The study on the main mode of the death of red blood cells in grass carp (Ctenopharyngodon idella)

"Eryptosis" of mature mammalian red blood cells (RBCs) is widely acknowledged. As compared to mammalians, the teleost RBCs possess a nucleus and other cellular organelles, but the mode of death of teleost RBCs remains unclear. We isolated the RBCs from circulating blood of grass carp (Ctenopharyngodon idella) and cultured in saline solution (to simulate the cells lacking of nutrients) and normal L15 medium (to simulate the cells in circulation), respectively, to investigate the changes in cell morphology during death, which showed the death of RBCs were accompanied the presence of autolysosome analogues. Flow cytometry analysis revealed that the prolonged exposure time in saline solution or L15 medium increased the accumulation of reactive oxygen species and nitric oxide in the cells, promoted cell death. To further explore which death model occurred in the RBCs, four death inhibitors were used, which showed the ferroptosis inhibitor, Fer-1, to play a key role in rescuing the viability of RBCs, indicated ferroptosis to be the main player in the death of RBCs. The results of quantitative real-time PCR demonstrated that the exposure time modulated the expression of iron metabolism-related genes in RBCs. In addition, prolonged exposure of RBCs was accompanied by an increase in lipid peroxidation and decreased GSH-PX activity. Taken together, the above data suggest that ferroptosis may be the main mode of death of RBCs in grass carp, shedding new light on the biological characteristics of teleost RBCs.