Fig. 4

P2RX7 knockdown can reduce ferroptosis and oxidative stress in vitro. A Based on the GSE143560 database (6422 single nuclei after quality control), a tSNE plot of each cell based on the ferroptosis activity score as calculated by AUCell in the control and epilepsy groups. Cells with high-ferroptosis-scoring are highlighted in red. The 6422 single nuclei were divided into Low-Ferro score (Low-FS) (5029 cells) and High-Ferro score (High-FS) (1393 cells) expression groups. Meanwhile, the 6422 single nuclei were divided into control group (3467 cells) (B) and an epilepsy group (2955 cells) (C). Among 1393 cells, the proportion of cells with high ferroptosis activity in the epilepsy group (675/2955, 22.84%) (C) was greater than that in the control group (718/3467, 20.71%) (B). D Representative quantification of relative mRNA levels of GPX4 in blood samples from epilepsy patients and non-diseased controls. GPX4 was downregulated in epilepsy group compared to control group. E, F SH-SY5Y cells were transfected with P2RX7 (100 nM) for 48 h. Representative quantification of relative protein levels of P2RX7 in control, si-P2RX7 and si-Con groups. P2RX7 was decreased in the si-P2RX7 group compared to the control and si-Con groups. G–I Western blots and quantification of the protein levels of GPX4 and HO-1 in the control, Erastin, si-P2RX7 + Erastin and si-Con + Erastin groups. J, K Representative quantification of the relative protein and mRNA levels of GPX4 and HO-1 in the control, Erastin, si-P2RX7 + Erastin and si-Con + Erastin groups. L–N MDA, SOD and GSH assays to detect lipid peroxidation levels in the control, Erastin, si-P2RX7 + Erastin and si-Con + Erastin groups. O, P Representative images of SH-SY5Y cells by DHE (O) and semiquantitative analysis of relative fluorescence intensity (P). Q, R Representative images of SH-SY5Y cells by HO/PI staining (Q) and semiquantitative analysis of the percentage of cell death (R). All data are expressed as the mean ± SD. *p < 0.05, **p < 0.01