| 【Objective】Spinal cord injury(SCI)is a severe traumatic disease of the central nervous system with high-incidence and refractory.Although there are various treatment methods,none of them have achieved satisfactory clinical effects and there is no effective treatment strategy currently.Drug therapy runs through the entire diagnosis and treatment process,while the treatment on SCI facing the dilemma of no medicine available with methylprednisolone is no longer recommended by the guidelines due to its non-ignorable complications.Neurotropin(NTP)is clinically used to treat low back pain,symptomatic neuralgia with its wide-ranging pharmacological effects.Studies in vivo and in vitro have reported that NTP could reduce memory damage and neuroinflammation and probably has certain effects in nerve repairing.The purpose of this study is to explore the effectiveness and safety of SCI treatment with NTP and its internal mechanism.Our ultimate goal is accelerating NTP clinical transformation,and bring more drug options for the repairing of SCI.【Method】1.Behavioral observation after NTP treatment on SCI.The motor function recovery of rats after NTP treatment was evaluated and the optimal treatment dose was determined through BBB score.Inclined plate test and Catwalk was detected to clarify the effectiveness of NTP treatment on SCI at the behavioral level.2.Histological observation after NTP treatment on SCI.The improvement of spinal cord and bladder tissue after NTP treatment was evaluated by HE,NISSL staining and gross observation to determine the effectiveness of NTP treatment on SCI at the histological level.3.Electrophysiological and MRI after NTP treatment on SCI.The changes of SEP,MEP and injury degree after NTP treatment were evaluated by electrophysiological and MRI,so as to clarify the effectiveness of NTP treatment on SCI at the electrophysiological and imaging levels.4.Safety evaluation after NTP treatment.The safety of NTP treatment was evaluated by HE staining and liver and kidney function tests.5.Mechanism analysis of NTP treatment on SCI.RNA-sequencing and protein chip built before and after the NTP treatment on SCI to show the genes and proteins expression spectrum,and then investigate the main biological processes,molecular function,cell composition,critical path and the key factors by GO,KEGG and PPI protein interaction network analysis of SCI pathological changes and NTP repairing process.6.Effects of NTP treatment on astrocyte activation,neurons and apoptosis after SCI.Immunofluorescence was detected to show the activation and neuronal protection of astrocytes and flow cytometry and Western Blot was detected to show the apoptosis after NTP treatment.7.Effectiveness and mechanism of NTP treatment on pathologic neuralgia caused by SCI.Mechanical tenderness,heat pain and cold tenderness was detected to show the effectiveness of NTP on pathologic neuralgia after SCI and Western Blot was detected to evaluate the intrinsic mechanism.【Result】1.NTP treatment promoted behavioral recovery after SCI.BBB score,inclined plate test and Catwalk all suggested that NTP could improve motor function and exercise posture of rats,and 50 NU/kg was the optimal treatment dose of NTP.2.Spinal cord and bladder histology improved by SCI with NTP.Spinal cord tissue structure was significantly damaged with unclear boundaries,tissue cavities formed in the injured center,irregular cell distribution and relatively large nucleus after SCI.NTP treatment could significantly improve tissue structure,reduce tissue cavities,and the internal cell distribution was relatively uniform.The bladder volume increased significantly,the villi in the inner wall of the bladder increased and became thinner with more inflammatory cells infiltration after SCI.After NTP treatment,the bladder volume decreased and the bladder tissue structure was improved significantly.3.NTP improves SEP and MEP after SCI and reduces the degree of tissue damage.The incubation periods of SEP and MEP were significantly prolonged with their amplitude significantly reduced after SCI.While NTP treatment could shorten the incubation periods and increase the amplitude.The intensity of signal value increased significantly under T2-weighted images after SCI,while NTP treatment could reduce the intensity of signal value.4.NTP treatment has a good safety.The histological observation of liver,kidney,lung,heart and spleen showed that the tissue morphology of organs was normal with no structural damage,no significant heterogenous changes in cytoplasm and nucleus and no pathological changes such as edema,bleeding and inflammatory infiltration after NTP treatment.Liver and kidney function showed that the indexes of glutamic-oxalacetic transaminase,glutamic-pyruvic transaminase and creatinine were normal with no significant difference between each group.5.Analysis of the mechanism of NTP treatment on SCI.Gene expression profiles were constructed by RNA high-throughput sequencing before and after NTP treatment on SCI.It was found that DEGs were enriched in apoptosis,inflammatory response and related pathways,especially JAK-STAT,TNF and NF-κB signaling pathways.PPI network analysis showed that IL-6,KIF2,PLK1 and CD68 were key nodes.DEGs were enriched in inflammatory immune response,apoptosis and related pathways,especially JNK and NF-κB signaling pathways after NTP treatment.PPI network analysis showed that RPLP1,TNF,COX1,CXCL10 and IL-1 were the key nodes.Protein chip analysis showed that IL-3,IL-13,IL-22,Adiponectin,Activin A,CCL27,Neuroplin-1,HGF,CD48,NGF were up-regulated,while RAGE,CXCL5,IL-1β,IL-1α,IL-6,TNG-α,MCP-1 were down-regulated after NTP treatment.Further experiments confirmed the expression of key factors,such as,the expression of inflammatory factors TNF-α,IL-6 and IL-1β were down-regulated,and the expression of neurotrophic factors BDNF,NGF and NT-3 were up-regulated after NTP treatment.Further experiments confirmed that NF-κB and JNK pathways were activated by SCI,while NTP could inhibit these two pathways.6.NTP treatment could inhibit the activation of astrocytes and apoptosis and protect neurons.NTP treatment could inhibit the activation of astrocytes and protect neurons.Otherwise,NTP treatment could inhibit apoptosis,and significantly down-regulated the expression of pro-apoptotic factor Caspase-3,while upregulated the expression of anti-apoptotic factor Bcl-2.7.NTP improved hyperalgesia symptoms such as mechanical tenderness,heat pain and cold tenderness after SCI.Further investigation into its mechanism of action revealed that NTP treatment could activate the 5-HT pain suppression system,upregulate the expression of BDNF and activate the Trk B signaling pathway,thus promoting the expression of GABA and exerting its analgesic effect.【Conclusion】1.NTP treatment could promote the recovery of motor function and the tissue structure of spinal cord and bladder after SCI,and showed good safety.2.NTP treatment could repair SCI through improving inflammatory and nutritional microenvironment and inhibiting apoptosis.3.NTP-targeted 5-HT pain suppression system and BDNF-Trk B-GABA pathway to improve pathologic neuralgia after SCI. |
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