Ethanol-induced Liver Injury In Mice Research Articles

Differential contribution of complement receptor C5aR in myeloid and non-myeloid cells in chronic ethanol-induced liver injury in mice.

Complement is implicated in the development of alcoholic liver disease. C3 and C5 contribute to ethanol-induced liver injury; however, the role of C5a receptor (C5aR) on myeloid and non-myeloid cells to progression of injury is not known. C57BL/6 (WT), global C5aR-/-, myeloid-specific C5aR-/-, and non-myeloid-specific C5aR-/- mice were fed a Lieber-DeCarli diet (32%kcal EtOH) for 25 days. Cultured hepatocytes were challenged with ethanol, TNFα, and C5a. Chronic ethanol feeding increased expression of pro-inflammatory mediators in livers of WT mice; this response was completely blunted in C5aR-/- mice. However, C5aR-/- mice were not protected from other measures of hepatocellular damage, including ethanol-induced increases in hepatic triglycerides, plasma alanine aminotransferase and hepatocyte apoptosis. CYP2E1 and 4-hydroxynonenal protein adducts were induced in WT and C5aR-/- mice. Myeloid-specific C5aR-/- mice were protected from ethanol-induced increases in hepatic TNFα, whereas non-myeloid-specific C5aR-/- displayed increased hepatocyte apoptosis and inflammation after chronic ethanol feeding. In cultured hepatocytes, cytotoxicity induced by challenge with ethanol and TNFα was completely eliminated by treatment with C5a in cells from WT, but not C5aR-/- mice. Further, treatment with C5a enhanced activation of pro-survival signal AKT in hepatocytes challenged with ethanol and TNFα. Taken together, these data reveal a differential role for C5aR during ethanol-induced liver inflammation and injury, with C5aR on myeloid cells contributing to ethanol-induced inflammatory cytokine expression, while non-myeloid C5aR protects hepatocytes from death after chronic ethanol feeding.

Dendrobium huoshanense polysaccharide prevents ethanol-induced liver injury in mice by metabolomic analysis

The prevalence of alcohol consumption has increased in modern dietary life and alcoholic liver injury can follow. Dendrobium huoshanense polysaccharide (DHP) is a homogeneous polysaccharide isolated from Dendrobium huoshanense, which possesses hepatoprotection function. In this study, we investigated the metabolic profiles of serum and liver tissues extracts from control, ethanol-treated and DHP\\ethanol-treated mice using a UHPLC/LTQ Orbitrap XL MS-based metabolomics approach. Our results indicated that DHP alleviated early steatosis and inflammation in liver histology and the metabolomic analysis of serum and hepatic tissue revealed that first, ethanol treatment mainly altered phosphatidylcholines (PCs) including PC (13:0) and phosphocholine, arachidonic acid metabolites including 20-ethyl PGF2α and amino acids including l-Proline; Second, DHP supplementation ameliorated the altered metabolic levels particularly involved in phosphocholine and l-Proline. These data suggested that DHP might restore the perturbed metabolism pathways by ethanol exposure to prevent the progression of alcoholic liver injury.

646 sIgM Contributes to Complement Activation and Injury in Ethanol-Induced Liver Injury in Mice

Background: The activation of classical complement pathway contributes to ethanol-induced liver injury. Ethanol feeding in mice activates complement pathway via C1q binding to apoptotic cells in the liver and increases downstream activation of C3b. However, the mechanism by which ethanol activates C1q is unclear. This knowledge is critical to understanding liver injury-repair mechanisms. Molecules targeting the C1q pathway to decrease inflammation would be expected to preserve complement activation via the lectin and alternative pathways, thus maintaining the role of complement in resistance to infections. Secretory IgM (sIgM) is the primary isotype of natural antibodies that arise in the absence of exposure to foreign antigens. In addition to the anti-microbial functions, there is accumulating evidence that sIgM has critical housekeeping functions by facilitating clearance of apoptotic cells through activation of C1q. Therefore, we hypothesized that ethanol exposure activates C1q through sIgM binding to apoptotic hepatocytes. The presence of sIgM-C1q complex further exacerbates apoptosis in a feed forward mechanism. Further, C1INH (C1 esterase inhibitor) targeted to decrease the sIgM-C1q activation may prevent complement activation and decrease apoptosis in ethanol-induced liver injury.Methods: Two experimental protocols were used: 1) Female C57BL/6J wild type mice and sIgM-/mice were fed ethanol containing diets for 2 weeks, at a maximal concentration of 4% (vol/vol) ethanol (22% calories) or pair-fed control diets. 2) C57BL/6 mice were fed ethanol containing diets for 4 days, at a maximal concentration of 6%(vol/vol) ethanol (32% of calories). Mice were treated with C1INH or vehicle via tail vein injection at 24 and 48 hours prior to euthanasia. Results: Ethanol exposure increased hepatic IgM, C3b and C1q deposition as measured by IHC. C1q co-localized with IgM in liver. sIgM-/mice had decreased hepatic C1q, C3b deposition and TUNEL positive nuclei after ethanol exposure. AST/ALT, as well as MCP-1 mRNA, was decreased in sIgM-/mice compared to wild type mice after ethanol feeding. Importantly, C1INH decreased hepatic C3b deposition and TUNEL positive nuclei after ethanol feeding. Conclusion: The data indicate that sIgM contributes to activation of classical complement pathway in ethanol-induced liver injury via binding of sIgM to C1q. The presence of sIgMC1q complex is associated with cell death and may further enhance cell death and injury. Blocking of the activation via C1INH decreased C3b deposition and apoptosis. Taken together, the data suggest that sIgM is critical in ethanol-induced complement activation and that therapies targeting the classical complement pathway may be beneficial in ethanolinduced liver injury. The study is supported in part by ABMRF/The Foundation for Alcohol Research to DJC and NIH grant 5R37 AA011876 to LEN.

Increased activity of the complement system in the liver of patients with alcoholic hepatitis

Inflammation has been suggested as a mechanism underlying the development of alcoholic hepatitis (AH). The activation of the complement system plays an important role in inflammation. Although it has been shown that ethanol‐induced activation of the complement system contributes to the pathophysiology of ethanol‐induced liver injury in mice, whether ethanol consumption activates the complement system in the human liver has not been investigated. Using antibodies against C1q, C3, and C5, the immunoreactivity of the complement system in patients with AH was examined by immunohistochemistry and quantified by morphometric image analysis. The immunoreactivity intensity of C1q, C3, and C5 in patients with AH was significantly higher than that seen in normal controls. Further, the gene expression of C1q, C3, and C5 was examined using real‐time PCR. There were increases in the levels of C1q and C5, but not C3 mRNA in AH. Moreover, the immunoreactivity of C5a receptor (C5aR) also increased in AH. To explore the functional implication of the activation of the complement system in AH, we examined the colocalization of C5aR in Mallory‐Denk bodies (MDBs) forming balloon hepatocytes. C5aR was focally overexpressed in the MDB forming cells. Collectively, our study suggests that alcohol consumption increases the activity of the complement system in the liver cells, which contributes to the inflammation‐associated pathogenesis of AH.

Inhibition of miR122a by Lactobacillus rhamnosus GG Culture Supernatant Increases Intestinal Occludin Expression and Protects Mice from Alcoholic Liver Disease

Alcoholic liver disease (ALD) has a high morbidity and mortality. Chronic alcohol consumption causes disruption of intestinal microflora homeostasis, dysfunction of the intestinal tight junction barrier, increased endotoxemia and eventually liver steatosis/steatohepatitis and injury. Probiotics supernatant supplementation has been shown to be a potential strategy for the treatment and prevention of ALD via restoring epithelial barrier function. Little is known about how probiotic supernatant works to increase intestinal tight junction proteins, thereby contributing to a protective effect in ALD. Here we show that chronic ethanol exposure increases intestinal miR122a expression, which decreases occludin expression leading to increased intestinal permeability, and Lactobacillus rhamnosus GG culture supernatant (LGGs) supplementation decreases ethanol‐elevated miR122a level and attenuates ethanol‐induced liver injury in mice. Similar to the effect of ethanol exposure, overexpression of miR122a in Caco‐2 monolayers markedly decreases occludin protein level. LGGs supplementation functions in intestinal integrity by inhibition of miR122a, leading to occludin restoration in mice exposed to chronic ethanol treatment.

Inhibition of miR122a by Lactobacillus rhamnosus GG culture supernatant increases intestinal occludin expression and protects mice from alcoholic liver disease

Alcoholic liver disease (ALD) has a high morbidity and mortality. Chronic alcohol consumption causes disruption of intestinal microflora homeostasis, intestinal tight junction barrier dysfunction, increased endotoxemia, and eventually liver steatosis/steatohepatitis. Probiotic Lactobacillus rhamnosus GG (LGG) and the bacteria-free LGG culture supernatant (LGGs) have been shown to promote intestinal epithelial integrity and protect intestinal barrier function in ALD. However, little is known about how LGGs mechanistically works to increase intestinal tight junction proteins. Here we show that chronic ethanol exposure increased intestinal miR122a expression, which decreased occludin expression leading to increased intestinal permeability. Moreover, LGGs supplementation decreased ethanol-elevated miR122a level and attenuated ethanol-induced liver injury in mice. Similar to the effect of ethanol exposure, overexpression of miR122a in Caco-2 monolayers markedly decreased occludin protein levels. In contrast, inhibition of miR122a increased occludin expression. We conclude that LGGs supplementation functions in intestinal integrity by inhibition of miR122a, leading to occludin restoration in mice exposed to chronic ethanol.

Curcumin attenuates chronic ethanol-induced liver injury by inhibition of oxidative stress via mitogen-activated protein kinase/nuclear factor E2-related factor 2 pathway in mice.

Objective:This study aimed to investigate the protective effect of curcumin on chronic ethanol-induced liver injury in mice and to explore its underlying mechanisms.Materials and Methods:Ethanol-exposed Balb/c mice were simultaneously treated with curcumin for 6 weeks. Liver injury was evaluated by biochemical and histopathological examination. Lipid peroxidation and anti-oxidant activities were measured by spectrophotometric method. Anti-oxidative genes expression such as NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), and superoxide dismutase (SOD) were determined by real-time polymerase chain reaction. The nuclear factor E2-related factor 2 (Nrf2) and the phosphorylation states of specific proteins central to intracellular signaling cascades were measured by western blotting.Results:Curcumin treatment protected liver from chronic ethanol-induced injury through reducing serum alanine aminotransferase and aspartate aminotransferase activities, improving liver histological architecture, and reversing lipid disorders indicated by decrease of triglyceride, total cholesterol and low-density lipoprotein-cholesterol levels and increase of High-density lipoprotein-cholesterol levels. Meanwhile, curcumin administration attenuated oxidative stress via up-regulating SOD and glutathione peroxidase activities, leading to a reduction of lipid hydroperoxide production. In addition, curcumin increased Nrf2 activation and anti-oxidative genes expressions such as NQO1, HO-1, and SOD through inducing extracellular signal-regulated kinase (ERK) and p38 phosphorylation.Conclusion:Our data suggested that curcumin protected the liver from chronic-ethanol induced injury through attenuating oxidative stress, at least partially, through ERK/p38/Nrf2-mediated anti-oxidant signaling pathways.

Protective effects of Penthorum chinense Pursh against chronic ethanol-induced liver injury in mice

Ethnopharmacological relevancePenthorum chinense Pursh (Penthoraceae) has been used as a Miao ethnomedicine for the treatment of jaundice, cholecystitis, edema, infectious hepatitis and anti-drunk hangover in China. The aim of present study is to investigate the possible protective effects of Penthorum chinense against chronic ethanol-induced liver injury. Material and methodsMice were fed a Lieber-DeCarli liquid diet containing alcohol or isocaloric maltose dextrin as control diet with or without aqueous extract of Penthorum chinense (PCP, 5.15 and 10.30g/kg/BW) for 4 weeks. Silymarin (86mg/kg) was used as positive control to compare the efficacy of PCP against chronic ethanol-induced hepatotoxicity. ResultsTreatment with PCP (10.30g/kg) significantly reduced the increases in serum ALT and AST levels, hepatic lipid accumulation and inflammatory cytokines (i.e. TNF-α, IL-6), which were induced by chronic ethanol exposure. PCP was also found to attenuate reactive oxygen species (ROS) generation and malondialdehyde (MDA) level, restore the glutathione (GSH) depletion, and increase the superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities. In addition, PCP supplementation (10.30g/kg) inhibited the induction of hepatic cytochrome P450 2E1 (CYP2E1), a major contributor to ethanol-mediated oxidative stress, and up-regulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream anti-oxidant protein heme oxygenase-1 (HO-1) in ethanol-treated mice. ConclusionsThese results indicate that the co-treatment with aqueous extract of Penthorum chinense (10.30g/kg) protects against chronic ethanol-induced liver injury, possibly through suppressing CYP2E1-mediated oxidative stress and enhancing the oxidant defense systems via the activation of Nrf2/HO-1 pathway.

Supplementation of Saturated Long-Chain Fatty Acids Maintains Intestinal Eubiosis and Reduces Ethanol-induced Liver Injury in Mice

Alcoholic liver disease is a leading cause of mortality. Chronic alcohol consumption is accompanied by intestinal dysbiosis, and development of alcoholic liver disease requires gut-derived bacterial products. However, little is known about how alterations to the microbiome contribute to pathogenesis of alcoholic liver disease. We used the Tsukamoto-French mouse model, which involves continuous intragastric feeding of isocaloric diet or alcohol for 3 weeks. Bacterial DNA from the cecum was extracted for deep metagenomic sequencing. Targeted metabolomics assessed concentrations of saturated fatty acids in cecal contents. To maintain intestinal metabolic homeostasis, diets of ethanol-fed and control mice were supplemented with saturated long-chain fatty acids (LCFA). Bacterial genes involved in fatty acid biosynthesis, amounts of lactobacilli, and saturated LCFA were measured in fecal samples of nonalcoholic individuals and patients with active alcohol abuse. Analyses of intestinal contents from mice revealed alcohol-associated changes to the intestinal metagenome and metabolome, characterized by reduced synthesis of saturated LCFA. Maintaining intestinal levels of saturated fatty acids in mice resulted in eubiosis, stabilized the intestinal gut barrier, and reduced ethanol-induced liver injury. Saturated LCFA are metabolized by commensal Lactobacillus and promote their growth. Proportions of bacterial genes involved in fatty acid biosynthesis were lower in feces from patients with active alcohol abuse than controls. Total levels of LCFA correlated with those of lactobacilli in fecal samples from patients with active alcohol abuse but not in controls. In humans and mice, alcohol causes intestinal dysbiosis, reducing the capacity of the microbiome to synthesize saturated LCFA and the proportion of Lactobacillus species. Dietary approaches to restore levels of saturated fatty acids in the intestine might reduce ethanol-induced liver injury in patients with alcoholic liver disease.

Increased activity of the complement system in the liver of patients with alcoholic hepatitis

Inflammation has been suggested as a mechanism underlying the development of alcoholic hepatitis (AH). The activation of the complement system plays an important role in inflammation. Although it has been shown that ethanol-induced activation of the complement system contributes to the pathophysiology of ethanol-induced liver injury in mice, whether ethanol consumption activates the complement system in the human liver has not been investigated. Using antibodies against C1q, C3, and C5, the immunoreactivity of the complement system in patients with AH was examined by immunohistochemistry and quantified by morphometric image analysis. The immunoreactivity intensity of C1q, C3, and C5 in patients with AH was significantly higher than that seen in normal controls. Further, the gene expression of C1q, C3, and C5 was examined using real-time PCR. There were increases in the levels of C1q and C5, but not C3 mRNA in AH. Moreover, the immunoreactivity of C5a receptor (C5aR) also increased in AH. To explore the functional implication of the activation of the complement system in AH, we examined the colocalization of C5aR in Mallory–Denk bodies (MDBs) forming balloon hepatocytes. C5aR was focally overexpressed in the MDB forming cells. Collectively, our study suggests that alcohol consumption increases the activity of the complement system in the liver cells, which contributes to the inflammation-associated pathogenesis of AH.

Protective role of HO-1 and carbon monoxide in ethanol-induced hepatocyte cell death and liver injury in mice

Alcoholic liver disease is associated with inflammation and cell death. Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with anti-apoptotic and anti-inflammatory properties. Here we tested the hypothesis that induction of HO-1 or treatment with a carbon monoxide releasing molecule (CORM) during chronic ethanol exposure protects and/or reverses ethanol-induced liver injury. Female C57BL/6J mice were allowed free access to a complete liquid diet containing ethanol or to pair-fed control diets for 25days. Mice were treated with cobalt protoporphyrin (CoPP) to induce HO-1 expression during ethanol feeding or once liver injury had been established. Mice were also treated with CORM-A1, a CO-releasing molecule (CORM), after ethanol-induced liver injury was established. The impact of HO-1 induction on ethanol-induced cell death was investigated in primary cultures of hepatocytes. Induction of HO-1 during or after ethanol feeding, as well as treatment with CORM-A1, ameliorated ethanol-induced increases in AST and expression of mRNAs for inflammatory cytokines. Treatment with CoPP or CORM-A1 also reduced hepatocyte cell death, indicated by decreased accumulation of CK18 cleavage products and reduced RIP3 expression in hepatocytes. Exposure of primary hepatocyte cultures to ethanol increased their sensitivity to TNFα-induced cell death; this response was attenuated by necrostatin-1, an inhibitor of necroptosis, but not by caspase inhibitors. Induction of HO-1 with CoPP or CORM-3 treatment normalized the sensitivity of hepatocytes to TNFα-induced cell death after ethanol exposure. Therapeutic strategies to increase HO-1 and/or modulate CO availability ameliorated chronic ethanol-induced liver injury in mice, at least in part by decreasing hepatocellular death.

Dietary Zinc Deficiency Exaggerates Ethanol-Induced Liver Injury in Mice: Involvement of Intrahepatic and Extrahepatic Factors

Clinical studies have demonstrated that alcoholics have a lower dietary zinc intake compared to health controls. The present study was undertaken to determine the interaction between dietary zinc deficiency and ethanol consumption in the pathogenesis of alcoholic liver disease. C57BL/6N mice were subjected to 8-week feeding of 4 experimental liquid diets: (1) zinc adequate diet, (2) zinc adequate diet plus ethanol, (3) zinc deficient diet, and (4) zinc deficient diet plus ethanol. Ethanol exposure with adequate dietary zinc resulted in liver damage as indicated by elevated plasma alanine aminotransferase level and increased hepatic lipid accumulation and inflammatory cell infiltration. Dietary zinc deficiency alone increased hepatic lipid contents, but did not induce hepatic inflammation. Dietary zinc deficiency showed synergistic effects on ethanol-induced liver damage. Dietary zinc deficiency exaggerated ethanol effects on hepatic genes related to lipid metabolism and inflammatory response. Dietary zinc deficiency worsened ethanol-induced imbalance between hepatic pro-oxidant and antioxidant enzymes and hepatic expression of cell death receptors. Dietary zinc deficiency exaggerated ethanol-induced reduction of plasma leptin, although it did not affect ethanol-induced reduction of white adipose tissue mass. Dietary zinc deficiency also deteriorated ethanol-induced gut permeability increase and plasma endotoxin elevation. These results demonstrate, for the first time, that dietary zinc deficiency is a risk factor in alcoholic liver disease, and multiple intrahepatic and extrahepatic factors may mediate the detrimental effects of zinc deficiency.

Argininosuccinate synthase conditions the response to acute and chronic ethanol-induced liver injury in mice

Argininosuccinate synthase (ASS) is the rate-limiting enzyme in both the urea and the L-citrulline/nitric oxide (NO·) cycles regulating protein catabolism, ammonia levels, and NO· generation. Because a proteomics analysis identified ASS and nitric oxide synthase-2 (NOS2) as coinduced in rat hepatocytes by chronic ethanol consumption, which also occurred in alcoholic liver disease (ALD) and in cirrhosis patients, we hypothesized that ASS could play a role in ethanol binge and chronic ethanol-induced liver damage. To investigate the contribution of ASS to the pathophysiology of ALD, wildtype (WT) and Ass(+/-) mice (Ass(-/-) are lethal due to hyperammonemia) were exposed to an ethanol binge or to chronic ethanol drinking. Compared with WT, Ass(+/-) mice given an ethanol binge exhibited decreased steatosis, lower NOS2 induction, and less 3-nitrotyrosine (3-NT) protein residues, indicating that reducing nitrosative stress by way of the L-citrulline/NO· pathway plays a significant role in preventing liver damage. However, chronic ethanol-treated Ass(+/-) mice displayed enhanced liver injury compared with WT mice. This was due to hyperammonemia, lower phosphorylated AMP-activated protein kinase alpha (pAMPKα) to total AMPKα ratio, decreased sirtuin-1 (Sirt-1) and peroxisomal proliferator-activated receptor coactivator-1α (Pgc1α) messenger RNAs (mRNAs), lower fatty acid β-oxidation due to down-regulation of carnitine palmitoyl transferase-II (CPT-II), decreased antioxidant defense, and elevated lipid peroxidation end-products in spite of comparable nitrosative stress but likely reduced NOS3. Partial Ass ablation protects only in acute ethanol-induced liver injury by decreasing nitrosative stress but not in a more chronic scenario where oxidative stress and impaired fatty acid β-oxidation are key events.

Tetrahydroxystilbene glucoside protects against ethanol-induced liver injury in mice by inhibition of expression of inflammation-related factors

AIM:To investigate the protective effects of tetrahydroxystilbene glucoside(TSG) against acute ethanol-induced liver injury in mice and to explore the possible mechanisms involved.METHODS:Kunming mice were randomly divided into five groups:normal control group,liver injury model group,low-(30mg/kg) and high-dose(60mg/kg) TSG groups,and Silibinin(50mg/kg) group.Liver histopathology was measured by light microscopy.The contents of serum tumor necrosis factor-α(TNF-α),interleukin 1β(IL-1β),and IL-6 were assayed by ELSIA,and plasma endotoxin level was measured by LAL chromogenic endpoint assay.The mRNA expression of TNF-α and iNOS in liver tissue was detected by real-time PCR.The phospho-IκBα level and NF-κB nuclear translocation in hepatic tissue were measured by Western blot.RESULTS:Compared to the control group,the model group showed significant hydropic degeneration and steatosis of hepatocytes,increased contents of serum TNF-α,IL-1β and IL-6 and plasma level of endotoxin(all P0.01),up-regulated mRNA expression of TNF-α and iNOS in liver tissue(TNF-α:128.5pg/mL±20.7pg/mL vs 45.2pg/mL±14.2pg/mL;IL-1β:71.8pg/mL ±9.1 pg/mL vs 33.1pg/mL±9.5pg/mL;IL-6:59.8pg/mL±13.1pg/mL vs 23.7pg/mL±5.9pg/mL;endotoxin:0.35EU/mL±0.09EU/mL vs 0.11EU/mL±0.03EU/mL.TNF-α mRNA:1.33±0.11 vs 0.63±0.10;iNOS mRNA:0.85±0.09 vs 0.40±0.07;phospho-IκB-α:2.02±0.14 vs 0.92±0.19;NF-κB P65:1.10±0.14 vs 0.44±0.13,P0.05,P0.01),and significantly increased phospho-IκBα level and NF-κB nuclear translocation in hepatic tissue(both P0.01).Compared to the model group,serum levels of TNF-α,IL-1β and IL-6 and plasma endotoxin level significantly decreased(all P0.05 or 0.01),the mRNA expression of TNF-α and iNOS was down-regulated,and phospho-IκBα level and NF-κB nuclear translocation in hepatic tissue were significantly inhibited in the TSG groups(TSG 60 mg/kg group:TNF-α:65.9pg/mL±13.9pg/mL vs 128.5pg/mL±20.7pg/mL;IL-1β:43.0pg/mL±7.1pg/mL vs 71.8pg/mL±9.1pg/mL;IL-6:36.3pg/mL±10.1pg/mL vs 59.8pg/mL±13.1pg/mL;endotoxin:0.20EU/mL±0.05EU/mL vs 0.35EU/mL±0.09EU/mL;TNF-α mRNA:0.79±0.09 vs 1.33±0.11;iNOS mRNA:0.53±0.10 vs 0.85±0.09;phospho-IκB-α:1.35±0.32 vs 2.02±0.14;NF-κB P65:0.62±0.05 vs 1.10±0.14,all P0.05 or 0.01).CONCLUSION:TSG has a protective effect on acute alcoholic liver injury in mice possibly by decreasing endotoxin level and NF-κB nuclear translocation and attenuating the trigger of inflammation-related cascade amplification.

Complement and Alcoholic Liver Disease: Role of C1q in the Pathogenesis of Ethanol-Induced Liver Injury in Mice

Complement is involved in the development of alcoholic liver disease in mice; however, the mechanisms for complement activation during ethanol exposure have not been identified. C1q, the recognition subunit of the first complement component, binds to apoptotic cells, thereby activating the classical complement pathway. Because ethanol exposure increases hepatocellular apoptosis, we hypothesized that ethanol-induced apoptosis would lead to activation of complement via the classical pathway. Wild-type and C1qa-/- mice were allowed free access to ethanol-containing diets or pair-fed control diets for 4 or 25 days. Ethanol feeding for 4 days increased apoptosis of Kupffer cells in both wild-type and C1qa-/- mice. Ethanol-induced deposition of C1q and C3b/iC3b/C3c was colocalized with apoptotic Kupffer cells in wild-type, but not C1qa-/-, mice. Furthermore, ethanol-induced increases in tumor necrosis factor-alpha and interleukin-6 expression at this early time point were suppressed in C1q-deficient mice. Chronic ethanol feeding (25 days) increased steatosis, hepatocyte apoptosis, and activity of serum alanine and aspartate aminotransferases in wild-type mice. These markers of hepatocyte injury were attenuated in C1qa-/- mice. In contrast, chronic ethanol (25 days)-induced increases in cytochrome P450 2E1 expression and oxidative stress did not differ between wild-type and C1qa-/- mice. For the first time, these data indicate that ethanol activates the classical complement pathway via C1q binding to apoptotic cells in the liver and that C1q contributes to the pathogenesis of ethanol-induced liver injury.

Differential Contribution of Apoptosis and Necrosis to Ethanolinduced Liver Injury in Mice

Differential Contribution of Apoptosis and Necrosis to Ethanolinduced Liver Injury in Mice

Role of C1q in ethanol-induced liver injury in mice

Role of C1q in ethanol-induced liver injury in mice

Formation of γ-ketoaldehyde–protein adducts during ethanol-induced liver injury in mice

Ethanol metabolism promotes the formation of a variety of reactive aldehydes in the liver. These aldehydes can rapidly form covalent protein adducts. Accumulating evidence indicates that these protein adducts may contribute to ethanol-mediated liver injury. Overproduction of γ-ketoaldehydes, levuglandins (LGs) and isolevuglandins, is implicated in the pathogenesis of several chronic inflammatory diseases. γ-Ketoaldehydes can form protein adducts orders of magnitude more quickly than 4-hydroxynonenal (4-HNE) or malondialdehyde. We hypothesized that ethanol-induced oxidative stress in vivo results in overproduction of LGE 2– and iso[4]LGE 2–protein adducts in mouse liver. Female C57BL/6 mice were allowed free access to an ethanol-containing diet for up to 39 days or pair-fed control diets. Pathological markers of ethanol-induced hepatic injury including serum alanine aminotransferase, hepatic triglyceride, and CYP2E1 were elevated in response to ethanol feeding. Ethanol-induced formation of iso[4]LGE 2–, LGE 2–, and 4-HNE–protein adducts in mouse liver was dependent on both dose and duration of ethanol feeding. Deficiency of cyclooxygenase 1 or 2 did not prevent ethanol-induced iso[4]LGE 2 or LGE 2 adducts in the liver, but adduct formation was reduced in both TNFR1- and CYP2E1-deficient mice. In summary, ethanol feeding enhanced γ-ketoaldehyde–protein adduct production via a TNFR1/CYP2E1-dependent, but cyclooxygenase-independent, mechanism in mouse liver.

An early complement-dependent and TLR-4–independent phase in the pathogenesis of ethanol-induced liver injury in mice #

The innate immune system has been implicated in the pathogenesis of alcoholic liver disease. Although innate immunity is usually considered an early response to injury, previous work implicating innate immunity in ethanol-induced liver injury focuses primarily on long-term ethanol exposure. We investigated the early period of ethanol exposure to determine whether there were temporal associations between activation of innate immune responses and known correlates of liver injury. Female C57BL/6 mice were allowed free access to an ethanol-containing Lieber-DeCarli diet or were pair-fed a control diet. Within 4 days of ethanol exposure, we observed a striking spike in expression of hepatic proinflammatory cytokines-including tumor necrosis factor alpha (TNF-alpha), interleukin-6, and interferon-gamma-prior to hepatic triglyceride accumulation or increased plasma alanine aminotransferase activities, as well as before the induction of cytochrome P450 2E1 or oxidative stress. This early spike in inflammatory cytokines coincided with deposition of C3b-iC3b/C3c (C3b) in the liver. This deposition, resulting from the cleavage of the third component of the complement system (C3), is evidence for activation of complement in response to ethanol. C3(-/-) mice were protected from the early, ethanol-induced increase in hepatic TNF-alpha expression. Ethanol increased C3b deposition in mice deficient in C3a receptor or C5a receptor, as well as in wild-type mice depleted of hepatic macrophages; however, there was no increase in hepatic TNF-alpha in the absence of C3a receptor, C5a receptor, or hepatic macrophages. In contrast, the absence of Toll-like receptor 4 (TLR-4) had no effect on the early, ethanol-induced increase in either C3b or TNF-alpha. We have identified a complement- and macrophage-dependent, but TLR-4 independent, phase in the pathogenesis of ethanol-induced liver injury.

L‐Buthionine (S,R) Sulfoximine Depletes Hepatic Glutathione But Protects Against Ethanol‐Induced Liver Injury

L-Buthionine (S,R) sulfoximine (BSO) is an inhibitor of glutathione biosynthesis and has been used as an effective means of depleting glutathione from cells and tissues. Here we investigated whether treatment with BSO enhanced ethanol-induced liver injury in mice. Female C57Bl/6 mice were pair fed with control and ethanol-containing liquid diets in which ethanol was 29.2% of total calories. During the final 7 days of pair feeding, groups of control-fed and ethanol-fed mice were given 0, 5 or 7.6 mM BSO in the liquid diets. Compared with controls, ethanol given alone decreased total liver glutathione. This effect was exacerbated in mice given ethanol with 7.6 mM BSO, causing a 72% decline in hepatic glutathione. While ethanol alone caused no decrease in mitochondrial glutathione, inclusion of 7.6 mM BSO caused a 2-fold decline compared with untreated controls. L-Buthionine (S,R) sulfoximine did not affect ethanol consumption, but serum ethanol levels in BSO-treated mice were nearly 6-fold lower than in mice given ethanol alone. The latter decline in serum ethanol was associated with a significant elevation in the specific activities of cytochrome P450 2E1 and alcohol dehydrogenase in livers of BSO-treated animals. Ethanol consumption caused a 3.5-fold elevation in serum alanine aminotransferase levels but the enzyme fell to control levels when BSO was included in the diet. L-Buthionine (S,R) sulfoximine administration also attenuated ethanol-induced steatosis, prevented the leakage of lysosomal cathepsins into the cytosol, and prevented the ethanol-elicited decline in proteasome activity. L-Buthionine (S,R) sulfoximine, administered with ethanol, significantly depleted hepatic glutathione, compared with controls. However, despite the decrease in hepatic antioxidant levels, liver injury by ethanol was alleviated, due, in part, to a BSO-elicited acceleration of ethanol metabolism.