Reactive oxygen species (ROS) often promote acute brain injury after stroke, but their roles in the recovery phase have not been well studied. We tested the hypothesis that ROS activity mediated by NADPH oxidase 2 (NOX2) contributes to acute brain injury but promotes functional recovery during the delayed phase, which is linked with neuroinflammation, autophagy, angiogenesis, and the PI3K/Akt signaling pathway. We used the NOX2 inhibitor apocynin to study the role of NOX2 in brain injury and functional recovery in a middle cerebral artery occlusion (MCAO) stroke mouse model. Infarct size, neurological deficits and behavior were evaluated on days 3, 7, 10 and 14 after reperfusion. In addition, dynamic NOX2-induced ROS levels were measured by dihydroethidium (DHE) staining. Autophagy, inflammasomes, and angiogenesis were measured by immunofluorescence staining and western blotting. RNA sequencing was performed, and bioinformatics technology was used to analyze differentially expressed genes (DEGs), as well as the enrichment of biological functions and signaling pathways in ischemia penumbra at 7 days after reperfusion. Then, Akt pathway-related proteins were further evaluated by western blotting. Our results showed that apocynin injection attenuated infarct size and mortality 3 days after stroke but promoted mortality and blocked functional recovery from 5 to 14 days after stroke. DHE staining showed that ROS levels were increased at 3 days after reperfusion and then gradually declined in WT mice, and these levels were significantly reduced by the NOX2 inhibitor apocynin. RNA-Seq analysis indicated that apocynin activated the immune response under hypoxic conditions. The immunofluorescence and western blot results demonstrated that apocynin inhibited the NLRP3 inflammasome and promoted angiogenesis at 3 days but promoted the NLRP3 inflammasome and inhibited angiogenesis at 7 and 14 days after stroke, which was mediated by regulating autophagy activation. Furthermore, RNA-Seq and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that apocynin injection resulted in PI3K–Akt signaling pathway enrichment after 7 days of MCAO. We then used an animal model to show that apocynin decreased the protein levels of phosphorylated PI3K and Akt and NF-κB p65, confirming that the PI3K–Akt–NF-κB pathway is involved in apocynin-mediated activation of inflammation and inhibition of angiogenesis. NOX2-induced ROS production is a double-edged sword that exacerbates brain injury in the acute phase but promotes functional recovery. This effect appears to be achieved by inhibiting NLRP3 inflammasome activation and promoting angiogenesis via autophagy activation.

中文翻译：

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NOX2 介导的活性氧是小鼠局灶性脑缺血的双刃剑

活性氧 (ROS) 通常会促进中风后的急性脑损伤，但它们在恢复阶段的作用尚未得到很好的研究。我们检验了由 NADPH 氧化酶 2 (NOX2) 介导的 ROS 活性有助于急性脑损伤但在延迟期促进功能恢复的假设，这与神经炎症、自噬、血管生成和 PI3K/Akt 信号通路有关。我们使用 NOX2 抑制剂夹竹桃麻素来研究 NOX2 在大脑中动脉闭塞 (MCAO) 中风小鼠模型中脑损伤和功能恢复中的作用。在再灌注后第 3、7、10 和 14 天评估梗死面积、神经功能缺损和行为。此外，通过二氢乙锭 (DHE) 染色测量动态 NOX2 诱导的 ROS 水平。自噬、炎症小体、通过免疫荧光染色和蛋白质印迹测量血管生成。进行RNA测序，利用生物信息学技术分析差异表达基因（DEGs），以及再灌注后7天缺血半影区生物学功能和信号通路的富集情况。然后，通过蛋白质印迹进一步评估 Akt 通路相关蛋白。我们的研究结果表明，夹竹桃麻注射液可在中风后 3 天减轻梗死面积和死亡率，但在中风后 5 至 14 天会促进死亡率并阻止功能恢复。DHE染色显示，再灌注后3天，WT小鼠的ROS水平升高，然后逐渐下降，而NOX2抑制剂夹竹桃苷显着降低了这些水平。RNA-Seq 分析表明夹竹桃麻素在缺氧条件下激活免疫反应。免疫荧光和蛋白质印迹结果表明，夹竹桃苷在 3 天抑制 NLRP3 炎性体并促进血管生成，但在卒中后 7 天和 14 天促进 NLRP3 炎性体并抑制血管生成，这是通过调节自噬激活介导的。此外，RNA-Seq 和京都基因和基因组百科全书 (KEGG) 分析表明，在 MCAO 7 天后，夹竹桃麻注射液导致 PI3K-Akt 信号通路富集。然后我们使用动物模型显示夹竹桃苷降低了磷酸化 PI3K 和 Akt 以及 NF-κB p65 的蛋白质水平，证实 PI3K-Akt-NF-κB 通路参与了夹竹桃苷介导的炎症激活和血管生成抑制。NOX2 诱导的 ROS 产生是一把双刃剑，在急性期会加剧脑损伤，但会促进功能恢复。这种效果似乎是通过抑制 NLRP3 炎性体激活和通过自噬激活促进血管生成来实现的。