The Basic Research On The Mechanism Of MiR-124-3p Affecting Blood-brain Barrier Through Adjusting Brain Microvascular Endothelial Cell Autophagy After TBI

ObjectiveThe main pathological changes caused by Traumatic Brain Injury(TBI)include nerve injury,intracranial hemorrhage and Blood-Brain Barrier(BBB)destruction.These pathological changes interact with each other to cause secondary brain injury,in which the nerve injury,brain edema and even brain hernia caused by secondary BBB destruction are serious.Therefore,the improvement of secondary BBB injury has important clinical and scientific significance for reducing the pathological changes and improving the prognosis after TBI.The previous study of our group found that the increase of miR-124-3p in microglia exosomes was the most significant after TBI,which could inhibit the activity of mTOR signal by directly targeting PDE4 B,thus inhibiting the development of neuroinflammation.And some studies have shown that autophagy plays an important role in the activity of vascular endothelial cells.Based on the above research results,we intend to establish an in vitro model of TBI and apply the techniques of transfection and hopping probe ion conductance microscopy to observe the changes of autophagy,apoptosis and tight junction protein of in brain microvascular endothelial cells(BMVECs)by regulating the expression of miR-124-3p,and then explore its effect on the function of BBB and its mechanism,so as to provide a theoretical basis for the new strategy of miR-124-3p in the treatment of TBI.Methods 1.b End.3 cells cultured in vitro were divided into five experimental groups: uninjured cells(Control),injured cells(Injury),injured cells treated with miR-124-3p mimics(I+miR-124-3p),injured cells treated with negative control(I+Negative Control),injured cells treated with miR-124-3p mimics and chloroquine(I+miR-124-3p+CQ).Mi R-124-3p mimics was transfected into b End.3 cells by cell transfection technique,and the transfection efficiency was detected by immunofluorescence staining and RT-PCR.2.The apoptosis of BMVECs was detected by CD31 and TUNEL fluorescence double staining,the expression of tight junction proteins Occludin and ZO-1 was detected by Western Blot,and the growth of tight junction was observed by hopping probe ion conductance microscopy to explore the effect of miR-124-3p on the survival of BMVECs and the function of BBB.3.Western Blot was used to detect the expression of autophagy-related proteins LC3-II/I,Beclin1 and p62 in TBI model,and to explore the effect of miR-124-3p differential expression BMVECs on autophagy in TBI model.4.CD31 and TUNEL fluorescence double staining was used to detect apoptosis in injured cells treated with miR-124-3p mimics(I+miR-124-3p),injured cells treated with miR-124-3p mimics and chloroquine(I+miR-124-3p+CQ)to explore the effect of autophagy on BMVECs apoptosis in TBI model.5.The changes of mTOR signal pathway related proteins mTOR and PDE4 B were detected by Western Blot,in order to explore the effect of miR-124-3p differential expression of mTOR signal pathway in TBI model.Results 1.Overexpression of miR-124-3p inhibits TBI induced apoptosis in BMVECs.2.Overexpression of miR-124-3p can reduce BBB damage induced by TBI.3.MiR-124-3p protects BBB by activating BMVECs autophagy after TBI.4.MiR-124-3p promotes BMVECs autophagy by inhibiting mTOR signal activity.ConclusionUp regulating the expression of miR-124-3p in BMVECs can inhibit the apoptosis of injured cells in TBI model in vitro.Further studies have found that miR-124-3p promotes BMVECs autophagy by reducing the expression of PDE4 B and inhibiting mTOR signal pathway,thus protecting BBB after traumatic brain injury.Therefore,miR-124-3p may become a potential therapeutic target for intervention of secondary BBB injury after TBI,so as to provide a theoretical basis for a new strategy of miR-124-3p in the treatment of TBI.