Graphene quantum dot antioxidant and proautophagic actions protect SH-SY5Y neuroblastoma cells from oxidative stress-mediated apoptotic death

Abstract

We investigated the ability of graphene quantum dot (GQD) nanoparticles to protect SH-SY5Y human neuroblastoma cells from oxidative/nitrosative stress induced by iron-nitrosyl complex sodium nitroprusside (SNP). GQD reduced SNP cytotoxicity by preventing mitochondrial depolarization, caspase-2 activation, and subsequent apoptotic death. Although GQD diminished the levels of nitric oxide (NO) in SNP-exposed cells, NO scavengers displayed only a slight protective effect, suggesting that NO quenching was not the main protective mechanism of GQD. GQD also reduced SNP-triggered increase in the intracellular levels of hydroxyl radical (^•OH), superoxide anion (O₂^•-), and lipid peroxidation. Nonselective antioxidants, ^•OH scavenging, and iron chelators, but not superoxide dismutase, mimicked GQD cytoprotective activity, indicating that GQD protect cells by neutralizing ^•OH generated in the presence of SNP-released iron. Cellular internalization of GQD was required for optimal protection, since a removal of extracellular GQD by extensive washing only partly diminished their protective effect. Moreover, GQD cooperated with SNP to induce autophagy, as confirmed by the inhibition of autophagy-limiting Akt/PRAS40/mTOR signaling and increase in autophagy gene transcription, protein levels of proautophagic beclin-1 and LC3-II, formation of autophagic vesicles, and degradation of autophagic target p62. The antioxidant activity of GQD was not involved in autophagy induction, as antioxidants N-acetylcysteine and dimethyl sulfoxide failed to stimulate autophagy in SNP-exposed cells. Pharmacological inhibitors of early (wortmannin, 3-methyladenine) or late stages of autophagy (NH₄Cl) efficiently reduced the protective effect of GQD. Therefore, the ability of GQD to prevent the in vitro neurotoxicity of SNP depends on both ^•OH/NO scavenging and induction of cytoprotective autophagy.

Keywords: Autophagy; Graphene quantum dots; Hydroxyl radical; Neurotoxicity; Nitric oxide; Oxidative stress; Sodium nitroprusside.