Abstract:
Objective To investigate the anti-inflammatory and neuroprotective effects of PF-06650833 on peripubertal mice with spinal cord injury (SCI) by inhibiting interleukin-1 receptor-associated kinase 4(IRAK4)/
transforming growth factor-β-activated kinase 1 (TAK1) pathway and its mechanism. Methods A total of 54 C57BL/6 mice, male, 6 weeks old, with a body weight of 18-20 g, were selected. The mice were divided into control group (Sham group, n=12), SCI group (n=21) and PF-06650833 group (n=21) by computer-generated random number table. The modified Allen's impact method was used to make the SCI mice model. Motor function of mice was evaluated using Basso Mouse Scale (BMS) at 1, 3, 7, 14, 21, and 28 days post-injury. At 3 days post-injury, mice from all three groups were anesthetized and sacrificed. Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression levels of the inflammatory cytokines interleukin (IL)-1β and IL-10. Western blotting was used to detect the protein expression levels of phosphorylated-IRAK4 (p-IRAK4) and phosphorylated-TAK1 (p-TAK1). At 7 days post-injury, spinal cord tissues from the SCI and PF-06650833 groups were collected, fixed with paraformaldehyde, dehydrated
in graded sucrose solutions, and prepared as 30 μm frozen sections for immunofluorescence staining. At 28
days post-injury, mice from all three groups underwent the tail suspension test and the sucrose preference test. Statistical analysis was performed by one-way analysis of variance (ANOVA) and t-test. For the changes of BMS score over time among groups, the Mann-Whitney U test was used along with Bonferroni correction.
Results There were no statistically significant differences in BMS scores between the SCI and PF-06650833
groups at 1, 3, 7 or 14 days post-injury (all P>0.05). However, at 21 and 28 days post-injury, BMS scores in
the PF-06650833 group were significantly higher than those in the SCI group [3.5 (3.0, 4.0), 4.0 (4.0, 4.3)
points vs 2.0 (2.0, 2.3), 3.0 (2.0, 3.0) points, respectively; U values were 1.500 and <0.001, respectively, all
P < 0.05]. At 3 days post-injury, compared with the SCI group, the PF-06650833 group showed significantly
higher levels of the anti-inflammatory cytokine IL-10 [(577.94±12.01) pg/mL vs (492.97±20.06) pg/mL],
significantly lower levels of the pro-inflammatory cytokine IL-1β [(84.11±14.81) pg/mL vs (123.40±14.61)
pg/mL)], and significantly lower relative protein expression levels of p-IRAK4 (1.61±0.19 vs 3.35±0.29)
and p-TAK1 (1.14±0.15 vs 2.35±0.19), with all differences being statistically significant (t values were
6.295, -3.271, -8.693, -8.658, respectively; all P<0.05). At 7 days post-injury, compared with the SCI group,
the PF-06650833 group exhibited significantly fewer glial fibrillary acidic protein (GFAP)-positive cells
(167.67±6.51 vs 225.33± 7.77) and significantly more neuronal nuclei (NeuN)-positive cells (104.00±8.19
vs 62.33±6.43, t values were -9.858, 6.934, respectively; all P<0.05)]. At 28 days post-injury, compared with
the SCI group, the PF-06650833 group showed a significantly higher sucrose preference [(64.99±1.63)% vs
(51.85±2.63)%], and a significantly shorter immobility time in the tail suspension test [(172.06±7.14) s vs
(215.00±16.72) s, t values were 10.398, -5.786, respectively; all P<0.05]. Conclusions PF-06650833 exerts
anti-inflammatory and neuroprotective effects on peripubertal mice with SCI by inhibiting IRAK4/TAK1
pathway.
2026, Vol. 14 
