Effects Of RAP1 Signaling Pathway On Blood-brain Barrier After Traumatic Brain Injury

Objective: Traumatic craniocerebral injury(TBI)is a serious injury to the nervous system,which can directly lead to neuronal necrosis,severe damage of the blood-brain barrier(BBB)structure,and secondary cause a series of fatal injuries such as brain edema.At present,the therapeutic effect of protecting the BBB after traumatic brain injury is not satisfactory,so it is urgent to find new therapeutic targets.Rap1 is a small GTPase protein with high homology to Ras protein,which circulates between the active GTP-binding state and the inactive GDP binding state using the small GTP-enzyme as a molecular switch.Previous studies have found that Rap1 signaling pathway mainly mediates and participates in cell adhesion,cell connection formation and endothelial barrier protection,etc.Afadin plays an important role in cell-cell connection as a downstream effector of Rap1.Therefore,we focused our research on the effect of Rap1 signaling pathway on the blood-brain barrier after traumatic brain injury.The purpose of this study was to explore the effect of activation of Rap1 signaling pathway on BBB after traumatic brain injury and its mechanism,and to understand whether activation of Rap1 can improve BBB permeability and reduce brain edema after traumatic brain injury,so as to serve as a new target to improve BBB permeability after traumatic brain injury.Methods: 1.Model building: Adult male C57BL6 mice were selected and the model of traumatic brain injury mouse model was made by using pneumatic craniocerebral trauma apparatus after anesthesia.2.After protein extraction from the brain tissue of the wound site,Western blotting was used to detect the differences in the expressions of Afadin and BBB-related junction proteins between the brain tissues of normal mice and the brain tissues of TBI at different time points.3.The changes of BBB permeability of mice in different time points were detected by Evans Blue test;4.Water content in brain tissues of mice in different time points was detected by dry-wet specific gravity method;5.The activation of Rap1 in mice of each group was detected by GTP-Rap1 pull down test;6.Blood brain barrier related junction proteins were detected by immunofluorescence technique in each group;7.After Afadin was knocked out,the expression of BBB-related junction proteins was detected by Western blotting and immunofluorescence.Results: 1.The results of Western blotting showed that the expression levels of BBB connective proteins such as Afadin,ZO-1 and claudin-5 in the experimental group and the trauma group were decreased compared with the control group after TBI(P<0.05),and the expression levels were the lowest on the third day.However,the protein expression in the experimental group was higher than that in the trauma group(P<0.05).2.The results of Evans Blue experiment showed that the content of EB in the brain tissue of mice in the experimental group and the trauma group was significantly higher than that in the control group(P<0.01).The content of EB in the brain tissue of mice in the experimental group was significantly lower than that in the trauma group(P<0.01),although higher than that in the control group.3.The results of dry-wet specific gravity method showed that the water content of brain tissue of mice after TBI was significantly higher than that of control mice(P<0.01),indicating that the brain tissue edema of mice after TBI was obvious,but the brain tissue edema of mice in the experimental group was lighter than that in the control group(P<0.01).4.The results of GTP-Rap1 pull down test showed that the expression of activated Rap1(GTP-Rap1)in the brain tissues of the experimental group was higher than that of the trauma group(P<0.01).5.The results of immunofluorescence test showed that the blood-brain barrier of mice was significantly damaged after TBI,and the expression of connectin in the experimental group was significantly lower than that in the control group(P<0.05),but the expression of connectin in the experimental group was higher than that in the trauma group(P<0.05).6.After Afadin knockdown,the expression level of BBB related junction protein was significantly decreased by Western blotting and immunofluorescence(P<0.01),but there was no significant difference in the protein expression between the trauma group and the experimental group(P>0.05).Conclusion: Rap1 signaling pathway plays an important role in the protection of the blood-brain barrier after traumatic brain injury.After activation of Rap1,endothelial cell connective proteins were recruited through downstream Afadin,which increased the expression of BBB connective proteins after TBI,thereby reducing BBB permeability.Deduction of Afadin protein blocks the protective effect of Rap1 activation on BBB and increases BBB permeability.