Autophagic Neuroprotection Of ? Opioid Peptide DADLE Against Cerebral Ischemic Stroke

Ischemic stroke has become a major disease threatening human health around the world,and clinical therapies for the disease are still limited until now.In recent years,much more interests have been increasingly drawn on the delta opioid receptor(DOR)-mediated neuroprotection against hypoxic/ischemic injury.Our previous studies revealed that DOR activation with [d-Ala2,d-Leu5] enkephalin(DADLE),a selective DOR agonist,could alleviate ischemia-reperfusion injury and improve cognitive impairment after ischemia.Meanwhile,it could significantly promote neuronal survival and astrocytic activation,and reduce damaged astrocytes in the rat hippocampus on day 3 after ischemia.However,the mechanism of DOR activation with DADLE improving ischemic neuronal survival is still unclear.In vitro study showed that DOR activation with DADLE induced autophagy in astrocytes against OGD injury.Evidence is mounting that autophagy plays an important role in cerebral ischemic injury,while the regulation of ULK1 by AMPK and mTOR has impressively impact on autophagy.Thus,we hypothesized that DADLE-induced DOR activation enhanced autophagy to improve neuronal survival against ischemic injury via AMPK/mTOR/ULK1 signaling pathway.The present study in SD rats and SH-SY5 Y cells revealed that DADLE-induced DOR activation had effect on neuronal autophagy by AMPK-dependent signaling after ischemia.The study is divided into two parts,and the specific contents are as follows.1.Effect of DOR activation on autophagy in rats after global cerebral ischemia/reperfusion injury and its relevant molecular mechanismThe rat model of global cerebral ischemia/reperfusion injury was constructed to simulate clinical ischemia/reperfusion injury.The different drugs were used in different groups by ischemia postconditioning according to the experimental groups.The effect of DADLE on DOR after ischemia was detected by Western blotting.The changes of autophagy-related proteins LC3 and P62 were detected by Western blotting,reflecting the effect of DOR activation on autophagy after ischemic injury.To explore the effect of autophagy on DOR-mediated neuroprotection,the survival of rat hippocampal neurons was observed by NeuN-labeled neurons.The astrocytic activation in hippocampal CA1 region was observed by GFAP-labeled astrocytes,and the effect of autophagy on DOR-regulated astrocytic activation after ischemia was investigated.The mechanism of DOR-mediated autophagy after ischemia was studied by immunofluorescence and Western blotting.The experimental results showed that: 1)DADLE treatment significantly increased the DOR level after ischemia.2)DOR activation enhanced autophagy after ischemia,indicated by the elevated LC3 II/LC3 I level and the reduced P62 level.The combination of DADLE and 3-MA abolished the DOR-mediated protection on improving the survival of ischemic CA1 neurons and regulating the astrocytic activation after ischemia.3)DOR activation activated AMPK/mTOR/ULK1 signaling pathway to enhance autophagy after ischemia.The combination of DADLE and Compound C erased the protective effect of DOR activation on the survival of ischemic CA1 neurons.2.Effect of DOR activation on autophagy in SH-SY5 Y cells after OGD/R injury and its relevant molecular mechanismBased on the results of animal experiments,cell experiments were further conducted.The OGD/R model was constructed to simulate ischemia/reperfusion injury in vitro.The cell viability assay assessed the effects of SH-SY5 Y cells exposing to gradient concentration of DADLE or Naltrindole treatment,and the effect of DOR activation after OGD/R injury was investigated.The changes of LC3 and P62 were detected by Western blotting,and the changes of autophagic flow were observed by mCherry-GFPLC3 B adenovirus infection to investigate the effect of DOR activation on autophagy after OGD/R injury.The mechanism of DOR-mediated autophagy was investigated by Monodansylcadaverine(MDC)staining and Western blotting.The experimental results showed that: 1)DOR activation with DADLE attenuated OGD/R injury.2)DADLE significantly elevated the DOR level after OGD/R injury.3)DOR activation enhanced autophagy after OGD/R injury,indicated by the increased LC3 II/LC3 I level and the decreased P62 level.DOR activation improved the autophagic flux dysfunction induced by OGD/R injury.4)OGD/R injury promoted autophagy by AMPK/mTOR/ULK1 signaling pathway,while DOR activation reinforced it by promoting AMPK/mTOR/ULK1 signaling pathway.DADLE combined with Compound C inhibited the effect of DOR-mediated autophagy.The present study revealed that DOR activation with DADLE enhanced neuronal autophagy through the AMPK/mTOR/ULK1 signaling pathway to improve ischemic neurons survival,and exerted neuroprotective effects.It provided new experimental evidence for DOR-mediated neuroprotection,and revealed for the first time the effects of DOR-mediated autophagy on ischemic neurons,deepening understanding of DORmediated downstream molecular pathways,and further expanded DOR-mediated neuroprotective mechanisms in ischemic injury.The AMPK-dependent autophagy by DOR activation provided new ideas and drug targets to find new solutions for cerebral ischemia treatment.