DADLE promotes autophagic flux and depresses necroptosis by inhibiting cytosolic phospholipase A2 mediated lysosomal membrane permeabilization after spinal cord injury

Abstract Programmed cell death plays a critical role in the progression of spinal cord injury (SCI). Autophagy is a protective factor for controlling neuronal damage, while necroptosis promotes further cell death and neuroinflammation after SCI. DADLE (d-Ala2, d-Leu5) is a selective agonist for delta opioid receptor (DOR) and has been identified as a promising drug for its neuroprotective effects. Our present work aims to investigate the therapeutic effect of DADLE on locomotive function recovery following SCI and its concrete mechanism. By establishing a mouse model of spinal cord contusion injury and using functional behavioural assessment, our results showed that DADLE promoted functional recovery after SCI. Through experimental methods such as western blotting and immunofluorescence, we found that DADLE promoted autophagic flux and inhibited necroptosis. Then, analysis of the enzyme activity of NAG and related protein expression of CTSD and CTSB in lysosomes and cytoplasm revealed that DADLE decreased lysosomal membrane permeabilization (LMP). The autophagy inhibitor CQ reversed the protective effect of inhibiting necroptosis. Further analysis identified that DADLE decreased phosphorylated cPLA2, and network pharmacology analysis revealed that the AMPK (Adenosine monophosphate-activated protein kinase) signalling pathway may be involved in the therapeutic effect of DADLE. Finally, blocking the interaction between DOR and DADLE by using naltrindole abolished the anti-phosphorylation effect of DADLE on cPLA2 and p38, resulting in a decrease in autophagic markers and an increase in necroptosis and LMP markers. Altogether, our study indicated that DADLE promotes autophagic flux and inhibits necroptosis by decreasing LMP by interacting with DOR and then activating the AMPK/SIRT1/P38/cPLA2 pathway after SCI, which may have potential clinical application value in the future.