Trypsin-mediated sensitization to ferroptosis increases the severity of pancreatitis in mice.

Background & aims Pancreatitis is characterized by acinar cell death and persistent inflammation. Ferroptosis is a type of lipid peroxidation-dependent necrosis, which is negatively regulated by GPX4. We studied how trypsin, a serine protease secreted by pancreatic acinar cells, affects the contribution of ferroptosis to triggering pancreatitis. Methods In vitro, the mouse pancreatic acinar cell line 266-6 and mouse primary pancreatic acinar cells (mPACs) were used to investigate the effect of exogenous trypsin on ferroptosis sensitivity. Short hairpin RNAs were designed to silence gene expression, whereas a library of 1080 approved drugs was used to identify new ferroptosis inhibitors in 266-6 cells. In vivo, a Cre/LoxP system was utilized to generate mice with a pancreas-specific knockout of Gpx4 (Pdx1-Cre;Gpx4flox/flox mice). Acute or chronic pancreatitis was induced in these mice (Gpx4flox/flox mice served as controls) by cerulein injections or a Lieber-DeCarli alcoholic liquid diet. Pancreatic tissues, acinar cells, and serum were collected and analyzed by histology, immunoblot, qPCR, ELISA, or immunohistochemical analyses. Results Supraphysiological doses of trypsin (500 or 1000 ng/ml) alone did not trigger significant cell death in 266-6 cells and mPACs, but did increase the sensitivity of these cells to ferroptosis upon treatment with cerulein, L-arginine, alcohol, erastin, or RSL3. PSMD4-dependent lipid peroxidation caused ferroptosis in pancreatic acinar cells by promoting the proteasomal degradation of GPX4. The drug screening campaign identified the antipsychotic drug olanzapine as an antioxidant inhibiting ferroptosis in pancreatic acinar cells. Mice lacking pancreatic Gpx4 developed more severe pancreatitis after cerulein infection or ethanol feeding than control mice. Conversely, olanzapine administration protected against pancreatic ferroptotic damage and experimental pancreatitis in Gpx4-deficient mice. Conclusions Trypsin-mediated sensititization to ferroptotic damage increases the severity of pancreatitis in mice, and this process can be reversed by olanzapine.