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.