Effect Of Rapamycin To Mitochondrial Damage After Cerebral Ischemia Reperfusion

Objective:1. To explore dynamic change of mitophagy, mitochondriaoxidative stress, mitochondrial dysfunction at different point after cerebralischemia-reperfusion.2. To evalute the effect that rapamycin pretreatment onmitochondria damage after cerebral ischemia-reperfusion and its specificmechanism.Methods:1, Stroke was induced in the rats by MCAO. We isolated themitochondria from ischemic cortical tissue at6h,24h,48h,72h after cerebralischemia-reperfusion.We detected the mitophagy expression with transmissionelectron microscope, LC3immunohistochemical staining and LC3western-blotmethods. We determine MDA level, ATP level, JC-1level of isolatedmitochondria at6h,24h,48h,72h after cerebral ischemia-reperfusion. Wedetect mtDNA copy numbers with real-time PCR at6h,24h,48h,72h aftercerebral ischemia-reperfusion.2, Intracerebroventricular (icv) injections wereperformed with2μl of a400ng/ml solution prepared in0.1%DMSO at30minutes before MCAO. We evaluated the neuroprotective effect of RAPpretreatmen by TTC stainning, Neurological Deficit Score, TUENL staining. Wemeasured mitophagy level, mitochondria number, and mitochondria MDA, ATP,JC-1level at24h after cerebral ischemia-reperfusion. We detected theexpression of beclin1, p62in mitochondrial fractions proteins and detected theexpression of p-Erk1/2in brain tissue. We evaluated cytochrome c release andBax mitochondria translocation, it is executed with measuring the different expression of Cyt c, Bax between the mitochondrial fractions and cytosolicfractions.Results:1.We observed the significant increasement of autophagosomesand mitophagy structure under transmission electron microscope after cerebralischemia-reperfusion. Western blot results showed mitophagy levels wasobserved as early as6h post-IR, reached the peak as early as24hours post-IR,and lasted for3days. IR resulted in the increase of mitochondrial MDA leveland the decrease of mitochondrial membrane potential. Mitochondrial ATP leveland mtDNA copy number decreased at6hours post-IR, but they recoverdpartly at24h post-IR（p＞0.05）.2, RAP pretreatment reduced infarct volumeand improved neurological function (p＜0.05）. It reduced the MDA level ofmitochondrial (p＜0.05）and improved mitochondrial function（ATP level andJC-1level, p＜0.01）.3, RAP pretreatment increased mitophagy level,recoverdmitochondrial numbers and mtDNA copy numbers(p＜0.05）.4, RAPpretreatment enhanced the expression of beclin1、p62in mitochondrial fractions.RAP pretreatment inhibited p-ERK activation at24h post-IR. RAP pretreatmentreduced cytochrome c release and inhibite Bax translocation to mitochondriawhich inhibited cell apoptosis induced by cerebral ischemia–reperfusion.Conclusion:1, Mitophagy increases significantly after cerebral ischemiareperfusion, which is survival path against oxidative stress damage induced bycerebral ischemia-reperfusion.2, RAP pretreatment has a neruoprotectiveeffect on ischemic tissue injury, which is related to enhanced mitophagy.Enhanced mitophagy can reduce the mitochondrial ROS damage, improvemitochondrial function,and increase mitochondrial number.3, RAPpretreatment can increase the expression of Beclin1、p62in the mitochondrialwhich result in enhanced mitophagy.4, RAP pretreatment also can inhibit intrinsic apoptotic mitochondrial pathway, which was linked to inhibitinntracellular Erk1/2phosphorylation activation.