Experiment Study Of Inhibition Of TNFR/RIPK Signaling Pathway In The Neurological Functional Recovery After Acute Spinal Cord Injury

Objective: Studies have shown that TNFR/RIPK signaling pathway was up-regulated after spinal cord injury(SCI), the pathway may played an important role in functional recovery after SCI. By making acute spinal cord injury model in rats, the research group intervened experimental rats using specific inhibitors of RIPK1, Necrostatin-1(Nec-1), to observe the expression of apoptosis-related proteins and recovery situation of rats motor and sensory function, to investigate the impact on neuronal necroptosis by inhibition TNFR/RIPK signaling pathway, further improve the new mechanism of neuronal necroptosis after SCI.Methods: Healthy Clean male 120 S-D rats weighing 0.25-0.30 kg were randomized equally into four groups: the sham(only laminectomy, I group, Sham group, n=30), sham+Nec-1 group(II group, Sham+Nec-1group, n=30), SCI+Dimethyl Sulfoxide(given DMSO after SCI, III group, DMSO group, n=30), SCI + Nec-1(given inhibitor of necroptosis after SCI, Ⅳgroup, Nec-1 group, n=30). Each group was divided five subgroups: 6h、12h、24h、3d and 7d after SCI, each subgroup with six rats.Spinal cord clamp compression method was used to make rat acute spinal cord injury model, and PE-0402 was placed in the subdural to inject drugs topically. Nec-1 will be dubbed the 25μg/μl solution; sham group(I group) without any treatment, II and Ⅳ group injected 1ul Nec-1 using a micro-syringe at 30 minute after SCI, III group injected the same amount of DMSO solution in the same time after injury. Giving penicillin to prevent infection, 40,000 units/day for 3 days, rehydration when necessary. Postoperative bladder massage twice daily to help them urination until their micturition reflex restored.Before SCI, 6h, 12 h, 24 h, 3d and 7d after injury, the rats were rated by BBB scoring to evaluate functional recovery. 6 rats were sacrificed in each group at a predetermined time randomly, then removed spinal cord were taken HE staining, Nissl staining to observe nerve tissue pathological changes; PI staining and TUNEL to detect cell apoptosis, Western Blot to detect the expression level of Bcl-2, RIPK1 and RIPK3 protein at 24 h after SCI. The data were analyzed by SPSS19.0.Results: Within 6 hours after SCI, rats hind legs completely paralyzed, motor function began to recover after 24 hours in DMSO group, while recover after 12 hours in Nec-1 group, hind limb motor function BBB scores were significantly higher in Nec-1 group than DMSO group after 24h(P<0.05). HE and Nissl staining showed spinal cord tissue structural integrity, nerve cells with normal morphology in Sham and Sham+Nec-1 group within 7 days after operation. The degree of SCI and the number of neuronal survival in Nec-1 group were better than DMSO group at each points, the difference was statistically significant at 24h(P<0.05). PI staining showed severe structural damage, cell disintegration, and PI stained red cells increased significantly in SCI zone in DMSO group. After the Nec-1 intervention, PI stained red cells and neuronal disintegration were significantly reduced compared with DMSO group(P<0.05). Increasing apoptotic bodies were observed in DMSO group at 24 hour after SCI, and the apoptotic bodies significantly reduced after intervention with Nec-1, the difference was statistically significant(P <0.05). The expression of Bcl-2 was stable in the Sham and Sham+Nec-1 group, while expression upregulated when giving Nec-1 in SCI group. The expression level of RIPK1 and RIPK3 was fairly low in Sham group, raised in spinal cord injury, and downregulated after giving Nec-1, the difference was statistically significant(P<0.05).Conclusions:(1) TNFR/RIPK signal pathway mediated necroptosis participated in neuronal programmed cell death.(2) RIPK1 specific inhibitor, Nec-1, could promoted motor function recovery in rats, reduced the degree of spinal cord injury, increased the number of surviving neurons, downregulated the expression of apoptosis-related proteins RIPK1 and RIPK3, enhanced nerve functional recovery.(3) TNFR/RIPK signaling pathway played an important role in necroptosis after acute SCI process, provided a new strategy for the prevention and treatment of acute SCI.