Gut-derived endotoxin is a critical factor in the development and progression of alcoholic liver disease (ALD). Probiotics can treat alcohol-induced liver injury associated with gut leakiness and endotoxemia in animal models, as well as in human ALD; however, the mechanism or mechanisms of their beneficial action are not well defined. We hypothesized that alcohol impairs the adaptive response-induced hypoxia-inducible factor (HIF) and that probiotic supplementation could attenuate this impairment, restoring barrier function in a mouse model of ALD by increasing HIF-responsive proteins (eg, intestinal trefoil factor) and reversing established ALD. C57BJ/6N mice were fed the Lieber DeCarli diet containing 5% alcohol for 8 weeks. Animals received Lactobacillus rhamnosus GG (LGG) supplementation in the last 2 weeks. LGG supplementation significantly reduced alcohol-induced endotoxemia and hepatic steatosis and improved liver function. LGG restored alcohol-induced reduction of HIF-2α and intestinal trefoil factor levels. In vitro studies using the Caco-2 cell culture model showed that the addition of LGG supernatant prevented alcohol-induced epithelial monolayer barrier dysfunction. Furthermore, gene silencing of HIF-1α/2α abolished the LGG effects, indicating that the protective effect of LGG is HIF-dependent. The present study provides a mechanistic insight for utilization of probiotics for the treatment of ALD, and suggests a critical role for intestinal hypoxia and decreased trefoil factor in the development of ALD. Gut-derived endotoxin is a critical factor in the development and progression of alcoholic liver disease (ALD). Probiotics can treat alcohol-induced liver injury associated with gut leakiness and endotoxemia in animal models, as well as in human ALD; however, the mechanism or mechanisms of their beneficial action are not well defined. We hypothesized that alcohol impairs the adaptive response-induced hypoxia-inducible factor (HIF) and that probiotic supplementation could attenuate this impairment, restoring barrier function in a mouse model of ALD by increasing HIF-responsive proteins (eg, intestinal trefoil factor) and reversing established ALD. C57BJ/6N mice were fed the Lieber DeCarli diet containing 5% alcohol for 8 weeks. Animals received Lactobacillus rhamnosus GG (LGG) supplementation in the last 2 weeks. LGG supplementation significantly reduced alcohol-induced endotoxemia and hepatic steatosis and improved liver function. LGG restored alcohol-induced reduction of HIF-2α and intestinal trefoil factor levels. In vitro studies using the Caco-2 cell culture model showed that the addition of LGG supernatant prevented alcohol-induced epithelial monolayer barrier dysfunction. Furthermore, gene silencing of HIF-1α/2α abolished the LGG effects, indicating that the protective effect of LGG is HIF-dependent. The present study provides a mechanistic insight for utilization of probiotics for the treatment of ALD, and suggests a critical role for intestinal hypoxia and decreased trefoil factor in the development of ALD. Alcohol consumption causes fatty liver, which can in some cases progress to inflammation, fibrosis, cirrhosis, and even liver cancer.1Beier J.I. McClain C.J. Mechanisms and cell signaling in alcoholic liver disease.Biol Chem. 2010; 391: 1249-1264Crossref PubMed Google Scholar, 2Frazier T.H. Stocker A.M. Kershner N.A. Marsano L.S. McClain C.J. Treatment of alcoholic liver disease.Therap Adv Gastroenterol. 2011; 4: 63-81Crossref PubMed Scopus (112) Google Scholar, 3McClain C.J. Song Z. Barve S.S. Hill D.B. Deaciuc I. Recent advances in alcoholic liver disease IV. Dysregulated cytokine metabolism in alcoholic liver disease.Am J Physiol Gastrointest Liver Physiol. 2004; 287: G497-G502Crossref PubMed Scopus (228) Google Scholar, 4Kirpich I.A. Solovieva N.V. Leikhter S.N. 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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 (489) Google Scholar Although the exact mechanism by which endotoxins cause liver injury is still not clear, lipopolysaccharide stimulation of tumor necrosis factor-α and other inflammatory cytokines in ALD leading to liver injury is one likely pathway. Elimination of bacteria to prevent endotoxin-induced liver injury has been used in clinical practice and experimental animal models. For example, the use of antibiotics to sterilize the gut to reduce endotoxin production prevented experimental alcohol-induced liver injury.7Adachi Y. Moore L.E. Bradford B.U. Gao W. Thurman R.G. Antibiotics prevent liver injury in rats following long-term exposure to ethanol.Gastroenterology. 1995; 108: 218-224Abstract Full Text PDF PubMed Scopus (596) Google Scholar Moreover, treatment with probiotics and prebiotics to alter the gut flora and reduce the Gram-negative bacteria population has been successfully used in several studies of alcohol-induced liver injury in rodents.8Forsyth C.B. Farhadi A. Jakate S.M. Tang Y. Shaikh M. Keshavarzian A. Lactobacillus GG treatment ameliorates alcohol-induced intestinal oxidative stress, gut leakiness, and liver injury in a rat model of alcoholic steatohepatitis.Alcohol. 2009; 43: 163-172Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 9Yan A.W. Fouts E. Brandl J. Starkel P. Torralba M. Schott E. Tsukamoto H. Nelson E. Brenner A. Schnabl B. Enteric dysbiosis associated with a mouse model of alcoholic liver disease.Hepatology. 2011; 53: 96-105Crossref PubMed Scopus (533) Google Scholar, 10Mutlu E. Keshavarzian A. 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The intestinal epithelium forms an essential barrier to gut luminal contents. The barrier function of intestinal epithelium is provided by paracellular apical junction complexes, including tight junctions and adherens junctions,11Hartsock A. Nelson W.J. Adherens and tight junctions: structure, function and connections to the actin cytoskeleton.Biochim Biophys Acta. 2008; 1778: 660-669Crossref PubMed Scopus (971) Google Scholar located at the apical end of epithelial cells, and a thick mucus gel layer secreted by the intestinal mucosa. This structure provides a dynamic and regulated barrier to the flux of the luminal contents to the lamina propria. The barrier function of the intestinal epithelium is regulated by the availability of oxygen.12Laukoetter M.G. Bruewer M. Nusrat A. Regulation of the intestinal epithelial barrier by the apical junctional complex.Curr Opin Gastroenterol. 2006; 22: 85-89Crossref PubMed Scopus (203) Google Scholar, 13Taylor C.T. Colgan S.P. Hypoxia and gastrointestinal disease.J Mol Med. 2007; 85: 1295-1300Crossref PubMed Scopus (233) Google Scholar, 14Turner J.R. Intestinal mucosal barrier function in health and disease.Nat Rev Immunol. 2009; 9: 799-809Crossref PubMed Scopus (2293) Google Scholar Intestinal epithelial cells function within a uniquely steep physiological oxygen gradient. Under stress conditions, the gradient shifts toward hypoxia, using more oxygen-independent glycolysis for energy production. This oxygen adaptation process is characterized by the expression of a master transcription factor, hypoxia-inducible factor (HIF). HIF is a heterodimer consisting of an α-subunit and a β-subunit.15Wang G.L. Jiang B.H. Rue E.A. Semenza G.L. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.Proc Natl Acad Sci USA. 1995; 92: 5510-5514Crossref PubMed Scopus (5030) Google Scholar HIF-β is constitutively expressed and translocates into the nucleus, whereas stabilization and nuclear accumulation of HIF-α are induced by hypoxia or hypoxic mimics. Under normoxic conditions, the HIF-α subunit is degraded through a process mediated by hydroxylation of two proline residues in HIF-α through three HIF hydroxylases (prolyl hydroxylases).16Kaelin Jr, W.G. Ratcliffe P.J. Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway.Mol Cell. 2008; 30: 393-402Abstract Full Text Full Text PDF PubMed Scopus (2192) Google Scholar, 17Semenza G. Signal transduction to hypoxia-inducible factor 1.Biochem Pharmacol. 2002; 64: 993-998Crossref PubMed Scopus (744) Google Scholar, 18Semenza G.L. 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Epithelial hypoxia-inducible factor-1 is protective in murine experimental colitis.J Clin Invest. 2004; 114: 1098-1106Crossref PubMed Scopus (492) Google Scholar and this HIF-dependent protection affects overall tissue integrity, rather than only tight junction proteins. Probiotics are microorganisms that can alter the gut microbiota profile, resulting in improved barrier integrity. Lactobacillus rhamnosus Gorbach Goldin (LGG) is a widely studied probiotic. Although probiotics have several beneficial effects on intestinal function, including ameliorating diarrhea and prolonging remission in ulcerative colitis and pouchitis (these effects are generally attributed as anti-inflammatory and as reducing oxidative stress), the precise mechanisms by which probiotics attenuate alcohol-induced disruption of intestinal integrity and subsequent liver injury remain to be elucidated. To determine whether LGG can attenuate established alcohol-induced intestinal barrier disruption, endotoxemia, and liver injury, we investigated the effect of LGG on epithelial cell permeability and severity of hepatic steatosis using in vivo (mouse) and in vitro (epithelial cell culture) models. We hypothesized that LGG would potentiate HIF function, increase epithelial protective gene expression, and preserve barrier function, thus reducing liver injury in the mouse model. We showed that LGG treatment in mice with established hepatic steatosis increases HIF-mediated signaling in intestinal epithelium, reduces endotoxemia, normalizes barrier function, and ameliorates alcohol-induced liver injury. LGG was purchased from the American Type Culture Collection (accession 53103; ATCC, Rockville, MD) and was cultured in Lactobacillus de Man, Rogosa, and Sharpe broth (Difco MRS broth; BD Biosciences-Advanced Bioprocessing, Sparks, MD) at 37°C in accordance with ATCC guidelines. Bacteria were harvested from MRS broth by centrifugation, and colony forming units (CFU) were counted by dilution and streaking on MRS agar plates (Difco) at 37°C overnight. To prepare supernatant, LGG culture broth was centrifuged and filtered through a 0.22-μm filter. The supernatant was stored at 4°C for later use. Male C57BL/6N mice were obtained from Harlan Laboratories (Indianapolis, IN). Mice were pair-fed liquid diets (Lieber DeCarli) containing 17% of energy as protein, 40% as corn oil, 7% as carbohydrate, and 35% as either alcohol (alcohol-fed, AF) or as an isocaloric maltose-dextrin (pair-fed, PF) in the following groups: PF, AF, AF+LGG, and an overall control group of normal chow (13% of energy from fat, N). LGG culture broth (109 CFU/mouse per day) was added into the diet in last 2 weeks of the experiment. Mice were maintained on the treatments for a total of 8 weeks. At the end the of experiment, the mice were anesthetized with Avertin (2,2,2-tribromoethanol) after overnight fasting. Plasma and tissue samples were collected for assays. All mice were treated according to the protocols reviewed and approved by the Institutional Animal Care and Use Committee of the University of Louisville. Formalin-fixed, paraffin-embedded tissue sections were processed for staining with H&E and then were studied by light microscopy. Caco-2 cells obtained from the ATCC were cultured in Eagle's minimal essential medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum at 37°C in a 5% CO2 environment for 21 days. Culture medium was changed every 2 days. Caco-2 cells were subcultured after partial digestion with 0.25% trypsin-EDTA. For probiotic treatment, LGG cultural supernatant (LGG-s) from cultural broth at the density of 109 CFU/mL was prepared and added into Caco-2 cell medium at 1% (v/v; LGG-s/medium) concentration. Caco-2 cells grown on chamber slides (LabTek, Naperville, IL) were used for fluorescent staining of tight junction proteins ZO-1, occludin and claudin-1, whereas Caco-2 cells grown on 24-well plates were used for immunoblotting analysis. For measurement of epithelial barrier function, Caco-2 cells were seeded and cultured on 24-well inserts (pore size 0.4 μm; BD Biosciences, San Jose, CA) for 21 days, and then were treated with 5% ethanol in the presence or absence of the LGG-s for 24 hours before measurement. The transepithelial electrical resistance (TEER) of the filter-grown Caco-2 monolayers was measured with an epithelial volt ohmmeter (World Precision Instruments, Sarasota, FL). TEER was recorded with three consecutive measurements after subtracting the resistance value of the filters alone. For determination of paracellular permeability, fluorescein isothiocyanate-dextran-4 (FD-4) was added to the apical compartment of Caco-2 cells at a concentration of 10 mg/mL in Eagle's minimal essential medium. After 90 minutes of incubation, the medium was collected and the FD-4 that penetrated to the medium was measured using a microplate fluorescence reader with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Blood samples from control and alcohol-treated mice were drawn from the dorsal vena cava. Plasma was obtained by centrifuging the blood at 1560 × g for 30 minutes at 4°C. Lipopolysaccharide levels were measured with a Limulus amebocyte lysate test kit (Lonza, Walkersville, MD) according to the manufacturer's instructions. Plasma alanine aminotransferase (ALT) was measured using an ALT Infinity enzymatic assay kit (Thermo Scientific, Waltham, MA). Liver tissue triglyceride, free fatty acid, and cholesterol concentrations were measured using Infinity kits (Thermo Scientific). Hepatic triglyceride levels were determined as described previously,4Kirpich I.A. Solovieva N.V. Leikhter S.N. Shidakova N.A. Lebedeva O.V. Sidorov P.I. Bazhukova T.A. Soloviev A.G. Barve S.S. McClain C.J. Cave M. Probiotics restore bowel flora and improve liver enzymes in human alcohol-induced liver injury: a pilot study.Alcohol. 2008; 42: 675-682Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar using a triglyceride reagent (Thermo Fisher Scientific, Middletown, VA). The mRNA levels were assessed by real-time quantitative RT-PCR. In brief, the total RNA was isolated with TRIzol reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA) and was reverse-transcribed using a GeneAmp RNA PCR kit (Applied Biosystems, Foster City, CA). Primer sequences are given in Table 1. Real-time quantitative RT-PCR was performed on an ABI 7500 real-time PCR thermocycler with SYBR Green PCR master mix (Applied Biosystems). The relative quantities of target transcripts were calculated from duplicate samples after normalization of the data against the housekeeping gene β-actin. Dissociation curve analysis was performed after PCR amplification to confirm the specificity of the primers. Relative mRNA expression was calculated using the ΔΔCT method.21Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123392) Google ScholarTable 1Primer Sequences for Real-Time RT-PCRGeneForward sequenceReverse sequenceTJP1 (alias ZO-1)5′-ATCCCTCAACAAGGGCCATTC-3′5′-CACTTGTTTTGCCAGGTTTA-3′OCLN5′-CCAATGTGCAGGAGTGGG-3′5′-CGCTGCTGTAACGAGGCT-3′CLDN15′-AAGTGCTTGGAAGACGATGA-3′5′-CTTGGTGTTGGGTAAAGAGGTT-3′MUC15′-GTGCCCCCTAGCAGTACCG-3′5′-GACGTGCCCCTACAAGTTGG-3′MUC25′-ACTGCACATTCTTCAGCTGC-3′5′-ATTCATGAGGACGGTCTTGG-3′CD735′-ATTGCAAAGTGGTTCAAAGTCA-3′5′-ACACTTGGCCAGTAAAATAGGG-3′ITF5′-TGGTCCTGGCCTTGCTGT-3′5′-GGCACACTGGTTTGCAGACA-3′VEGF5′-TTACTGCTGTACCTCCACC-3′5′-ACAGGACGGCTTGAAGATG-3′HIF5′-GCAAGCCCTGAAAGCG-3′5′-GGCTGTCCGACTTTGA-3′ACTB5′-GAGACCTTCAACACCCC-3′5′-ATAGCTCTTCTCCAGGGAGG-3′ Open table in a new tab SiRNAs targeting human HIF-1α/2α and a negative mismatched control were designed and synthesized by Ambion (Austin, TX). The Caco-2 monolayers cultured for 21 days were transfected with 100 nmol/L HIF-1α/2α or negative mismatched siRNA using Lipofectamine 2000 transfection agent (Invitrogen) according to the manufacturer's instruction. Nuclear extracts were prepared as described previously,22Feng W. Ye F. Xue W. Zhou Z. Kang Y.J. Copper regulation of hypoxia-inducible factor-1 activity.Mol Pharmacol. 2009; 75: 174-182Crossref PubMed Scopus (171) Google Scholar with minor modifications. In brief, cells were washed once with ice-cold PBS. Ice-cold buffer (10 mmol/L Tris-HCl, pH 7.8, 1.5 mmol/L MgCl2, and 10 mmol/L KCl) containing freshly added 0.4 mmol/L phenylmethylsulfonyl fluoride, 0.5 mmol/L dithiothreitol, and 1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) was overlaid on cells in the well and incubated for 10 minutes. The cells were then harvested and lysed by Dounce homogenization. Nuclei were pelleted by centrifugation and then resuspended in ice-cold buffer (20 mmol/L Tris-HCl, pH 7.8, 420 mmol/L KCl, 1.5 mmol/L MgCl2, and 20% glycerol) containing freshly added 0.4 mmol/L phenylmethylsulfonyl fluoride, 0.5 mmol/L dithiothreitol, 1% protease inhibitor cocktail, and 1 mmol/L Na3VO4 and incubated for 30 minutes on ice with occasional tapping. The extracts were clarified by centrifugation at 12,000 × g for 15 minutes at 4°C, placed in aliquots, and stored at −80°C. Tissues were homogenized, and Caco-2 cell monolayers were lysed on ice for 30 minutes in radioimmunoprecipitation assay buffer (50 mmol/L Tris · HCl, pH 7.4, 150 mmol/L NaCl, 2 mmol/L EDTA, 4 mmol/L Na3VO4, 40 mmol/L NaF, 1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, 1% protease inhibitor cocktail) and centrifuged at 14,000 × g for 10 minutes. The supernatant was collected. Aliquots of tissue and cell lysates and nuclear fractions prepared as above containing 10 to 30 μg protein were loaded onto a 4% to 15% SDS-polyacrylamide gel. After electrophoresis, proteins were transferred to polyvinylidene fluoride or nitrocellular membrane. The membrane was probed with antibody against HIF-1α (BD Biosciences-Advanced Bioprocessing), HIF-2α (Novus, Littleton, CO), and ITF, VEGF, claudin-1, occludin, or β-actin (Santa Cruz Biotechnology, Santa Cruz, CA). The membrane was then processed with HRP-conjugated IgG. The protein bands were visualized by an enhanced chemiluminescence detection system (GE Healthcare, Piscataway, NJ) and quantified by densitometry analysis. Cryostat sections of the small intestines, and Caco-2 cells on chamber slides were fixed with cold methanol for 15 minutes at −20°C. They were then incubated with polyclonal rabbit anti-claudin-1, occludin, or ZO-1 antibodies (Zymed Laboratories, South San Francisco, CA) overnight at 4°C, followed by incubation with a Cy3-conjugated antibody (Invitrogen) or fluorescein isothiocyanate-conjugated antibody (Invitrogen) for 30 minutes at room temperature. ROS accumulation in the small intestine and Caco-2 cells was examined by dihydroethidium fluorescence microscopy.23Xue W. Liu Q. Cai L. Wang Z. Feng W. Stable overexpression of human metallothionein-IIA in a heart-derived cell line confers oxidative protection.Toxicol Lett. 2009; 188: 70-76Crossref PubMed Scopus (28) Google Scholar Nonfluorescent dihydroethidium is oxidized by ROS to yield the red fluorescent product ethidium, which binds to nucleic acids, staining the nucleus a bright fluorescent red. Cryostat sections of ileum or Caco-2 cell chamber slides were incubated with 5 μmol/L dihydroethidium (Molecular Probes, Eugene, OR) for 30 minutes at 37°C in the dark. The ROS-catalyzed ethidium red fluorescence was examined under fluorescence microscopy. The relative fluorescence intensity was quantified with SigmaScan Pro 5 software (Systat Software, San Jose, CA). All data are expressed as means ± SEM or as indicated. The data were analyzed by analysis of variance and Newman-Keuls multiple-comparison test. Differences between groups were considered significant at P <0.05. Alcohol exposure for 8 weeks produced a lower mouse body weight compared with the PF controls (Table 2). LGG supplementation in AF mice in the last 2 weeks did not affect body weight, compared with the AF mice without LGG. Alcohol exposure significantly increased plasma ALT level, and this elevation was attenuated by LGG supplementation. Plasma endotoxin level (lipopolysaccharide) was elevated in alcohol exposure group, and the rise was reduced in the LGG supplementation group. Alcohol exposure increased liver triglyceride, free fatty acid, and cholesterol levels. LGG supplementation significantly attenuated these increases. Alcohol exposure and LGG supplementation did not change the intestine/body weight ratio (data not shown).Table 2Characteristics and Biochemical Changes in LGG-Treated MiceCharacteristicPFAFAF + LGGBody weight (g)28.1 ± 0.6425.96 ± 0.72⁎P < 0.05 versus PA;26.68 ± 0.48Plasma ALT (U/L)28.88 ± 2.9343.66 ± 2.02⁎P < 0.05 versus PA;32.74 ± 2.67⁎⁎P < 0.05 versus AF.Plasma LPS (mg/L)0.13 ± 0.040.37 ± 0.17⁎P < 0.05 versus PA;0.17 ± 0.04⁎⁎P < 0.05 versus AF.Liver TG (mg/dL)39.93 ± 5.59100.2 ± 8.17⁎P < 0.05 versus PA;89.7 ± 3.87⁎⁎P < 0.05 versus AF.Free fatty acid (mE/g liver)0.14 ± 0.040.21 ± 0.08⁎P < 0.05 versus PA;0.13 ± 0.03Cholesterol (mmol/g liver)12.75 ± 0.7817.33 ± 0.99⁎P < 0.05 versus PA;14.9 ± 0.46Values are reported as means ± SD.AF, alcohol-fed; ALT, alanine aminotransferase; LGG, L. rhamnosus GG; LPS, lipopolysaccharide; PF, pair-fed; TG, triglyceride. P < 0.05 versus PA; P < 0.05 versus AF. Open table in a new tab Values are reported as means ± SD. AF, alcohol-fed; ALT, alanine aminotransferase; LGG, L. rhamnosus GG; LPS, lipopolysaccharide; PF, pair-fed; TG, triglyceride. Representative photomicrographs depicting liver pathology (H&E staining) are presented in Figure 1. Pair-feeding caused hepatic steatosis, compared with normal chow control, but no inflammation was observed after the 8-week feeding in the PF group. However, alcohol feeding increased hepatic damage, with necroinflammatory foci detectable microscopically. Two weeks of LGG supplementation in AF mice remarkably reduced the number and size of lipid droplets and inflammatory foci in the liver. Consistent with the serum ALT and liver triglyceride levels, LGG supplementation attenuated alcohol-induced liver pathology alterations, which include inflammatory cell infiltration and cell death. We next sought to elucidate possible mechanisms for the observed protection by LGG in intestines and livers of the alcohol-treated mice by evaluating the effects of LGG on intestinal protective gene expression. Intestinal trefoil factor (ITF) and vascular endothelial growth factor (VEGF) play important roles in epithelial protection. Alcohol exposure caused significant reduction in ITF and VEGF protein levels in the ileum, which was normalized with LGG supplementation (Figure 2). Notably, expression of hypoxia-inducible factor 2α (HIF-2α), which is an important transcription factor for ITF and VEGF, was almost completely abrogated by alcohol exposure, but LGG supplementation restored the HIF-2α protein levels. The isoform HIF-1α, however, was not detected. Tight junction proteins play a critical role in gut permeability. Immunofluorescent staining revealed that alcohol exposure caused a reduction in distribution of both ZO-1 and claudin-1 between the adjacent epithelial cells in some parts of ileal epithelium, and LGG treatment normalized tight junction protein distribution (Figure 3A). Western blotting indicated a nonsignificant decrease in occludin protein level after alcohol exposure; LGG treatment attenuated this reduction (Figure 3C). Alcohol exposure decreased but LGG supplementation increased the mRNA levels of ZO-1, claudin-1, and occludin (Figure 3B). To gain additional mechanistic insights, we used Caco-2 cells to evaluate the effect of LGG on intestinal epithelial integrity. Caco-2 cells are colon carcinoma cells that differentiate into intestine-like epithelial cells after 21 days of culture. Alcohol exposure significantly reduced the ITF protein level in Caco-2 cells. Because HIFs are transcription factors of ITF, we examined the protein levels of HIFs. HIF-1α, and HIF-2α are barely detected under normoxic conditions, but alcohol decreased both HIF-1a and HIF-2a protein levels induced by hypoxia (see Supplemental Figure S1 at http://ajp.amjpathol.org). Alcohol exposure also reduced the tight junction proteins claudin-1 and occludin. LGG supernatant (LGG-s) treatment of alcohol-exposed cells increased these protein levels (Figure 4A). Alcohol treatment did not affect the mRNA levels of tight junction proteins, whereas a significant reduction of mRNA level of ITF by alcohol treatment was observed (Figure 4B). LGG-s treatment increased the gene expression of ITF and the tight junction proteins under both control and alcohol-treated conditions (Figure 4B). Immunofluorescent staining showed that alcohol exposure disrupted tight junction protein ZO-1, occludin, and claudin-1 distribution in Caco-2 cells, and LGG-s treatment prevented this effect (Figure 5A). We further analyzed the transcripts of other HIF-targeting genes in response to alcohol and LGG-s treatments. There were no changes in mRNA levels observed in alcohol-treated cells for genes involved in epithelial mucus protection [MUC1, MUC2, and NT5E (alias CD73)], but LGG treatment increased the expression of these genes (Figure 5B).Figure 5Effects of LGG on the protein distribution and mRNA levels of tight junction proteins and mucus protecting proteins in Caco-2 cells treated with 5% alcohol for 24 hours. A: Immunofluorescent staining of ZO-1, occludin, and claudin-1. Magnification: ×400 B: mRNA levels of Muc1, Muc-2, and CD73. *P < 0.05 versus control; **P < 0.05 versus EtOH.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next, we investigated whether the alteration in barrier-protective proteins led to a change in epithelial permeability. Alcohol exposure caused a significant decrease in the epithelial TEER (Figure 6). Consistent with these results, the paracellular permeability to FD-4 was significantly increased by alcohol exposure. LGG-s treatment did not affect epithelial TEER and FD-4 measurements under control conditions, but normalized the alcohol-induced changes in TEER and FD-4. Oxidative stress in the ileum and in Caco-2 cells was assessed by measuring ROS accumulation with ethidium fluorescence microscopy (Figure 7A) and image quantification (Figure 7B). Only trace amounts of ROS were detected in the ileum in the PF mice, but alcohol exposure caused ROS accumulation (as indicated by increased red fluorescence intensity; Figure 7). The alcohol-induced ROS formation was also detected in Caco-2 cells. LGG treatment attenuated the ROS accumulation
