Atmospheric pressure plasma preconditioning reduces oxygen and glucose deprivation induced human neuronal SH-SY5Y cells apoptosis by activating protective autophagy and ROS/AMPK/mTOR pathway

Abstract

Reactive oxygen species (ROS)/reactive nitrogen species (RNS) exert a “double edged” effect on the occurrence and development of ischemic stroke. We previously indicate that atmospheric pressure plasma (APP) shows a neuroprotective effect in vitro based on the ROS/RNS generations. However, the mechanism is still unknown. In this work, SH-SY5Y cells were treated with oxygen and glucose deprivation (OGD) injuries for stimulating the ischemic stroke pathological injury process. A helium APP was used for SH-SY5Y cell treatment for evaluating the neuroprotective impacts of APP preconditioning against OGD injuries with the optimized parameters. During the preconditioning, APP significantly raised the extracellular and intracellular ROS/RNS production. As a result, APP preconditioning increased SH-SY5Y cell autophagy by elevating LC3-II/LC3-I ratio and autophagosome formation. Meanwhile, APP preconditioning reduced cell apoptosis caused by OGD with the increased APP treatment time, which was abolished by pretreatment with autophagy inhibitor 3-methyladenine (3-MA). The ROS scavenger N-acetyl-L-cysteine (NAC) alone or combined with NO scavenger carboxy-PTIO abolished the APP preconditioning induced SH-SY5Y autophagy and the cytoprotection, whereas the NO scavenger alone did not. In addition, we observed the elevated phosphorylation of AMP-activated protein kinase (AMPK) and decreased phosphorylation of mammalian target of rapamycin (mTOR) in APP treated SH-SY5Y cells. This effect was attenuated by AMPK inhibitor Compound C (CC), the ROS scavenger NAC and autophagy inhibitor 3-MA. Furthermore, the cytoprotective effect of APP was preliminarily confirmed in the rats of middle cerebral artery occlusion (MCAO) model. Results showed that APP inhalation by rats during MCAO process could improve neurological functions, reduce cell apoptosis in brain tissues and decrease cerebral infarct volume. Our data suggested that ROS produced by APP preconditioning played a vital role in the neuroprotective effect of SH-SY5Y cells against OGD injuries by activating autophagy and ROS/AMPK/mTOR pathway.