Protective role of Sestrin2 overexpression in hypoxia and re-oxygenation injury of hippocampal neurons and its mechanism in rats

Abstract

Objective To investigate the protective role of Sestrin2 overexpression in hypoxia and re-oxygenation injury (H/R) injury of hippocampal neurons and its mechanism in rats. Methods Neurons were enzymatically isolated from hippocampi of newborn Sprague-Dawley rats (less than 24 h old) and cultured in vitro. These neurons were randomly assigned into 4 groups (n=20) using a random number table: control group, H/R group, vector group and Sestrin2 overexpression group. The hippocampal neurons were seeded in 6-well plates at a density of 2×104 cells/mL; neurons in the latter two groups were transfected with lentiviruses containing empty vector and Sestrin2 overexpressed genes, respectively; the hippocampal neurons in the later three groups were subjected to oxygen-glucose deprivation (OGD) for 6 h followed by restoration of O2 supply for 20 h. The reactive oxygen species (ROS) content was detected by Reactive Species Assay Kit, and the ATP concentration was detected by ATP Assay Kit. Cell apoptosis rates were measured by flow cytometry. The levels of Sestrin2, dynamin-related protein 1 (Drp1), Fis1, and apoptosis-related proteins Bcl-2, Bax and cytochrome C (Cyt C) were measured by Western blotting. The ratio of Bcl-2 to Bax was calculated. The ultrastructure of mitochondria was observed by transmission electron microscopy. Results As compared with control group, H/R group had significantly lower ATP concentration, Bcl-2 protein expression and ratio of Bcl-2 to Bax ([11.15±0.42] nmol/mg protein vs. [5.30±0.39] nmol/mg protein; 2.20±0.26 vs. 0.91±0.02; 6.46±0.41 vs. 1.04±0.05), statistically higher average fluorescence intensity of ROS and cell apoptosis rate (152.41±17.38 vs. 1530.00±14.69; 3.77%±0.74% vs. 56.57%±1.35%), and significantly higher protein levels of Sestrin2, Drp1, Fis1, Bax and Cyt C (0.66±0.06 vs. 1.11±0.20; 0.48±0.03 vs. 1.16±0.07; 1.14±0.09 vs. 2.47±0.09; 0.34±0.03 vs. 0.88±0.04; 0.17±0.03 vs. 0.30±0.03, P<0.05); what's more, the structure of mitochondria was obviously destroyed in I/R group. As compared with H/R group, Sestrin2 overexpression group had significantly increased ATP concentration, Sestrin2 and Bcl-2 protein expressions and ratio of Bcl-2 to Bax ([8.95±0.27] nmol/mg protein; 2.67±0.07; 1.80±0.19; 3.95±0.28), significantly lower average fluorescence intensity of ROS and cell apoptosis rate (337.27±15.32; 10.33%±2.60%), and statistically lower protein levels of Drp1, Fis1, Bax and Cyt C (0.43±0.02; 1.11±0.08; 0.45±0.02; 0.17±0.02, P<0.05); the structure of mitochondria was relatively completed in Sestrin2 overexpression group. Conclusion Sestrin2 overexpression can inhibit mitochondrial fission, reduce accumulation of reactive oxygen species, improve mitochondrial energy metabolism and block mitochondria mediated apoptosis pathway, thereby alleviating I/R injury of rat hippocampal neurons. Key words: Sestrin2; Hypoxia and re-oxygenation injury; Neuron; Cerebral ischemia-reperfusion injury; Mitochondrial fission