We have previously demonstrated the significance of endothelial cell-expressed α5β1 integrin in ischemic stroke, having shown that α5β1 integrin endothelial cell-selective knockout mice are significantly resistance to ischemic stroke injury via preservation of the tight junction protein claudin-5 and subsequent stabilization of the blood–brain barrier (BBB). In addition, inhibition of α5β1 by the small peptide noncompetitive integrin α5 inhibitor, ATN-161, is beneficial in a mouse model of ischemic stroke through reduction of infarct volume, edema, stabilization of the BBB, and reduced inflammation and immune cell infiltration into the brain. In continuation with our previous findings, we have further evaluated the mechanistic role of ATN-161 in vitro and found that oxygen and glucose deprivation and reperfusion (OGD/R)-induced inflammation, oxidative stress, apoptosis, mitochondrial depolarization, and fibrosis attenuate tight junction integrity via induction of α5, NLRP3, p-FAK, and p-AKT signaling in mouse brain endothelial cells. ATN-161 treatment (10 µM) effectively inhibited OGD/R-induced extracellular matrix (ECM) deposition by reducing integrin α5, MMP-9, and fibronectin expression, as well as reducing oxidative stress by reducing mitochondrial superoxide radicals, intracellular ROS, inflammation by reducing NLRP3 inflammasome, tight junction loss by reducing claudin-5 and ZO-1 expression levels, mitochondrial damage by inhibiting mitochondrial depolarization, and apoptosis via regulation of p-FAK and p-AKT levels. Taken together, our results further support therapeutically targeting α5 integrin with ATN-161, a safe, well-tolerated, and clinically validated peptide, in ischemic stroke.

我们先前已经证明内皮细胞表达的α5β1整合素在缺血性中风中的重要性，表明α5β1整合素内皮细胞选择性敲除小鼠通过保留紧密连接蛋白claudin-5和随后的血液稳定来显着抵抗缺血性中风损伤-脑屏障（BBB）。此外，小肽非竞争性整合素 α5 抑制剂 ATN-161 对 α5β1 的抑制作用通过减少梗塞体积、水肿、稳定 BBB 以及减少炎症和免疫细胞浸润到缺血性中风的小鼠模型中是有益的。脑。继续我们之前的研究结果，我们进一步评估了 ATN-161 在体外的机制作用，发现氧气和葡萄糖剥夺和再灌注 (OGD/R) 诱导的炎症，氧化应激、细胞凋亡、线粒体去极化和纤维化通过诱导小鼠脑内皮细胞中的 α5、NLRP3、p-FAK 和 p-AKT 信号传导减弱紧密连接的完整性。ATN-161 处理 (10 µM) 通过减少整合素 α5、MMP-9 和纤连蛋白的表达，以及通过减少线粒体超氧化物自由基、细胞内 ROS、炎症来减少氧化应激，有效抑制 OGD/R 诱导的细胞外基质 (ECM) 沉积通过减少 NLRP3 炎性体，通过降低 claudin-5 和 ZO-1 表达水平导致紧密连接丢失，通过抑制线粒体去极化导致线粒体损伤，通过调节 p-FAK 和 p-AKT 水平导致细胞凋亡。总之，我们的结果进一步支持用 ATN-161 治疗靶向 α5 整合素，ATN-161 是一种安全、耐受性良好且经过临床验证的肽，