Journal: Redox Biology
Article Title: A spinal USP7-Bach1 positive feedback loop drives NOX4-mediated ferroptosis in neuropathic pain
doi: 10.1016/j.redox.2026.104153
Figure Lengend Snippet: FER-1 attenuates neuropathic pain and spinal ferroptosis in the SNI model. (A) Mechanical and thermal hyperalgesia were evaluated by the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) in Sham + Vehicle, SNI + Vehicle, and SNI + FER-1 groups. Behavioral tests were conducted at day 0 and on days 6, 7, 8, 9, 10, and 11 post-surgery (n = 9 per group). (B) Representative Western blot images and quantitative analysis of 4-HNE in spinal cord lysates 11 days post-SNI (n = 6 per group). (C) Representative double immunofluorescence images showing co-localization of 4-HNE (red) with the neuronal marker NeuN (green) in the spinal dorsal horn. Scale bar: 100 μm. White boxes indicate higher magnification views of representative neurons (n = 3 per group). (D-G) Representative Western blot images and quantitative analysis of ferroptosis-related proteins (ACSL4, SLC7A11 and GPX4) in the spinal cord. β-actin served as the loading control (n = 6 per group). (H–I) Biochemical quantification of Fe 2+ and MDA levels in spinal cord tissues (n = 6 per group). (J) Representative transmission electron microscopy (TEM) images and quantitative analysis of mitochondrial morphology in spinal neurons. Red arrowheads indicate classical shrunken mitochondria with increased electron density of the bilayer membrane, accompanied by decreased mitochondrial cristae and mitochondrial membrane rupture; blue arrowheads indicate reduced or vanishing cristae; and black arrowheads indicate outer mitochondrial membrane rupture. Scale bar: 500 nm (n = 3 per group). Significance was determined using one-way ANOVA or two-way ANOVA followed by Bonferroni's post-hoc tests. Data are expressed as mean ± SEM. (∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 vs. Sham + Vehicle group; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 vs. SNI + Vehicle group). The value of n represents the number of independent biological samples. All molecular experiments were independently repeated at least three times with consistent results.
Article Snippet: In in vivo experiments, the ferroptosis inhibitor FER-1 (10 mg/kg, i.p., Hy-100579) [ ], the Bach1 inhibitor Bach1-IN-1 (also known as HPPE, 10 mg/kg, i.g., HY-153040 , MCE, China), the NOX4 inhibitor GLX351322 (5 mg/kg, i.p., HY-100111, MCE, China), and the USP7 inhibitor P005091 (15 mg/kg, i.p., HY-15667 , MCE, China) were administered once daily for 5 consecutive days.
Techniques: Western Blot, Immunofluorescence, Marker, Control, Transmission Assay, Electron Microscopy, Membrane