脊髓损伤
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Figure 2｜Hemin treatment attenuates the protective effect of rhFGF21 in rats after SCI.
Based on the expression patterns of FGF21, HO-1, GPX4, and 4HNE, we chose the 2nd day after SCI as the time point for Experiment 2. The extent of neurodegeneration and iron accumulation were assessed by Nissl staining and Perls staining, respectively. The Nissl results revealed that normal neurons were round and darkly stained, while atrophied and mauve neurons appeared after SCI (P < 0.0001; Figure 2A and B). Both Fer-1 and rhFGF21 treatments improved the contracted morphology of neurons. In contrast, hemin treatment aggravated the morphological changes of neurons in the rhFGF21 + hemin group compared with the rhFGF21 group (Fer-1 vs. SCI: P < 0.0001; rhFGF21 vs. SCI: P < 0.0001; rhFGF21 + hemin vs. rhFGF21: P < 0.001; Figure 2A and B). Perls staining of spinal cord sections showed that the SCI group had significantly more iron-positive cells than the Sham group (P < 0.0001; Figure 2A and C). In the Fer-1 and rhFGF21 groups, iron-positive cells were significantly decreased, while the rhFGF21 + hemin group had more iron-positive cells than the rhFGF21 group (Fer-1 vs. SCI: P < 0.0001; rhFGF21 vs. SCI: P < 0.0001; rhFGF21 + hemin vs. rhFGF21: P < 0.0001; Figure 2A and C).
The BBB scores of the rhFGF21 group increased more rapidly than the SCI group on day 28 (P < 0.0001; Figure 2D). The rhFGF21 + hemin group had a lower BBB score than the rhFGF21 group on day 28 (P < 0.0001; Figure 2D). In the incline plate test, we observed that rats in the rhFGF21 group could hold their posture on the tilt plate at higher angles than the SCI group on day 28 (P < 0.0001; Figure 2E). The angles were lower in the rhFGF21 + hemin group than rhFGF21 group (P < 0.0001; Figure 2E).
Figure 4｜Mitochondrial pathological changes and ferroptotic process in rats after SCI.
For quantitative analysis of the ferroptotic process in spinal cord tissue of all groups, we measured the concentration of iron, reduced GSH, and lipid peroxide (MDA) (Figure 4). In the rhFGF21 group, iron and MDA content significantly decreased and GSH content increased compared with the SCI group (iron: P < 0.0001; MDA: P < 0.0001; GSH: P < 0.0001; Figure 4B–D). In contrast, iron and MDA content increased and GSH levels decreased in the rhFGF21 + hemin group compared with the rhFGF21 group (iron: P < 0.0001; MDA: P < 0.0001; GSH: P < 0.0001; Figure 4B–D). Transmission electron microscopic images revealed structural pathological changes in mitochondria of spinal cord neurons in the SCI group and rhFGF21 + hemin group. These changes included shrinkage of mitochondria and reduction or disappearance of mitochondrial cristae. Fer-1 and rhFGF21 treatments attenuated these pathological changes. The structure and morphology of mitochondria improved in the Fer-1 group and rhFGF21 group (Figure 4A). These results also indicate that FGF21 inhibited ferroptosis after SCI in rats, while HO-1 exacerbated it.
Figure 5｜Immunofluorescence of HO-1 and GPX4 expression in the spinal cord anterior horn of rats after SCI.
We observed differences in HO-1 and GPX4 expression in the spinal cord anterior horn by immunofluorescence staining of spinal cord tissue sections from each group (Figure 5A). Similar to the WB and qRT-PCR results, HO-1 expression was decreased in the Fer-1 group and rhFGF21 group compared with the SCI group. Meanwhile, HO-1 expression was increased in the rhFGF21 + hemin group compared with the rhFGF21 group (Fer-1: P < 0.0001; rhFGF21: P < 0.0001; rhFGF21+hemin: P < 0.0001; Figure 5B and C). The change in GPX4 expression was opposite to that of HO-1, being increased in the Fer-1 group and rhFGF21 group and decreased in the rhFGF21 + hemin group (Fer-1: P < 0.0001; rhFGF21: P < 0.0001; rhFGF21 + hemin: P < 0.0001; Figure 5B and C). A cartoon schematic of the spinal cord anterior horn was showed in Figure 5D. These findings also indicate that FGF21 inhibited SCI-induced ferroptosis by suppressing HO-1 expression in the spinal cord anterior horn.