Preconditioning of endoplasmic reticulum stress protects against acrylonitrile-induced cytotoxicity in primary rat astrocytes: The role of autophagy.

ABSTRACT

This study explored the protective effects of endoplasmic reticulum (ER) stress preconditioning induced by 2-deoxy-d-glucose (2-DG) or oxidized dithiothreitol (DTTox) on acrylonitrile (AN)-induced cytotocity in primary rat astrocytes. Cells were pretreated with 2-DG or DTTox for different times at various concentration. Next, astrocytes were treated with 2.5mM AN for an additional 12h. Cell viability and cytotoxicity were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) leakage, respectively. Reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) were determined. Expression of glucose-regulated protein 78 (GRP78), phosphorylated-eukaryotic translation initiation factor 2α (p-eIF2α), microtubule-associated protein light chain 3 (LC3), P62, and Beclin1 were used to assess autophagy. In addition, 3-methyadenine (3-MA), an autophagy-specific inhibitor, was used to assess the role of autophagy in ER stress preconditioning-induced protection against AN cytotoxicity. The results showed that AN alone significantly decreased astrocytic viability and enhanced cytotoxicity. Compared to the AN-alone group, preconditioning with 2-DG or DTTox significantly increased cell viability and reduced cytotoxicity to indistinguishable levels. Decreased ROS generation and increased ΔΨm were also inherent to ER stress preconditioning with these compounds. Furthermore, autophagy was activated by both 2-DG and DTTox. Blockage of autophagy attenuated the protection afforded by 2-DG or DTTox preconditioning in AN-treated astrocytes. These results establish that ER stress preconditioning affords cellular protection against AN, and that activation of autophagy mediates the cytoprotection. Modulation of ER stress and resultant activation of autophagy may be a novel target for to ameliorate AN toxicity.