3.5 Knockdown Nrf2 abolishes the effect of leonurine on ROS generation, lipid peroxidation and ferroptosis
Considering the role of Nrf2 in preventing lipid peroxidation and ferroptosis, we attempted to investigate the potential for Nrf2 to mediate the protective effect of leonurine on ferroptosis. Control or Nrf2 siRNA was transiently transfected into cells, and Nrf2 protein expression was shown to be significantly inhibited in the Nrf2 siRNA group (Fig. 5A-B). As shown in Fig. 5C, the protective effect of leonurine on RSL3-induced cell death was alleviated in Nrf2 knockdown cells. Furthermore, the protective effects of leonurine against ROS generation, MDA production and GSH depletion were nearly abolished in Nrf2 knockdown cells (Fig. 5D-F). The inhibitory effect of leonurine on cellular iron levels was almost eliminated in Nrf2 knockdown cells as determined by calcein-AM detection (Fig. 5G). Next, we examined the protein expression of key factors regulating cellular iron homeostasis and ferroptosis. As shown in Fig. 5H-J, the regulatory effects of leonurine on TFR, FTH1, GPX4 and XCT are nearly eliminated in Nrf2 knockdown cells.