Reoxygenation speed and its implication for cellular injury responses in hypoxic RAW 264.7 cells

Ischemia/reperfusion injury is characterized by excess generation of reactive oxygen species (ROS). The purpose of this study is to test the effect of reoxygenation speed on ROS production and the cellular injury responses in hypoxic macrophages RAW 264.7 cells and its potential mechanisms for the generation of ROS.

After hypoxic exposure of RAW 264.7 cells for 20 h, reoxygenation was performed for 6 h by stepwise increase in oxygen concentration (0.8% increase of oxygen every 15 min) in the slow reoxygenation (SRox) group or by moving the culture flasks quickly to a normoxic incubator in the rapid reoxygenation (RRox) group. To identify the potential effect of reoxygenation speed on the generation of ROS, the cells were pretreated with apocynin, VAS2870, and MitoTEMPO before the induction of hypoxia.

SRox significantly decreased cell death and cytotoxicity compared with RRox (P < 0.05). RRox resulted in significantly more generation of ROS, interleukin-1β, interleukin-6, and nitric oxide than SRox (P < 0.05). SRox also increased the expression of prosurvival proteins and decreased apoptosis. In cells pretreated with VAS2870 or MitoTEMPO, the reduced ROS generation by SRox was maintained. However, pretreatment with apocynin abolished the effect of reoxygenation speed on ROS generation.

SRox compared with RRox decreased cellular injury in hypoxic RAW 264.7 cells by decreasing ROS and inflammatory cytokine production and decreasing apoptosis.