MiR-149-5p Alleviates Blood-brain Barrier Permeability By Targeting S1PR2 Of Pericytes After Acute Cerebral Ischemia/reperfusion Injury In Rats

Objective Blood–brain barrier(BBB)disruption caused by reperfusion injury after ischemic stroke is an intractable event conducive to further injury.Brain pericytes play a vital role in maintaining BBB integrity by interacting with other components of the BBB.This study targets pericytes to explore new strateges for assessment and treatment for BBB injury induced by cerebral ischemia/reperfusion.Methods In this study,transient cerebral infarction model(tMCAO)of Sprague-Dawley(SD)rats and glucose/oxygen deprivation/reoxygenation model(OGD/R)were used to simulate ischemia/reperfusion in vivo and in vitro.Intracerebroventricular injection of micro RNA(miR)-149-5p analogue 149-agomir and inhibitor 149-antagomir were used to up-regulate and down-regulate miR-149-5p in vivo,respectively.Intraperitoneal injection of sphingosine-1-phosphate receptor(S1PR)2 selective receptor antagonist JTE-013 inhibited S1PR2 activity.A modified neurological severity scale(m NSS)was used to assess the neurological deficits of rats.MRI scans and Evans blue extravasation assay were performed to assess BBB permeability in vivo.And transendothelial electrical resistance(TEER)and FITC-Dextran permeability assessed the permeability of the in vitro BBB model.S1pr2 gene expression was knocked down by S1PR2 si RNA.Wound healing and Transwell migration assay were used to detect the migration ability of pericytes in vitro.Real-time quantitative PCR(q RT-PCR)was used to detect miR-149-5p levels and related gene m RNA expressions.Simultaneously,Western Blot assay was used to detect the expression levels of various proteins,and immunofluorescence confocal microscopy was used to locate S1PR2 and N-cadherin expression in pericytes.Results In this study,we found that S1PR2 expressed in pericytes was significantly up-regulated after ischemia/reperfusion in vivo and in vitro.By using the S1PR2 antagonist JTE-013,we showed that S1PR2 plays a critical role in the induction of BBB permeability of transientmiddle cerebral artery occlusion(t MCAO)rats and the in vitro BBB model.Furthermore,we discovered that S1PR2 may decrease N-cadherin expression and increase pericyte migration via NF-k B p65 signal.We also found that S1PR2 could be regulated by miR-149-5p negatively,which was decreased in the ischemic boundary zone(IBZ),peripherial blood and cultured pericytes after ischemia/reperfusion.Overexpression of miR-149-5p in cultured pericytes substantially increased N-cadherin expression and decreased pericyte migration,which decreased BBB leakage in the in vitro model.Up-regulating miR-149-5p by intracerebroventricular injection of agomir-149-5p attenuated BBB permeability and improved the outcomes of t MCAO rats significantly.Conclusions miR-149-5p could alleviate BBB permeability by targeting S1PR2 of pericytes after acute cerebral ischemia/reperfusion injury in rats.Our data suggest that miR-149-5p may serve as a potential target for assessment and treatment of BBB disruption after ischemic stroke.