Fig 2: Inhibition of GDF15 aggravated neurological damage and neuroinflammation. (A) Neuronal death determined by TUNEL assay at 7 dpi in Sham, SCI, and SCI + KD mice (Scale bar = 200 μm). (B) Quantitative analysis of TUNEL-positive neurons. (C) Representative immunofluorescence labeling of neurofilaments for NF200 (green) and myelin sheath for MBP (red) and obtained from longitudinal sections centered around the injured core 1.5 mm at 28 dpi (Scale bar = 300 μm). (D) Quantitative analysis of NF200 positive area at 28 dpi (n = 6). (E) Quantitative analysis of MBP positive area at 28 dpi (n = 6). (F) Double immunofluorescence labeling of microglia for IBA-1 (green) and astrocytes for GFAP (red) obtained from longitudinal sections centered around the injured core 3 mm at 7 dpi (Scale bar = 300 μm). (G) Quantitative analysis of IBA-1 positive area at 7 dpi. (H) Quantitative analysis of GFAP positive area at 7 dpi. The error bars represent the SD. **p < 0.01, ***p < 0.001, vs. Sham group; #p < 0.05, ##p < 0.01, ###p < 0.001, vs. SCI group by t-test, one-way ANOVA followed by Tukey’s post hoc analysis.

Fig 3: Knockdown of GDF15 interferes locomotor recovery by aggravated neuronal loss in SCI mice. (A) H&E staining images of cords centered around the injured core 3 mm obtained at 7 and 28 dpi (Scale bar = 300 μm). (B) Quantitative analysis of the defected area at 7 and 28 dpi (n = 6). (C) Representative images for Nissl staining obtained from longitudinal sections centered around the injured core 1.5 mm at 7 and 28 dpi in Sham, SCI, and SCI + KD mice (Scale bar = 200 μm). (D) Quantitative analysis of the amounts of survived neurons at 7 and 28 dpi (n = 6). (E) The BMS score post SCI in Sham, SCI, and SCI + KD mice. (F,G) Photographs of Swimming at 28 dpi, showing the worse trunk instability and uncoordinated action in SCI mice, and statistical analysis of the Louisville Swim Scale over a period of 28 days (n = 6). ***p < 0.001, vs. Sham group; #p < 0.05, ##p < 0.01, ###p < 0.001, vs. SCI group by two-way ANOVA followed by Tukey’s post hoc analysis.

Fig 4: GDF15 mitigates Hemin-induced ROS production and ferroptosis in neurons through the p62-Keap1-Nrf2 signaling pathway. (A) Relative mRNA level of p62 after knockdown (n = 6). (B) Western blotting performed for p62, Keap1, Nrf2, HO-1, ACSL4, FTH1, and GPX4 in Hemin-activated primary neurons after adding rGDF15 or transfection of sh-p62 (n = 3). β-Tubulin was used as the control. (C–I) Bar graph showing quantitative analysis of p62, Keap1, Nrf2, HO-1, ACSL4, FTH1, and GPX4 (n = 3). (J) Representative immunofluorescence labeling images for p62 (red) and Nrf2 (green) in Hemin-activated primary neurons after adding rGDF15 or transfection of sh-p62 (Scale bar = 200 μm). (K) Bar graph showing quantitative analysis of ROS expression (n = 6). (L) The value of ROS was determined (n = 6). The error bars represent the SD. **p < 0.01, ***p < 0.001, vs. control group; ##p < 0.01, ###p < 0.001, vs. Hemin group; &p < 0.05, &&p < 0.01, &⁣&&p < 0.001, vs. Hemin + rGDF15 group by one-way ANOVA followed by Tukey’s post hoc analysis.

Fig 5: GDF15 alleviates SCI-induced neuronal ferroptosis by regulating the p62-Keap1-Nrf2 signaling pathway. After SCI, cracked blood vessels at the injured cords result in the lysis and destruction of erythrocytes, which release a large amount of iron. Excessive iron accumulated in neurons produces superfluous ROS and causes neuronal ferroptosis, which promotes the activation of the p62-Keap1-Nrf2 signaling pathway. Besides, GDF15 in neurons further facilitates the activation of the p62-Keap1-Nrf2 pathway by stabilizing p62 and increased Nrf2 and HO-1 inhibit the accumulation of ROS and thus alleviate neuronal ferroptosis. GDF15 is suggested as a potential target on mitigating nervous tissue loss and promoting locomotor recovery post SCI.