Abrogation of Nuclear Factor-κB Activation Is Involved in Zinc Inhibition of Lipopolysaccharide-Induced Tumor Necrosis Factor-α Production and Liver Injury
2004; Elsevier BV; Volume: 164; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)63713-3
ISSN1525-2191
AutoresZhanxiang Zhou, Lipeng Wang, Zhenyuan Song, Jack T. Saari, Craig J. McClain, Y. James Kang,
Tópico(s)Alcohol Consumption and Health Effects
ResumoEndotoxin (lipopolysaccharide, LPS)-induced tumor necrosis factor-α (TNF-α) release from Kupffer cells is critically involved in the pathogenesis of alcohol-induced liver injury. We recently reported that inhibition of alcohol-induced plasma endotoxin elevation contributes to the protective action of zinc against alcoholic hepatotoxicity. The present study was undertaken to determine whether zinc interferes with the endotoxin-TNF-α signaling pathway, and possible mechanism(s) by which zinc modulates the endotoxin-TNF-α signaling. Administration of LPS to metallothionein (MT)-knockout (MT-KO) mice and 129/Sv wild-type (WT) controls at 4 mg/kg induced hepatic TNF-α elevation at 1.5 hours, followed by liver injury at 3 hours. Zinc pretreatment (two doses at 5 mg/kg) attenuated TNF-α production and liver injury in both MT-KO and WT mice, indicating a MT-independent action of zinc. Immunohistochemical detection of the phosphorylation of I-κB and nuclear factor (NF)-κB in the liver of MT-KO mice demonstrated that zinc pretreatment abrogated LPS-induced NF-κB activation in the Kupffer cells. Fluorescent microscopy of superoxide by dihydroethidine and of zinc ions by Zinquin in the liver of MT-KO mice showed that zinc pretreatment increased the intracellular labile zinc ions and inhibited LPS-induced superoxide generation. These results demonstrate that zinc inhibits LPS-induced hepatic TNF-α production through abrogation of oxidative stress-sensitive NF-κB pathway, and the action of zinc is independent of MT. Thus, zinc may be beneficial in the treatment of LPS-induced liver injuries, such as sepsis and alcoholism. Endotoxin (lipopolysaccharide, LPS)-induced tumor necrosis factor-α (TNF-α) release from Kupffer cells is critically involved in the pathogenesis of alcohol-induced liver injury. We recently reported that inhibition of alcohol-induced plasma endotoxin elevation contributes to the protective action of zinc against alcoholic hepatotoxicity. The present study was undertaken to determine whether zinc interferes with the endotoxin-TNF-α signaling pathway, and possible mechanism(s) by which zinc modulates the endotoxin-TNF-α signaling. Administration of LPS to metallothionein (MT)-knockout (MT-KO) mice and 129/Sv wild-type (WT) controls at 4 mg/kg induced hepatic TNF-α elevation at 1.5 hours, followed by liver injury at 3 hours. Zinc pretreatment (two doses at 5 mg/kg) attenuated TNF-α production and liver injury in both MT-KO and WT mice, indicating a MT-independent action of zinc. Immunohistochemical detection of the phosphorylation of I-κB and nuclear factor (NF)-κB in the liver of MT-KO mice demonstrated that zinc pretreatment abrogated LPS-induced NF-κB activation in the Kupffer cells. Fluorescent microscopy of superoxide by dihydroethidine and of zinc ions by Zinquin in the liver of MT-KO mice showed that zinc pretreatment increased the intracellular labile zinc ions and inhibited LPS-induced superoxide generation. These results demonstrate that zinc inhibits LPS-induced hepatic TNF-α production through abrogation of oxidative stress-sensitive NF-κB pathway, and the action of zinc is independent of MT. Thus, zinc may be beneficial in the treatment of LPS-induced liver injuries, such as sepsis and alcoholism. Tumor necrosis factor-α (TNF-α) is a cytokine involved in alcoholic liver disease.1McClain CJ Hill D Schmidt J Diehl AM Cytokines in alcoholic liver disease.Semin Liver Dis. 1993; 13: 170-182Crossref PubMed Scopus (240) Google Scholar, 2McClain CJ Barve S Barve S Deaciuc I Hill D Tumor necrosis factor and alcoholic liver disease.Alcohol Clin Exp Res. 1998; 22: 248S-252SCrossref PubMed Scopus (72) Google Scholar, 3Thurman RG Mechanisms of hepatic toxicity. II. Alcoholic liver injury involves activation of Kupffer cells by endotoxin.Am J Physiol. 1998; 275: G605-G611PubMed Google Scholar The production of TNF-α by exposure to alcohol has been repeatedly demonstrated both in animal models and in patients with alcoholic hepatitis.4McClain C Cohen DA Increased tumor necrosis factor production by monocytes in alcoholic hepatitis.Hepatology. 1989; 9: 349-351Crossref PubMed Scopus (485) Google Scholar, 5Nanji AA Jokelainen K Rahemtulla A Miao L Fogt F Matsumoto H Tahan SR Su GL Activation of nuclear factor kappa B and cytokine imbalance in experimental alcoholic liver disease in the rat.Hepatology. 1999; 30: 934-943Crossref PubMed Scopus (195) Google Scholar, 6Hill DB Barve S Joshi-Barve S McClain C Increased monocyte nuclear factor-kappaB activation and tumor necrosis factor production in alcoholic hepatitis.J Lab Clin Med. 2000; 135: 387-395Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar These studies often reported elevations in serum TNF-α protein and hepatic TNF-α mRNA levels. Neutralizing TNF-α with a polyclonal antibody resulted in suppression of hepatic necrosis and inflammation caused by chronic alcohol exposure.7Iimuro Y Gallucci RM Luster MI Kono H Thurman RG Antibodies to tumor necrosis factor alpha attenuate hepatic necrosis and inflammation caused by chronic exposure to ethanol in the rat.Hepatology. 1997; 26: 1530-1537Crossref PubMed Scopus (444) Google Scholar Blocking TNF-α signaling in a TNF-α receptor-1 knockout mouse model also led to attenuation of alcohol-induced liver injury.8Yin M Wheeler MD Kono H Bradford BU Gallucci RM Luster MI Thurman RG Essential role of tumor necrosis factor alpha in alcohol-induced liver injury in mice.Gastroenterology. 1999; 117: 942-952Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar, 9Yin M Gabele E Wheeler MD Connor H Bradford BU Dikalova A Rusyn I Mason R Thurman RG Alcohol-induced free radicals in mice: direct toxicants or signaling molecules?.Hepatology. 2001; 34: 935-942Crossref PubMed Scopus (72) Google Scholar These reports strongly suggest that TNF-α plays a critical role in alcohol-induced liver injury. Kupffer cells are the main source of TNF-α after alcohol exposure. Endotoxin has been shown to trigger TNF-α production in alcohol-induced liver injury. Previous investigations demonstrated that endotoxin activates Kupffer cells by binding to the CD14/Toll-like receptor 4 on Kupffer cells, whose activation leads to increased activity of NADPH oxidase, nuclear factor (NF)-κB activation, and, eventually, TNF-α production.10Wheeler MD Kono H Yin M Nakagami M Uesugi T Arteel GE Gabele E Rusyn I Yamashina S Froh M Adachi Y Iimuro Y Bradford BU Smutney OM Connor HD Mason RP Goyert SM Peters JM Gonzalez FJ Samulski RJ Thurman RG The role of Kupffer cell oxidant production in early ethanol-induced liver disease.Free Radic Biol Med. 2001; 31: 1544-1549Crossref PubMed Scopus (214) Google Scholar, 11McClain CJ Hill DB Song Z Deaciuc I Barve S Monocyte activation in alcoholic liver disease.Alcohol. 2002; 27: 53-61Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 12Su GL Lipopolysaccharides in liver injury: molecular mechanisms of Kupffer cell activation.Am J Physiol. 2001; 283: G256-G265Google Scholar Thus, preventing endotoxin-induced Kupffer cell activation and TNF-α production may be an important strategy in the prevention of alcohol-induced liver injury. Zinc is an essential trace element involved in many physiological functions, including catalytic and structural roles in metalloenzymes, as well as regulatory roles in diverse cellular processes such as synaptic signaling and gene expression. Many reports indicate that zinc acts as an effective hepatoprotective agent under a variety of toxic conditions.13Dhawan D Goel A Further evidence for zinc as a hepatoprotective agent in rat liver toxicity.Exp Mol Pathol. 1995; 63: 110-117Crossref PubMed Scopus (42) Google Scholar, 14Liu J Liu Y Hartley D Klaassen CD Shehin-Johnson SE Lucas A Cohen SD Metallothionein-I/II knockout mice are sensitive to acetaminophen-induced hepatotoxicity.J Pharmacol Exp Ther. 1999; 289: 580-586PubMed Google Scholar, 15Kimura T Fujita I Itoh N Muto N Nakanishi T Takahashi K Azuma J Tanaka K Metallothionein acts as a cytoprotection against doxorubicin toxicity.J Pharmacol Exp Ther. 2000; : 299-302PubMed Google Scholar These reports showed that the action of zinc is associated with metallothionein (MT) induction. MT has been known as the major protein responsible for zinc homeostasis. MT has one-third cysteine residues and functions as an antioxidant. It has been long debated whether protection by zinc treatment results from zinc per se or from MT induction. Our recent studies with a MT-transgenic and MT-knockout (MT-KO) mouse models demonstrated that zinc, independent of MT, provides effective protection against acute alcohol-induced liver injury.16Zhou Z Sun X Kang YJ Metallothionein protection against alcoholic liver injury through inhibition of oxidative stress.Exp Biol Med. 2002; 227: 214-222Google Scholar, 17Zhou Z Sun X Lambert JC Saari JT Kang YJ Metallothionein-independent zinc protection from alcoholic liver injury.Am J Pathol. 2002; 160: 2267-2274Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar Furthermore, it was found that zinc prevented alcohol-induced alterations in gut permeability, which was involved in zinc inhibition of alcohol-induced endotoxin release from gut to blood stream.18Lambert JC Zhou Z Wang L Song Z McClain CJ Kang YJ Prevention of alterations in intestinal permeability is involved in zinc inhibition of acute ethanol-induced liver damage in mice.J Exp Phamarcol Ther. 2003; 305: 880-886Crossref PubMed Scopus (113) Google Scholar However, it is unclear whether zinc can modulate the endotoxin-TNF-α signaling in the liver. Therefore, the present study was undertaken to determine whether zinc interferes with the endotoxin-TNF-α signaling pathway and the possible mechanism(s) by which zinc interacts with the endotoxin-TNF-α signaling. Homozygous MT-KO mice were obtained from Jackson Laboratories (Bar Harbor, ME) and were produced on the 129/Sv genetic background. The MT-KO mice lacking MT-I and MT-II, the major mouse hepatic MT isoforms, were produced by a gene-targeting technique.19Masters BA Kelly EJ Quaife CJ Brinster RL Palmiter RD Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium.Proc Natl Acad Sci USA. 1994; 91: 584-588Crossref PubMed Scopus (562) Google Scholar Both MT-KO and 129/Sv wild-type (WT) controls were housed in the animal quarters at the University of Louisville Research Resources Center. They were maintained at 22°C with a 12-hour light/dark cycle and had free access to rodent chow and tap water. The experimental procedures were approved by the Institutional Animal Care and Use Committee, which is certified by the American Association for the Accreditation of Laboratory Animal Care. Nine-week-old male mice (23 to 25 g body weight) were used in the following experiments. In experiment 1 the effects of zinc pretreatment on lipopolysaccharide (LPS)-induced TNF-α production and liver injury were studied. MT-KO and WT mice were divided into eight groups by a 2 × 2 × 2 factorial design (+/−MT, +/−zinc, +/−LPS). For zinc treatment, two doses of zinc sulfate in saline at 5 mg of zinc ion/kg were administrated intraperitoneally at 36 hours and 12 hours before LPS (from E. Coli Serotype 0111: B4, Sigma, St. Louis, MO) treatment (4 mg/kg). Saline was used for controls for both zinc and LPS treatments. To assess intrahepatic TNF-α level, liver samples were harvested at 1.5 hours after LPS administration. To evaluate liver injury, plasma and liver samples were harvested at 3 hours after LPS administration. In experiment 2 the effects of zinc pretreatment on LPS signal transduction in the liver were studied. MT-KO mice were divided into four groups by a 2 × 2 factorial design (+/−zinc, +/−LPS). Two doses of zinc sulfate in saline at 5 mg of zinc ion/kg were administrated intraperitoneally at 36 hours and 12 hours before LPS treatment (4 mg/kg). Saline was used for controls of both zinc and LPS treatments. Dihydroethidine (Molecular Probes, Eugene, OR) at 5 mg/kg and Zinquin ethyl ester (Sigma) at 2.5 mg/kg were injected via tail vein immediately after LPS challenge for in situ detections of superoxide and zinc ions in the liver. Liver samples were harvested at 1 hour after LPS administration. At the end of each experiment, the mice were anesthetized with sodium pentobarbital (0.05 mg/g body weight). Blood was drawn using a heparinized syringe from the dorsal vena cava, and plasma was obtained by centrifugation using a plasma separator tube. The liver was perfused with saline and tissue samples were processed for both pathological and biochemical analysis. Liver samples for TNF-α assay were prepared according to a previous report20Wolf D Schumann J Koerber K Kiemer AK Vollmar AM Sass G Papadopoulos T Bang R Klein SD Brune B Tiegs G Low-molecular-weight hyaluronic acid induces nuclear factor-kappaB-dependent resistance against tumor necrosis factor alpha-mediated liver injury in mice.Hepatology. 2001; 34: 535-547Crossref PubMed Scopus (51) Google Scholar with some minor modifications. Briefly, liver samples were disintegrated in 4 vol of ice-cold Ripa buffer.20Wolf D Schumann J Koerber K Kiemer AK Vollmar AM Sass G Papadopoulos T Bang R Klein SD Brune B Tiegs G Low-molecular-weight hyaluronic acid induces nuclear factor-kappaB-dependent resistance against tumor necrosis factor alpha-mediated liver injury in mice.Hepatology. 2001; 34: 535-547Crossref PubMed Scopus (51) Google Scholar After incubation on ice for 30 minutes, samples were centrifuged twice at 25,000 × g for 15 minutes at 4°C. The resulting supernatants were used for the assay. The TNF-α levels in the liver were detected by immunoassay using a murine kit (BioSource Int., Camarillo, CA) and were expressed as pg/g liver tissue. Plasma ALT (EC 2.6.1.2.) activity was colorimetrically measured by using a Sigma Diagnostic kit (procedure no. 505, Sigma) following the instructions provided by the manufacture. Liver tissues were fixed with 10% neutral formalin and embedded in paraplast. Tissue sections of 5 μm were stained by hematoxylin and eosin. Tissue MT concentrations were determined by a cadmium-hemoglobin affinity assay. Briefly, liver tissues were homogenized in 4 vol of 10 mmol/L Tris-HCl buffer, pH 7.4, at 4°C. After centrifugation of the homogenate at 10,000 × g for 15 minutes, 200 μl of supernatant were transferred to microtubes for MT analysis, as described previously.21Kang YJ Chen Y Yu A Voss-McCowan M Epstein PN Overexpression of metallothionein in the heart of transgenic mice suppresses doxorubicin cardiotoxicity.J Clin Invest. 1997; 100: 1501-1506Crossref PubMed Scopus (227) Google Scholar Hepatic zinc concentrations were determined by inductively coupled argon-plasma emission spectroscopy (model 1140; Jarrel-Ash, Waltham, MA) after lyophilization and digestion of the tissues with nitric acid and hydrogen peroxide.22Nielsen FH Zimmerman TJ Shuler TR Interactions among nickel, copper, and iron in rats: liver and plasma contents of lipid and trace elements.Biol Trace Elem Res. 1982; 4: 1225-1243Crossref Scopus (74) Google Scholar Zinc concentrations in the liver were expressed as μg/g dry tissue. Cryostat liver sections were cut at 5 μm, air-dried, and fixed in acetone for 20 minutes at −20°C. For p-I-κB and p-NF-κB immunoperoxidase staining, endogenous peroxidase activity was quenched by incubating sections in 3% H2O2. Nonspecific binding sites were blocked by 5% normal goat serum for 30 minutes. Sections were incubated with polyclonal rabbit anti-TNF-α (BioSource), rabbit anti-phospho-I-κB (p-I-κB; Cell Signaling Technology, Beverly, MA), or rabbit anti-phospho-NF-κB (p-NF-κB, Cell Signaling) overnight at 4°C, followed by incubation with DAKO EnVision+ horseradish peroxidase-conjugated goat anti-rabbit IgG (DAKO, Carpinteria, CA) for 30 minutes. The antibody binding sites were visualized by incubation with a diaminobenzidine-H2O2 solution. For double-immunofluorescence staining of Kupffer cells and TNF-α, TNF-α staining was first performed by incubation with rabbit anti-TNF-α and Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), and Kupffer cells were then stained by incubation with monoclonal rat anti-mouse pan tissue-fixed macrophages (clone EM8; Research Diagnostics, Flanders, NJ) and fluorescein isothiocyanate-conjugated donkey anti-rat IgG (Jackson ImmunoResearch Laboratories). For double-immunofluorescence staining of TNF-α and p-NF-κB, p-NF-κB was first stained by incubation with rabbit anti-p-NF-κB and Cy3-conjugated donkey anti-rabbit IgG, and TNF-α staining was then followed by incubation with monoclonal rat anti-mouse TNF-α (BD PharMingen, San Diego, CA) and fluorescein isothiocyanate-conjugated donkey anti-rat IgG. All of the reactions of primary antibodies were conducted at 4°C overnight, and the fluorescence conjugates were conducted at room temperature for 1 hour. Dihydroethidine was used for in situ detection of superoxide production in the liver. Dihydroethidine is oxidized to ethidine (red fluorescence) selectively by superoxide, but not by other reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radicals, or peroxynitrite.23Becker LB vanden Hoek TL Shao ZH Li CQ Schumacker PT Generation of superoxide in cardiomyocytes during ischemia before reperfusion.Am J Physiol. 1999; 277: H2240-H2246PubMed Google Scholar, 24Murakami K Kondo T Kawase M Li Y Sato S Chen SF Chan PH Mitochondrial susceptibility to oxidative stress exacerbates cerebral infarction that follows permanent focal cerebral ischemia in mutant mice with manganese superoxide dismutase deficiency.J Neurosci. 1998; 18: 205-213Crossref PubMed Google Scholar Intracellular zinc ions were detected by Zinquin. Zinquin is a membrane-permeable, blue fluorescent probe that fluoresces on binding to zinc ions.25Zalewski PD Forbes IJ Betts WH Correlation of apoptosis with a change in intracellular labile Zn, using Zinquin, a new specific fluorescent probe for Zn.Biochem J. 1993; 296: 403-408Crossref PubMed Scopus (450) Google Scholar, 26Coyle P Zalewski PD Philcox JC Forbes IJ Ward AD Lincoln SF Mahadevan I Rofe AM Measurement of zinc in hepatocytes by using a fluorescent probe, Zinquin: relationship to metallothionein and intracellular zinc.Biochem J. 1994; 303: 781-786Crossref PubMed Scopus (135) Google Scholar For simultaneous in situ detection of superoxide and zinc ions in the liver, dihydroethidine (Molecular Probes) at 5 mg/kg and Zinquin ethyl ester (Sigma) at 2.5 mg/kg were injected via tail vein 1 hour before tissue harvest, as described above. Cryostat sections of liver were cut at 5 μm and mounted on glass slides. The fluorescence was detected with a Nikon 2000S fluorescent microscope. All data are expressed as mean ± SD (n = 4 to 6). The data were analyzed by analysis of variance and Newman-Keuls multiple-comparison test. Experiments involved in factorial designs were analyzed accordingly. Differences between groups were considered significant at P < 0.05. To determine the role of zinc in inhibition of LPS-induced TNF-α production and liver injury, WT mice were treated with zinc before LPS administration. TNF-α was measured at 1.5 hours and liver injury was assessed at 3 hours after LPS administration based on our preliminary time-course study at 1.5, 3, 6, and 12 hours, in which TNF-α production was peaked at 1.5 hours and liver injury at 3 hours after LPS administration (data not shown). As shown in Figure 1A, LPS administration induced a 5-fold increase in TNF-α concentrations in the liver. Zinc pretreatment significantly inhibited LPS-induced TNF-α production in the liver. Plasma ALT activity also was significantly elevated by LPS administration, whereas zinc pretreatment attenuated LPS-induced increase in plasma ALT activity (Figure 1B). Corresponding to plasma ALT elevation, histopathological observation demonstrated that zinc pretreatment suppressed LPS-induced necrotic cell death in the liver (Figure 2). To determine whether zinc inhibition of LPS-induced liver injury is mediated by MT production, MT-KO mice were treated in the same manner as the WT mice. As shown in Figure 1A, zinc significantly inhibited LPS-induced TNF-α production in the MT-KO mice, although the value was higher than in WT mice. Furthermore, the inhibitory effect of zinc on LPS-induced liver injury in the MT-KO mice was comparable to that observed in the WT mice (Figure 1B, Figure 2).Figure 2Effect of zinc on LPS-induced histopathological changes in the liver. MT-KO and WT mice were administrated with two doses of zinc sulfate at 5 mg of zinc ion/kg, followed by one dose of LPS at 4 mg/kg. Histopathological changes were observed at 3 hours after LPS challenge. LPS-induced necrotic damages (arrows) in the liver were inhibited by zinc pretreatment in both WT and MT-KO mice. CV, Central vein. H&E. Original magnifications, ×260.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To confirm the MT-independent action of zinc in hepatic protection from LPS damage, MT and zinc concentrations in the liver of WT and MT-KO mice were compared. As shown in Figure 3A, zinc treatment induced more than a 15-fold increase in hepatic MT concentrations in WT mice with or without LPS treatment. However, LPS per se did not affect MT concentrations in the liver. When the same treatment was applied to MT-KO mice, only trace amounts of hepatic MT were detected in all groups of MT-KO mice. On the other hand, zinc treatment elevated hepatic zinc concentrations by 2-fold in the WT mice and 1.5-fold in the MT-KO mice (Figure 3B). LPS per se did not cause significant change in hepatic zinc concentrations in either WT or MT-KO mice. To determine the molecular mechanism by which zinc inhibits LPS-induced TNF-α production, the effects of zinc on NF-κB activation were monitored by immunohistochemical staining of p-I-κB and p-NF-κB at 1 hour after LPS challenge to WT mice. As shown in Figure 4, there were no p-I-κB immunoreactive cells that were detected in the livers of control or zinc-treated mice. In the LPS-treated mice, there were numerous positive cells found in the liver, and the positive staining was apparently in the Kupffer cells that localize on the sinusoid wall. In the zinc/LPS-treated mice, there were only a few weakly stained cells found in the liver. Correspondingly, a large number of Kupffer cells in the LPS-treated mice showed immunoreactivity to p-NF-κB, and the positive staining was localized mainly in the nuclei. Zinc pretreatment abrogated LPS-induced NF-κB activation; there were only a few Kupffer cells showing weak staining. To elucidate the link between TNF-α production and NF-κB activation in the Kupffer cells, double-immunofluorescence staining of Kupffer cell/TNF-α and p-NF-κB/TNF-α were performed in the liver of MT-KO mice. As shown in Figure 5A, TNF-α and p-NF-κB were co-localized in the Kupffer cells. To understand the mechanism by which zinc abrogates LPS-induced NF-κB activation, in situ detections of superoxide by dihydroethidine and zinc ions by Zinquin in the liver of MT-KO mice were performed at 1 hour after LPS challenge. As shown in Figure 5B, only weak ethidine fluorescence was found in the control and zinc-treated livers. However, strong ethidine fluorescence was detected in the livers of LPS-treated mice. This LPS-induced ethidine fluorescence was attenuated by zinc pretreatment. The Zinquin fluorescence was slightly increased by LPS treatment, whereas strong Zinquin fluorescence was found in the livers of zinc- and zinc/LPS-treated mice. Co-localization analysis of ethidine and Zinquin fluorescence demonstrated that the increased availability of intracellular zinc ions correlates with decreased superoxide accumulation after LPS administration. The results presented in the present study demonstrated that zinc pretreatment provided effective protection against LPS-induced liver injury through inhibition of TNF-α production, and the protective action of zinc was independent of MT. The mechanistic link through in situ detection of p-I-κB, p-NF-κB, superoxide, and zinc ions was found to be likely that zinc pretreatment increased the availability of intracellular zinc ions and abrogated LPS-induced oxidative stress and NF-κB activation in the Kupffer cells. Our results are the first to demonstrate that zinc protection, independent of MT, against LPS-induced hepatotoxicity is mediated via abrogating NF-κB activation and TNF-α production in the Kupffer cells. TNF-α has been repeatedly reported to be specifically responsible for LPS-induced liver injury.12Su GL Lipopolysaccharides in liver injury: molecular mechanisms of Kupffer cell activation.Am J Physiol. 2001; 283: G256-G265Google Scholar A convinced evidence is that TNF-α neutralization with an anti-TNF-α antibody prevented liver injury in LPS-treated rat.27Eastin CE McClain CJ Lee EY Bagby GJ Chawla RK Choline deficiency augments and antibody to tumor necrosis factor-alpha attenuates endotoxin-induced hepatic injury.Alcohol Clin Exp Res. 1997; 21: 1037-1041Crossref PubMed Scopus (47) Google Scholar This mechanism has been demonstrated in alcoholic liver disease.1McClain CJ Hill D Schmidt J Diehl AM Cytokines in alcoholic liver disease.Semin Liver Dis. 1993; 13: 170-182Crossref PubMed Scopus (240) Google Scholar, 2McClain CJ Barve S Barve S Deaciuc I Hill D Tumor necrosis factor and alcoholic liver disease.Alcohol Clin Exp Res. 1998; 22: 248S-252SCrossref PubMed Scopus (72) Google Scholar, 3Thurman RG Mechanisms of hepatic toxicity. II. Alcoholic liver injury involves activation of Kupffer cells by endotoxin.Am J Physiol. 1998; 275: G605-G611PubMed Google Scholar, 4McClain C Cohen DA Increased tumor necrosis factor production by monocytes in alcoholic hepatitis.Hepatology. 1989; 9: 349-351Crossref PubMed Scopus (485) Google Scholar, 5Nanji AA Jokelainen K Rahemtulla A Miao L Fogt F Matsumoto H Tahan SR Su GL Activation of nuclear factor kappa B and cytokine imbalance in experimental alcoholic liver disease in the rat.Hepatology. 1999; 30: 934-943Crossref PubMed Scopus (195) Google Scholar, 6Hill DB Barve S Joshi-Barve S McClain C Increased monocyte nuclear factor-kappaB activation and tumor necrosis factor production in alcoholic hepatitis.J Lab Clin Med. 2000; 135: 387-395Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 10Wheeler MD Kono H Yin M Nakagami M Uesugi T Arteel GE Gabele E Rusyn I Yamashina S Froh M Adachi Y Iimuro Y Bradford BU Smutney OM Connor HD Mason RP Goyert SM Peters JM Gonzalez FJ Samulski RJ Thurman RG The role of Kupffer cell oxidant production in early ethanol-induced liver disease.Free Radic Biol Med. 2001; 31: 1544-1549Crossref PubMed Scopus (214) Google Scholar, 11McClain CJ Hill DB Song Z Deaciuc I Barve S Monocyte activation in alcoholic liver disease.Alcohol. 2002; 27: 53-61Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar Alcohol administration increases intestinal permeability, thereby causing elevation of blood LPS.28Keshavarzian A Holmes EW Patel M Iber F Fields JZ Pethkar S Leaky gut in alcoholic cirrhosis: a possible mechanism for alcohol-induced liver damage.Am J Gastroenterol. 1999; 94: 200-207Crossref PubMed Scopus (285) Google Scholar, 29Parlesak A Schafer C Schutz T Bode JC Bode C Increased intestinal permeability to macromolecules and endotoxemia in patients with chronic alcohol abuse in different stages of alcohol-induced liver disease.J Hepatol. 2000; 32: 742-747Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar, 30Mathurin P Deng QG Keshavarzian A Choudhary S Holmes EW Tsukamoto H Exacerbation of alcoholic liver injury by enteral endotoxin in rats.Hepatology. 2000; 32: 1008-1017Crossref PubMed Scopus (246) Google Scholar Because the LPS/TNF-α signaling pathway is critically involved in the pathogenesis of alcoholic liver injury, abrogating this pathway should attenuate the progression of alcoholic liver injury. In vitro studies have generated strong evidence that LPS activates the inflammatory response in Kupffer cells through oxidative stress.31Landmann R Scherer F Schumann R Link S Sansano S Zimmerli W LPS directly induces oxygen radical production in human monocytes via LPS binding protein and CD14.J Leukoc Biol. 1995; 57: 440-449PubMed Google Scholar A number of antioxidants and herbal extracts have been reported to suppress LPS-induced TNF-α production from Kupffer cells and macrophages, including N-acetyl-l-cysteine,32Fox ES Brower JS Bellezzo JM Leingang KA N-acetylcysteine and alpha-tocopherol reverse the inflammatory response in activated rat Kupffer cells.J Immunol. 1997; 158: 5418-5423PubMed Google Scholar, 33Bellezzo JM Leingang KA Bulla GA Britton RS Bacon BR Fox ES Modulation of lipopolysaccharide-mediated activation in rat Kupffer cells by antioxidants.J Lab Clin Med. 1998; 131: 36-44Abstract Full Text PDF PubMed Scopus (53) Google Scholar, 34Haddad JJ Land SC Redox/ROS regulation of lipopolysaccharide-induced mitogen-activated protein kinase (MAPK) activation and MAPK-mediated TNF-alpha biosynthesis.Br J Pharmacol. 2002; 135: 520-536Crossref PubMed Scopus (132) Google Scholar α-tocopherol,32Fox ES Brower JS Bellezzo JM Leingang KA N-acetylcysteine and alpha-tocopherol reverse the inflammatory response in activated rat Kupffer cells.J Immunol. 1997; 158: 5418-5423PubMed Google Scholar, 33Bellezzo JM Leingang KA Bulla GA Britton RS Bacon BR Fox ES Modulation of lipopolysaccharide-mediated activation in rat Kupffer cells by antioxidants.J Lab Clin Med. 1998; 131: 36-44Abstract Full Text PDF PubMed Scopus (53) Google Scholar dilinoleoylphosphatidylcholine,35Cao Q Mak KM Lieber CS Dilinoleoylphosphatidylcholine de
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