Role of PI3K/Akt/GSK-3β signaling pathway in hydrogen-induced inhibition of neuronal apoptosis induced by focal cerebral ischemia-reperfusion in rats

Abstract

Objective To investigate the role of phosphatidylinositol 3-kinase/protein kinase B/glycogen synthase kinase-3β (PI3K/Akt/GSK-3β) signaling pathway in hydrogen-induced inhibition of neuronal apoptosis induced by focal cerebral ischemia-reperfusion (I/R)in rats. Methods One hundred and twenty healthy adult male Sprague-Dawley rats, weighing 200-220 g, were divided into 5 groups (n=24 each) using a random number table: sham operation group (group S); cerebral I/R group (group I/R); hydrogen group (group H2); LY294002 (specific PI3K inhibitor) group (group LY); LY294002+ hydrogen group (group LY+ H2). Focal cerebral I/R was induced by occlusion of the middle cerebral artery for 1 h followed by 24 h reperfusion.In H2 and H2+ LY groups, the animals inhaled 67% H2+ 33% O2 for 2 h starting from onset of reperfusion, and then inhaled H2 for 2 h every 6 h. In LY and LY+ H2 groups, LY294002 (10 mmol/L) 10 μl was injected into the lateral cerebral ventricle at 10 min before reperfusion.Neurologic deficit was evaluated and scored (NDS) at 24 h of reperfusion.The rats were then sacrificed, and the brains were removed for measurement of the cerebral infarct size (by TTC staining) and apoptosis in cortical neurons (by TUNEL) and for determination of the expression of Akt in the ischemic cerebral cortex, phosphorylated Akt (p-Akt), GSK-3β and phosphorylated GSK-3β (p-GSK-3β) and Bcl-2 and Bax positive cell count in the ischemic cerebral cortex (by immuno-histochemistry). The apoptosis index (AI), p-Akt/Akt ratio and p-GSK-3β/GSK3-β ratio were calculated. Results Compared with group S, the NDS, cerebral infarct size, AI, p-Akt/Akt ratio, p-GSK-3β/GSK-3β ratio and Bax positive cell count were significantly increased, and the Bcl-2 positive cell count was significantly decreased in group I/R (P 0.05). Compared with group H2, the NDS, cerebral infarct size, AI and Bax positive cell count were significantly increased, and the p-Akt/Akt ratio, p-GSK-3β/GSK-3β ratio and Bcl-2 positive cell count were significantly decreased in group LY+ H2(P<0.05). Conclusion The mechanism by which hydrogen inhibits focal cerebral I/R-induced neuronal apoptosis is associated with the activation of PI3K/Akt/GSK-3β signaling pathway in rats. Key words: Hydrogen; 1-Phosphatidylinositol 3-kinase; Protein-serine-threonine kinases; Glycogen synthase kinase 3; Apoptosis; Reperfusion injury; Brain