Zileuton protects pancreatic stellate cells from oxidative stress and ferroptosis by modulating the Nrf2 pathway and mitochondrial activity.

Abstract

Lipoxygenases exhibit a leading role in cancer development and growth. Additionally, pancreatic stellate cells (PSCs) are pivotal in the formation of fibrotic tissue in pancreatic tumors. In this work, we have studied the effects of the 5-lipoxygenase (5-LOX) inhibitor, zileuton, on rat PSCs. Cell viability was analyzed by employing crystal violet. Reactive oxygen species (ROS) generation, reduced (GSH) and oxidized (GSSG) levels of glutathione and protein and lipid oxidation were analyzed by fluorescence and colorimetric methods respectively. The phosphorylation of nuclear factor erythroid 2-related factor (Nrf2) and the expression of superoxide dismutase (SOD) was studied by western blotting. The effect of zileuton on erastin-induced ferroptosis was assessed by colorimetric methods. Mitochondrial metabolic activity was studied employing Alamar Blue^®. Mitochondrial membrane potential was monitored by fluorescence techniques. The results show that the 5-LOX inhibitor protected PSCs against oxidation evoked by hydrogen peroxide, which induced an increase in CM-H₂DCFDA-derived fluorescence, a decrease in the reduced/oxidized glutathione ratio and increases in the detection of lipid and protein oxidation. In the presence of zileuton, increases in the phosphorylation of nuclear factor erythroid 2-related factor and in the expression of the antioxidant enzymes catalase, glutamate-cysteine ligase,,heme oxygenase-1, NAD(P)H quinone oxidoreductase 1, and superoxide dismutases 1 and 2 were observed. Additionally, zileuton diminished erastin-evoked ferroptosis. Finally, a drop in the reduction of resazurin by mitochondria and a slowing of the Krebs cycle flow rate were observed, whereas no detectable changes in mitochondrial membrane potential were noted in the presence of zileuton. We conclude that the inhibition of 5-LOX by zileuton places cells in a state of antioxidant readiness coupled with a protected metabolic deceleration.