FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2009490402> ?p ?o ?g. }

• W2009490402 endingPage "54" @default.
• W2009490402 startingPage "43" @default.
• W2009490402 abstract "Experimental alcohol-induced liver injury is exacerbated by a high polyunsaturated fat diet rich in linoleic acid. We postulated that bioactive oxidized linoleic acid metabolites (OXLAMs) play a critical role in the development/progression of alcohol-mediated hepatic inflammation and injury. OXLAMs are endogenous ligands for transient receptor potential vanilloid 1 (TRPV1). Herein, we evaluated the role of signaling through TRPV1 in an experimental animal model of alcoholic liver disease (ALD). Chronic binge alcohol administration increased plasma OXLAM levels, specifically 9- and 13-hydroxy-octadecadienoic acids. This effect was associated with up-regulation of hepatic TRPV1. Exposure of hepatocytes to these OXLAMs in vitro resulted in activation of TRPV1 signal transduction with increased intracellular Ca2+ levels. Genetic depletion of TRPV1 did not blunt hepatic steatosis caused by ethanol, but prevented hepatic injury. TRPV1 deficiency protected from hepatocyte death and prevented the increase in proinflammatory cytokine and chemokine expression, including tumor necrosis factor-α, IL-6, macrophage inflammatory protein-2, and monocyte chemotactic protein 1. TRPV1 depletion markedly blunted ethanol-mediated induction of plasminogen activator inhibitor-1, an important alcohol-induced hepatic inflammation mediator, via fibrin accumulation. This study indicates, for the first time, that TRPV1 receptor pathway may be involved in hepatic inflammatory response in an experimental animal model of ALD. TRPV1-OXLAM interactions appear to play a significant role in hepatic inflammation/injury, further supporting an important role for dietary lipids in ALD. Experimental alcohol-induced liver injury is exacerbated by a high polyunsaturated fat diet rich in linoleic acid. We postulated that bioactive oxidized linoleic acid metabolites (OXLAMs) play a critical role in the development/progression of alcohol-mediated hepatic inflammation and injury. OXLAMs are endogenous ligands for transient receptor potential vanilloid 1 (TRPV1). Herein, we evaluated the role of signaling through TRPV1 in an experimental animal model of alcoholic liver disease (ALD). Chronic binge alcohol administration increased plasma OXLAM levels, specifically 9- and 13-hydroxy-octadecadienoic acids. This effect was associated with up-regulation of hepatic TRPV1. Exposure of hepatocytes to these OXLAMs in vitro resulted in activation of TRPV1 signal transduction with increased intracellular Ca2+ levels. Genetic depletion of TRPV1 did not blunt hepatic steatosis caused by ethanol, but prevented hepatic injury. TRPV1 deficiency protected from hepatocyte death and prevented the increase in proinflammatory cytokine and chemokine expression, including tumor necrosis factor-α, IL-6, macrophage inflammatory protein-2, and monocyte chemotactic protein 1. TRPV1 depletion markedly blunted ethanol-mediated induction of plasminogen activator inhibitor-1, an important alcohol-induced hepatic inflammation mediator, via fibrin accumulation. This study indicates, for the first time, that TRPV1 receptor pathway may be involved in hepatic inflammatory response in an experimental animal model of ALD. TRPV1-OXLAM interactions appear to play a significant role in hepatic inflammation/injury, further supporting an important role for dietary lipids in ALD. Alcohol consumption remains one of the most common and important causes of liver disease in the United States and worldwide. Alcoholic liver disease (ALD) ranges from steatosis and steatohepatitis to advanced injury, such as fibrosis, cirrhosis, and hepatocellular carcinoma. It has been estimated that 15% to 30% of heavy drinkers develop advanced ALD.1Kim W.R. Brown Jr., R.S. Terrault N.A. El-Serag H. Burden of liver disease in the United States: summary of a workshop.Hepatology. 2002; 36: 227-242Crossref PubMed Scopus (490) Google Scholar, 2Hassan M.M. Hwang L.Y. Hatten C.J. Swaim M. Li D. Abbruzzese J.L. Beasley P. Patt Y.Z. Risk factors for hepatocellular carcinoma: synergism of alcohol with viral hepatitis and diabetes mellitus.Hepatology. 2002; 36: 1206-1213Crossref PubMed Scopus (631) Google Scholar, 3Kung H.C. Hoyert D.L. Xu J. Murphy S.L. Deaths: final data for 2005.Natl Vital Stat Rep. 2008; 56: 1-120PubMed Google Scholar Despite the significant progress made on ALD pathogenesis, the specific mechanism(s) responsible for ALD development and progression remain poorly understood. Due, in part, to this incomplete understanding of the mechanisms by which alcohol damages the liver, there is still no Food and Drug Administration–approved therapy for this common and often devastating disease. Understanding the molecular mechanisms involved in the pathogenesis of alcohol-induced liver injury may, therefore, lead to the development of new therapeutic options and/or preventive interventions.Dietary fat is an important determinant of ALD development and progression.4Ronis 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 (94) Google Scholar, 5Nanji A.A. Role of different dietary fatty acids in the pathogenesis of experimental alcoholic liver disease.Alcohol. 2004; 34: 21-25Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 6Kirpich 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 (94) Google Scholar, 7Kirpich 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: 257-264Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar Recent publications have shown that experimental and clinical alcohol-induced liver steatosis and injury were associated with elevated oxidized linoleic acid metabolites (OXLAMs), specifically 9- and 13-hydroxy-octadecadienoic acids (9- and 13-HODEs).8Raszeja-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, 9Yang 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 It has been reported that 9- and 13-HODEs are natural endogenous ligands for the transient receptor potential vanilloid 1 (TRPV1).10Patwardhan A.M. Akopian A.N. Ruparel N.B. Diogenes A. Weintraub S.T. Uhlson C. Murphy R.C. Hargreaves K.M. Heat generates oxidized linoleic acid metabolites that activate TRPV1 and produce pain in rodents.J Clin Invest. 2010; 120: 1617-1626Crossref PubMed Scopus (186) Google Scholar, 11Patwardhan 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 (173) Google Scholar The TRPV1 receptor is a ligand-gated nonselective cation channel with high permeability for Ca2+,12Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway.Nature. 1997; 389: 816-824Crossref PubMed Scopus (6938) Google Scholar which is expressed in many cells and tissues, including liver.13Li L. Chen J. Ni Y. Feng X. Zhao Z. Wang P. Sun J. Yu H. Yan Z. Liu D. Nilius B. Zhu Z. TRPV1 activation prevents nonalcoholic fatty liver through UCP2 upregulation in mice.Pflugers Arch. 2012; 463: 727-732Crossref PubMed Scopus (48) Google Scholar, 14Miao X. Liu G. Xu X. Xie C. Sun F. Yang Y. Zhang T. Hua S. Fan W. Li Q. Huang S. Wang Q. Liu G. Zhong D. High expression of vanilloid receptor-1 is associated with better prognosis of patients with hepatocellular carcinoma.Cancer Genet Cytogenet. 2008; 186: 25-32Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 15Rychkov G.Y. Barritt G.J. Expression and function of TRP channels in liver cells.Adv Exp Med Biol. 2011; 704: 667-686Crossref PubMed Scopus (25) Google Scholar, 16Vriens J. Janssens A. Prenen J. Nilius B. Wondergem R. TRPV channels and modulation by hepatocyte growth factor/scatter factor in human hepatoblastoma (HepG2) cells.Cell Calcium. 2004; 36: 19-28Crossref PubMed Scopus (88) Google Scholar, 17Li X.H. McGrath K.C. Tran V.H. Li Y.M. Mandadi S. Duke C.C. Heather A.K. Roufogalis B.D. Identification of a calcium signalling pathway of S-[6]-gingerol in HuH-7 cells.Evid Based Complement Alternat Med. 2013; 2013: 951758PubMed Google Scholar, 18Caterina M.J. Rosen T.A. Tominaga M. Brake A.J. Julius D. A capsaicin-receptor homologue with a high threshold for noxious heat.Nature. 1999; 398: 436-441Crossref PubMed Scopus (1233) Google Scholar, 19Zhang L.L. Yan Liu D. Ma L.Q. Luo Z.D. Cao T.B. Zhong J. Yan Z.C. Wang L.J. Zhao Z.G. Zhu S.J. Schrader M. Thilo F. Zhu Z.M. Tepel M. Activation of transient receptor potential vanilloid type-1 channel prevents adipogenesis and obesity.Circ Res. 2007; 100: 1063-1070Crossref PubMed Scopus (323) Google Scholar, 20Akiba Y. Kato S. Katsube K. Nakamura M. Takeuchi K. Ishii H. Hibi T. Transient receptor potential vanilloid subfamily 1 expressed in pancreatic islet beta cells modulates insulin secretion in rats.Biochem Biophys Res Commun. 2004; 321: 219-225Crossref PubMed Scopus (156) Google Scholar, 21Heiner I. Eisfeld J. Halaszovich C.R. Wehage E. Jungling E. Zitt C. Luckhoff A. Expression profile of the transient receptor potential (TRP) family in neutrophil granulocytes: evidence for currents through long TRP channel 2 induced by ADP-ribose and NAD.Biochem J. 2003; 371: 1045-1053Crossref PubMed Scopus (156) Google Scholar, 22Saunders C.I. Kunde D.A. Crawford A. Geraghty D.P. Expression of transient receptor potential vanilloid 1 (TRPV1) and 2 (TRPV2) in human peripheral blood.Mol Immunol. 2007; 44: 1429-1435Crossref PubMed Scopus (83) Google Scholar The TRPV1 is a polymodal molecular detector of multiple stimuli responding to a large variety of physical (eg, noxious heat), and chemical (eg, H+ ions) stimuli. In addition to HODEs, several exogenous and endogenous TRPV1 agonists have been identified, including capsaicin,12Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway.Nature. 1997; 389: 816-824Crossref PubMed Scopus (6938) Google Scholar cannabinoids,23Smart D. Gunthorpe M.J. Jerman J.C. Nasir S. Gray J. Muir A.I. Chambers J.K. Randall A.D. Davis J.B. The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1).Br J Pharmacol. 2000; 129: 227-230Crossref PubMed Scopus (674) Google Scholar retinoids,24Yin S. Luo J. Qian A. Du J. Yang Q. Zhou S. Yu W. Du G. Clark R.B. Walters E.T. Carlton S.M. Hu H. Retinoids activate the irritant receptor TRPV1 and produce sensory hypersensitivity.J Clin Invest. 2013; 123: 3941-3951Crossref PubMed Scopus (44) Google Scholar and metabolites of arachidonic acid.25Hwang S.W. Cho H. Kwak J. Lee S.Y. Kang C.J. Jung J. Cho S. Min K.H. Suh Y.G. Kim D. Oh U. Direct activation of capsaicin receptors by products of lipoxygenases: endogenous capsaicin-like substances.Proc Natl Acad Sci U S A. 2000; 97: 6155-6160Crossref PubMed Scopus (960) Google ScholarAccumulating evidence suggests an important role of TRPV1 in several diseases and pathological conditions, including chronic pain,26Julius D. TRP channels and pain.Annu Rev Cell Dev Biol. 2013; 29: 355-384Crossref PubMed Scopus (706) Google Scholar neurogenic inflammation,27Richardson J.D. Vasko M.R. Cellular mechanisms of neurogenic inflammation.J Pharmacol Exp Ther. 2002; 302: 839-845Crossref PubMed Scopus (407) Google Scholar diabetes,28Gram D.X. Hansen A.J. Wilken M. Elm T. Svendsen O. Carr R.D. Ahren B. Brand C.L. Plasma calcitonin gene-related peptide is increased prior to obesity, and sensory nerve desensitization by capsaicin improves oral glucose tolerance in obese Zucker rats.Eur J Endocrinol. 2005; 153: 963-969Crossref PubMed Scopus (75) Google Scholar, 29Gram D.X. Ahren B. Nagy I. Olsen U.B. Brand C.L. Sundler F. Tabanera R. Svendsen O. Carr R.D. Santha P. Wierup N. Hansen A.J. Capsaicin-sensitive sensory fibers in the islets of Langerhans contribute to defective insulin secretion in Zucker diabetic rat, an animal model for some aspects of human type 2 diabetes.Eur J Neurosci. 2007; 25: 213-223Crossref PubMed Scopus (121) Google Scholar metabolic syndrome and obesity,19Zhang L.L. Yan Liu D. Ma L.Q. Luo Z.D. Cao T.B. Zhong J. Yan Z.C. Wang L.J. Zhao Z.G. Zhu S.J. Schrader M. Thilo F. Zhu Z.M. Tepel M. Activation of transient receptor potential vanilloid type-1 channel prevents adipogenesis and obesity.Circ Res. 2007; 100: 1063-1070Crossref PubMed Scopus (323) Google Scholar, 30Marshall N.J. Liang L. Bodkin J. Dessapt-Baradez C. Nandi M. Collot-Teixeira S. Smillie S.J. Lalgi K. Fernandes E.S. Gnudi L. Brain S.D. A role for TRPV1 in influencing the onset of cardiovascular disease in obesity.Hypertension. 2013; 61: 246-252Crossref PubMed Scopus (73) Google Scholar and liver diseases.13Li L. Chen J. Ni Y. Feng X. Zhao Z. Wang P. Sun J. Yu H. Yan Z. Liu D. Nilius B. Zhu Z. TRPV1 activation prevents nonalcoholic fatty liver through UCP2 upregulation in mice.Pflugers Arch. 2012; 463: 727-732Crossref PubMed Scopus (48) Google Scholar, 31Li Q. Li L. Wang F. Chen J. Zhao Y. Wang P. Nilius B. Liu D. Zhu Z. Dietary capsaicin prevents nonalcoholic fatty liver disease through transient receptor potential vanilloid 1-mediated peroxisome proliferator-activated receptor delta activation.Pflugers Arch. 2013; 465: 1303-1316Crossref PubMed Scopus (53) Google Scholar, 32Avraham Y. Zolotarev O. Grigoriadis N.C. Poutahidis T. Magen I. Vorobiav L. Zimmer A. Ilan Y. Mechoulam R. Berry E.M. Cannabinoids and capsaicin improve liver function following thioacetamide-induced acute injury in mice.Am J Gastroenterol. 2008; 103: 3047-3056Crossref PubMed Scopus (33) Google Scholar To the best of our knowledge, there are no data assessing the role of TRPV1 in ALD. The present study evaluates the role of TRPV1 in the development of ethanol-induced liver steatosis, inflammation, and injury using an experimental animal model of ALD. Our findings collectively indicate that the genetic deficiency of TRPV1 protects against alcohol-induced liver inflammation and injury but not steatosis. Our data point toward a role for TRPV1-OXLAM receptor-ligand interactions as a potentially relevant pathway contributing to alcohol-mediated steatohepatitis.Materials and MethodsAnimal Model of ALDAnimals were housed in a pathogen-free barrier facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, and the study protocol was approved by the University of Louisville (Louisville, KY) Institutional Animal Care and Use Committee. Eight-week-old male TRPV1 knockout mice (B6.129X1-TRPV1tm1Jul/J, 11th backcross generation) and their genetically unaltered wild-type (WT; C57Bl6/J) counterparts were obtained from the Jackson Laboratory (Bar Harbor, ME). Animals were fed Lieber-DeCarli control (isocaloric maltose-dextrin) or ethanol (5% w/v) liquid diets ad libitum for 10 days plus a single binge ethanol administration (5 g/kg, body weight, 20% ethanol) by gavage, whereas mice in control groups were gavaged with isocaloric dextrin maltose.33Bertola 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 (561) Google Scholar Both diets were prepared fresh daily. In the control group diet, the levels of protein, carbohydrate, and fat were held constant at 17%, 43%, and 40% of total energy, respectively. In the alcohol diet, ethanol (35% of total calories) was substituted for carbohydrate energy. The diet was enriched in corn oil containing a high amount of polyunsaturated linoleic fatty acid, and purchased from Research Diet (New Brunswick, NJ). At the conclusion of the experiment, the mice were anesthetized; and blood and tissue samples were obtained. Plasma was stored at −80°C. Portions of liver tissue were frozen immediately in liquid nitrogen, whereas others were fixed in 10% neutral-buffered formalin or embedded in frozen specimen medium (Tissue-Tek OCT compound; Sakura Finetek, Torrance, CA).Blood and Liver Biochemical AnalysisPlasma alanine transaminase (ALT) and aspartate transaminase (AST) activity, cholesterol, triglycerides (TGs), glucose, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very LDL (VLDL) were determined by Lipid Panel Plus using the Piccolo Xpress chemistry analyzer (Abaxis, Union City, CA). Blood alcohol levels were measured using nicotinamide adenine dinucleotide-alcohol dehydrogenase (NAD-ADH) Reagent Multiple Test (Sigma, St. Louis, MO), according to the manufacturer's instructions. Plasma endotoxin levels were measured with the Limulus Amoebocyte Lysate kit (Lonza, Walkersville, MD). For the determination of hepatic lipid levels, hepatic lipids were extracted with an aqueous extract from chloroform and methanol. Hepatic TGs were measured, as previously described,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: 38-45Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar using TG reagent (Thermo Fisher Scientific Inc., Middletown, VA). Liver cholesterol was assayed using reagents from Sigma.Liver Histological Examination and StainingFor histological analysis, liver sections were fixed in 10% buffered formalin and embedded in paraffin. Tissue sections (5 μm thick) were prepared and stained with hematoxylin and eosin. Oil-Red-O staining was performed to evaluate hepatic fat accumulation. Apoptotic cells were identified by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay using the ApopTag Peroxidase In Situ Apoptosis Detection kit (Millipore, Billerica, MA), according to the manufacturer's instructions. Neutrophil accumulation in the livers was assessed by chloroacetate esterase (CAE) staining using a commercially available kit (Sigma), according to the manufacturer's instructions. Immunofluorescence detection of hepatic fibrin deposition was performed in frozen tissue, as previously described.35Beier J.I. Guo L. von Montfort C. Kaiser J.P. Joshi-Barve S. Arteel G.E. New role of resistin in lipopolysaccharide-induced liver damage in mice.J Pharmacol Exp Ther. 2008; 325: 801-808Crossref PubMed Scopus (39) Google ScholarHepatic Caspase-3 Activity AssessmentCaspase-3 activity was determined using 200 μg whole liver protein with the caspase-3 colorimetric kit (Abcam, Cambridge, MA), according to the manufacturer's instructions.RNA Isolation and Real-Time RT-PCR AssayTotal liver RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. Reverse transcription was performed with qScript cDNA Supermix (Quanta Biosciences, Gaithersburg, MD) and quantitative real-time RT-PCR (RT-qPCR) with Perfecta SYBR Green FastMix (Quanta Biosciences) using an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA). The reverse and forward specific primers are presented in Table 1. Primers were designed using Primer3 software version 4.0.0 (http://bioinfo.ut.ee/primer3-0.4.0/primer3).36Untergrasser A. Cutcutache I. Koressaar T. Ye J. Faircloth B.C. Remm M. Rozen S.G. Primer3–new capabilities and interfaces.Nucleic Acids Research. 2012; 40: e115Crossref PubMed Scopus (5571) Google Scholar All primer pairs were validated by demonstrating high-amplification efficiency, consistent single-peak dissociation patterns, and the presence of single products of the expected size on agarose gels. The relative gene expression was normalized with 18s rRNA as the internal control, and calculated using the 2−ΔΔCT method.Table 1Primer Sequences for the Targeted Mouse Gene RT-qPCR AssayPrimer setForward sequenceReverse sequence18s5′-CTCAACACGGGAAACCTCAC-3′5′-CGCTCCACCAACTAAGAACG-3′TRPV15′-TGGACAGCTACAGTGAGATACTTTTC-3′5′-CCATGGAAGCCACATACTCC-3′IL-65′-TGGAAATGAGAAAAGAGTTGTGC-3′5′-CCAGTTTGGTAGCATCCATCA-3′TNF-α5′-GTGATCGGTCCCCAAAGG-3′5′-GGTGGTTTGCTACGACGTG-3′MIP-25′-GCGCCCAGACAGAAGTCATA-3′5′-TCCAGGTCAGTTAGCCTTGC-3′MCP-15′-GGCTCAGCCAGATGCAGT-3′5′-TGAGCTTGGTGACAAAAACTACAG-3′PAI-15′-TCAATGACTGGGTGGAAAGG-3′5′-AGGCGTGTCAGCTCGTCTAC-3′IL-1b5′-TTCATCTTTGAAGAAGAGCCCAT-3′5′-TCGGAGCCTGTAGTGCAGTT-3′IL-1a5′-CAAGCAACGGGAAGATTCTG-3′5′-CTGATCTGGGTTGGATGGTC-3′LCN25′-ATGTCACCTCCATCCTGGTC-3′5′-ACCTGAGGATACCTGTGCAT-3′ Open table in a new tab Western Blot AnalysisWestern blot analysis was performed to evaluate the phospho–extracellular signal-regulated kinase (ERK) 1/2 (p42/44) mitogen-activated protein kinase (MAPK) and nuclear phospho–NF-κB p65 protein levels using commercially available primary antibody from Cell Signaling (Danvers, MA). Equal amounts of proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. Immunoreactive signals were visualized using enhanced chemiluminescence light detection reagents (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Band intensities were quantified using ImageJ software version 1.49j (NIH, Bethesda, MD). The protein content was normalized to total NF-κB p65 (for pNF-κB p65) and β-actin [for pERK 1/2 (p42/44) MAPK]. The results were expressed as the ratio of protein of interest/NF-κB p65 or β-actin.Plasma OXLAM MeasurementLipid extraction from plasma and quantification of 9- and 13-HODEs were performed as previously described.37Feldstein 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 (203) Google Scholar, 38Zein 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 (125) Google Scholar Briefly, plasma samples with antioxidant solution, internal standards [synthetic 9(s)-HODE-d4; 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. Quantification of oxidized fatty acids was done on a triple quadrupole mass spectrometer (model API 365; Applied Biosystems, Foster City, CA) with Ionics EP 10þ upgrade (Ionics Mass Spectrometry, Concord, ON, Canada) using stable isotope dilution methods and multiple reaction monitoring with characteristic parent-to-daughter ion transitions.Cell Culture and TreatmentsHepG2, a human hepatoma cell line obtained from ATCC (Manassas, VA), was used for in vitro experiments. Cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37°C in a humidified 5% CO2, 95% air atmosphere. Cells were plated in 96-well plates at the density of 25,000 cells per well. Cells were treated with 9-HODE (10 μmol/L), 13-HODE (10 μmol/L), and capsaicin (10 μmol/L) for 24 hours. The HODE concentration was chosen on the basis of the observation that serum OXLAM levels were up to 1000 nmol/L in our experimental model of ALD, and the knowledge that HODEs can be found in human blood in the low μmol/L range.39Willker W. Leibfritz D. Lipid oxidation in blood plasma of patients with neurological disorders.Brain Res Bull. 2000; 53: 437-443Crossref PubMed Scopus (7) Google Scholar HODEs were purchased from Cayman Chemical Company (Ann Arbor, MI), capsaicin from Sigma, and the dyes for Cellomics assays from Invitrogen. By using the cell viability MTT assay, we have confirmed that concentrations of up to 25 μmol/L of HODEs cause minimal cell death in HepG2 cells (data not shown). After treatment, cells were incubated for 1 hour in growth media containing the following dyes: i) Hoechst (for nuclear fluorescence), ii) Fluo-4 (for free calcium), and iii) TOTO-3 (for cell membrane permeability). Cellomics analysis was performed using a Thermo Scientific Array Scan VTI HCS Reader (Thermo Fisher Scientific Inc., Waltham, MA), as described by the manufacturer. Cellomics Array Scan 60 software version 7.6.2.1-1.00x (Thermo Fisher Scientific Inc.) was used to determine fluorescence intensities of the dyes. Well averages, as well as individual cell data, were recorded and analyzed.Statistical AnalysisThe data were expressed as means ± SEM. A Student's t-test (two tailed) was performed to evaluate significant differences between alcohol- and pair-fed animals. Two-way analysis of variance, followed by the Tukey's multiple-comparison test, was used to evaluate significant differences between experimental groups (WT–pair fed, WT-ethanol, TRPV1−/−–pair fed, and TRPV1−/−-ethanol). P < 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism version 5.01 for Windows (GraphPad Software, Inc., La Jolla, CA).ResultsChronic Binge Ethanol Administration Increases Circulating OXLAM Levels and Induces Hepatic TRPV1 ExpressionRecent clinical and experimental studies have demonstrated that alcohol-induced liver inflammation and injury are associated with elevated levels of bioactive OXLAMs.8Raszeja-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, 9Yang 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 Indeed, chronic binge ethanol administration significantly elevated plasma OXLAM levels, specifically 9- and 13-HODEs, compared with their pair-fed controls (Figure 1A). A similar trend was found for 9- and 13-oxo-octadecadenoic acids (oxoODEs) (data not shown). OXLAMs, specifically 9- and 13-HODEs, have been reported as endogenous activators/agonists of TRPV1,10Patwardhan A.M. Akopian A.N. Ruparel N.B. Diogenes A. Weintraub S.T. Uhlson C. Murphy R.C. Hargreaves K.M. Heat generates oxidized linoleic acid metabolites that activate TRPV1 and produce pain in rodents.J Clin Invest. 2010; 120: 1617-1626Crossref PubMed Scopus (186) Google Scholar, 11Patwardhan 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 (173) Google Scholar a ligand-gated nonselective cation channel with high permeability for Ca2+.12Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway.Nature. 1997; 389: 816-824Crossref PubMed Scopus (6938) Google Scholar On the basis of these observations, we examined TRPV1 expression in the livers of control pair- and ethanol-fed animals. We observed that ethanol exposure increased hepatic TRPV1 mRNA expression in parallel with the increase in circulating OXLAMs in ethanol but not in control pair-fed animals (Figure 1B). Next, we performed in vitro studies using HepG2 cells as a prototype for liver hepatocytes to determine whether OXLAMs were able to activate TRPV1 and thereby increase intracellular Ca2+. We found that both 9- and 13-HODE exposure increased intracellular Ca2+ levels (Figure 1, C and D), analogous to capsaicin, a classic TRPV1 agonist (Figure 1E).Metabolic Characteristics of TRPV1−/− Mice in Response to Chronic Binge Ethanol FeedingTo determine whether increased expression of TRPV1 plays a role in alcohol-induced liver injury, and to examine the potential role of OXLAM/TRPV1 interactions, we next evaluated the effects of TRPV1 deletion in an experimental animal model of ALD. Both WT and TRPV1−/− animals tolerated the experimental protocol, and no mortality was observed. The effects of genotype and ethanol on body weight and clinical chemistry variables are presented in Table 2. Food consumption was similar in WT and TRPV1−/− mice fed an alcohol-containing diet, and there were no significant differences in body weight between the experimental groups. Ethanol exposure significantly increased liver/body weight ratios, which were not affected by mouse strain. At the end of ethanol feeding, 9 hours after a single ethanol gavage, elevated blood alcohol levels were observed in WT compared with" @default.
• W2009490402 created "2016-06-24" @default.
• W2009490402 creator A5012100988 @default.
• W2009490402 creator A5040494327 @default.
• W2009490402 creator A5049669101 @default.
• W2009490402 creator A5053295611 @default.
• W2009490402 creator A5054025613 @default.
• W2009490402 creator A5081543826 @default.
• W2009490402 creator A5083426937 @default.
• W2009490402 date "2015-01-01" @default.
• W2009490402 modified "2023-10-15" @default.
• W2009490402 title "Transient Receptor Potential Vanilloid 1 Gene Deficiency Ameliorates Hepatic Injury in a Mouse Model of Chronic Binge Alcohol-Induced Alcoholic Liver Disease" @default.
• W2009490402 cites W1495409864 @default.
• W2009490402 cites W1511198581 @default.
• W2009490402 cites W178476257 @default.
• W2009490402 cites W1956927897 @default.
• W2009490402 cites W1965189515 @default.
• W2009490402 cites W1968708605 @default.
• W2009490402 cites W1973196413 @default.
• W2009490402 cites W1975752601 @default.
• W2009490402 cites W1980483650 @default.
• W2009490402 cites W1987329700 @default.
• W2009490402 cites W1990247687 @default.
• W2009490402 cites W2002873638 @default.
• W2009490402 cites W2007094035 @default.
• W2009490402 cites W2007123357 @default.
• W2009490402 cites W2012621699 @default.
• W2009490402 cites W2016034906 @default.
• W2009490402 cites W2021812983 @default.
• W2009490402 cites W2022819833 @default.
• W2009490402 cites W2022862946 @default.
• W2009490402 cites W2024933805 @default.
• W2009490402 cites W2028023737 @default.
• W2009490402 cites W2030781339 @default.
• W2009490402 cites W2032106211 @default.
• W2009490402 cites W2037600884 @default.
• W2009490402 cites W2043839229 @default.
• W2009490402 cites W2045737598 @default.
• W2009490402 cites W2049265775 @default.
• W2009490402 cites W2057681671 @default.
• W2009490402 cites W2061721602 @default.
• W2009490402 cites W2062266980 @default.
• W2009490402 cites W2066400512 @default.
• W2009490402 cites W2070745672 @default.
• W2009490402 cites W2075306179 @default.
• W2009490402 cites W2075865355 @default.
• W2009490402 cites W2076295447 @default.
• W2009490402 cites W2078504972 @default.
• W2009490402 cites W2084496974 @default.
• W2009490402 cites W2090859289 @default.
• W2009490402 cites W2091115040 @default.
• W2009490402 cites W2094100492 @default.
• W2009490402 cites W2098539191 @default.
• W2009490402 cites W2098620454 @default.
• W2009490402 cites W2098712238 @default.
• W2009490402 cites W2101682092 @default.
• W2009490402 cites W2102179496 @default.
• W2009490402 cites W2102266054 @default.
• W2009490402 cites W2105746545 @default.
• W2009490402 cites W2111187697 @default.
• W2009490402 cites W2113981125 @default.
• W2009490402 cites W2122778205 @default.
• W2009490402 cites W2124141753 @default.
• W2009490402 cites W2128865950 @default.
• W2009490402 cites W2130243588 @default.
• W2009490402 cites W2140880966 @default.
• W2009490402 cites W2146125855 @default.
• W2009490402 cites W2149839520 @default.
• W2009490402 cites W2152170298 @default.
• W2009490402 cites W2154881563 @default.
• W2009490402 cites W2158585862 @default.
• W2009490402 cites W2161526491 @default.
• W2009490402 cites W2162098634 @default.
• W2009490402 cites W2167191939 @default.
• W2009490402 cites W2167875991 @default.
• W2009490402 cites W2171717653 @default.
• W2009490402 cites W2236615393 @default.
• W2009490402 cites W2276006697 @default.
• W2009490402 cites W2301131921 @default.
• W2009490402 cites W2326523156 @default.
• W2009490402 cites W2395286598 @default.
• W2009490402 cites W4233770333 @default.
• W2009490402 cites W4254723952 @default.
• W2009490402 doi "https://doi.org/10.1016/j.ajpath.2014.09.007" @default.
• W2009490402 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4278357" @default.
• W2009490402 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/25447051" @default.
• W2009490402 hasPublicationYear "2015" @default.
• W2009490402 type Work @default.
• W2009490402 sameAs 2009490402 @default.
• W2009490402 citedByCount "22" @default.
• W2009490402 countsByYear W20094904022016 @default.
• W2009490402 countsByYear W20094904022017 @default.
• W2009490402 countsByYear W20094904022018 @default.
• W2009490402 countsByYear W20094904022019 @default.
• W2009490402 countsByYear W20094904022021 @default.
• W2009490402 countsByYear W20094904022022 @default.
• W2009490402 countsByYear W20094904022023 @default.
• W2009490402 crossrefType "journal-article" @default.

• next