| Objectives 1 To explore the effects of Hypoxic Postconditioning on expression of LC3 in hippocampus after transient global cerebral ischemia in rats. 2 To explore effects of Hypoxic Postconditioning on expression of Sirt1/Fox O1 pathway in hippocampus after global cerebral ischemia in rats.Methods Adult male SD rats(n=120) were divided into four groups randomly: shamoperation group, global cerebral ischemia group, hypoxic postconditioning group, and inhibitor group(Sirt1 inhibitor EX527). Transient global cerebral ischemic model of rat was induced by improved four-vessel occlusion as described according to the description of Pulsinelli’s method. Each group was divided into three subgroups-1d, 3d and 7d. Hypoxic Postconditioning of global cerebral was achieved as follows: at the end of 20 min of ischemia, the myocytes were initially transferred to the normoxic incubator for 24 h and then to the hypoxic incubator for an additional 2h(8% O2). Sirt1 inhibitor EX527 was used before the ischemia. The Morris water maze was applied to investigate spatial learning and memory in laboratory rats. Both morphological changes in the hippocampus and protein expression(Sirt1, Fox O1 and LC3) were analysed from hippocampus tissue.Results 1 Morris water maze results: compared with sham-operation group, the time of escape latency to find the platform increased and the number of platform crossings decrease significantly in the global cerebral ischemia group(1d、3d、7d)(P < 0.05); compared with global cerebral ischemia group, the time of escape latency to find the platform decreased and the number of platform crossings increased significantly in the hypoxic postconditioning group(1d 、 3d 、 7d)(P < 0.05); compared with hypoxic postconditioning group, the time of escape latency to find the platform increased and the number of platform crossings decrease significantly in the inhibitor group(1d、3d、7d)(P < 0.05). 2 The result of morphological changes(HE staining results): Structural changes in hippocampus neurons were not detected by microscope in sham-operation group. Compared with sham-operation group, global cerebral ischemia group each time point(1 d, 3 d, 7 d) nerve cell mortality rate increased significantly, the difference was statistically significant(P < 0.05); Compared with global cerebral ischemia group, hypoxic postconditioning group 1 d nerve cells mortality was decreased, 1 d, 3 d, 7 d nerve cells mortality significantly reduced, the difference was statistically significant(P < 0.05); Compared with hypoxic postconditioning group, inhibitor group of nerve cell were damaged, 1 d, 3 d, 7 d nerve cell mortality rate increased significantly, the difference was statistically significant(P < 0.05). 3 Determination of LC3: The LC3 was detected by immunohistochemistry and Western blot. The immunoreactivity of LC3 was evaluated according to the intensity and percentage of positively stained cells. Compared with shamoperation group, the LC3 immunoreactivity and protein expression levels in hippocampus were significantly higher in the global cerebral ischemia group(1, 3, 7 day)(P < 0.05); compared with global cerebral ischemia group, the LC3 immunoreactivity and protein expression levels were further increased in the hypoxic postconditioning group(1, 3, 7 day)(P < 0.05); but decrease significantly in the inhibitor group compared with postconditioning group(1, 3, 7 day)(P < 0.05). 4 Determination of Sirt1: The Sirt1 was detected by immunohistochemistry and Western blot analysis. The expression of Sirt1 began to increase within 24 h following the end of the ischemia, and keep the highest level after 3 days, and even 7days. Compared with sham-operation group, the Sirt1 immunoreactivity and protein expression levels in hippocampus were significantly higher in the global cerebral ischemia group(1, 3, 7 day)(P < 0.05); compared with global cerebral ischemia group, the Sirt1 immunoreactivity and protein expression levels were further increased in the hypoxic postconditioning group(1, 3, 7 day)(P < 0.05); but decrease significantly in the inhibitor group compared with postconditioning group(1, 3, 7 day)(P < 0.05). 5 Determination of Fox O1(forkhead turn green factor 1): The Fox O1 was detected by immunohistochemistry and Western blot analysis. The immunoreactivity of Fox O1 was evaluated according to the intensity and percentage of positively stained cells, which located in the cytoplasm and mainly expressed in neurons and glial cells. Compared with sham-operation group the Fox O1 immunoreactivity and protein expression levels was significantly higher in the global cerebral ischemia group(1, 3, 7 day)(P < 0.05); Compared with global cerebral ischemia group the Fox O1 levels were further increased in the hypoxic postconditioning group(1, 3, 7 day)(P < 0.05); Compared with hypoxic postconditioning group but decrease significantly in the inhibitor group compared(1, 3, 7 day)(P < 0.05). The expression of Fox O1 began to increase within 24 h following the end of the ischemia, and keep the highest level after 3 days, and even 7days.Conclusions 1. Hypoxic postconditioning can promote the autophagy activation in hippocampus after global cerebral ischemia in rats, and relieve nervous injury. 2 Hypoxic postconditioning can increase the expression of t Sirt1, Fox O1 in hippocampus, improve the activity of Sirt1 / Fox O1 pathways, and reduce nerve cells mortality after global cerebral ischemia in rats. |
PDF Full Text Request