Experimental Study On The Signal Pathway Of PI3K/Akt-eNOS-No For Ischemic Postconditioning Of Rats

Objective:Using the middle cerebral artery occlusion thread model on rats, the study is to explore the protective mechanisms of ischemic postconditioning on easing ischemia/reperfusion injury and prolong thrombolysis time window, and the protective effect of the signal pathway of PI3K/Akt-eNOS-NO in it.Method:There were two parts of the experiment. It used68adult male Sprague Dawley rats with healthy, and established ischemia/reperfusion model of rats through occluding middle cerebral artery by thread.1. The study on the therapeutic time window of thrombolysis:20SD rats were randomly divided into the group of reperfusion after2h for ischemia and the group of postconditioning after4.5h for ischemia, and each group had10rats. After24h for ischemia reperfusion, the rats were used to make four types of neurological score, and the brain tissues of the rats were removed and are made the TTC staining for the determination of the infarct volume and HE staining for observing the pathological changes of them.2. The study on the protective mechanism of ischemic postconditioning:48SD rats were randomly divided into the group of sham operation, the group of reperfusion after2h for ischemia and the group of postconditioning after4.5h for ischemia, and every group had16rats. After24h for ischemia reperfusion, the brain tissues of the rats were removed. Eight of every group were made the contents of NO, eNOS, iNOS, tNOS by chemical method and others of every group were made the contents of eNOS, p-eNOS, Akt, p-Akt by Western Blot.Result:1. When24h in I/R, there are no significant difference on neurological scores (Ludmila12points test, Zea Longa5points test and over hanging experiments) and volume of cerebral infarction between the group of reperfusion after2h for ischemia and the group of postconditioning after4.5h for ischemia(P>0.05).2. Compared with the group of reperfusion after2h for ischemia, when24h in I/R, the pathological changes of the brain tissues in HE staining were not obviously aggravated.3. Compared with the groups of reperfusion after2h for ischemia and sham operation, when24h in I/R, the contents of NO and eNOS were obviously increased in the group of postconditioning after4.5h for ischemia(P<0.05). There were no significant difference on the contents of iNOS and tNOS in three groups(P>0.05), but compared with other groups, their contents in the group of postconditioning after4.5h for ischemia had a slight increase.4. Compared with the groups of reperfusion after2h for ischemia and sham operation, when24h in I/R, the contents of eNOS, p-eNOS and p-Akt were obviously increased in the group of postconditioning after4.5h for ischemia(P<0.05).Conclusion:1. Cerebral protective effect of ischemic postconditioning could prolong thrombolysis time window to4.5h in rats with MCAO, and it provided a new idea for the clinical treatment of ischemic stroke;2. Ischemic postconditioning could improve the nerve function damage and reduce the infarct volume after ischemic stroke.3. Ischemic postconditioning could increase the expression of NO in the brain in the early stage after ischemic stroke, and then NO played a protective role for brain tissue.4. Ischemic postconditioning could activate the signaling pathway of PI3K/Akt-eNOS-NO, and could make a neuroprotective effect by increasing levels of p-Akt, p-eNOS, eNOS and NO, so the pathway might be one of the protective mechanism of ischemic postconditioning.