Preconditioning Combined With Ischemic Postconditioning Enhances The Neuroprotection Effect Of Cerebral Ischemia/reperfusion Injury In Mice

Objective:To observe differences in protective effects of spontaneous running wheel preconditioning,ischemic postconditioning and their combination on cerebral ischemia-reperfusion(IR)injury in mice,and to explore the potential regulatory mechanism by combining the detection of oxidative stress indexes and apoptotic factors.Methods:The experiment consisted of three parts.In the first part,experimental mice were divided into five groups:1.Sham-operation group(Sham);2.Model group of cerebral I/R injury(IR);3.Spontaneous running wheel preconditioning+IR group(IR+RW);4.Ischemic postconditioning group+IR group(IR+IP);and 5.Combined conditioning group;combining spontaneous running wheel preconditioning with ischemic postconditioning+IR group(IR+RW+IP).The second part experiment is based on the first part's results.It was established into three groups:1.Sham;2.IR+IP;and 3.IR+RW+IP.The third part experiment was divided animals into four groups,in order to verify the inferences of first and second parts,1.IR+IP;2 IR+RW+IP;3.IR+RW+IP-PI3K inhibitor group(IR+RW+IP-PI3K);and 4.IR+RW+IP-STAT3 inhibitor group(IR+RW+IP-STAT3).The Neuro Deficit Score was used to detect the neurological deficit score of mice.Morris water maze assessed the cognitive function.TTC test was used to measure the cerebral infarction volume.Molecular biological series experiments were applied to measure biochemical indicators of oxidative stress and neuronal apoptosis.The changes in cellular immunity were evaluated by detecting the apoptotic expression and the signal transduction pathway-related factors by using Western blot.Results:In the first part of the experiment:Compared with the IR group,there was no obvious effect of IR+RW on improving the neurological function,cognitive function and reducing volume of cerebral infarction in mice after cerebral IR injury.By contrast,IR+IP improved neurological function,cognitive function and reduced the volume of cerebral infarction after cerebral IR injury in mice.Significantly,the effect of IR+RW+IP on improving neurological function,cognitive function and reducing the volume of cerebral infarction in mice after cerebral IR injury was evidently stronger than those of preconditioning or postconditioning alone.Furthermore,preconditioning resulted in slight increase in the activity of superoxide dismutase(SOD)and decrease in the content of malondialdehyde(MDA)than those in the cerebral ischemia reperfusion group.Meanwhile,IR+IP could significantly increase SOD activity and decrease MDA content in brain tissue.While combined intervention of running wheel preconditioning and postconditioning increased SOD activity in brain tissue and decreased MDA content,which was remarkably stronger than those of running wheel preconditioning or postconditioning alone.In addition,compared with the model group,the number of neuronal apoptosis in ischemic brain tissue was not significantly reduced by IR+RW,and IR+IP could decrease the number of neuronal apoptosis,the effect of decrease the number of neuronal apoptosis was obviously enhanced after combined intervention of cerebral IR injury when compared to that of intervention alone.Western blot was used to detect the protein expression of apoptosis-related factors,compared with IR,the protein expression of Bax,caspase-3 of apoptosis promoting factors was decreased after IR+RW+IP,and the expression of IR+RW or IR+IP was not statistically significant.The protein expression of bcl-2,which is anti-apoptotic factors,was enhanced by IR+RW+IP,while the expression of IR+RW or IR+IP was not statistically significant.In the second part of the experiment:Western blot was used to detect the expression of signal transduction pathway related factors,in which PI3K and STAT3 were expressed,and PI3K and STAT3 activities were increased after ischemic postconditioning.Meanwhile,combined intervention of running wheel preconditioning and postconditioning indicated a stronger impact on the activity of PI3K and STAT3 pathways than that of postconditioning alone.In the third part of the experiment:After separate application of PI3K inhibitor LY294002 and STAT3 inhibitor SH454,the cerebral protective effect of running wheel preconditioning combined with postconditioning was significantly weakened,which was manifested as decreased neurological score,reduced cognitive function,increased cerebral infarction volume,increased number of apoptotic cells detected by TUNEL,increased protein expression of Bax,Caspase-3 and cytochrome C in mitochondria of pro-apoptotic factors,and suppressed expression of Bcl-2 and cytochrome C in cytoplasm of anti-apoptotic factors.Conclusion:1.Postconditioning has protective effect on cerebral IR injury in mice.The intervention of preconditioning using spontaneous running wheel combined with postconditioning can further enhance the above effects.2.The enhancement protectived effect of preconditioning using spontaneous running wheel combined with postconditioning on cerebral ischemia/reperfusion injury in mice may depends on activating antioxidant and anti-apoptotic PI3K and STAT3 signaling pathways.3.The cerebral protective effect of preconditioning using spontaneous running wheel combined with postconditioning is significantly weakened after separate application of PI3K inhibitor and STAT3 inhibitor.