Date on Master's Thesis/Doctoral Dissertation

5-2023

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, PhD

Committee Chair

Kirpich, Irina

Committee Co-Chair (if applicable)

Hong, Kyung

Committee Member

Hong, Kyung

Committee Member

Clark, Barbara

Committee Member

Palmer, Kenneth

Committee Member

Watson, Walter

Author's Keywords

Alcohol-associated liver disease; soluble epoxide hydrolase; TUCB; pharmacology; transcriptomics; proteomics

Abstract

Introduction Alcohol-associated liver disease (ALD) is a common chronic liver disease and healthcare burden, and a spectrum of pathologies ranging from steatosis to steatohepatitis, fibrosis, and alcohol-associated hepatitis (AH). Despite its prevalence, there are limited effective therapeutic options for the treatment of ALD/AH. Previous evidence suggested that inhibition of soluble epoxide hydrolase (sEH), a lipid metabolism enzyme which degrades beneficial metabolites of dietary polyunsaturated fatty acids, may be a beneficial strategy. The central hypothesis of the current dissertation is that sEH inhibition will ameliorate alcohol-associated liver injury and inflammation in mouse models of ALD. Methods To determine the efficacy of sEH inhibition in ALD and uncover mechanisms, we leveraged multiple mouse models including the acute-on-chronic “NIAAA” model and the 8-week chronic feeding model. In each, mice were administered either an ethanol-containing liquid diet or isocaloric control diet. Mice were further randomized to receive either an sEH inhibitor (t-TUCB or liver-targeting t-TUCB liposomes, 3 mg/kg-day) or a vehicle control orally for the duration of the study. Liver injury, inflammation, and cell death were assessed. Liver epoxy- and dihydroxy-fatty acids were measured, and individual lipids were explored in vitro. Hepatic mRNA expression (global and spatial), miRNA expression, epitranscriptomic RNA modifications, and proteins/phosphopeptides were measured by sequencing and metabolomics. Results Pharmacological sEH inhibition altered the abundance of epoxy- and dihydroxy-fatty acids and ameliorated ethanol-associated liver injury, inflammation, and cell death. In vitro, 17,18-EpETE and 19,20-EpDPA decreased hepatocyte cell death and macrophage cytokine production. Multi-omics revealed numerous alterations associated with sEH inhibition including acute phase response, interleukin signaling, and phagocytosis. mRNA-miRNA networks were identified based on differentially expressed miRNAs such as miR-34, among others, and epitranscriptomic marks such as m6A were identified as altered with t-TUCB. Proteomics and phosphoproteomics revealed functional-level changes in proteins related to metabolism and peroxisomal function which may be influenced by differential phosphorylation. Conclusions Our data supported the hypothesis that sEH inhibition will ameliorate alcohol-induced liver injury and inflammation in mice. Future work will continue to explore sEH inhibition-associated signaling pathways in vivo and in vitro in support of the clinical translation of this therapy for individuals with ALD/AH.

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