Rho Kinase Inhibitor Y27632 Improves Recovery After Spinal Cord Injury by Shifting Astrocyte Phenotype and Morphology via the ROCK/NF-κB/C3 Pathway

Abstract

Spinal cord injury (SCI) usually results in loss or reduction in motor and sensory functions. Despite extensive research, no available therapy can restore the lost functions after SCI. Reactive astrocytes play a pivotal role in SCI. Rho kinase inhibitors have also been shown to promote functional recovery of SCI. However, the role of Rho kinase inhibitors in reactive astrocytic phenotype switch within SCI remains largely unexplored. In this study, astrocytes were treated with proinflammatory cytokines and/or the Rho kinase inhibitor Y27632. Concomitantly the phenotype and morphology of astrocytes were examined. Meanwhile, the SCI model of SD rats was established, and nerve functions were evaluated following treatment with Y27632. Subsequently, the number of A1 astrocytes in the injured area was observed and analyzed. Eventually, the expression levels of nuclear factor kappa B (NF-κB), C3, and S100A10 were measured. The present study showed that the Rho kinase inhibitor Y27632 improved functional recovery of SCI and elevated the proliferation and migration abilities of the astrocytes. In addition, Y27632 treatment initiated the switch of astrocytes morphology from a flattened shape to a process-bearing shape and transformed the reactive astrocytes A1 phenotype to an A2 phenotype. More importantly, further investigation suggested that Y27632 was actively involved in promoting the functional recovery of SCI in rats by inhabiting the ROCK/NF-κB/C3 signaling pathway. Together, Rho kinase inhibitor Y27632 effectively promotes the functional recovery of SCI by shifting astrocyte phenotype and morphology. Furthermore, the pro-regeneration event is strongly associated with the ROCK/NF-κB/C3 signal pathway.

Keywords: Astrocyte; C3; NF-κB; Rho kinase inhibitor; S100A10; Spinal cord injury.

Fig. 1

The primary culture and purification of astrocytes. (a) Bright field of astrocytes with primary culture. (b) A schematic diagram depicting the method of purification by hand cross shaking at RT. (c) Bright field of astrocytes post-purification. (d) Immunofluorescence identification of astrocytes (GFAP+) showed that the percentage of GFAP + cells post-purification was higher than primary culture (P < 0.0001). Scale bars, 100μm

Fig. 2

Y27632 induced the shift of morphology of astrocytes. (a) Phase-contrast microscope showed the cells of control groups had a flattened shape, while the cells treated with Y27632 exhibited a process-bearing shape. (b) 96.67 ± 0.01% of the cells treated with Y27632 developed more than two processes that were ≥ 100μm. (c) Immunofluorescence showed that the processes of the cells treated with Y27632 were GFAP⁺/S100A10⁺. Scale bars, 100μm

Fig. 3

Y27632 increased the proliferation and migration abilities of the astrocytes. (a) The CCK8 assay showed that the Y27632 improved the proliferation of astrocytes. (b) The auxiliary lines assisted to the wound scratch. (c) A schematic diagram depicting the scratch assay. (d) The astrocytes were scraped by pipette tip (200 µL), and the final scratch areas (24h later) were calculated. (e) The migration ratio of astrocytes treated with Y27632 was higher compared to the control group (P < 0.01). Scale bars, 100μm

Fig. 4

Y27632 transformed the naive astrocytes and the A1 reactive astrocytes into A2 reactive astrocytes. (a) Double fluorescent staining (GFAP in red, C3 in green) showed that astrocytes treated with IL-1α + TNFα + C1q were C3-positive, while the rest astrocytes were C3-negative. (b) Double fluorescent staining (GFAP in red, S100A10 in green) showed that astrocytes treated with Y27632 were S100A10-positvie, while the rest astrocytes were S100A10-nagative. DAPI stained all cell nuclei blue; Scale bars, 100μm

Fig. 5

The BBB scores for rats in each group changed over time. There were no statistically significant differences in BBB scores among each recorded time point (1d, 3 d, 7 d, 14 d, 21 d and 28 d) in the sham group (P > 0.05) after operation. On day 28, the Y27632 group score was significantly higher than that of the SCI group (P < 0.01)

Fig. 6

Y27632 promoted the recovery of neurological function by decreasing the number of A1 astrocytes in SCI rats. (a) Rats were anesthetized and perfused, then the spinal cord contained the damage area were harvested. (b) Double fluorescent staining(GFAP in purple, C3 in green)of spinal cord sections of the three group (Sham, Y27632 and SCI). (c) Immunochemical stained sections showed positive C3 cells in the three groups. (d) Quantification of the fluorescence intensity of C3. (e) The strong positive staining rate of C3 + cell in the three groups. Scale bars: 100μm

Fig. 7

Possible molecular mechanism of Y27632 on the transformation of reactive astrocyte phenotypes in vitro. (a) Western blotting detected protein levels of C3, ROCK2, NF-κB, and phosphorylated NF-κB in astrocytes with different treatments. (b-e). Quantification of the relative expression of C3, ROCK2, NF-κB, and phosphorylated NF-κB of astrocytes in each group