Ferroptosis inhibitor protected against cytotoxicity induced by CoNPs in Balb/3T3 cells Cells were treated with different concentrations of CoNPs for 4, 24, or 48 hours and the effect of CoNPs on Balb/3T3 cell survival was determined by CCK-8 assay. The percentage of survived cells strikingly decreased with the increase of CoNPs concentration (Figure 1A). The IC50 value was 400 μmol/L 24 hours later, respectively. Cells cultured in the medium with varying concentrations of DFP showed a dose-dependent survival rate. When the concentration of DFP increased, the viability of Balb/3T3 cells increased. When cells were cultured in 400 μmol/L CoNPs and different concentrations of DFP, the viability of cells was significantly higher than the cells treated with CoNPs (Figure 1B)."

Ferroptosis inhibitor reduced inflammatory cytokines in Balb/3T3 cells The results are shown in Figure 2. The levels of TNF-α, IL-1β, and IL-6 in the CoNPs group were noticeably higher than those in the control group. Compared with the CoNPs group, DFP treatment had a significant protective effect and significantly reduced TNF-α (P < 0.05), IL-1β (P < 0.05), and IL-6 (P < 0.05) induced by cobalt nanoparticles expression. However, there was no significant difference between the single DFP group and the CoNPs group."

The oxidative damage induced by ROS was considered an important cause of the toxicity of cobalt nanoparticles. ROS was also the main incentive of ferroptosis. In this study, as shown in Figure 3, after 400 μmol/L CoNPs were treated for 24 hours, the ROS level of Balb/3T3 cells increased significantly (P < 0.05), while DFP (25 μmol/L) could significantly inhibit this increase (P < 0.05), and the difference was statistically significant (P < 0.05) (Figure 3A, B). GSH is the key cellular endogenous antioxidant that can scavenge toxic free radicals, and depletion of GSH appears to promote intracellular ROS accumulation, leading to apoptosis. Altered GSH levels represent the increased cellular response to oxidative stress. The results suggested that CoNPs treatment decreased GSH levels (P < 0.05), and DFP inhibited this decrease (P < 0.05) as measured by the GSH assay kit (Figure 3C).  ROS promotes lipid peroxidation, leading to damage to the cell membrane and eventual cell death. When the concentration of iron ions in the cell increases, the likelihood of ferroptosis is significantly heightened. Conversely, reducing the intracellular free iron content or using iron chelators can effectively inhibit or slow down the process of ferroptosis[25-26]. Our results showed that CoNPs could lead to significant increases in Fe2+ ion and total iron concentration in Balb/3T3 cells compared with the control group. However, the increase was inhibited when cells were exposed to DFP (Figure 3D). These results indicate that CoNPs can induce the occurrence of ferroptosis in Balb/3T3 cells by stimulating oxidative stress."

Apoptosis assay in Balb/3T3 cells To investigate the apoptosis rate of cells treated with 400 μmol/L CoNPs and 25 μmol/L DFP, the AnnexinV-FITC assay kit was used. Incubation with 400 μmol/L CoNPs for 24 hours mildly increased the cell apoptosis rate compared with the control group. When DFP was added as a treatment before exposure to CoNPs, the apoptosis was obviously alleviated (Figure 4A, B)."

Ferroptosis inhibitor exerted the positive regulation on GPX4 in Balb/3T3 cells GPX4 is the central regulator of ferroptosis, and the decline of GPX4 is often used as a marker of ferroptosis[27-28]. SLC7A11 is a unit of the glutamate-cystine antiporter Xc-, resulting in enhanced lipid oxidation and ferroptosis. Western blot assay showed that CoNPs noticeably decreased the protein amount of GPX4 and SLC7A11. However, the decrease in GPX4 and SLC7A11 levels was inhibited when cells were treated with 25 μmol/L DFP and 400 μmol/L CoNPs for 24 hours. These results suggest that CoNPs might induce ferroptosis and the ferroptosis inhibitor DFP exerts therapeutic action against CoNPs (Figure 5)."