Title

Mechanisms of Lipocalin-2 (Lcn2) in Mediating Progression of Acetaminophen-Induced Acute Liver Failure

Date of Award

6-2018

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Vishhakha Bhave, BS, PhD

Second Advisor

Francis Jenney, PhD

Third Advisor

Vicky Mody, PhD

Fourth Advisor

Rangaiah Shashidharamurthy, PhD

Fifth Advisor

Richard White, PhD

Abstract

Acetaminophen (APAP) overdose is the most common cause of Acute Liver Failure (ALF) in United States. Toxicity is initiated by the conversion of APAP to its reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) by CYP2El/CYP3A4. · NAPQI binds covalently to mitochondrial proteins forming APAP-cys. This generates initial hepatotoxicity in the form of centrilobular necrosis and sterile inflammation. We have demonstrated that Lipocalin-2 (Lcn2) is pro-inflammatory and elevated during APAP overdose in a mouse model. Lcn2 knockout (Lcn2KO) mice exhibited protection from APAP overdose while wild-type (WT) littermates suffered significantly higher liver injury and inhibited liver regeneration response. Mechanisms underlying this phenomenon are not fully understood. Necrotic hepatocytes release Damage Associated Molecular Pattern molecules (DAMPS), like High Mobility Group Box 1 (HMGB1), that further exacerbate the inflammatory process. The roles of Lcn2 and HMGB1 in APAP overdose have not been investigated. The present study investigates the molecular mechanism of Lcn2 during the progression of acute liver injury in APAP overdose. We hypothesize that during APAP induced ALF, Lcn2 induces the expression and translocation of HMGB1 which in turn mediates inflammation and progression of injury. An in vitro model of HepG2 cells as well as an in vivo model of WT and Lcn2KO mice were utilized. HepG2 cells were treated with recombinant Lcn2 to investigate whether exogenous Lcn2 can cause induction and translocation of HMGB1 using immunofluorescence. HepG2 cells were also treated with recombinant HMGB1 to investigate if HM GB I can affect the expression of Lcn2. Tissue samples of WT and Lcn2KO mice subjected to APAP overdose were assessed for expression ofHMGBl. APAP-cys formation was measured in WT and Lcn2KO using a Chromatography-Mass Spectrometry (LC-MS) method. Lastly, histopathology was performed to assess liver samples from both. Current results have not shown that Lcn2 is able to induce translocation of HMGB1. However, results have indicated that liver injury was more prominent in WT mice than Lcn2KO mice. Findings also demonstrate that both WT and Lcn2KO mice undergo similar bioactivation-based initial injury as assessed by APAP-cys formation. Taken together, Lcn2 mediates progression of acute liver injury. Further studies are required to investigate the mechanism of its involvement.

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