Occludin Degradation Makes Brain Microvascular Endothelial Cells More Vulnerable To Reperfusion Injury In Vitro

[Objective]Intracerebral hemorrhage is the most dangerous complication in tPA thrombolytic therapy for ischemic stroke,which occurs as a consequence of endothelial cell death at the blood brain barrier(BBB)during thrombolytic reperfusion.We have previously shown that cerebral ischemia induced rapid occludin degradation and BBB disruption.Here we demonstrated an important role of occludin degradation in facilitating the evolution of ischemic endothelial cells towards death.[Methods]Cultured brain microvascular endothelial cells(b End.3 cells)were exposed to oxygen-glucose deprivation(OGD)or incubated with occludin siRNA or occludin AAV to achieve an occludin deficiency or overexpression status before exposing to reoxygenation(R)or TNF-? treatment.Cell death was assessed by measuring lactate dehydrogenase(LDH)release,TUNEL staining and flow cytometry analysis.[Results]Our data show that:(1)Inhibition of occludin degradation alleviates OGD/R-induced apoptosis in endothelial cells;(2)Knockdown of occludin promotes TNF-?-induced endothelial cell apoptosis;(3)Knockdown of occludin exacerbates TNF-? induced pyroptosis and inflammation in endothelial cells;(4)Over-expression of occludin inhibits TNF-?-induced endothelial cell apoptosis and pyroptosis;(5)Inhibition of occludin degradation or overexpression of occludin led to c-Yes upregulation and the activation of AKT and ERK1/2 in bEnd.3 cells.[Conclusion]These data indicate that occludin degradation inflicted during ischemia makes BBB endothelial cells more vulnerable to reperfusion-associated stress stimuli.