| Convulsions is the most common critical disease in the infants, status convulsions can produce serious nervous system injury, such as the influence of intelligence development, health of body and mind, and even endanger the lives of children. Growth period eclampsia can inhibit the growth of brain development, change the neural circuits, improve the neuronal excitability, aggravating brain damage, leading to visual can, space can, learning and memory and affective disorder, if not timely intervention, will be serious harm to the patient’s life and forward quality of life, to the family and the society brings the heavy burden. A large population in the national condition, it faces the difficulty of bed than other countries more severe. Therefore, to explore development period convulsive happen development mechanism and the development of new anticonvulsants become our at present is urgent needs to solve medical problem.This study with our development period repeated convulsion rats model, and carry out the system of convulsions related research. Trifluoroethyl ether suction to jing is the classical period convulsion model. In the previous experiments, we use TaqMan MicroRNA Arrays to period after repeated convulsion rats in the hippocampus miRNA expression spectrum detection, in766known miRNA were convulsion rats after in the hippocampus with expression differences of miRNA, the results showed that, with normal control group comparison, growth period after repeated convulsion rat hippocampal there were20known miRNA expression by0.5times or more,9miRNA were obviously up-regulated more than two times. Additionally, the expression of miR-34b-5p and miR-204were upregulated at2-24h in both hippocampus and cerebral cortex with a different magnitude, followed by downregulation at72h-7d post-seizures compared with the control group. On the contrary, miR-672and miR-582-3p expressions were decreased significantly at2h-7d post-seizures in hippocampus, whereas upregulated in cerebral cortex compared with the control. Moreover, our data also demonstrated strong correlations between miR-34b-5p and miR-204expression in hippocampus and cerebral cortex at different time-points post-seizures, but the correlation between miR-672and miR-582-3p in these two tissues were poor. Additionally, the expression of miR-204was increased significantly in PC12cells after100ug/ml KA incubation compared with the control. For its expression is rich in rat hippocampus, we select miR-204as the research object, to further explore the convulsion cerebral damage the effect and its related mechanism, we choose starBase (http://starbase.sysu.edu.cn/browse ClipSeqIntersection. PHP) of three kinds of target gene prediction software (miRanda, PITA and PicTar) results are comprehensive analysis, choose the at the same time appear in three kinds of prediction software of target gene, and further were involved in the growth period convulsion cerebral damage process of target genes. The results showed that, in the3’UTR-SIRT1may exist and miR-204binding site. Therefore, we speculate that SIRT1may be miR-204control development period convulsion cerebral injury point of sites. Next we conducted binding site missing luciferase activity analysis, the result further confirmed in miR-204can combine the SIRT1in3’-UTR, participate in the regulation of SIRT1expression.We used Western blot analysis to detect the expression of GAP-43, SIRT1and p53in rat hippocampus after recurrent seizures2h,6h,24h,72h and7d induced by inhalation of trifluoro ethyl. As a result, we found that the developmental seizures could induced an enhanced neurons apoptosis compared with the control group in hippocampus, and the expression of GAP-43was decreased at6h after seizures, and reached the minimum at72h post-seizures, then recovered to normal at7d in hippocampus, but there was no significantly difference at2h post-seizures compared with the control group. On the other hand, the expression of SIRT1protein and mRNA were both decreased at2h post-seizures in hippocampus, and reached the minimum at72h post-seizures, then recovered to normal at7d. On contrary, the expression of p53protein was increased significantly at2h post-seizures in hippocampus, and reached the maximum at72h post-seizures, then recovered to normal at7d. Additionally, we found that KA induced the apoptosis of PC12cells in a dose-dependent manner, and reach the maximum at the concentration of250μmol/1. What’s more, KA incubation could decreased the expression of GAP-43and SIRT1at24h in PC12cells, while increased the levels of p53protein compared with the control group. These results demonstrated that recurrent seizures could induced the apoptosis of neurons and the alteration of synaptic plasticity, and SIRT1, a neuron protective factor, could regulated the process of seizures in through the regulation of p53protein level.Previous study had demonstrated that SIRT1expression was regulated by specific miRNAs in a series of diseases, here we use bioinformatics analysis to detecet if miR-204could change the expression of SIRT1to regulate the event of brain injury induced by recurrent seizures. First of all, we changed the expression of miR-204through the transfection of miR-204mimics/inhibitors in PC12cells. The results showed that the expression level of miR-204was increased after transfection of miR-204mimics, while decreased significantly after incubation of miR-204inhibitors, suggesting success to transfection. Then we detected that the levels of SIRT1protein was decreased after miR-204transfection, while increased after miR-204inhibitors incubation, but there were few effects on mRNA levels. On contrary, miR-204mimics transfection resulted in an enhanced level of p53protein, and decreased after incubation of miR-204. In order to observe the effects of miR-204on the apoptosis of PC12cells and the expression of GAP-43after KA treatment, we used TUNEL arrays to detect. As a result, we found that the overexpression of miR-204aggravated cells apoptosis, while miR-204inhibitors could confront PC12cells from apoptosis. On the other hand, miR-204mimics transfection resulted to a decreased level of GAP-43, while miR-204inhibitors led to an enhance GAP-43protein levels. To decreased the expression of SIRT1in PC12cells, we transfected cells with SIRT1siRNA, then Western blot analysis were utilized to detect the transfection efficiency. Finally, we measured the effect of miR-204inhibitors on the expression of p53after SIRT1scilence through Western blot, and we found that the level of p53protein was decreased compared with the KA incubation group.In conclusion, miR-34b-5p,-204,-582-3p and-672might be involved in the process of developmental recurrent seizures, which have a different expression manner in rat hippocampus and cerebral cortex post-seizures. MiR-204has a rich expression in both hippocampus and PC12cells, and increased after sezures in vivo and in vitro, which could be mediated by negatively regulation the expression of SIRT1protein, then inhibited the level of p53, finally decreased neuron apoptosis and accelerated the growth of axon, alleviated the brain injury induced by recurrent seizures. Therefore, intervention the expression of miR-204might protect neuron from apoptosis, and improve synaptic plasticity in vivo, then broad implications for the field of neurology by suggesting another layer of controls of gene expression to modulate pathways and mechanisms that will provide new insights for seizures research. |
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