Pd146176

A clinically relevant acute-on-chronic alcohol exposure model was used to test the therapeutic potential of ALOX15 inhibitor, PD146176. Ten-week-old male C57BL/6 J mice were pair-fed with the Lieber-DeCarli alcohol (4% ethanol, w/v) or control diet for 2 weeks with one ethanol oral gavage at 4 g/kg body weight each week (n = 8). PD146176 (Tocris, Avonmouth, Bristol, UK), a specific ALOX15 inhibitor, was dissolved in DMSO, diluted with PBS, and given to mice at a dose of 10 mg/kg body weight (in a final volume of 300 µl) once a day for the second week via intraperitoneal injection. Control mice were given same amount of DMSO.

To prevent 4HNE-mediated proteolysis of proAKAP4 and AKAP4 in male germ cells and mature spermatozoa, the cells were treated with several broad-spectrum pharmacological suppressors of proteolytic activity (Complete Mini Protease Inhibitor Cocktail; Roche), or with a selective inhibitor of proteasomal activity (MG132). Alternatively, cells were treated with a selective inhibitor of arachidonate 15-lipoxygenase (PD146176; Tocris Bioscience, Bristol, United Kingdom), an enzyme involved in the propagation of oxidative stress via the metabolism of polyunsaturated fatty acids to 4HNE (Walters et al., 2018b (link)). The concentrations of each inhibitor (Complete Mini Protease Inhibitor Cocktail at 1× working concentration; MG132, 10–25 μM; PD146176, 1 μM), were selected based on published IC₅₀ values and from our previous use of these reagents to effectively block the loss of other 4HNE targeted proteins from the germ cell proteome (Bromfield et al., 2017b (link); Walters et al., 2018a (link)). Germ cells and mature spermatozoa were pre-treated with each inhibitor for 15 min prior to exposure to 4HNE (protease inhibitor cocktail and MG132) or H₂O₂ (in the case of PD146176) and the inhibitor was retained for the duration of treatment thereafter to ensure adequate inhibition of each target. A DMSO vehicle control (1 μM) was also included in these experiments.