The Protective Effects And Related Mechanisms Of Combined Treatment With Preconditioning And Postconditioning On Cerebral Ischemia/Reperfusion Injury In Rats

Objective:To investigate the protective effects of ischemic preconditioning, postconditioning and combined treatment with preconditioning and postconditioning on cerebral ischemia/reperfusion injury in rats, the protein and gene expression of HSP70, and the activities of PI3K/Akt and ERK1/2signaling pathways in the ischemic brain..Methods:120SD rats were randomly divided into5groups: Sham-operated control group, cerebral ischemia/reperfusion (I/R) group, ischemic preconditioning alone group, ischemic postconditioning alone group and ischemic preconditioning combined with postconditioning group. The model of focal cerebral ischemia/reperfusion injury was established by middle cerebral artery occlusion for120minutes and reperfusion for48hours. Ischemic preconditioning was induced by3cycles of a15-second ischemia/30-second reperfusion24and1hours before middle cerebral artery occlusion respectively; Ischemic postconditioning was induced by3cycles of a30-second reperfusion/15- second ischemia at the beginning of48hours reperfusion. At the end of48hours reperfusion, the neurological deficits and the volumes of cerebral infarction of rats were evaluated; the biochemical indicators of oxidation injury and the apoptosis cell numbers in the ischemic brain were also measured.50SD rats were randomly divided into5groups, and the methods for grouping, inducing ischemia/reperfusion injury, preconditioning and postconditioning were the same as the Chapter I mentioned. After48-h reperfusion, the protein and gene expression of HSP70were determined by immunohistochemistry and Real-time PCR respectively. The the level of phosphorylation of Akt (p-Akt) and ERK1/2(p-ERK1/2) were analyzed by Western Blot technique.Results:Compared with I/R group, both ischemic pre-and postconditioning significantly reduced the neurological deficits scores and the infarct volumes of the rat models. Further, ischemic pre-and postconditioning have equivalent effect on improving neurological function and reducing infarct volume. The treatment of ischemic preconditioning combined with postconditioning had a more powerful effect on improving neurological function and reducing infarct volume than ischemic pre-and postconditioning alone. The biochemical indicators of oxidation injury in rat brain tissues showed that superoxide dismutase (SOD) activities were decreased and malondialdehyde (MDA) levels were increased markedly after I/R. Ischemic preconditioning only showed increase SOD activities and decrease MDA levels a little, but, ischemic postconditioning showed a strong ability to increase SOD activities and decrease MDA levels. The treatment of ischemic preconditioning combined with postconditioning showed an enhanced effect on increasing SOD activities and decreasing MDA levels, compared with ischemic pre-and postconditioning alone. Compared with the I/R group, both ischemic pre-and postconditioning significantly reduced the apoptosis cell numbers, and then the effect of postconditioning on inhibiting apoptosis was better than preconditioning. The treatment of ischemic preconditioning combined with postconditioning was more effective on antiapoptosis than both ischemic pre-and postconditioning alone. A very low level expression of HSP70protein was seen in the sham-operated group. I/R group exhibited a low level expression and preconditioning or postconditioning exhibited a middle expression of HSP70protein repectively, compared with the sham-operated control group. However, the group of combination of pre-and postconditioning exhibited a high expression of HSP70protein. We examined the mRNA expression of HSPA8, HSPA1A and HSPA12B in the cerebral cortex, subcortex and hippocampus, the result showed that three genes of HSP70subtype were slightly increased; these three genes of HSP70subtype increased significantly after ischemic pre-and postconditioning, in which HSPA1A expressed evidently after preconditioning and HSPA8after postconditioning; the increasing level of HSPA12B is not significant between pre-and postconditioning. The expression level of above mentioned three genes of HSP70were all increased exceeding the pre-or postconditioning alone. In the Western Blot analysis, the p-Akt and p-ERK1/2expressions were increased after I/R, compared with sham-operated control group. Preconditioning can slightly increase p-Akt and remarkably inhibit p-ERK1/2expression, while postconditioning can remarkably increase p-Akt and slightly inhibit p-ERK1/2expression. In the combination of pre-and postconditioning group, the effects of both increasing p-Akt and inhibiting p-ERK1/2exceeded pre-or postconditioning alone.Conclusions:1. Both ischemic pre-and postconditioning have protection on cerebral ischemia/reperfusion injury in rats, the combination of those has more effective protection.2. Both pre-and postconditioning can upregulate HSP70expression, but they influence different HSP70subtypes.3. Both preconditioning and postconditioning can raise the p-Akt and restrain the p-ERK1/2. The over expression of HSPA1A can protect neuroepithelial cells from oxidative damage.