3.3 Leonurine inhibits iron accumulation and ferroptosis in HK-2 cells
HK-2 cells were treated with different doses of erastin or RSL3, classical inducers of ferroptosis, to determine the exact concentrations that inhibited cell growth. As shown in Fig. 3A and 3B, erastin (30 μM) and RSL3 (0.4 μM) inhibited cell viability in a dose-dependent manner. Importantly, leonurine treatment significantly ameliorated the effects of erastin and RSL3 on cell death (Fig. 3C-D). Then, we used a fluorescent probe calcein-AM, to detect changes in cellular labile iron in cells based on its ability to be rapidly quenched when bound to cellular iron. As shown in Fig. E, RSL3 treatment markedly decreased the fluorescence signals, indicating that cellular iron levels were increased by RSL3 treatment but reversed by leonurine treatment. We next detected Fe2+ in HK-2 cells using FerroOrange. As shown in Fig. 3F, the Fe2+ content was increased after RSL3 treatment but decreased after leonurine treatment. We further evaluated the protein expression of key factors regulating cellular iron homeostasis. As shown in Figure 3G and 3H, RSL3 significantly increased the protein levels of TFR, FTH-1 and FTL, and these trends were reversed by leonurine treatment.