Glibenclamide protects neurovascular units in rats with cerebral ischemia/reperfusion injury by regulating inflammatory signaling pathway HSP70/p-Akt/MMP-9/COX-2

Objective To investigate the protective effect of glibenclamide on neurovascular units (NVUs) and its possible mechanism in cerebral ischemia/reperfusion injury models. Methods One hundred and twenty healthy male SD rats were randomly divided into sham-operated group, model group, and glibenclamide (GBC) treatment group (n=40). Two h reperfusion models of acute focal middle cerebral artery occlusion were prepared by thread occlusion in rats of the latter two groups; rats in the model group were treated with 0.05% DMSO saline solution two h after ischemia, and rats in the GBC treatment group were given intraperitoneal injection of 10 μg/kg GBC with single dose. Immunofluorescence and Western blotting were used to detect the protein levels of sulfonylurea receptor 1 (SUR1) and transient receptor potential cation channel subfamily M member 4 (TRPM4) 8 h after reperfusion, and ELISA was used to detect the plasma level of matrix metalloproteinase 9 (MMP-9). At 24 h after reperfusion, Zea Longa scale was used to determine the neurological deficits; water content in the brain tissues was detected by dry and wet weight method, and blood-brain barrier (BBB) permeability was detected by Evans blue (EB) staining; Nissl staining was employed to detect the survival neurons; ionized calcium bindingadaptor molecule-1 (Iba-1) and cyclooxygenase-2 (COX-2) positive cells and IgG seepage quantity were detected by immumohistochemical staining to assess the neuro-vascular inflammation; the expressions of heat shock protein 70 (HSP70), phosphorylated protein kinase B (p-Akt), phosphorylated c-jun amino-terminal kinase (p-JNK), and phosphatidylinositol-3 kinase (PI3K) were detected by Western blotting. Results (1) At 8 h after reperfusion, the protein expressions of SUR1 and TRPM4 in the brain tissues of the model group were significantly increased as compared with those of the sham-operated group (P 0.05). As compared with the sham-operated group, the model group had significantly higher MMP-9 level (P<0.05); as compared with the model group, the GBC treatment group had significantly lower MMP-9 level (P<0.05). (2) At 24 h after reperfusion, as compared with the sham-operated group, the model group had significantly increased Zea Longa scale scores, statistically increased brain water content, significantly increased EB permeability, significantly increased IgG seepage quantity, significantly smaller number of Nissl's staining-positive neurons, significantly larger number of Iba-1, COX-2 positive cells, and significantly decreased protein expressions of HSP70 and p-Akt (P<0.05); as compared with the model group, the GBC treatment group had significantly decreased Zea Longa scores, statistically decreased brain water content, significantly decreased EB permeability, significantly decreased IgG seepage quantity, significantly larger number of Nissl's staining-positive neurons, significantly smaller number of Iba-1, COX-2 positive cells, and significantly increased protein expressions of HSP70 and p-Akt (P<0.05). Conclusion SUR1-TRPM4 expression is increased after cerebral ischemia/reperfusion injury, and inhibition of SUR1-TRPM4 with GBC shows a protective role in NVUs after cerebral ischemia/reperfusion injury, possibly by regulating HSP70/p-Akt/MMP-9/COX-2 inflammatory signal pathway. Key words: Sulfonylurea receptor 1; Transient receptor potential cation channel subfamily M4; Glibenclamide; Ischemia/reperfusion injury; Heat shock protein 70; Protein kinase B; Matrix metalloproteinase 9; Cyclooxygenase-2; Neurovascular unit