| Neuronal death as a result of neuronal injury following hypoxic/ischemic insults, such as stroke, is an irreversible process that leads to long term neurological deficit. The prevention of neuronal injury is therefore critical in rescuing the brain from neurological disaster. However, clinical strategies that may help mitigate the effects of hypoxic/ischemic injury are still very limited.δ-opioid receptor (DOR) is a guanine nucleotide-binding regulatory protein (G protein)-coupled receptor that is widely distributed in different mammalian cells. It is well established in recent years that stimulating delta-opioid receptor (DOR) with its specific agonists elicits neuroprotection against hypoxia/ischemia. However, the underlying mechanisms are not well understood yet.Mitochondria are key players in apoptosis regulation during hypoxia/ischemia. Inadequate oxygen supply perturbs mitochondrial membrane potentials, diminishes their ability to buffer cytosolic calcium, and increases formation of free radicals. Irreversible mitochondrial dysfunction further leads to the opening of mitochondrial permeability transition pore and the release of apoptosis-inducing factors, which constitute a point of no return in cell commitment to death. And suppressing mitochondrial dysfunction helps greatly to prevent neurons from entering mitochondria-dependent cell death pathways during hypoxia/ischemia.DOR is an oxygen-sensitive membrane protein, whose expression is severely suppressed by hypoxia/ischemia. The mechanisms underlying are unclear, though it is suggested that hypoxic/ischemic insults might promote epigenetic reprogramming of DOR expression in neurons.In this investigation, we studied the effects of DOR activation on mitochondrial injury and mitochondria-dependent cell death pathways in rat primary cortical neuronal cultures exposed to sodium azide (NaN3), and explore the possible mechanisms underlying. The exposure of primary cortical neuronal cultures to NaN3 has been widely adopted as a mitochondrial dysfunction model to investigate hypoxia/ischemia-induced injuries and neuroprotection against these injuries in vitro.Here we show that selective DOR activation reverses mitochondrial dysfunction in primary rat cortical neuronal cultures treated with 10 mM NaN3 for 30 min, including mitochondrial membrane depolarization, mitochondrial Ca2+ overload and reactive oxygen species generation. Short-term stimulation of DOR also inhibits cytochrome c release and caspase-3 activation in neurons, and attenuates the severe neuronal injury caused by NaN3 insults. Further study on the mechanisms underlying indicates that DOR activation prevents mitochondrial dysfunction, cytochrome c release and caspase-3 activation induced by NaN3 treatment for 30 min mainly through PKC/mitochondrial ERK pathway.We also find that selectively stimulating DOR attenuates cell damage in primary rat cortical neurons exposed with 80μM NaN3 for 2 days. DOR protein levels in neurons are down-regulated during NaN3 treatment, which is reversed by prolonged DOR activation and may contribute greatly to its neuroprotective effects, since DOR expression is a critical determinant of neuronal survival during prolonged stress. DOR stimulation also reverses the down-regulation of Bcl-2 caused by NaN3 treatment for 2 days, which prevents cytochrome c release and ensuing neuronal death. Further investigation on the mechanisms underlying demonstrates that DOR activation suppresses neuronal injury induced by NaN3 exposure for 2 days mainly through PI3K/Akt/NF-KB pathway. Prolonged DOR stimulation prevents the down-regulation of DOR and Bcl-2 by increasing the binding of NF-κB to their promoters and promoting histone acetylation. |
