Anagliptin Protected against Hypoxia/Reperfusion-Induced Brain Vascular Endothelial Permeability by Increasing ZO-1.

Abstract

Background and purpose: Cerebral ischemia-reperfusion injury is commonly induced during the treatment of ischemic stroke and is reported to be related to the blood–brain barrier destruction and brain vascular endothelial cell dysfunction. Anagliptin is a novel antidiabetic agent recently reported to protect neurons from oxidative stress. In the present study, we aim to investigate the protective property of anagliptin against oxygen–glucose deprivation and reperfusion (OGD/R)-induced injury on endothelial cells and clarify the potential underlying mechanism. Methods: OGD/R modeling was established on bEnd.3 brain endothelial cells. Cell viability was detected using the MTT assay, and the mitochondrial reactive oxygen species (ROS) level was measured using the mitoses red staining assay. The endothelial monolayer permeability was determined using an FITC-dextran permeation assay. The expression levels of NOX-4 and ZO-1 were evaluated using qRT-PCR and Western blot assays. The expressions of MLC-2, p-MLC-2, and myosin light chain kinase (MLCK) were determined using Western blot. Results: First, the decreased cell viability, upregulated NOX-4, and elevated mitochondrial ROS level in the endothelial cells induced by OGD/R were reversed by treatment with anagliptin. Second, the enlarged endothelial permeability and the decreased expression level of ZO-1 in the endothelial cells induced by OGD/R were alleviated by anagliptin. Third, the downregulation of ZO-1 and enlarged brain endothelial monolayer permeability induced by OGD/R were ameliorated by an MLCK inhibitor, ML-7. Lastly, the elevated expressions of MLCK and p-MLC-2 induced by OGD/R were suppressed by anagliptin. Conclusion: Anagliptin protected against hypoxia/reperfusion-induced brain vascular endothelial permeability by increasing the expression ZO-1, mediated by inhibition of the MLCK/MLC-2 signaling pathway.