Inhibiting a spinal cord signaling pathway protects against ischemia injury in rats.

Objective: The aim of the study was to examine whether the cannabinoid agonist WIN55212-2 could attenuate ischemic spinal cord injury (SCI) in rats through inhibition of GAPDH/Siah1 signaling.

Methods: Male Sprague-Dawley rats were distributed randomly into 5 groups: (1) sham group that received no aortic occlusion and injected intraperitoneally (i.p.) with vehicle control after reperfusion; (2) control group that received a 12-minute aortic occlusion and injected i.p. with vehicle control after reperfusion; (3) WIN55212-2 group (WIN) that received the aortic occlusion and injected i.p. with 1 mg/kg of WIN55212-2 after reperfusion; and (4) WIN55212-2 plus AM251 group and (5) WIN55212-2 plus AM630 group that received the same surgical operation as the WIN group, except that 1 mg/kg of AM251 or AM630 was injected i.p. 30 min before each dose of WIN55212-2 injection, respectively. Neurologic function was assessed 48 hours after reperfusion. Histopathologic examination was performed to determine the number of normal neurons in anterior spinal cord. Protein expression of active caspase-3, total caspase-3, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), inducible nitric oxide synthase (iNOS), nuclear factor kappa light chain enhancer of activated B cells (NF-κB), Siah1, tumor necrosis factor α, and interleukin 1β were determined with Western blot and enzyme-linked immunosorbent assay; coimmunoprecipitation assays were also used to determine GAPDH/Siah1 complexing. Finally, terminal deoxynucleotidyl transferase dUTP nick end labeling staining was used to determine neuronal apoptosis in the lumbar spinal cord.

Results: The nuclear translocation of GAPDH and Siah1 in the spinal cord was initiated after ischemic spinal cord injury (SCI) along with the increased formation of GAPDH/Siah1 complexes. However, the activation of GAPDH/Siah1 was blocked by WIN. In addition, the treatment of WIN55212-2 promoted neuronal survival in the spinal cord, reduced apoptosis and inflammation, and improved neurologic scores. Furthermore, these beneficial effects of WIN55212-2 were abolished by the combined treatment of the CB2 antagonist AM630, but not the CB1 antagonist AM251.

Conclusions: Our findings reveal GAPDH/Siah1 signaling cascades as a novel therapeutic target for ischemic SCI and identify WIN55212-2 with the potential to treat ischemic SCI by targeting this pathway.