Antiapoptotic effects of delta opioid peptide [D-Ala2, D-Leu5]enkephalin in brain slices induced by oxygen-glucose deprivation

Background

Delta opioid peptide [D-Ala2, D-Leu5]enkephalin (DADLE) has been shown to play a role in neuronal protection against hypoxic ischemia. However, the cellular mechanisms of these actions of DADLE on neurons are not totally clear. Being an in vitro model of brain ischemia, oxygen-glucose deprivation (OGD) injury in rat brain slices has the advantages of both in vivo and in vitro models, and therefore can imitate damages induced by brain I/R injury in intact animals. In the present study, we examined the protective mechanism(s) of DADLE against apoptosis using a rat brain slice model. In addition, we determined whether delta-opioid receptors are unique and have a specific role in neuroprotection against OGD injury by activating the MAPK pathway, specifically through delta2-opioid receptors.

Methods

The brain slices were incubated with different concentrations of DADLE after injury by OGD. Selective delta2-opioid antagonist or selective inhibitor of ERK kinase was co-incubated with or without DADLE. The effects of DADLE against apoptosis in neurons were measured by the following biochemical and morphological assays: LDH release, RT-PCR, western blot, and TUNEL staining.

Results

OGD caused LDH release in brain slices. DADLE inhibited OGD-induced LDH release. Naloxone, and naltriben methane sulfonate, partially suppressed the survival of brain slices promoted by 10 μM DADLE. DADLE at lower concentrations (10^-6 and 10^-5 M) significantly suppressed Bax mRNA expression and enhanced Bcl-2 mRNA expression. But with the increased concentrations of DADLE, the ability to regulate apoptotic gene expression was attenuated. DADLE induced ERK phosphorylation in brain slices with the increase of concentrations. The withdrawal of oxygen and glucose per se decreased the ERK phosphorylation. In the absence of oxygen and glucose, DADLE at 10^-5 M concentrations could, however, increase the ERK phosphorylation. Naltrindole reduced the degree of ERK phosphorylation induced by 10^-5 M DADLE. PD98059 abolished the DADLE-induced phosphorylation of ERK in the condition of OGD. PD98059 not only abolished the cell survival promoted by DADLE, but also apparently led to a higher level of LDH release when compared with the DADLE control.

Conclusions

Our results suggest, therefore, that endogenous opioid peptides may, at low concentrations, promote cell survival via the MEK–ERK pathway, perhaps through delta2-opioid receptors.