Miao Shu-Hao, Gao Sheng-Qing, Li Hui-Xin, Zhuang Yun-Song, Wang Xue, Li Tao, Gao Chao-Chao, Han Yan-Ling, Qiu Jia-Yin, Zhou Meng-Liang
Department of Neurosurgery, Jinling Hospital, Jinling School of Clinical Medicine, Nanjing Medical University, Nanjing, China.
Department of Neurosurgery, Jinling Hospital, Medical School of Nanjing University, Nanjing, China.
Front Mol Neurosci. 2023 Mar 30;16:1121944. doi: 10.3389/fnmol.2023.1121944. eCollection 2023.
Endothelial nitric oxide synthase (eNOS) uncoupling plays a significant role in acute vasoconstriction during early brain injury (EBI) after subarachnoid hemorrhage (SAH). Astrocytes in the neurovascular unit extend their foot processes around endothelia. In our study, we tested the hypothesis that increased nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) expression in astrocytes after SAH leads to eNOS uncoupling.
We utilized laser speckle contrast imaging for monitoring cortical blood flow changes in mice, nitric oxide (NO) kits to measure the level of NO, and a co-culture system to study the effect of astrocytes on endothelial cells. Moreover, the protein levels were assessed by Western blot and immunofluorescence staining. We used CCK-8 to measure the viability of astrocytes and endothelial cells, and we used the HO kit to measure the HO released from astrocytes. We used GSK2795039 as an inhibitor of NOX2, whereas lentivirus and adeno-associated virus were used for dihydrofolate reductase (DHFR) knockdown and .
The expression of NOX2 and the release of HO in astrocytes are increased, which was accompanied by a decrease in endothelial DHFR 12 h after SAH. Moreover, the eNOS monomer/dimer ratio increased, leading to a decrease in NO and acute cerebral ischemia. All of the above were significantly alleviated after the administration of GSK2795039. However, after knocking down DHFR both and , the protective effect of GSK2795039 was greatly reversed.
The increased level of NOX2 in astrocytes contributes to decreased DHFR in endothelial cells, thus aggravating eNOS uncoupling, which is an essential mechanism underlying acute vasoconstriction after SAH.
内皮型一氧化氮合酶（eNOS）解偶联在蛛网膜下腔出血（SAH）后早期脑损伤（EBI）期间的急性血管收缩中起重要作用。神经血管单元中的星形胶质细胞在血管内皮周围伸出其足突。在我们的研究中，我们检验了以下假设：SAH后星形胶质细胞中烟酰胺腺嘌呤二核苷酸磷酸氧化酶2（NOX2）表达增加会导致eNOS解偶联。
我们利用激光散斑对比成像监测小鼠皮质血流变化，使用一氧化氮（NO）检测试剂盒测量NO水平，并使用共培养系统研究星形胶质细胞对内皮细胞的影响。此外，通过蛋白质印迹和免疫荧光染色评估蛋白质水平。我们使用CCK-8测量星形胶质细胞和内皮细胞的活力，并使用HO检测试剂盒测量星形胶质细胞释放的HO。我们使用GSK2795039作为NOX2抑制剂，而慢病毒和腺相关病毒用于敲低二氢叶酸还原酶（DHFR）。
SAH后12小时，星形胶质细胞中NOX2的表达和HO的释放增加，同时内皮细胞中DHFR减少。此外，eNOS单体/二聚体比例增加，导致NO减少和急性脑缺血。给予GSK2795039后，上述所有情况均得到显著缓解。然而，在敲低DHFR后，GSK2795039的保护作用大大逆转。
星形胶质细胞中NOX2水平升高导致内皮细胞中DHFR减少，从而加重eNOS解偶联，这是SAH后急性血管收缩的重要机制。
