Role of SIRT1/NF-κB signaling pathway in oxygen-glucose deprivation and restoration injury to hippocampal neurons of mice

Abstract

Objective To evaluate the role of sirtuin 1 (SIRT1)/nuclear factor kappa B (NF-κB) signaling pathway in oxygen-glucose deprivation and restoration (OGD/R) injury to hippocampal neurons of mice. Methods The HT22 hippocampal neurons were seeded in a culture plate (96-well plate, 100 μl/well; 6-well plate, 2 ml/well) at the density of 5×104 cells/ml or in a culture dish (6 cm in diameter), and then divided into 4 groups (n=24 each) using a random number table method: control group (group C), OGD/R group (group OGD), SIRT1 inhibitor EX-527 preconditioning group (group EX) and SIRT1 agonist SRT1720 preconditioning group (group SRT). Neurons were cultured in normal culture atmosphere at 37 ℃ in group C. In OGD, EX and SRT groups, the culture medium was replaced with oxygen-poor fluid, and neurons were exposed to 5% CO2-95% N2 for 12 h in an incubator at 37 ℃, oxygen-poor fluid was replaced with the culture medium, and neurons were cultured for 24 h in normal culture atmosphere at 37 ℃.SIRT1 inhibitor EX-527 1 μmol/L and SIRT1 agonist SRT1720 10 μmol/L were added at 12 h before OGD in EX and SRT groups, respectively.The cell viability was measured by CCK8 assay, the activity of LDH was detected by chemical colorimetry, cell apoptosis rate was determined by flow cytometry, the expression of SIRT1, NF-κB, IκBα, Bcl-2 and Bax was detected by Western blot, and the Bcl-2/Bax ratio was calculated. Results Compared with group C, the cell viability was significantly decreased, LDH activity and cell apoptosis rate were increased, the expression of SIRT1, IκBα and Bcl-2 was down-regulated, the expression of NF-κB and Bax was up-regulated, and the Bcl-2/Bax ratio was decreased in OGD, EX and SRT groups (P<0.05). Compared with group OGD, the cell viability was significantly increased, the LDH activity and cell apoptosis rate were decreased, the expression of SIRT1, IκBα and Bcl-2 was up-regulated, the expression of NF-κB and Bax was down-regulated, and the Bcl-2/Bax ratio was increased in group SRT, and the cell viability was significantly decreased, LDH activity and cell apoptosis rate were increased, the expression of SIRT1, IκBα and Bcl-2 was down-regulated, the expression of NF-κB and Bax was up-regulated, and the Bcl-2/Bax ratio was decreased in group EX (P<0.05). Conclusion SIRT1/NF-κB signaling pathway inhibition is involved in OGD/R injury to hippocampal neurons of mice. Key words: SIRT1; NF-κB; Reperfusion injury; Cell hypoxia; Hippocampal neurons