| BackgroundSpinal cord injury (SCI) occurred almost in 20-40 years old young people. Statistics show:millions of patients in China with spinal cord injury, including traffic accidents, product accidents, sports injuries caused by spinal cord injury, more than 70 thousand of spinal cord injuries caused by traffic accidents, more than 50 thousand of spinal cord injuries caused by product accidents and sports injuries. According to a new survey shows that in recently years, the incidence of spinal cord injury in China showed an increasing trend year by year, the survey show that:the incidence of spinal cord injury is 6.7/100 million. Among them, traffic accident is the primary cause of spinal cord injury, which is 46.9% of the total number of spinal cord injury. The following reason is falling, crushing, squeezing, which is 33.1% of the total number. SCI seriously affect the patient’s motor function. SCI caused a series of inflammatory response mediated by cytokines, including tumor necrosis factor (TNF-a), interleukin (IL-6)IL-β, and these factors lead to second injury of spinal cord. Previous research shows that:after SCI, mRNA of TNF-a、IL-6、 IL-and IL-β was significantly up-regulated. Tyrosine kinase (JAK) and signal transducer and activator of transcription (STAT) signal pathway is an important way for signal from the cell surface to the nucleus, Signals from the phosphorylated STAT then translocate into the nucleus, followed by the transcription of target genes. The JAK/STAT pathway may be involved with neuronal response to SCI.JAK/STAT signaling pathway is closely related to the growth, survival, development and differentiation of cells. As a kind of evolutionary protection of eukaryotes, it has been reported in previous central nervous system injury. JAK/STAT signaling pathway is directly related to the nerve growth and glial scar formation in the area of spinal cord injury. The activation and over expression of STAT3 is important for the protection of neurons after axonal injury. JAK/STAT signaling pathway is an intracellular signal transduction pathway involved in the activation of the two protein family:JAK and STAT. JAK kinase is a class of four cytoplasmic tyrosine protein.Kinases including JAK1、JAK2、JAK3 and TYK2; STAT family contains seven transcription factors STAT2、STAT3、STAT4、 STAT5A、STAT5B、STAT6、STAT1. JAK/STAT pathway is a major and effective regulation system for regulating gene expression. This pathway involves the activation of the receptor by growth factors, hormones or cytokines, which induce the activation of JAK in the cell membrane. JAK Protein tyrosine phosphorylation is an important biochemical mechanism regulated by growth factors or cytokines. JAKs tyrosine phosphorylation with the receptor binding in the cell membrane. Subsequently, cytoplasmic STATs was activated in cytoplasmic via tyrosine phosphorylation, and then STATs dimmers, The STAT dimers translocate to the nucleus in the cell nucleus, they bind to specificcis acting elements, resulting in the transcription of different target genesSTAT signaling pathway has been extensively studied in the peripheral nerve injury in mice on facial nerve and hypoglossal regeneration after nerve injury, Researches show that:JAK1、JAK2、JAK3、STAT1、STAT3、STAT5, mRNA transient increases only STAT3 increased significantly and continued by situ hybridization and semi quantitative polymerase chain reaction. Peripheral nerve injury activates the JAK/STAT pathway of the microglia in the dorsal spinal cord. In the SCI study, the STAT3 signaling pathway plays an important role in the formation of astrocytes and glial scar formation in SCI. Okada et al confirmed that STAT3 plays a key role in regulating the response of astrocytes in the process of recovery of motor function and damage after SCI. Western blot showed JAK1/STAT3 was significantly activated in all kinds of cells 6 hours later of SCI, The phosphorylation of STAT3 in nerve neurons after SCI contribute to protection of nerve, while phosphorylation of STAT3 in astrocytes and microglia activateion in the chronic phaseOkada et al showed that STAT1 was related to the death of spinal neurons and ischemia in the spinal cord region, but the mechanism was not completely clear. Spinal cord injury can be caused by both primary and secondary factors, and seriously affect the quality of patients’ life. In the primary spinal cord injury, spinal cord injury occurs in a very short of time, but the secondary spinal cord injury involves a series of pathological changes, including tissue ischemia, edema and inflammation. In myocardial ischemia/reperfusion injury, STAT1 was found to induce apoptosis, and STAT1 was activated after cerebral ischemia, which caused ischemic injury in the brain. The mechanism is to inhibit the PI3-kinase /AKT pathway of the nerve protective effect and increase the expression of the apoptotic receptor Fas. The mechanism of Free radical scavenger and antioxidant is inhibition of STAT1 phosphorylation, STAT1 deficient rats showed volume of cerebral ischemia reduced and the STAT1 deficient cells can resist TNF-a interferon -β mediated apoptosis.On the basis of these results, the proposed neural apoptosis may be due to the activation of STAT1 after SCI.Oxidative damage is the cause of many diseases, including:Parkinson, Alzheimer’s disease, amyotrophic lateral sclerosis, the oxidative stress associated with the activation of JAK/STAT pathway. It was reported that the activation of STAT1 in subarachnoid hemorrhage by oxidative stress, these studies suggest that JAK1/STAT1 signaling pathway may induce oxidative stress reaction and death in SCI neurons. JAK1/STAT3 signaling pathway is activated in neurons after cerebral ischemia. STAT3 plays an important role in neural protection through the bcl-2 gene, inhibition of STAT3 can increase the area of cerebral infarction. Previous studies suggest that inhibition of interleukin -6 leads to phosphorylation ofObjectiveThe time dependent JAK/STAT signal pathway was activated after SCI and the expression of JAK/STAT was associated with SCI, Based on the model of acute SCI in rats, the expression changes of JAK1/STAT1 was detected after SCI by Western blot analysis at different time points. JAK1/STAT1 activity was detected at different time points after Intraperitoneal injection of STAT1 inhibitor or STAT1 siRNA, expression of various inflammatory cytokines, apoptosis related protein, behavior score, demyelination and histological analysis were also detected, which was helpful to clinical treatment.MethodsSTAT3 and reduce cerebral ischemia, activation of STAT3 increased the survival of ischemic myocardium. STAT3 deficient rats showed cerebral infarction area increase and the cardiac function decrease after myocardial ischemia. JAK1/STAT3 activation contributes to neural protection. After STAT1 gene knockout or STAT1 inhibition, TNF-a, IL-6, IL-β expression decreased, ischemia reperfusion injury reduced, ischemia tolerance of cells increased. After spinal cord injury, However, the roles of STAT1 in SCI remains incompletely understood. So, the purpose of this study was to investigate the effect of the selective STAT1 inhibition, using small interfering, RNA or STAT inhibitor S1941 on mouse SCI model. JAK and STAT changes detected by Western blot analysis at different time points, observe its effect on spinal cord injury at different time points.Observe the changes of the expression of inflammatory cytokines behavior scores, spinal cord demyelination. Show the effect of STAT1 on the early treatment of spinal cord injury, provide help for clinical treatment.1. The rat spinal cord injury model was established. After surgery, the rats were randomly divided into sham operation group, SCI group and STATlinhibitor group. The motor dysfunction of the rats was evaluated by behavioral scores. The degree of spinal cord injury was detected by HE.2. The rat spinal cord injury model was established. JAK 1/STAT1 phosphorylation levels was detected by Western blot and immunofluorescence, Histological changes of spinal cord was also detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL).3 The rat spinal cord injury model was established. and the rats were randomly divided into sham operation group, SCI+control siRNA group and SCI+STAT1 siRNA group. The motor dysfunction of the rats was evaluated by behavioral scores, Histological changes of spinal cord by HE staining; inflammatory factors changes was detected by ELISA; spinal cord motor neurons of the forefoot was detected by Nissl staining. apoptosis and demyelination was detected by blot Western.Result1.The pathological sections confirmed the injury of spinal cord. After operation, the funtion of the bilateral hind limbs of rats were lost, and spinal cord injury model was established successfully. the motor function of the two groups recovered a fraction With the time, the movement function of the STATlinhibitor group were higher than that of SCI group in Each test point2. The results of CBS score and BBB score were similar, the limb sensory nerve function of the STATlinhibitor group after operation was higher than that of in group SCI in each test point.3. After SCI, p-JAK1 and p-STAT1 expression increased gradually over time, and reached the peak in 48 hours. The total protein levels of JAK1 and STAT1 were little changed. Immunofluorescence was performed, and the results were the same with that of blot western.4. It was found that the MPO concentration was significantly higher in the SCI group compared with the Sham group, while the MPO concentration in the STATlinhibitor group was significantly lower than that in the SCI group. The proinflammatory cytokine monocyte chemoattractant protein-1 (MCP-1) and inducible nitric oxide synthase (iNOS) and myeloperoxidase (MPO) had similar change.5. For histological detection after spinal cord injury in rats, the white matter edema phenomenon is obvious in SCI group, and the white matter edema was significantly reduced in STAT1siRNA group.6. Apoptosis positive cells in SCI group were significantly higher than Sham group by TUNEL test, and STAT1 inhibitor could significantly decrease the apoptosis of spinal cord tissue after the intervention.7. STATlsiRNA group can significantly reduce TNF-α, IL-1β and IL-6, but change of IL-10 was opposite to them.8. Through the Western blot test, STATlsiRNA intervention can significantly reduce the expression of STAT1, NF-κ Bp65 protein was significantly increased in SCI+controlsiRNA group, but NF-κ Bp65 protein can be significantly curbed after STATlsiRNA intervention. Bcl-2 protein was significantly reduced in SCI+controlsiRNA group, but Bcl-2 levels were significantly increased after STAT1 siRNA intervention, Whereas the Bax was opposite to Bcl-2.9. The number of neurons in the anterior horn of spinal cord in SCI group was obviously decreased by Nissl staining, and the number of motor neurons after STAT1siRNA intervention was significantly decreased.10. By Western blot test, the expression level of Sham group of myelin associated inhibitory factor MAG and Nogo-A was lower, and the expression of these factors was significantly increased in SCI+controlsiRNA group, after the intervention of STATlsiRNA, the expression of these factors has decreased significantly.Conclusion1. After spinal cord injury, motor funtion of rats was damaged, JAK1/STAT1 signaling pathway involved in the development of spinal cord injury.2. After spinal cord injury, Activation of JAK1/STAT1 signaling pathway can induce proinflammatory cytokine, neuronal apoptosis and demyelination, thereby aggravating spinal cord injury.3. Blocking the JAK1/STAT1 signaling pathway can effectively reverse the secondary spinal cord injury, which is helpful for the treatment of spinal cord injury. |
