Role of PI3K signaling pathway in NGF-β-produced mitigation of cell apoptosis induced by endoplasmic reticulum stress during hypoxia-reoxygenation in rat cardiomyocytes

Abstract

Objective To evaluate the role of 1-phosphatidylinositol 3-kinase(PI3K)signaling pathway in nerve growth factor-beta(NGF-β)-produced mitigation of cell apoptosis induced by endoplasmic reticulum stress during hypoxia-reoxygenation(H/R)in rat cardiomyocytes. Methods H9c2 cells were seeded in 96-well plates at a density of 5×105 cells/ml(100 μl/well). The wells were randomly divided into 5 groups(n = 15 each)using a random number table: control group(group C); group H/R; NGF-β group(group N); NGF-β+ NGF receptor trkA antagonist K252a group(group N+ K); NGF-β+ PI3K inhibitor LY294002 group(group N+ L). The cells were exposed to 95% N2-5%CO2 for 4 h in an anaerobic incubator, followed by reoxygenation in a standard incubator for 4 h in H/R, N, N+ K and N+ L groups.In addition, the cells in N, N+ K and N+ L groups were incubated in a standard incubator containing NGF-β, the mixture of NGF-β and K252a and the mixture of NGF-β and LY294002, respectively, during reoxygenation, and the final concentrations of NGF-β, K252a and LY294002 were 50 ng/ml, 100 nmol/L and 50 μmol/L, respectively.The cell viability was detected by using CCK-8 assay, and the cell survival rate was calculated.The cell apoptosis was examined by flow cytometry, and apoptosis rate was calculated.The expression of glucose-regulated protein 78(GRP78), C/EBP homologous protein(CHOP), caspase-12, Akt and phosphorylated Akt(p-Akt)was detected by Western blot.The ratio of p-Akt to Akt was calculated. Results Compared with group C, the cell survival rate was significantly decreased, and the apoptosis rate was increased in H/R and N groups, and the expression of GRP78, CHOP and caspase-12 was significantly up-regulated in group H/R, and p-Akt/Akt was significantly increased in group N(P 0.05). Compared with group N, the cell survival rate was significantly decreased, the apoptosis rate was increased, the expression of GRP78, CHOP and caspase-12 was up-regulated, and p-Akt/Akt was decreased in N+ K and N+ L groups(P<0.05). Conclusion NGF-β can mitigate the cell apoptosis induced by endoplasmic reticulum stress during H/R, and activation of PI3K signaling pathway is involved in the mechanism. Key words: 1-Phosphatidylinositol 3-kinase; Nerve growth factor; Endoplasmic reticulum; Stress; Apoptosis; Oxygen; Cell hypoxia