The Mechanism Of SNAP-25 In Rats With Contusion Spinal Cord Injury

Objective This study was aim to study the level of SNAP-25 m RNA and protein in spinal cord, and explore the effect of SNAP-25 on sensory function and motor function in rats with consution spinal cord injury(SCI), and explore the mechanism of SNAP-25.Methods One hundred and twenty four SD rats were randomly divided into sham group and spinal cord injury group, which contain 1 day operation group(1d), 3 days operation group(3d), 7 days operation group(7d), 14 days operation group(14d) and 28 days operation group(28d) subgroups. Firstly, the animals were performed spinal cord contusion by Allen’s method. Gene array technology was used to detect and fliter significant expression genes. Standard enrichment computing method was used to GO analysis. Secondly, using q RT-PCR and Western Blot to detect the level of SNAP-25 m RNA and protein in rats. Using immunohistochemistry to determine the location of SNAP-25 in spinal cord. Furthermore, Thermalgesia test and BBB scores was use to access the change of sensory and locomotor movement of rats with SCI. Finally, prediected the relative signal pathway of SNAP-25 by Bioinformatics.Results 1. The selection of difference gene following SCI Compared to sham group, there are 262 genes were significantly difference changes by using gene array in SCI group. About 249 of them were down-regulation after spinal cord injury. According to the level and biological ways of genes, SNAP-25, PEBP1, SNCB and AQP4 were chosen to verify. 2. Difference gene verify The results of q RT-PCR showed that the level of SNAP-25, PEBP1, SNCB and AQP4 were significantly decreased in SCI group(P<0.05). Combination with biological analysis, SNAP-25 was chosen to study in this work. 3. The change of SNAP-25 level in spinal cord after SCI The results of q RT-PCR showed that the level of SNAP-25 m RNA were significantly down-regulated at 1d,3d, 7d,14 d and 28 d, when compared to sham group(P<0.05). The results of Western blot showed that the level of SNAP-25 protein were significant decreased at 1d, 3d, 14 d and 28 d when compared to sham group(P <0.05). 4. The location of SNAP-25 The results of immunohistochemistry showed that SNAP-25 were expression in the neuritis of neurogliocyte of posterior horn. The expression of SNAP-25 positive immune production in posterior horn were significantly decreased at each point of check time when compared to sham group(P<0.05). Moreover, SNAP-25 positive immune production also be found in the neuritis of neurogliocyte and neuron of anterior horn, and it is significantly reduced at 1d,3d, 14 d and 28d(P <0.05). 5. The change of sensory function in rats with SCI Thermalgesia test showed that the latent period of pain sensory in hind limbs were shorten after SCI. With the time going, the latent period of pain sensory were recovery in sham group and SCI group. However, the latent period of pain sensory at 7d,14 d,21d and 28 d were lower than sham group(P <0.05). 6. The change of locomotor function in rats with SCI BBB scores showed that the locomotor function of hind limbs were significantly lower at 1d,7d,14 d,21d and 28 d than sham group(P <0.001). 7. The signal pathway of SNAP-25 in rats with SCI Biological analysis showed that SNAP-25 have a relationship with STAT3, which indicated that SNAP-25 affect the sensory function and locomotor function mediated by STAT3 signal pathway. The confirmation of q RT-PCR and immunohistochemistry showed that the level of STAT3 gene decreased, STAT3 was located in the neuron of spinal cord. Datas indicated that STAT signial pathway play roles via mediated SNAP-25. SNAP-25 plays its role and mediated by STAT3 signal pathway.Conclusion 1. The SNAP-25 gene was get from the results of fliter and verify by gene array. 2. The level of SNAP-25 m RNA and protein were both significantly decreased in rats after SCI.3. Following SCI, SNAP-25 immunoreactivity products decreased in the neuron and neuroglia of posterior horn, and thermalgesia test showed that sensory dysfunction, which indicated that SNAP-25 affect the sensory function. 4. Following SCI, SNAP-25 immunoreactivity products were reduced in the neuron and neuroglia of anterior horn, and locomotor dysfunction occurred, which illustrated that SNAP-25 affect locomotor function of hind limbs. 5. The function of SNAP-25 on sensory function and motor function mediated by STAT 3 signal pathway.