver disease (ALD) is a common and serious threat to human health worldwide. In this study, the hepatoprotective effect of gastrodin against...     

CD14 plays no major role in shock induced by Staphylococcus aureus but down-regulates TNF-alpha production

Recent in vitro studies have suggested that CD14, a major receptor for LPS, may also be a receptor for cell wall components of Gram-positive bacteria and thus play a role in Gram-positive shock. To analyze the in vivo role of CD14 in responses to Gram-positive bacteria, CD14-deficient and control mice were injected with Staphylococcus aureus, and the effects on lethality, bacterial clearance, and production of cytokines were analyzed. Survival of CD14-deficient and control mice did not differ significantly after administration of various doses of either unencapsulated or encapsulated S. aureus; furthermore, mice in both groups displayed similar symptoms of shock. In addition, inflammatory cytokines such as TNF-alpha and IL-6 were readily detectable in the serum of CD14-deficient mice injected with live or antibiotic-killed S. aureus. Surprisingly, the serum concentration of TNF-alpha in CD14-deficient mice was at least threefold higher than in control mice after injection of either unencapsulated or encapsulated S. aureus, suggesting that CD14 down-regulates TNF-alpha. A similar increase in serum TNF-alpha occurred when CD14-deficient animals were injected with gentamicin-killed bacteria even though no symptoms of shock were observed. These studies indicate that CD14, in contrast to its key function in responses to the Gram-negative bacterium, Escherichia coli 0111, does not play a prominent role in septic shock induced by S. aureus, and that the symptoms of S. aureus shock are not due solely to TNF-alpha.     

Diallyl sulfide attenuates bleomycin-induced pulmonary fibrosis: critical role of iNOS, NF-kappaB, TNF-alpha and IL-1beta

Diallylsulfide (DAS), an antioxidant and anti-inflammatory agent was evaluated for its ability to repress lung fibrosis induced by bleomycin in Wistar rats. A single intra tracheal administration of bleomycin (6.5 U/kg BW) was administered to pulmonary fibrosis group, while DAS (120 mg/kg BW) was administered intraperitoneally throughout the experimental period. Fibrotic changes in the lungs were estimated by measuring lung hydroxyproline content. Bleomycin administration significantly (P<0.05) reduced the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in the lung tissues. Bleomycin caused a significant decrease in the level of reduced glutathione (GSH), which was accompanied with significant increase in lipid peroxidation (LPO) level, and myeloperoxidase (MPO) activity, in the lung tissues. An increase in the level of cell counts in bronchoalveolar lavage fluid (BALF) was observed in bleomycin induced group. DAS administration altered the levels of enzymic antioxidants, TBARS, MPO and GSH towards normal values. Histopathological analysis and picrosirius red staining showed an increased collagen deposition in rats receiving bleomycin alone that was decreased upon DAS treatment. Immunohistochemical studies revealed that DAS reduced the bleomycin-induced activation of inducible nitric oxide synthase (iNOS) and nuclear factor kappa-B (NF-kappaB) and decreased the augmented levels of the early inflammatory cytokines, tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), in the lung tissues. The present study provides evidence that DAS might serve as a novel target for the therapeutic treatment of lung fibrosis.     

Protective effects of C-phycocyanin on alcohol-induced subacute liver injury in mice

Excessive and long-term alcohol consumption leads to liver disease and low immunity. Extensive evidence suggests that C-phycocyanin (C-PC), a chromophore phycocyanobilin derived from Arthrospira (Spirulina) platensis, exerts protective effects against chemical-induced organ damage and improves immunity. In this study, we investigated whether C-PC could protect against ethanol-induced subacute liver injury and improve immunity. KM mice with ethanol-induced liver injury were established, and animals were divided into three groups that were treated with high, medium, and low doses of C-PC. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (CHOL), low-density lipoprotein (LDL), total bilirubin (TBIL), liver homogenate malondialdehyde (MDA), and superoxide dismutase (SOD) levels were measured. In addition, the number of thymus T cell subsets was assessed, and liver sections were examined pathologically. C-PC exhibited obvious inhibitory effects on serum ALT, AST, TG, CHOL, LDL, and MDA levels and increased SOD content significantly in the liver. C-PC also increased serum CD3+ and CD4+ cell activation and T cell proliferation significantly compared with the model group. The structure of the hepatic lobules was clear, the liver sinus returned to normal, and the liver cell cords were arranged in neat rows. Therefore, C-PC could protect against ethanol-induced subacute liver injury significantly.     

CD14: Biology and role in the pathogenesis of disease

Human monocyte differentiation antigen CD14 is a pattern recognition receptor (PRR) that enhances innate immune responses. CD14 was first identified as a marker of monocytes to signal intracellular responses upon bacterial encounters. Given the absence of an intracellular tail, CD14 was doubted to have the signaling capacities. Later CD14 was confirmed as the TLR co-receptor for the detection of pathogen-associated molecular patterns. However, CD14 has been revealed as a multi-talented receptor. In last decade, CD14 was identified to activate NFAT to regulate the life cycle of myeloid cells in a TLR4-independent manner and to transport inflammatory lipids to induce phagocyte hyperactivation. And its influences on multiple related diseases have been further considered. In this review, we summarize advancements in the basic biology of the CD14 including its structure, binding ligands, signaling pathways, and its roles in the pathogenesis of inflammation, atherosclerosis, tumor and metabolic diseases. We also discuss the therapeutic potential of targeting the CD14 in related diseases.     

Overexpression of manganese superoxide dismutase prevents alcohol-induced liver injury in the rat

Mitochondria are thought to play a major role in hepatic oxidative stress associated with alcohol-induced liver injury. Thus, the hypothesis that delivery of the mitochondrial isoform of superoxide dismutase (Mn-SOD) via recombinant adenovirus would reduce alcohol-induced liver injury was tested. Rats were given recombinant adenovirus containing Mn-SOD (Ad.SOD2) or beta-galactosidase (Ad.lacZ) and then fed alcohol enterally for 4 weeks. Mn-SOD expression and activity of Ad.SOD2 in liver mitochondria of infected animals was increased nearly 3-fold compared with Ad.lacZ-infected controls. Mitochondrial glutathione levels in Ad.lacZ-infected animals were decreased after 4 weeks of chronic ethanol, as expected, but were unchanged in Ad.SOD2-infected animals. Alanine aminotransferase was elevated significantly by ethanol, an effect that was prevented by Ad.SOD2. Moreover, pathology (e.g. the sum of steatosis, inflammation, and necrosis) was elevated dramatically by ethanol in Ad.lacZ-treated rats. This effect was also blunted in animals infected with Ad.SOD2. Neutrophil infiltration was increased about 3-fold in livers from both Ad.lacZ- and Ad.SOD2-infected rats by ethanol treatment. Moreover, ESR-detectable free radical adducts in bile were increased about 8-fold by ethanol. Using (13)C-labeled ethanol, it was determined that nearly 60% of total adducts were due to the alpha-hydroxyethyl radical adduct. This increase in radical formation was blocked completely by Ad.SOD2 infection. Furthermore, apoptosis of hepatocytes was increased about 5-fold by ethanol, an effect also blocked by Ad.SOD2. Interestingly, tumor necrosis factor-alpha mRNA was elevated to the same extent in both Ad.lacZ- and Ad.SOD2-infected animals follows ethanol exposure. These data suggest that hepatocyte mitochondrial oxidative stress is involved in alcohol-induced liver damage and likely follows Kupffer cell activation, cytokine production, and neutrophil infiltration. These results also support the hypothesis that mitochondrial oxidant production is a critical factor in parenchymal cell death caused by alcohol.