Role of GPR35 activation in apoptosis in cortical astrocytes in a rat model of oxygen-glucose deprivation and restoration

Abstract

Objective To evaluate the role of G protein-coupled receptor 35 (GPR35) activation in apoptosis in cortical astrocytes in a rat model of oxygen-glucose deprivation and restoration (OGD/R). Methods Primary cortical astrocytes of rats cultured in vitro were seeded in 6-well plates or 96-well plates and divided into 4 groups (n=30 each) using a random number table method: control group (group C), OGD/R group (group O), GPR35 inhibitor plus OGD/R group (group GO) and GPR35 inhibitor group (group G). Astrocytes were subjected to 95% N2-5% CO2 in an incubator at 37℃ for 6 h followed by restoration of O2-glucose supply for 24 h. GPR35 inhibitor CID2745687 3 μmol/L was added at 1 h before OGD in GO and G groups.At 24 h after treatment in each group, apoptosis rate was calculated using Annexin V-FITC/GFAP/DAPI immunofluorescent staining, the concentration of calcium in cytoplasm was determined by Fluo3/AM fluorescence, the expression of hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) mRNA was detected by real-time polymerase chain reaction, and the expression of Bax and Bcl-2 was detected by Western blot. Results Compared with group C, the apoptosis rate of cortical astrocytes were significantly increased, the concentration of calcium in cytoplasm was increased, the expression of HCN2 mRNA, cAMP and Bax was up-regulated, and the expression of Bcl-2 was down-regulated in O and GO groups (P 0.05). Conclusion GPR35 activation is involved in the process of apoptosis in cortical astrocytes after OGD/R in rats. Key words: Receptors, G-protein-coupled; Hypoxia, brain; Astrocytes; Apoptosis