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• W2754166039 abstract "Alcoholic liver disease is a major human health problem leading to significant morbidity and mortality in the United States and worldwide. Dietary fat plays an important role in alcoholic liver disease pathogenesis. Herein, we tested the hypothesis that a combination of ethanol and a diet rich in linoleic acid (LA) leads to the increased production of oxidized LA metabolites (OXLAMs), specifically 9- and 13-hydroxyoctadecadienoic acids (HODEs), which contribute to a hepatic proinflammatory response exacerbating liver injury. Mice were fed unsaturated (with a high LA content) or saturated fat diets (USF and SF, respectively) with or without ethanol for 10 days, followed by a single binge of ethanol. Compared to SF+ethanol, mice fed USF+ethanol had elevated plasma alanine transaminase levels, enhanced hepatic steatosis, oxidative stress, and inflammation. Plasma and liver levels of 9- and 13-HODEs were increased in response to USF+ethanol feeding. We demonstrated that primarily 9-HODE, but not 13-HODE, induced the expression of several proinflammatory cytokines in vitro in RAW264.7 macrophages. Finally, deficiency of arachidonate 15-lipoxygenase, a major enzyme involved in LA oxidation and OXLAM production, attenuated liver injury and inflammation caused by USF+ethanol feeding but had no effect on hepatic steatosis. This study demonstrates that OXLAM-mediated induction of a proinflammatory response in macrophages is one of the potential mechanisms underlying the progression from alcohol-induced steatosis to alcoholic steatohepatitis. Alcoholic liver disease is a major human health problem leading to significant morbidity and mortality in the United States and worldwide. Dietary fat plays an important role in alcoholic liver disease pathogenesis. Herein, we tested the hypothesis that a combination of ethanol and a diet rich in linoleic acid (LA) leads to the increased production of oxidized LA metabolites (OXLAMs), specifically 9- and 13-hydroxyoctadecadienoic acids (HODEs), which contribute to a hepatic proinflammatory response exacerbating liver injury. Mice were fed unsaturated (with a high LA content) or saturated fat diets (USF and SF, respectively) with or without ethanol for 10 days, followed by a single binge of ethanol. Compared to SF+ethanol, mice fed USF+ethanol had elevated plasma alanine transaminase levels, enhanced hepatic steatosis, oxidative stress, and inflammation. Plasma and liver levels of 9- and 13-HODEs were increased in response to USF+ethanol feeding. We demonstrated that primarily 9-HODE, but not 13-HODE, induced the expression of several proinflammatory cytokines in vitro in RAW264.7 macrophages. Finally, deficiency of arachidonate 15-lipoxygenase, a major enzyme involved in LA oxidation and OXLAM production, attenuated liver injury and inflammation caused by USF+ethanol feeding but had no effect on hepatic steatosis. This study demonstrates that OXLAM-mediated induction of a proinflammatory response in macrophages is one of the potential mechanisms underlying the progression from alcohol-induced steatosis to alcoholic steatohepatitis. Alcohol-associated health problems, including alcoholic liver disease (ALD), are major global health problems. ALD progresses through the course of several pathologies, including steatosis, alcoholic hepatitis, cirrhosis, and potentially hepatocellular carcinoma. Alcoholic hepatitis occurs in approximately 10% to 35% of chronic heavy drinkers, and severe alcoholic hepatitis accounts for significant morbidity and mortality, approaching 35% to 45%.1McClain C.M. Carithers R.L. Alcoholic liver disease.in: Feldman M. Friedman L. Brandt L. Sleisenger and Fordtran's Gastrointestinal and Liver Disease Pathophysiology/Diagnosis/Management. ed 8. Saunders Elsevier, Philadelphia, PA2006: 1771-1792Google Scholar Approximately 10% to 20% of heavy drinkers will eventually develop cirrhosis.2D'Amico G. Garcia-Tsao G. Pagliaro L. Natural history and prognostic indicators of survival in cirrhosis: a systematic review of 118 studies.J Hepatol. 2006; 44: 217-231Abstract Full Text Full Text PDF PubMed Scopus (1954) Google Scholar, 3Wakim-Fleming J. Mullen K.D. Long-term management of alcoholic liver disease.Clin Liver Dis. 2005; 9: 135-149Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar The specific mechanisms responsible for ALD development and progression are not fully understood, and there is no Federal Drug Administration–approved therapy for any stage of ALD. The link between diet, specifically dietary fat, and alcohol consumption in ALD pathogenesis has been increasingly appreciated. Findings from an epidemiological study that analyzed dietary fat intake in individuals from countries with similar per capita alcohol consumption revealed that intake of saturated fat (SF) was associated with lower mortality rates, whereas dietary intake of unsaturated fat (USF) was associated with a higher mortality from alcoholic cirrhosis.4Nanji A.A. French S.W. Dietary factors and alcoholic cirrhosis.Alcohol Clin Exp Res. 1986; 10: 271-273Crossref PubMed Scopus (79) Google Scholar The beneficial effects of dietary SF (predominantly rich in medium- or long-chain saturated fatty acids) and the damaging effects of dietary USF [primarily enriched in linoleic acid (LA), an ω-6 polyunsaturated fatty acid (PUFA)] on alcohol-induced liver injury have been demonstrated in numerous preclinical studies using rodent models of ALD.5Nanji A.A. Yang E.K. Fogt F. Sadrzadeh S.M. Dannenberg A.J. Medium chain triglycerides and vitamin E reduce the severity of established experimental alcoholic liver disease.J Pharmacol Exp Ther. 1996; 277: 1694-1700PubMed Google Scholar, 6Ronis M.J. Korourian S. Zipperman M. Hakkak R. Badger T.M. Dietary saturated fat reduces alcoholic hepatotoxicity in rats by altering fatty acid metabolism and membrane composition.J Nutr. 2004; 134: 904-912Crossref PubMed Scopus (98) Google Scholar, 7Kirpich I.A. Feng W. Wang Y. Liu Y. Barker D.F. Barve S.S. McClain C.J. The type of dietary fat modulates intestinal tight junction integrity, gut permeability, and hepatic toll-like receptor expression in a mouse model of alcoholic liver disease.Alcohol Clin Exp Res. 2012; 36: 835-846Crossref PubMed Scopus (98) Google Scholar, 8Chen P. Torralba M. Tan J. Embree M. Zengler K. Starkel P. van Pijkeren J.P. DePew J. Loomba R. Ho S.B. Bajaj J.S. Mutlu E.A. Keshavarzian A. Tsukamoto H. Nelson K.E. Fouts D.E. Schnabl B. Supplementation of saturated long-chain fatty acids maintains intestinal eubiosis and reduces ethanol-induced liver injury in mice.Gastroenterology. 2015; 148: 203-214.e16Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 9Zhong W. Li Q. Xie G. Sun X. Tan X. Sun X. Jia W. Zhou Z. Dietary fat sources differentially modulate intestinal barrier and hepatic inflammation in alcohol-induced liver injury in rats.Am J Physiol Gastrointest Liver Physiol. 2013; 305: G919-G932Crossref PubMed Scopus (41) Google Scholar LA is required for the development of experimental ALD, with the severity of liver pathology correlating to the amount of LA in the diet.10Nanji A.A. French S.W. Dietary linoleic acid is required for development of experimentally induced alcoholic liver injury.Life Sci. 1989; 44: 223-227Crossref PubMed Scopus (138) Google Scholar On a mechanistic level, the deleterious effects of USF in comparison to the protective effects of SF are thought to result from alterations in hepatic lipid homeostasis,6Ronis M.J. Korourian S. Zipperman M. Hakkak R. Badger T.M. Dietary saturated fat reduces alcoholic hepatotoxicity in rats by altering fatty acid metabolism and membrane composition.J Nutr. 2004; 134: 904-912Crossref PubMed Scopus (98) Google Scholar, 11Li Q. Zhong W. Qiu Y. Kang X. Sun X. Tan X. Zhao Y. Sun X. Jia W. Zhou Z. Preservation of hepatocyte nuclear factor-4alpha contributes to the beneficial effect of dietary medium chain triglyceride on alcohol-induced hepatic lipid dyshomeostasis in rats.Alcohol Clin Exp Res. 2013; 37: 587-598Crossref PubMed Scopus (26) Google Scholar, 12You M. Fischer M. Deeg M.A. Crabb D.W. Ethanol induces fatty acid synthesis pathways by activation of sterol regulatory element-binding protein (SREBP).J Biol Chem. 2002; 277: 29342-29347Crossref PubMed Scopus (423) Google Scholar, 13You M. Rogers C.Q. Adiponectin: a key adipokine in alcoholic fatty liver.Exp Biol Med (Maywood). 2009; 234: 850-859Crossref PubMed Scopus (92) Google Scholar, 14Nanji A.A. Role of different dietary fatty acids in the pathogenesis of experimental alcoholic liver disease.Alcohol. 2004; 34: 21-25Crossref PubMed Scopus (72) Google Scholar induction of hepatic lipid peroxidation and oxidative stress,6Ronis M.J. Korourian S. Zipperman M. Hakkak R. Badger T.M. Dietary saturated fat reduces alcoholic hepatotoxicity in rats by altering fatty acid metabolism and membrane composition.J Nutr. 2004; 134: 904-912Crossref PubMed Scopus (98) Google Scholar, 15Nanji A.A. Griniuviene B. Sadrzadeh S.M. Levitsky S. McCully J.D. Effect of type of dietary fat and ethanol on antioxidant enzyme mRNA induction in rat liver.J Lipid Res. 1995; 36: 736-744PubMed Google Scholar changes in the gut microbiota, impaired intestinal barrier integrity, and elevated endotoxemia with subsequent hepatic macrophage activation and increased production of hepatic proinflammatory cytokines.7Kirpich I.A. Feng W. Wang Y. Liu Y. Barker D.F. Barve S.S. McClain C.J. The type of dietary fat modulates intestinal tight junction integrity, gut permeability, and hepatic toll-like receptor expression in a mouse model of alcoholic liver disease.Alcohol Clin Exp Res. 2012; 36: 835-846Crossref PubMed Scopus (98) Google Scholar, 8Chen P. Torralba M. Tan J. Embree M. Zengler K. Starkel P. van Pijkeren J.P. DePew J. Loomba R. Ho S.B. Bajaj J.S. Mutlu E.A. Keshavarzian A. Tsukamoto H. Nelson K.E. Fouts D.E. Schnabl B. Supplementation of saturated long-chain fatty acids maintains intestinal eubiosis and reduces ethanol-induced liver injury in mice.Gastroenterology. 2015; 148: 203-214.e16Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 16Kirpich I.A. Petrosino J. Ajami N. Feng W. Wang Y. Liu Y. Beier J.I. Barve S.S. Yin X. Wei X. Zhang X. McClain C.J. Saturated and unsaturated dietary fats differentially modulate ethanol-induced changes in gut microbiome and metabolome in a mouse model of alcoholic liver disease.Am J Pathol. 2016; 186: 765-776Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 17Mutlu E. Keshavarzian A. Engen P. Forsyth C.B. Sikaroodi M. Gillevet P. Intestinal dysbiosis: a possible mechanism of alcohol-induced endotoxemia and alcoholic steatohepatitis in rats.Alcohol Clin Exp Res. 2009; 33: 1836-1846Crossref PubMed Scopus (257) Google Scholar LA is the most abundant ω-6 PUFA in human diets and in human plasma and membrane lipids.18Obinata H. Hattori T. Nakane S. Tatei K. Izumi T. Identification of 9-hydroxyoctadecadienoic acid and other oxidized free fatty acids as ligands of the G protein-coupled receptor G2A.J Biol Chem. 2005; 280: 40676-40683Crossref PubMed Scopus (129) Google Scholar The consumption of LA, which has dramatically increased during the past several decades,19Blasbalg T.L. Hibbeln J.R. Ramsden C.E. Majchrzak S.F. Rawlings R.R. Changes in consumption of omega-3 and omega-6 fatty acids in the United States during the 20th century.Am J Clin Nutr. 2011; 93: 950-962Crossref PubMed Scopus (597) Google Scholar is positively correlated with increasing prevalence of several pathological conditions, including obesity.20Alvheim A.R. Malde M.K. Osei-Hyiaman D. Lin Y.H. Pawlosky R.J. Madsen L. Kristiansen K. Froyland L. Hibbeln J.R. Dietary linoleic acid elevates endogenous 2-AG and anandamide and induces obesity.Obesity (Silver Spring). 2012; 20: 1984-1994Crossref PubMed Scopus (180) Google Scholar LA can be enzymatically converted to bioactive oxidized LA metabolites (OXLAMs), primarily via the actions of cellular lipoxygenases [arachidonate 15-lipoxygenase (ALOX15) and ALOX15B in humans, and ALOX15 (12/15-LO) in rodents] or nonenzymatically via free radical–mediated oxidation in response to oxidative stress. OXLAMs are involved in various intracellular signaling pathways and may induce a proinflammatory response in different cell types.21Hatley M.E. Srinivasan S. Reilly K.B. Bolick D.T. Hedrick C.C. Increased production of 12/15 lipoxygenase eicosanoids accelerates monocyte/endothelial interactions in diabetic db/db mice.J Biol Chem. 2003; 278: 25369-25375Crossref PubMed Scopus (98) Google Scholar, 22Hattori T. Obinata H. Ogawa A. Kishi M. Tatei K. Ishikawa O. Izumi T. G2A plays proinflammatory roles in human keratinocytes under oxidative stress as a receptor for 9-hydroxyoctadecadienoic acid.J Invest Dermatol. 2008; 128: 1123-1133Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 23Mabalirajan U. Rehman R. Ahmad T. Kumar S. Singh S. Leishangthem G.D. Aich J. Kumar M. Khanna K. Singh V.P. Dinda A.K. Biswal S. Agrawal A. Ghosh B. Linoleic acid metabolite drives severe asthma by causing airway epithelial injury.Sci Rep. 2013; 3: 1349Crossref PubMed Scopus (75) Google Scholar OXLAMs play a role in the development of different pathological conditions, such as inflammatory hyperalgesia and the metabolic syndrome.24Vangaveti V.N. Jansen H. Kennedy R.L. Malabu U.H. Hydroxyoctadecadienoic acids: oxidised derivatives of linoleic acid and their role in inflammation associated with metabolic syndrome and cancer.Eur J Pharmacol. 2016; 785: 70-76Crossref PubMed Scopus (67) Google Scholar, 25Patwardhan A.M. Scotland P.E. Akopian A.N. Hargreaves K.M. Activation of TRPV1 in the spinal cord by oxidized linoleic acid metabolites contributes to inflammatory hyperalgesia.Proc Natl Acad Sci U S A. 2009; 106: 18820-18824Crossref PubMed Scopus (180) Google Scholar OXLAMs, specifically 9- and 13-hydroxyoctadecadienoic acids (9- and 13-HODEs, respectively), were elevated in patients with nonalcoholic fatty liver disease,26Feldstein A.E. Lopez R. Tamimi T.A. Yerian L. Chung Y.M. Berk M. Zhang R. McIntyre T.M. Hazen S.L. Mass spectrometric profiling of oxidized lipid products in human nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.J Lipid Res. 2010; 51: 3046-3054Crossref PubMed Scopus (209) Google Scholar and a decrease in plasma OXLAM levels was correlated with hepatic histological improvement in these patients.27Zein C.O. Yerian L.M. Gogate P. Lopez R. Kirwan J.P. Feldstein A.E. McCullough A.J. Pentoxifylline improves nonalcoholic steatohepatitis: a randomized placebo-controlled trial.Hepatology. 2011; 54: 1610-1619Crossref PubMed Scopus (271) Google Scholar Increased levels of 9- and 13-HODEs were found in patients with alcoholic liver cirrhosis in parallel with the induction of hepatic ALOX15 and ALOX15B.28Raszeja-Wyszomirska J. Safranow K. Milkiewicz M. Milkiewicz P. Szynkowska A. Stachowska E. Lipidic last breath of life in patients with alcoholic liver disease.Prostaglandins Other Lipid Mediat. 2012; 99: 51-56Crossref PubMed Scopus (28) Google Scholar Elevated 9- and 13-HODE levels were also observed in experimental rodent models of ALD and were associated with ethanol-induced liver injury, steatosis, and inflammation.29Yang L. Latchoumycandane C. McMullen M.R. Pratt B.T. Zhang R. Papouchado B.G. Nagy L.E. Feldstein A.E. McIntyre T.M. Chronic alcohol exposure increases circulating bioactive oxidized phospholipids.J Biol Chem. 2010; 285: 22211-22220Crossref PubMed Scopus (46) Google Scholar, 30Liu H. Beier J.I. Arteel G.E. Ramsden C.E. Feldstein A.E. McClain C.J. Kirpich I.A. Transient receptor potential vanilloid 1 gene deficiency ameliorates hepatic injury in a mouse model of chronic binge alcohol-induced alcoholic liver disease.Am J Pathol. 2015; 185: 43-54Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar Based on these observations, we hypothesized that OXLAMs may play a pathogenic role in ALD development and/or progression; however, the underlying mechanisms remain to be determined. In the present study, we tested the hypothesis that the combination of ethanol and a diet with a high content of LA contributes to and exacerbates ethanol-induced liver injury through increased OXLAM production and OXLAM-mediated induction of hepatic proinflammatory responses. We postulated that both increased substrate availability (LA-rich dietary fat) and activation of metabolic pathways of OXLAM production, in particular the 12/15-LO–mediated pathway, contribute to an ethanol-mediated increase in OXLAMs. Therefore, we further examined whether genetic ablation of Alox15 (the gene encoding 12/15-LO) would decrease OXLAM levels and attenuate liver injury caused by ethanol and an LA-enriched diet. For our studies, we used a chronic-binge ethanol exposure animal model of ALD (NIH National Institute on Alcohol Abuse and Alcoholism model).31Bertola A. Mathews S. Ki S.H. Wang H. Gao B. Mouse model of chronic and binge ethanol feeding (the NIAAA model).Nat Protoc. 2013; 8: 627-637Crossref PubMed Scopus (600) Google Scholar This model recapitulates the drinking pattern in humans and induces a moderately severe alcoholic liver injury with increased hepatic neutrophil infiltration,31Bertola A. Mathews S. Ki S.H. Wang H. Gao B. Mouse model of chronic and binge ethanol feeding (the NIAAA model).Nat Protoc. 2013; 8: 627-637Crossref PubMed Scopus (600) Google Scholar which is often observed in alcoholic hepatitis patients.32Takahashi T. Kamimura T. Ichida F. Ultrastructural findings on polymorphonuclear leucocyte infiltration and acute hepatocellular damage in alcoholic hepatitis.Liver. 1987; 7: 347-358Crossref PubMed Scopus (36) Google Scholar Eight-week-old genetically unaltered wild-type (WT; C57BL/6J) and Alox15 gene knockout (B6.129S2-Alox15tm11Fun/J, 11th backcross generation) male mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Animals were housed in a specific pathogen-free barrier facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. A 10-day ethanol feeding plus a single binge of ethanol administration was used as an experimental model of ALD (NIH National Institute on Alcohol Abuse and Alcoholism model).31Bertola A. Mathews S. Ki S.H. Wang H. Gao B. Mouse model of chronic and binge ethanol feeding (the NIAAA model).Nat Protoc. 2013; 8: 627-637Crossref PubMed Scopus (600) Google Scholar Briefly, the mice were provided free access to a Lieber-DeCarli control or ethanol (5% w/v)-containing diet for 10 days. On day 11, the mice were gavaged with a single dose of ethanol solution (20% v/v) prepared in ultrapure water; the gavage volume was calculated to deliver 5 g ethanol/kg body weight. The mice in control groups received an isocaloric/isovolumetric maltodextrin solution. The mice were euthanized 9 hours after the gavage. The experimental paradigm is outlined in Figure 1A. Two separate experiments were performed: WT mice were provided a USF- or SF-enriched diet with or without ethanol; and WT and Alox15 knockout mice were fed a USF-enriched diet with or without ethanol. The USF diet was supplemented with corn oil, a rich source of LA, and the SF diet was supplemented with beef tallow and medium-chain triglycerides (Research Diets, New Brunswick, NJ) (Table 1). The detailed composition of these diets has been described previously.33Kirpich I.A. Feng W. Wang Y. Liu Y. Beier J.I. Arteel G.E. Falkner K.C. Barve S.S. McClain C.J. Ethanol and dietary unsaturated fat (corn oil/linoleic acid enriched) cause intestinal inflammation and impaired intestinal barrier defense in mice chronically fed alcohol.Alcohol. 2013; 47: 257-264Crossref PubMed Scopus (50) Google Scholar In the control group, the levels of protein, carbohydrate, and fat were held constant at 17%, 43%, and 40% of total energy, respectively. In the alcohol-containing diets, ethanol (35% of total calories) was substituted for carbohydrate energy. The diets were prepared fresh each day, and the food consumption was monitored daily. The control groups (the pair-fed mice) received the same amount of isocaloric food (maltodextrin-containing diets) that ethanol-fed animals consumed in the previous day (pair-feeding protocol). At the conclusion of the experiment, the mice were euthanized and portions of liver tissue were snap frozen in liquid nitrogen or were fixed in 10% neutral-buffered formalin. Blood was collected from the inferior vena cava via venipuncture using heparinized syringes. Plasma was obtained, aliquoted, and stored at −80°C for further analysis. The study protocol was approved by the University of Louisville Institutional Animal Care and Use Committee (Louisville, KY).Table 1Nutritional Composition of the SF and USF Experimental DietsIngredientsSF dietUSF dietProtein, kcal%1717Carbohydrate, kcal%4343Fat, kcal%4040 Saturated FA, %84.113.2 Monounsaturated FA, %9.325.0 Polyunsaturated FA, %6.661.8FA, fatty acid; SF, saturated fat; USF, unsaturated fat. Open table in a new tab FA, fatty acid; SF, saturated fat; USF, unsaturated fat. Plasma alanine transaminase (ALT; a marker of liver damage) activities were measured using the Piccolo Xpress chemistry analyzer (Abaxis, Union City, CA). Hepatic lipids were extracted with chloroform and methanol, and triglycerides were measured using Triglycerides Reagent (Thermo Fisher Scientific Inc., Middletown, VA).34Kirpich I.A. Gobejishvili L.N. Bon Homme M. Waigel S. Cave M. Arteel G. Barve S.S. McClain C.J. Deaciuc I.V. Integrated hepatic transcriptome and proteome analysis of mice with high-fat diet-induced nonalcoholic fatty liver disease.J Nutr Biochem. 2011; 22: 38-45Crossref PubMed Scopus (129) Google Scholar Liver thiobarbituric acid reactive substances (TBARSs) were measured in whole-liver homogenate using a commercially available kit (Cayman Chemical, Ann Arbor, MI). Plasma lipopolysaccharide (LPS) levels were measured with the Limulus Amoebocyte Lysate kit (Lonza, Walkersville, MD). Soluble CD14 (R&D Systems, Minneapolis, MN) and lipopolysaccharide binding protein (Hycult Biotech, Plymouth Meeting, PA) were measured by enzyme-linked immunosorbent assays, according to the manufacturers' instructions. Mouse liver tissues were collected, fixed in 10% neutral-buffered formalin solution, and embedded in paraffin. Hematoxylin and eosin staining was performed for liver tissue histological evaluation. Oil-Red-O staining on liver tissue cryostat sections was performed to evaluate hepatic fat accumulation. Apoptotic cells were identified by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay using the ApopTag Peroxidase In Situ Apoptosis Detection kit (Millipore, Billerica, MA). Hepatic neutrophil accumulation was assessed by chloroacetate esterase staining using a commercially available kit (Sigma, St. Louis, MO). Hepatic 4-hydroxy-2-nonenal levels were detected by immunohistochemistry using antibodies from Alpha Diagnostic International (San Antonio, TX). Total RNA was purified using Trizol reagent (ThermoFisher, Waltham, MA), as described by the manufacturer. To ensure there was no genomic DNA carryover, samples were digested with DNase I (ThermoFisher). cDNA was synthesized using qScript cDNA Supermix (Quanta Biosciences, Beverly, MA). The cDNA equivalent of 10 ng RNA was used in each real-time quantitative PCR (with the exception of 18S ribosomal RNA, where 0.1 ng was used). RT-PCR assays were performed with PerfeCTa SYBR Green Fast Mix (Quanta Biosciences) on the Applied Biosystems 7900HT platform (Foster City, CA) using primers listed in Table 2. All data were normalized to the expression of the 18S ribosomal RNA gene, and the data were calculated as described previously.35Livak 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 2Murine Primer Sequences for RT-PCRGeneForward primerReverse primer18s5′-CTCAACACGGGAAACCTCAC-3′5′-CGCTCCACCAACTAAGAACG-3′Tnfa5′-GTGATCGGTCCCCAAAGG-3′5′-GGTGGTTTGCTACGACGTG-3′Mip2a5′-GCGCCCAGACAGAAGTCATA-3′5′-TCCAGGTCAGTTAGCCTTGC-3′Mcp15′-GGCTCAGCCAGATGCAGT-3′5′-TGAGCTTGGTGACAAAAACTACAG-3′Il1b5′-TTCATCTTTGAAGAAGAGCCCAT-3′5′-TCGGAGCCTGTAGTGCAGTT-3′Nos25′-GAGACAGGGAAGTCTGAAGCAC-3′5′-CCAGCAGTAGTTGCTCCTCTTC-3′Arg-15′-CATTGGCTTGCGAGACGTAGAC-3′5′-GCTGAAGGTCTCTTCCATCACC-3′Tgfb15′-TGATACGCCTGAGTGGCTGTCT-3′5′-CACAAGAGCAGTGAGCGCTGAA-3′ Open table in a new tab Western blotting was performed to evaluate the levels of cytochrome p450 2E1 (CYP2E1) using a commercially available antibody (Abcam, Cambridge, MA). Liver tissue lysates were separated by SDS-PAGE and subsequently transferred to a polyvinylidene difluoride membrane. Blots were visualized using Amersham enhanced chemiluminescence Western blot detection (GE Health Care, Hercules, CA), and quantified using ImageJ software version 1.50i (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology, Dallas, TX) was used as a loading control. Lipid extraction from liver tissue and quantification of lipid metabolite levels were performed, as previously described,36Maddipati K.R. Zhou S.L. Stability and analysis of eicosanoids and docosanoids in tissue culture media.Prostaglandins Other Lipid Mediat. 2011; 94: 59-72Crossref PubMed Scopus (42) Google Scholar, 37Maddipati K.R. Romero R. Chaiworapongsa T. Zhou S.L. Xu Z. Tarca A.L. Kusanovic J.P. Munoz H. Honn K.V. Eicosanomic profiling reveals dominance of the epoxygenase pathway in human amniotic fluid at term in spontaneous labor.FASEB J. 2014; 28: 4835-4846Crossref PubMed Scopus (66) Google Scholar, 38Maddipati K.R. Romero R. Chaiworapongsa T. Chaemsaithong P. Zhou S.-L. Xu Z. Tarca A.L. Kusanovic J.P. Gomez R. Chaiyasit N. Honn K.V. Lipidomic analysis of patients with microbial invasion of the amniotic cavity reveals up-regulation of leukotriene B4.FASEB J. 2016; 30: 3296-3307Crossref PubMed Scopus (36) Google Scholar, 39Maddipati K.R. Romero R. Chaiworapongsa T. Chaemsaithong P. Zhou S.-L. Xu Z. Tarca A.L. Kusanovic J.P. Gomez R. Docheva N. Honn K.V. Clinical chorioamnionitis at term: the amniotic fluid fatty acyl lipidome.J Lipid Res. 2016; 57: 1906-1916Crossref PubMed Scopus (33) Google Scholar by the Wayne State University Lipidomic Core Facility (Detroit, MI). Briefly, tissue samples were homogenized in phosphate buffer, pH 7.2. Samples were spiked with internal standards and extracted for PUFA metabolites on C18 extraction columns, as described earlier.36Maddipati K.R. Zhou S.L. Stability and analysis of eicosanoids and docosanoids in tissue culture media.Prostaglandins Other Lipid Mediat. 2011; 94: 59-72Crossref PubMed Scopus (42) Google Scholar, 37Maddipati K.R. Romero R. Chaiworapongsa T. Zhou S.L. Xu Z. Tarca A.L. Kusanovic J.P. Munoz H. Honn K.V. Eicosanomic profiling reveals dominance of the epoxygenase pathway in human amniotic fluid at term in spontaneous labor.FASEB J. 2014; 28: 4835-4846Crossref PubMed Scopus (66) Google Scholar, 38Maddipati K.R. Romero R. Chaiworapongsa T. Chaemsaithong P. Zhou S.-L. Xu Z. Tarca A.L. Kusanovic J.P. Gomez R. Chaiyasit N. Honn K.V. Lipidomic analysis of patients with microbial invasion of the amniotic cavity reveals up-regulation of leukotriene B4.FASEB J. 2016; 30: 3296-3307Crossref PubMed Scopus (36) Google Scholar, 39Maddipati K.R. Romero R. Chaiworapongsa T. Chaemsaithong P. Zhou S.-L. Xu Z. Tarca A.L. Kusanovic J.P. Gomez R. Docheva N. Honn K.V. Clinical chorioamnionitis at term: the amniotic fluid fatty acyl lipidome.J Lipid Res. 2016; 57: 1906-1916Crossref PubMed Scopus (33) Google Scholar High-performance liquid chromatography was performed on a Prominence XR system (Shimadzu Corp., Kyoto, Japan) using a Luna C18 (3 μm, 2.1 × 150 mm) column. Mass spectra for each detected lipid mediator were recorded using the Enhanced Product Ion feature to verify the identity of the detected peak in addition to multiple reaction monitoring transition and retention time match with the standard. The data were collected using Analyst 1.6.2 software (SCIEX, Framingham, MA), and the multiple reaction monitoring transition chromatograms were quantitated by MultiQuant software version 3.0.2 (SCIEX). The internal standard signals in each chromatogram were used for normalization for recovery as well as relative quantitation of each analyte. Lipid extraction from plasma and quantification of 9- and 13-HODE and LA levels were performed as previously described.26Feldstein A.E. Lopez R. Tamimi T.A. Yerian L. Chung Y.M. Berk M. Zhang R. McIntyre T.M. Hazen S.L. Mass spectrometric profiling of oxidized lipid products in human nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.J Lipid Res. 2010; 51: 3046-3054Crossref PubMed Scopus (209) Google Scholar, 40Zein C.O. Lopez R. Fu X. Kirwan J.P. Yerian L.M. McCullough A.J. Hazen S.L. Feldstein A.E. Pentoxifylline decreases oxidized lipid products in nonalcoholic steatohepatitis: new evidence on the potential therapeutic mechanism.Hepatology. 2012; 56: 1291-1299Crossref PubMed Scopus (128) Google Scholar Briefly, plasma samples with antioxidant solution, internal standards [synthetic 9(S)-HODE-d4 or 13(S)-HODE-d4], and potassium hydroxide were added to glass test tubes and overlaid with argon. After hydrolysis under argon atmosphere, the released fatty acids were extracted twice into the hexane layer by liquid/liquid extraction. The combined hexane extracts were dried under nitrogen gas and resuspended in 85% methanol/water. Reconstituted lipid extracts were analyzed by high-performance liquid chromatography. Oxidized fatty acids were quantified on a triple quadrupole mass spectrometer (API 365; Applied Biosystems," @default.
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• W2754166039 date "2017-10-01" @default.
• W2754166039 modified "2023-10-18" @default.
• W2754166039 title "Dietary Linoleic Acid and Its Oxidized Metabolites Exacerbate Liver Injury Caused by Ethanol via Induction of Hepatic Proinflammatory Response in Mice" @default.
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• W2754166039 doi "https://doi.org/10.1016/j.ajpath.2017.06.008" @default.
• W2754166039 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5808136" @default.
• W2754166039 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/28923202" @default.
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