Effects of Dihydroxylated Fatty Acid Mediators on Ethanol‐Induced Liver Injury in a Mouse Model of Alcoholic Liver Disease

Abstract

BackgroundAlcoholic liver disease (ALD) is the most common chronic liver disease and a major contributor to liver disease burden worldwide, and a disease for which there is no FDA‐approved treatment. The mechanisms of ALD pathogenesis are not completely understood. Data from our group indicate that dihydroxylated fatty acids (dihydroxy‐FAs) are increased in the livers of human alcoholic hepatitis patients. Dihydroxy‐FA lipid mediators are produced via cytochrome P450/epoxide hydrolase‐mediated metabolism of polyunsaturated fatty acids and are generally pro‐inflammatory. Despite their increase in human ALD, the role of dihydroxy‐FAs in ALD has not been explored.HypothesisExogenous administration of dihydroxy‐FAs will exacerbate liver injury in a mouse model of ALD.MethodsMale C57BL/6 mice were provided ethanol (EtOH) using a chronic‐binge Lieber DeCarli ad libitum EtOH feeding model. Mice were administered either 8,9‐DiHETE (arachidonic acid metabolite), a combination of 9,10‐DiHOME and 12,13‐DiHOME (linoleic acid metabolites), or a vehicle control by i.p. injection once daily for two days prior to sacrifice. Liver injury, inflammation, and steatosis were measured. To further elucidate the effects of dihydroxy‐FAs on liver cells, primary mouse hepatocytes or HepG2 cells were treated with 8,9‐DiHETE, 9,10‐DiHOME, 12,13‐DiHOME, or a vehicle control. Effects on baseline or palmitic acid (PA)‐induced cell death were measured by MTT assay and apoptotic cell death was identified by Annexin‐V staining (cellomics).ResultsIn vivo, 8,9‐DiHETE treatment modestly while 9,10/12,13‐DiHOME significantly increased liver injury as determined by elevated plasma ALT levels (1.3‐fold and 2.2‐fold p=0.04, respectively). While 8,9‐DiHETE had no effect on steatosis, 9,10/12,13‐DiHOME decreased liver triglyceride/free fatty acid levels compared to vehicle control. Dihydroxy‐FA treatment decreased the hepatic expression of several pro‐inflammatory chemokines at the mRNA level, including Mip2a and Mcp1. In vitro, 8,9‐DiHETE treatment of HepG2 cells or primary mouse hepatocytes significantly exacerbated PA‐induced cell death as determined by MTT assay, whereas 9,10‐DiHOME, and 12,13‐DiHOME had no effect on PA‐induced or baseline cell death or apoptosis.ConclusionsDifferent dihydroxy‐FAs have diverse effects in vivo, in a mouse model of ALD, and in vitro. In vivo, dihydroxy‐FAs exacerbated liver injury and was associated with down‐regulated several pro‐inflammatory cytokines. In vitro, 8,9‐DiHETE increased hepatocyte cell death, while 9,10/12,13‐DiHOME showed no effects. Future studies are warranted to dissect the effects of individual dihydroxy‐FAs in ALD.Support or Funding InformationThis work was funded by grants from the National Institutes of Health and the Department of Veterans Affairs.