Influence of mTOR signaling pathway on ketamine-induced injuries in the hippocampal neurons of rats

Abstract

ABSTRACTObjective: To explore the influences of mammalian target of rapamycin (mTOR) signaling pathway on ketamine-induced apoptosis, oxidative stress and Ca2+ concentration in the hippocampal neurons of rats.Methods: The primary hippocampal neurons isolated from fetal Sprague Dawley rats were treated with ketamine (0, 50, 100 and 500 μM) for 4 days to observe its effect on mTOR signaling pathway and apoptosis of rat hippocampal neurons. Then, the hippocampal neurons were divided into C (Control), R (Rapamycin, an inhibitor of mTOR signaling pathway), K (Ketamine) and R + K (Rapamycin + Ketamine) groups to detect the apoptosis, reactive oxygen species (ROS) production, and Ca2+ concentration via the terminal transferase uridyl nick end labelling (TUNEL) assay, dichloro-dihydro-fluorescein diacetate (DCFH-DA) method and Fluo-3 acetoxymethyl ester (Fluo-3AM) staining, respectively. The expressions of mTOR signaling pathway and apoptosis-related proteins in hippocampal neurons were examined by qRT-PCR and Western blot.Results: Ketamine could dose-dependently promote the apoptosis of rat hippocampal neurons with upregulation of p-mTOR and its downstream regulators (p-4E-BP-1 and p-p70S6K). However, ketamine-induced apoptosis in hippocampal neurons was reversed significantly by the administration of rapamycin, as evident by the decrease in expressions of pro-apoptotic proteins (Bax and cleaved Caspase-3) and the increase in anti-apoptotic protein (Bcl-2). Meanwhile, the ROS generation and Ca2+ concentration was inhibited accompanied with reduced malonildialdehyde levels but elevated superoxide and glutathione peroxidase activities.Conclusion: Inhibition of mTOR signaling pathway protected rat hippocampal neurons from ketamine-induced injuries via reducing apoptosis, oxidative stress, as well as Ca2+ concentration.Abbreviations: mTOR: mammalian target of rapamycin; SD: Sprague-Dawley; SPF: Specific-pathogen free; ROS: reactive oxygen species; TUNEL: terminal transferase uridyl nick end labelling; DCFH-DA: Dichloro-dihydro-fluorescein diacetate; Fluo-3A: Fluo-3 acetoxymethyl ester; NMDAR: non-competitive N-methyl-D-aspartame glutamate receptor; 4E-BP1: 4E binding protein 1; p70S6K: p70 S6 Kinase; PCR: Polymerase chain reaction; MDA: malonildialdehyde; GSH-PX: glutathione peroxidase; ANOVA: One-way Analysis of Variance.