Effect Of SOX11 On Promoting Axon Regeneration After Traumatic Brain Injury And Its Mechanism

Traumatic brain injury has gradually become a major global public health problem,and its pathogenesis is a complicated pathophysiological process involving a series of cascade reactions,among which axonal injury is one of the most common and important pathological features in mild,moderate and severe traumatic brain injury,and is also the main driving factor for the fatality rate and disability rate of traumatic brain injury.How to regulate and realize the regeneration of injured axons is an important research direction in the treatment of traumatic brain injury.The limited axonal regeneration after mature central nervous system injury is caused by the deficiency of internal regeneration ability and external environmental inhibitory factors such as glial scar,among which the intrinsic regeneration ability of damaged axons is the decisive factor for axonal regeneration after central nervous system injury.The main factor causing this difference in axon regeneration ability is the difference in gene expression between the developing neurons and the mature neurons in the central nervous system.For this reason,we found a differentially expressed and significantly up-regulated gene SOX11 after traumatic brain injury by establishing a controlled cortical injury model and carrying out high-throughput sequencing on tissues around the injury in the early stage,and further research found that the role and mechanism of SOX11 on central nervous system injury,especially traumatic brain injury,has not been reported.Therefore,in order to study the effect of SOX11 on axonal regeneration of cortical neurons after traumatic brain injury,we first established a mouse model of controlled cortical injury,observed the axonal injury and regeneration in acute phase of traumatic brain injury,detected the expression of SOX11 in cortical brain tissues around the injury,and preliminarily analyzed the correlation between SOX11 expression level and axonal regeneration.Secondly,the primary cortical neuron culture of mice was used to establish a model of cortical neuron stretch injury.Through overexpressing and knocking down the expression level of SOX11,the regulation of SOX11 on axonal regeneration of cortical neuron injury was further determined.Finally,through targeted regulation of relevant signal pathways,the mechanism of SOX11 promoting axonal regeneration of cortical neurons after traumatic brain injury is preliminarily discussed.Part I Correlation between SOX11 expression and axonal regeneration after traumatic brain injuryObjective: To observe the axonal injury and regeneration after craniocerebral injury through a mouse model of controlled cortical injury,to explore the expression level of SOX11 in injured cortical brain tissue,and to preliminarily analyze the correlation between SOX11 expression level and axonal regeneration in injured cortical brain tissue.Methods: A mouse model of controlled cortical injury was established.HE staining,silver plating staining,and β-APP and GAP-43 immunohistochemical staining were performed on the injured peripheral cortical brain tissue at 1h,6h,12 h,24h,48 h and 72 h after injury.q RT-PCR and western blotting was used to detect the expression of SOX11 in the injured peripheral cortical brain tissue at different time points after injury.Pearson was used to analyze the correlation between the expression level of SOX11 protein and the proportion of GAP-43 positive cells was analyzed at different time points after injury.Results: HE staining showed progressive swelling,necrosis and infiltration of inflammatory cells in the injured cortex,while silver plating staining showed progressive thickening,swelling and fracture of axons in the injured cortex.Immunohistochemistry ofβ-APP showed that the number of β-APP positive cells in the cerebral cortex around the injury in the acute phase was gradually increasing.The results of immunohistochemistry of GAP-43 showed that the expression of GAP-43 gradually increased within 3 days of controlled cortical injury.The results of q RT-PCR and western blot about SOX11 showed that the expression of SOX11 gene and protein in cortex tissue was significantly increased,and showed an increasing trend.Moreover,the expression level of SOX11 protein and the expression level of GAP-43 protein in the cortex tissue are significantly positively correlated,which also shows that the expression of SOX11 is strongly correlated with axonal regeneration after head injury.Conclusion: A mouse model of controlled cortical injury has been successfully established.It was found that in the acute phase craniocerebral injury is accompanied by extensive axonal injury and self-repair of injured axon regeneration.SOX11 might participate in the repair reaction of neurons in the acute phase of craniocerebral injury and had a certain relationship with axonal regeneration.Part Ⅱ Effect of SOX11 on neuron axon regeneration after traumatic brain injuryObjective: To further explore the effect of SOX11 on axonal regeneration after craniocerebral injury and whether SOX11 gene expression regulation can be used as a targetto enhance axonal intrinsic regeneration.Methods: Primary cortical neurons of mice were cultured and observed under inverted phase contrast microscope.The purity of cortical neurons was identified by MAP2 and DAPI double fluorescence.The primary cortical neuron stretch injury model was established,and the survival rate was calculated by trypan blue staining at 1h,6h,12 h,24h and 48 h after injury.The amount of LDH release was detected and flow cytometry were used to evaluate the change of apoptosis rate.Through overexpressing and knocking down the expression level of SOX11 in cortical neuron,q RT-PCR and western blot were used to detect the expression of cortical neurons SOX11,βIII-tubulin and GAP-43 24 hours after stretch injury.βIII-tubulin immunofluorescence was used to measure the average length of cortical neuron axons in each group 24 hours after stretch injury.Results: The primary cortical neurons of mice were cultured for about 7 days.The cells were plump and the processes interwoven into neural networks,which gradually developed and matured.The purity of cortical neurons was more than 90%.The survival rate of cortical neurons decreased gradually at 1h,6h,12 h,24h and 48 h after stretch injury,and the release amount of LDH and the apoptosis rate of neurons increased gradually.The expression levels of SOX11,GAP-43 and βIII-tubulin in cortical neurons increased significantly after stretch injury.knocking down SOX11 can reduce the expression of GAP-43 and βIII-tubulin and the length of axon in cortical neurons after stretch injury.Overexpressing SOX11 can further increase the expression of GAP-43 and βIII-tubulin and the length of axon in cortical neurons after stretch injury.Conclusion: Primary cortical neurons of mice had been successfully cultured and the model of stretch injury of primary cortical neurons of mice had been established.The expression of SOX11 gene could be used as a target to enhance the intrinsic regeneration ability of axons.The regeneration of injured axons can be promoted by increasing the expression level of SOX11.Part Ⅲ The mechanism of SOX11 promoting neuron axon regeneration after traumatic brain injuryObjective: To further explore the mechanism of SOX11 promoting axonal regeneration of injured neurons.Methods: Based on the stretch injury model of primary cortical neurons in mice,lentivirus were used to overexpress and knock-down the expression of SOX11,and then theexpression of TANK,TRAF2,JNK and DCX in cortical neurons was detected by q RT-PCR and western bolt.Furthermore,cortical neurons were treated with knocking down of SOX11、overexpressing of TANK or SP600125.q RT-PCR and WB was used to detect the expression of JNK and DCX in cortical neurons.Finally,through overexpressing and knocking down the expression levels of DCX,q RT-PCR and Western bolt were used to detect the expressions ofβIII-tubulin and GAP-43 in cortical neurons,and βIII-tubulin immunofluorescence was used to measure the average length of axons of cortical neurons in each group after stretch injury.Results: q RT-PCR and WB results showed that overexpression of SOX11 could promote the expression of TANK,TRAF2,JNK and DCX in cortical neurons after stretch injury,while knocking down SOX11 could reduce the expression of TANK,TRAF2,JNK and DCX in cortical neurons after stretch injury.Further knocking down SOX11 and overexpressing TANK,q RT-PCR and WB results showed that the expressions of JNK and DCX in cortical neurons were significantly increased after stretch injury,while knocking down SOX11 and overexpressing TANK and adding JNK inhibitor SP600125 significantly decreased the expression of JNK and DCX in cortical neurons after stretch injury.In addition,the expression of βIII-tubulin and GAP-43 in cortical neurons of overexpressing DCX group was significantly increased,and the axon length was also significantly increased,while the expression of βIII-tubulin and GAP-43 in cortical neurons of knocking down DCX group was significantly decreased,and the axon length was also significantly decreased.Conclusion: SOX11 promotes axon regeneration of cortical neurons after stretch injury through TANK/TRAF2-JNK-DCX signaling pathway.SOX11 might promote the expression of TANK and TRAF2 in cortical neurons after stretch injury,activate JNK pathway,enhance the expression of DCX and promote axon regeneration.