ELUCIDATING THE ROLE OF HET0016 IN AMELIORATING 20-HETE INDUCED OXIDATIVE STRESS AND LIVER INJURY IN B-THALASSEMIA: A NOVEL THERAPEUTIC TARGET

Background: Reactive oxygen species (ROS) are one of the main contributors to pathological outcomes in β-thalassemia, with their generation being a consequence of iron overload and abnormal red blood cell metabolism. We have previously shown that CYP450 mediates ROS production in the liver of a mouse model of β-thalassemia through an increase in 20-hydroxyeicosatetraenoic acid (20-HETE) activity. Aim: The aim of this study is to assess the efficacy of N-Hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (HET0016), an inhibitor of 20-HETE formation, in reducing 20-HETE-mediated oxidative stress and injury in the livers of mice affected by β-thalassemia. Methods: Hbbth3/+ mice (The Jackson Laboratory) were used as a model of non-transfusion dependent thalassemia (NTDT) and were divided into 3 groups: a control group (N=11) treated intraperitoneally (IP) with a 1:1 solution of dimethyl sulfoxide (DMSO) and phosphate-buffered saline (PBS), Hbbth3/+ mice treated with a 1:1 solution of DMSO and PBS (N=14), and Hbbth3/+ mice treated with HET0016 (N=7). HET0016 was administered in the form of a daily IP injection at a dose of 5mg/kg/day for a total duration of four weeks. Enzymatic activity of NADPH oxidases was assessed using the NADPH oxidase assay. mRNA expression levels of two isoforms of CYP4A, as well as CYP4A protein levels, were measured in the liver using Real-time Polymerase Chain Reaction (PCR) and western blot, respectively. Degree of expression of CYP4A was also evaluated in the liver by immunohistochemistry (IHC). Formalin-fixed paraffin-embedded liver tissues were also sectioned and stained with Pearl’s Prussian blue stain to detect ferric iron (Fe3+). Sirius red stain and measurement of hepatocyte growth factor (HGF) mRNA expression levels were performed to detect signs of liver injury. To assess the degree of lipid peroxidation, an IHC staining for 4-hydroxynonenal (4-HNE) in addition to a malondialdehyde (MDA) colorimetric assay was performed. IHC staining for Caspase-3 was conducted to assess apoptosis. Finally, the effects of 20-HETE inhibition on the ferroptosis pathway were examined by measuring the mRNA and protein expression levels of glutathione peroxidase 4 (GPX4) in addition to measuring total glutathione (GSH) levels. Results: In Hbbth3/+ mice, there was an increase in intracellular ROS levels and NADPH oxidase enzymatic activity. At both mRNA level and protein level, in addition to IHC staining, a significant increase was observed in the expression of CYP4A in Hbbth3/+ mice compared to control. This significantly decreased after HET0016 administration. Moreover, Hbbth3/+ mice showed an increase in the lipid peroxidation markers MDA and 4-HNE, which decreased after HET0016 administration. HET0016 also ameliorated liver injury in Hbbth3/+ mice treated with HET0016 as shown by a significant decrease in HGF mRNA expression levels and collagen fiber content. Increased tissue iron levels were detected in the liver of Hbbth3/+ mice compared to controls and this decreased with HET0016 administration. Caspase 3 levels also decreased following treatment with HET0016. Finally, HET0016 treated Hbbth3/+ mice showed a significant increase in GPX4 at both mRNA and protein level in addition to increased GSH levels, when compared to their non-treated counterparts. Conclusion: Our preclinical study is the first to assess the efficacy of HET0016 in reducing 20-HETE-mediated oxidative stress and injury in the livers of mice affected by β-thalassemia. These findings lay the groundwork for future and further investigations on the role of 20-HETE induced oxidative stress in β-thalassemia and may pave the way for a new novel therapeutic target in the field.