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 toward death. Cultured brain microvascular endothelial cells (bEnd.3 cells) were exposed to oxygen‐glucose deprivation (OGD) or incubated with occludin siRNA or occludin AAV to achieve an occludin deficiency or over‐expression status before exposing to reoxygenation (R) or TNF‐α treatment. Cell death was assessed by measuring lactate dehydrogenase release, TUNEL staining, and flow cytometry analysis. Inhibition of OGD‐induced occludin degradation with SB‐3CT or over‐expression of occludin with occludin AAV both significantly attenuated OGD/R‐induced apoptosis and pyroptosis in bEnd.3 cells. Consistently, knockdown of occludin with siRNA potentiated TNF‐α‐induced apoptosis, supporting an important role of occludin integrity in endothelial cell survival. Similar results were observed for pyroptosis, in which occludin knockdown with siRNA led to a significant augmentation of cytokines secretion, inflammasome activation, and pyroptosis occurrence in TNF‐α‐treated bEnd.3 cells. Lastly, up‐regulation of c‐Yes, PI3K/AKT, and ERK concurrently occurred with occludin degradation after OGD/R or TNF‐α treatment, and the level of these proteins were further increased when inhibition of occludin degradation or over‐expression of occludin. These data indicate that occludin degradation inflicted during ischemia makes BBB endothelial cells more vulnerable to reperfusion‐associated stress stimuli.

中文翻译：

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闭合蛋白的降解使脑微血管内皮细胞在体外更容易受到再灌注损伤

脑出血是tPA溶栓治疗缺血性卒中最危险的并发症，这是溶栓再灌注过程中血脑屏障（BBB）处内皮细胞死亡的结果。先前我们已经表明，脑缺血可导致快速的occludin降解和BBB破坏。在这里，我们证明了occludin降解在促进缺血性内皮细胞向死亡的进化中的重要作用。将培养的脑微血管内皮细胞（bEnd.3细胞）暴露于氧葡萄糖剥夺（OGD）或与occludin siRNA或occludin AAV孵育以达到occludin缺乏或过表达状态，然后再暴露于复氧（R）或TNF-α治疗。通过测量乳酸脱氢酶释放，TUNEL染色和流式细胞仪分析评估细胞死亡。SB-3CT抑制OGD诱导的occludin降解或occludin AAV过量表达occludin均显着减弱了bEnd.3细胞中OGD / R诱导的凋亡和凋亡。一致地，用siRNA抑制occludin增强了TNF-α诱导的细胞凋亡，支持occludin完整性在内皮细胞存活中的重要作用。对于发烧，也观察到了相似的结果，其中siRNA封闭闭合蛋白导致在TNF-α处理的bEnd.3细胞中细胞因子分泌，炎症小体激活和发烧的显着增加。最后，在OGD / R或TNF-α处理后，c-Yes，PI3K / AKT和ERK的上调与occludin降解同时发生，并且当抑制occludin降解或过度表达时，这些蛋白的水平进一步升高。封端蛋白。