Abstract
BackgroundIschemic stroke poses a severe risk to human health worldwide, and currently, clinical therapies for the disease are limited. Delta opioid receptor (DOR)-mediated neuroprotective effects against ischemia have attracted increasing attention in recent years. Our previous studies revealed that DOR activation by [d-Ala2, d-Leu5] enkephalin (DADLE), a selective DOR agonist, can promote hippocampal neuronal survival on day 3 after ischemia. However, the specific molecular and cellular mechanisms underlying the DOR-induced improvements in ischemic neuronal survival remain unclear.ResultsWe first detected the cytoprotective effects of DADLE in an oxygen–glucose deprivation/reperfusion (OGD/R) model and observed increased viability of OGD/R SH-SY5Y neuronal cells. We also evaluated changes in the DOR level following ischemia/reperfusion (I/R) injury and DADLE treatment and found that DADLE increased DOR levels after ischemia in vivo and vitro. The effects of DOR activation on postischemic autophagy were then investigated, and the results of the animal experiment showed that DOR activation by DADLE enhanced autophagy after ischemia, as indicated by elevated LC3 II/I levels and reduced P62 levels. Furthermore, the DOR-mediated protective effects on ischemic CA1 neurons were abolished by the autophagy inhibitor 3-methyladenine (3-MA). Moreover, the results of the cell experiments revealed that DOR activation not only augmented autophagy after OGD/R injury but also alleviated autophagic flux dysfunction. The molecular pathway underlying DOR-mediated autophagy under ischemic conditions was subsequently studied, and the in vivo and vitro data showed that DOR activation elevated autophagy postischemia by triggering the AMPK/mTOR/ULK1 signaling pathway, while the addition of the AMPK inhibitor compound C eliminated the protective effects of DOR against I/R injury.ConclusionDADLE-evoked DOR activation enhanced neuronal autophagy through activating the AMPK/mTOR/ULK1 signaling pathway to improve neuronal survival and exert neuroprotective effects against ischemia.
Highlights
Ischemic stroke poses a severe risk to human health worldwide, and currently, clinical therapies for the disease are limited
Delta opioid receptor (DOR) expression is changed in response to I/R injury and DADLE treatment As we previously showed that DADLE treatment exerts neuroprotective effects in vivo in ischemic rats [7], we first assessed the role of DADLE in vitro in SH-SY5Y cells exposed to oxygen-glucose deprivation (OGD)/R injury in this study
The cell toxicity assays showed that the viability of SH-SY5Y cells treated with 1–50 μM DADLE was unchanged (Fig. 2a) but that the viability of oxygen–glucose deprivation/reperfusion (OGD/R)-exposed cells treated with 10–30 μM DADLE was markedly increased, with peak viability being observed at a dose of 20 μM (Fig. 2b)
Summary
Ischemic stroke poses a severe risk to human health worldwide, and currently, clinical therapies for the disease are limited. Our previous studies revealed that DOR activation by [d-Ala, d-Leu5] enkephalin (DADLE), a selective DOR agonist, can promote hippocampal neuronal survival on day 3 after ischemia. The specific molecular and cellular mechanisms underlying the DOR-induced improvements in ischemic neuronal survival remain unclear. According to a systematic countryspecific analysis on the lifetime risk of stroke, China has the highest estimated risk worldwide [1]. There are currently obvious limitations (e.g., a narrow therapeutic window and selective efficacy) on clinical therapies for ischemic stroke, partly due to the limited understanding of disease pathogenesis. Further studies on the pathological mechanisms of the disease and potential therapeutic strategies are necessary
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