The Mechanism Of Activation Of STAT3Signaling Pathway Contributes To The Development Of Central Neuropathic Pain After Spinal Cord Injury

Central neuropathic pain after spinal cord injury (central neuropathic pain, CNP) is a kind of pain occurs below the level of spinal cord injury, where pain have has disappeared. CNP is the intractable complication after spinal cord injury. It generates spontaneously or is induced by skin irritation,. Besides, these kind of pain is persistent and unbearable, bringing great suffering to patients and makes patients lose the ability of self-care and unable to perform therapeutic rehabilitation exercise program, which affects the quality of life of patients seriously. For the mechanism is not clear at present, clinical treatment of CNP is very difficult, traditional drugs and surgical treatments are often not significant effect. The pathogenesis of CNP after spinal cord injury is a very complex process, during which the immune mechanism is currently a hot research field of pain. Janus kinase (JAK)/signal transducer and activators of transcription (STAT)-mediated signaling pathway is an important signal channel, thatplays a dual role on signal transduction and gene activation. It mediates the process of intracellular signal transduction ofmultiple cytokines and growth factors, then activatesof the corresponding target genes, resulting in biological effects. STAT3is involved in cell proliferation and differentiation mediated by many cytokine and growth factor. STAT3is expressed mainly in microglia in the CNS. Research has shown that STAT3in microglia in spinal dorsal horn cells is the key transduction signal during the early formation of molecules neuropathic pain. In this study, we used gene interference to inhibit the expression of STAT3gene in rat spinal cord, Studying the role of JAK/STAT3signal transduction pathway on central neuropathic pain after spinal cord injury in rats, which provided a theoretical basis for use the JAK/STAT3signal transduction pathway as an analgesic drug target.Part Ⅰ The significance of STAT3expression in dorsal horn in the model of central neuropathic pain after spinal cord injuryObjective:To observe the changes of pain behavior of the animals after T10spinal cord blunt injury,the changes of STAT3expression in lumbar4-6spinal cord dorsal horn, the expression of the downstream cytokines of STAT3and also the activation of glia in spinal cord dorsal horn.Methods:Forty two female SD rats were selected and randomly divided into3groups:CNP group, sham operation group and control group. Rats in the CNP group were induced central neuropathic pain (below-level pain) by T10spinal cord blunt injury. Rats in tthe sham group were only exposed the spinal cord segment T10without blunt injury. we observed the changes of pain behavior index, such as he mechanical withdrawal threshold、thermal withdrawal duration and hindlimb function, on1day pre-operation and every interval7days after injury until the28th day.Besides, the animals were killed on day3th,7th,14th,21st,28th and then thelumbar4-6segments of spinal cord were taken. Glia activation and the changes of STAT3mRNA and protein expression in lumbar4～6spinal cord dorsal horn were detected by immunohistochemistry, double-labeling immunofluorescene.western-blotand qRT-PCR Relationship between the up-regulation of STAT3and time-related changes in pain threshold levels in the rats was also studied.Results:There was no significant difference of basic pain thresholdamong all groups before operation (P>0.05). Compared CNP group with the sham group, the pain threshold is observably decreased form the3rd day to the14th day, and until to the28th day, the decreases remained significantly (P<0.05. The immunohistochemistry result displayed that the expression of STAT3-positive cells at lumbar4-6spinal cord were mainly in the gray matter in CNP group, and we found that it has more expression in all gray matter layer, particularly high expression in the spinal cord dorsal horn. Compared with the sham group, the difference was statistically significant (P<0.05). The fluorescent double staining suggest that the STAT3positive cells in spinal cord dorsal horn were mainly microglia. Co-expression of STAT3and OX42starting from the1st dayafter injury and then increased markedly on the3rd day, thenup to the most on the14th day, and last to the28th day.Compared with the sham group, the difference was statistically significant (P<0.05); Results of western blot analysis were in coincidence with those of the immunohistochemistry showed that STAT3protein expression was significantly increased fromthe7th day tothe21st day andthe28th daySTAT3mRNA significantly increased after surgery and it up to the maximum on the14th day and until to the28th day, it was still significantly higher than the sham group (P<0.01). Corelation analysis indicated that the STAT3expression in dorsal angle in rats had a significant positive correlation relationship with the central neuropathic pain after spinal cord injury (P<0.05).Conclusion:The STAT3protein expression in microglia at lumbar spinal dorsal horn in the rats with central neuropathic pain were significantly increased after T10spinal cord injury, which paralleled with the changes of pain behavior in these rats. Part Ⅱ Construction and identification of lentiviral vector expressing siRNA of STAT3Objective:STAT3-shRNA lentivirus vectors targeting rat were built by small RNA interference technology and then identified their interference effects in vitro in rat microglia (HAPI cell strains).Methods:The target sequences interference with STAT3-shRNA and the negative control sequences were designed individually.BLAST was used to verified the gene homology. After annealing, these double strand DNAs were connected with pGPU6vector which was digested by BamHI/Xhol, then converted into DH5a competent cells, extracted plasmid and then sequencing DNA. The recombinant lentivirus vector was made by plasmid that digested by a single restriction endonuclease BamHI and cloned. The three-plasmid lentivirus packaging systems and hybrid recombinant lentivirus vector were mixed, then the virus was produced in293T cell. The series of gradient dilution method was used to detect the virus titer. Use the same method to constructed the negative interference lentivirus vector. The packaged virus infected with HAPI cells were divided into3groups interference groups, negative interference group and control group. The interference group and the negative interference group were added to construct the interference vectors and negative interference vector, while the control group was added the same amount culture medium without virus in. Ninety six hours after transfection, westem-blot was used to test the expression of STAT3protein to identified the interference effects of lentivirus vectors. HAPI cells were collected form the3experimental groups and drops, the polyclonal antibodies STAT3was used as a the first antibody, a FITC labeled goat anti-rabbit IgG as the secondary antibody,and the indirect immunofluorescence was used to observe the expression of STAT3.Results:DNA Sequencing demonstrated that, the STAT3-shRNA interference sequences were successfully inserted into pGPU6vector.The recombinant plasmid vector with pRsv-REV reorganization, transformation, resistance to ampicillin to screen positive clones. Digestion verified that the recombinant plasmid was success.Virus was packaged in293T cells. Series of gradient dilution method Showed that virus titer was measured4x108Tu/ml. Western Blot showed that the expression of STAT3protein was lower in the interference group than that in the negative interference group (P<0.05) and control group (p <0.05). The efficiency of inhibition is75%. The negative interference group and the control group showed a strong fluorescence of STAT3, while the STAT3fluorescence in the interference group was significantly decreasedConclusion:Construction of STAT3-shRNA lentivirus vector targeting rat LV-GFP-STAT3could effectively inhibit the expression of STAT3protein in HAPI cells. Part Ⅲ The intrathecal injection of STAT3siRNA can reduce central neuropathic pain after spinal cord injuryObjective:To observed the analgesic effect of lentivirus fragment carrying STAT3siRNA on central neuropathic pain after spinal cord injury.Methods:Thirty six adult female SD rats were selected to make the model of central neuropathic pain. One day after T10spinal cord blunt injury (CNP, below-level), the subarachnoid catheter was done, then the rats were randomly divided into3groups:saline group (intrathecal injection of10μl saline), transfection reagent group (intrathecal injection of recombinant lentiviral vector10μl) and negative control siRNA group (intrathecal injection of lentiviral empty10μ, vector). The changes of pain behavioral of each group on post-operation7d,14d,21d,28d were observed. Fluorescence microscopy was used to detect the expression of GFP (green fluorescent protein) in lumbar spinal cord. Real time-PCR was carried out to detect the mRNA levels of STAT3gene. Meanwhile, western blot analysis was used to detect the content of STAT3protein in order to observe the effects of RNA interference. Also real time-PCR was used to detect the expression level of OX-42, AFT-3and MCP-1. Histology was used to observe the injury and recovery of the spinal cord4weeks after. intrathecal injection.Results:It was showed that after the intrathecal injection of LV-GFP-STAT3lentivirus vector, the mechanical hyperalgesia and thermal hyperalgesia of CNP rats was significantly improved, but the injection can not completely prevent the progress of the development of pain. The intrathecal injection of LV-GFP-STAT3lentivirus vector could significantly reduce the expression of STAT3mRNA and protein in lumbar spinal cord after spinal cord injury. The expression of OX-42, AFT-3and MCP-1mRNA was also significantly reduced.(P<0.05). BBB score showed that the intrathecal injection of LV-GFP-STAT3lentivirus vector could greatly improve the recovery of the motor function.Conclution:The Intrathecal injection of STAT3siRNA can inhibit microglia activation and the expression of STAT3and its downstream cytokine in lumbar spinal in the rats with central neuropathic pain after T10spinal cord blunt injury. Thus the central nervous pain was alleviated, and also axon regeneration of spinal cord neurons were improved.