Figure 3.

DPA inhibits glutamate-induced neuronal ferroptosis in vitro. (A) Representative phase contrast images showing the effects of DPA by different concentrations (5, 10, 20, 40 and 80 μg/mL) on neuronal ferroptosis induced by 5 mmol/L of glutamate for 8 h in HT22 cells. DPA was treated 2 h prior to glutamate treatment for 8 h in HT22 cells. The optimal concentration of DPA for the inhibition of neuronal ferroptosis in HT22 cells after glutamate exposure was 10 μg/mL, which was employed for the subsequent experiments. Arrows indicate the dead neurons. Scale bar indicates 50 μm. (B–E) The effects of DPA on ferroptosis-associated indices including lipid ROS, Ptgs2 mRNA and protein expression levels of ACSL4 in glutamate-induced ferroptosis in HT22 neuronal cells. The concentrations of DPA and Fer-1 (a specific ferroptosis inhibitor) were 10 μg/mL and 12.5 μmol/L, respectively. Regarding the detections of lipid ROS and Ptgs2 mRNA, HT22 cells were treated with DPA or Fer-1 2 h prior to glutamate exposure for 8 h while the cell samples were collected for determination of ACSL4 protein expression following pretreatment with DPA for 2 h and glutamate challenge at different time points (2 h, 6 h and 8 h). All the data were expressed as mean ± SEM (n = 3 independent biological replicates). *** p < 0.001. Abbreviation: Glu, glutamate; DPA, D-penicillamine; Fer-1, ferrostatin-1; Ptgs2, prostaglandin-endoperoxide synthase 2; lipid ROS, lipid reactive oxygen species; ACSL4, acyl-coA synthetase long chain family member 4; BRs, biological replicates.