| Background:Disruption of the blood-brain barrier（BBB）integrity is an important mechanism of ischemic stroke,which further leads to damage to neurons and glial cells.Damage to endothelial cells,especially the destruction of tight junctions,is a very important part of the pathology of ischemic stroke.Nur77 is a member of the orphan nuclear receptor NR4 A family and plays a significant role in both ischemic stroke and vascular injury.In this study,we explored whether Nur77 ameliorates the damage of endothelial cell after cerebral ischemia and the destruction of the blood-brain barrier integrity,and finally plays a positive role in neural protection.In addition,we also explored the underlying molecular mechanism by which Nur77 improves endothelial tight junction injury.Methods:In the in vivo experiment,we first used the C57BL/6J mice and Nur77-/-mice objected to the right middle cerebral artery occlusion（MCAO）surgery.The MRI scan T2-weighted images and TTC staining were used to examine the infarct volume and edema after ischemic stroke between the two groups.The sensory and motor function of the two groups was tested by m NSS score,rotarod test,open field experiment and forelimb grip test.Moreover,the Evans Blue extravasation test and the FITC-Dextran extravasation test were used to detect the blood-brain barrier permeability of the two groups.Finally the release of MMPs and the expression of tight junction proteins were detected by immunofluorescence and Western Blot.In the in vitro experiment,we first established a model of extracorporeal blood-brain barrier using Transwell,and transfected endothelial cells with Nur77 knock-out and over-expressing virus.The effect of Nur77 knockout or overexpression on blood-brain barrier permeability was examined by transepithelial electrical residence（TEER）measurement and FITCDextran extravasation test.Western Blot was used to detect the effect of Nur77 knockdown or overexpression on tight junction protein expression in endothelial cells.To further explore the mechanism of action of Nur77 on endothelial injury,Western blot was used to detect the phosphorylation of ASK1 and its downstream molecules p38 and JNK on the MAPK pathway,and the corresponding inhibitors were used to observe their effects on the expression of tight junctions.Results:1)Infarct volume of Nur77-/-mice at 1 day,3 days,and 7 days after MCAO reperfusion was significantly increased compared with the sham group.At the same time,the results of m NSS score,open field test,rotarod test,and forelimb grip test showed that the sensory and motor function of Nur77-/-mice was significantly worse than that of the sham group.2)The permeability of the blood-brain barrier of Nur77-/-mice after MCAO was significantly increased compared with the sham group,and the release of MMPs in the brain was also increased.The expression of tight junction proteins in the cortical blood vessels of Nur77-/-mice after MCAO was significantly reduced compared with the sham group.3)In vitro OGD/R treatment of endothelial cells revealed that Nur77 knockout significantly increased the permeability of the blood-brain barrier model in vitro compared with the control group,while Nur77 overexpression partially reversed the increase in permeability of the blood-brain barrier model.The expression of tight junction proteins in Nur77 knockout endothelial cells was further reduced compared with the control group,while the expression of tight junction protein in Nur77 overexpressing endothelial cells was increased compared with the control group.4)The results of Western Blot showed that the phosphorylation levels of ASK1 and p38 in Nur77 knockout endothelial cells were further increased compared with the control group after OGD/R treatment of endothelial cells in vitro;inhibition of ASK1 and p38 phosphorylation reversed further destruction of endothelial tight junctions caused by Nur77 knockdown.Conclusion:Nur77 inhibits the destruction of endothelial cell tight junction after ischemiareperfusion injury,ameliorates the damage of blood-brain barrier integrity and finally plays a protective role in ischemic stroke.The protective effect of Nur77 on endothelial injury is related to the MAPK signal transduction pathway. |
