| Baekground and Objective:Exendin-4, a high-affinity GLP-1R agonist, is the first incretin mimetic to be approved by the FDA (in 2005). Compared with endogenous GLP-1, exendin-4 has some advantages such as long half-life, high potency, and long duration of action. It has potent effects on normalizing insulin signalling in T2DM by binding to specific receptors on the surface of pancreaticβcells in a cAMP/PKA-dependent manner. Intriguingly, GLP-1 receptors (GLP-1Rs) have also been detected throughout the brain which reveals the neurotrophic and neuroprotective functions of GLP-1s. Researches over the past decade have proved solid proof that GLPs also play important roles in the CNS, such as modulation of satiety, improvement of memory and learning, defence against insults. Nevertheless, little is known about their role in brain ischemia. Whether exendin-4 mediated protectiveaction is also applicable to brain ischemia still needs to be elucidated. In the present study, we used an oxygen-glucose deprivation/recovery (OGD/R) model in vitro to assess the effects of exendin-4 on cultured rat cortical neurons. Clearly, such potential neuroprotective effects of exendin-4 have important implications for treating both central and peripheral neurological diseases, especially patients with T2DM.Methods:Rat cortical neurons were cultured in vitro, identified by NES-immunohistological staining and immunofluorescence staining, and randomly divided into groups as follows:1) Exendin-4 group:cells treated with exendin-4, the final concentration of which was determined by MTT assay, during the phases of pre-OGD (2h), OGD (6h) and reperfusion; 2) OGD/R group:cells treated with equivalent volume of water; 3) Control group:cells received no any therapy. RT-PCR and ELISA were performed to establish the existence of active GLP-1R. Fluorescent DNA binding dye Hoescht 33258 and flow cytometry assay were used to analyze the anti-apoptotic capability of Exendin-4. CHOP and GADD34 mRNAs were detected by real-time PCR. Both immunofluorescence and immunoblotting were used to analyse the alternation of GRP78 and CHOP protein levels.Results:The apoptosis cell counting was increasing gradually with the prolongation of reperfusion (P< 0.05); the data given by both RT-PCR and ELISA confirmed the presence of the existence of active GLP-1Rs on primary cortical neurons; the relative survival ration after treatment with exendin-4 was elevated in a does-dependent manner in the range of 0.2-0.8μmol/1 (P< 0.01); the apoptosis rate induced by OGD/R was reduced by exendin-4 (0.4μg/ml) treating at 12 h after reperfusion (P< 0.01); the expression of GRP78 and CHOP were up-regulated by exendin-4 (0.4μg/ml) treating at 12 h after OGD/R (P< 0.01); levels of CHOP and GADD34 mRNA in cortical neurons increased since 4h and peaked at 12 h after OGD/R, while exendin-4 group showed a sharp elevation of levels of CHOP and GADD34 mRNA and descend at last (P< 0.05); the CHOP protein level were increased first,and later dropped below the level of the model groupd (P< 0.05); Exendin-4 also gradually up-regulated the expression of GRP78 after ischemia/reperfusion (P<0 .01).Conclusion:Exendin-4 has protective effect on rat cortical neurons injury induced by ischemia/reperfusion. The protective effect may be related to inhibit ESR-related neuron apoptosis caused by such injury via regulation of unfolded protein response.
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