Exendin-4 improved rat cortical neuron survival under oxygen/glucose deprivation through PKA pathway

Previous studies demonstrated that exendin-4 (Ex-4) may possess neurotrophic and neuroprotective functions in ischemia insults, but its mechanism remained unknown. Here, by using real-time PCR and ELISA, we identified the distribution of active GLP-1Rs in the rat primary cortical neurons. After establishment of an in vitro ischemia model by oxygen/glucose deprivation (OGD), neurons were treated with various dosages of Ex-4. The MTT assay showed that the relative survival rate increased with the dosage of Ex-4 ranging from 0.2 to 0.8 μg/ml (P < 0.001, vs. OGD group). The apoptosis rate was reduced from (49.47 ± 2.70)% to (14.61 ± 0.81)% after Ex-4 treatment (0.4 μg/ml) 12 h after OGD (P < 0.001). Moreover, immunofluorescence staining indicated that Ex-4 increased glucose-regulated proteins 78 (GRP78) and reduced C/EBP-homologous protein (CHOP). Western blot analysis demonstrated that, after neurons were treated with Ex-4, GRP78 was up-regulated over time (P < 0.01, vs. OGD group), while CHOP levels rose to a peak 8 h after OGD and then decreased (P < 0.05, vs. OGD group). This effect was changed by both the protein kinase A (PKA) inhibitor H89 (P < 0.01, P < 0.05, respectively, vs. Ex-4 group) and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (P < 0.01, P < 0.01, respectively, vs. Ex-4 group) but not by the mitogen-activated protein kinase (MAPK) inhibitor U0126. Our study also revealed that, compared with the Ex-4 group, inhibition of the PKA signaling pathway significantly decreased the survival rate of neurons, down-regulated the expression of B-cell lymphoma 2 (Bcl-2) and up-regulated the Bax expression 3 h after ODG (P < 0.05, P < 0.01, respectively), while neither PI3K nor MAPK inhibition exerted such effects. Furthermore, Western blotting exhibited that PKA expression was elevated in the presence or absence of OGD insults (P < 0.05). This study indicated that Ex-4 protected neurons against OGD by modulating the unfolded protein response (UPR) through the PKA pathway and may serve as a novel therapeutic agent for stroke.

► Oxygen/glucose deprivation can induce ER stress and the UPR.
► Exendin-4 improved rat cortical neuron survival under oxygen/glucose deprivation by modulating the UPR.
► The neuroprotective action was primarily mediated through PKA pathways.