The Delta Opioid Receptor Agonist DADLE Improves Autophagy And Reduces Apoptosis Of The Brain Microvascular Endothelial Cells To Relieve Hypoxia-Ischemic/Reperfusion Injury

Aim:Ischemic Stroke is one of the leading causes of death.[D-Ala~2,D-Leu~5]-enkephalin(DADLE)is a synthetic peptide and highly selective delta opioid receptor(δOR)agonist that has exhibited protective properties in ischemia.However,the specific target and mechanism are still unclear.The present study explores the expression ofδOR on brain microvascular endothelial cells(BMECs)and whether DADLE could alleviate I/R-induced injury by increasing their levels of autophagy and reducing apoptosis.Mesthods:After lateral ventricular injection of DADLE for the pretreatment of MCAO/R,the neurofunctional behavior score,and TTC staining were used to evaluate the protective effect of DADLE.Immunofluorescence was used to label different types of cells with apoptosis-positive signals to test the co-localization status.The formation of cellular autophagic vesicles was observed by using transmission electron microscopy.The activation levels of cellular autophagy-related proteins were examined by Western blot.Primary cultured BMECs were separated and treated with DADLE accompanied by OGD/R.The CCK-8 test was conducted to evaluate the cell viability and the Td T-mediated d UTP Nick-end Labelling(TUNEL)staining to test their apoptosis levels.The levels of apoptosis-and autophagy-related proteins were analysed by Western blot.Results:The co-localization results showed that BMECs,but not astrocytes,microglia,or neurons,presented mostly TUNEL-positive signals,especially in the Dentate gyrus(DG)area of the hippocampus.Activation ofδORs either on rats’brains or on primary BMECs mainly reduced the cellular apoptosis and relieves the injury.On the cell cultures,interference with the expressionδOR could block these effect.DADLE also significantly increased the levels of Bcl-2 and reduced levels of Bax.At the same time,DADLE increased the expression of autophagy proteins Beclin-1 and LC3II/Ⅰ,and improved the level of autophagy.δOR’s expression can be detected on the BMECs,but not on the HEK293 cells,by Western blot and IFC.Conclusion:DADLE exerts a cytoprotective effect primarily under hypoxia-ischemic injury/reperfusion conditions by increasing the autophagy levels and inhibit the apoptosis of BMECs.The molecular pathway ofδOR-mediated autophagy under ischemic conditions was subsequently investigated.In vitro and in vivo data showed that activation ofδORs increased the level of autophagy in post-ischemic cells possibly by reducing the binding ofβ-arrestins to GAPDH proteins.