Proteomics Research Of Cerebral Protection Induced By Remote Preconditioning

Background:The concept of cerebral ischemia preconditioning(PC) emerged that a brief sublethal ischemic challenge could increased tolerance against subsequent lethal ischemia of neuron, Recent studies have found that this protection phenomenon exists between different tissues and organs, this phenomenon is called remote preconditioning (RPC),which could be used to induce protection to vital organs such as brain or heart through ischemia of non-vital organs such as limbs. Although induced endogenous effects were considered as a mainly mechanisms in RPC, it has not yet fully understood. Here we used proteomic methods included 2D-DIGE, MALDI-TOF to study the proteomics of brain tissues from cerebral ischemia or protected brain tissues induced by RPC.Objective:To investigate the cerebral protemics differences between sham, ischemia and RPC induced group.Methods:30 SD male rats, weight 280-330 grams and 11-12 weeks old. These animals were divided into sham group(n=6),1 hour ischemia group(n=6),6 hours ischemia group(n=6),1 hour RPC group(n=6),6 hours RPC group(n=6).Sham group rats were anesthesia using isoflurane, sham surgery and without ischemia.Ischemia group rats were created cerebral ischemia using dMCAO model. RPC group rats were did cerebral ischemia as Ischemia group, before this,3 cycles --left femoral artery 15min ischemia and 15 min reperfusion did as RPC. All animals were perfused using cold 0.02M PBS(pH=7.4) 100ml through heart at every time point. Fresh brain tissues cut from ischemic core, ischemic pneumbra and contralateral were stored in -80℃refrigeratory. Tissue proteins were extracted by ultrasonication. After centrifugation, 2-D gel electroph--oresis were did to separate thousands of proteins. Fluorescence dyes(DIGE) included Cy2,Cy3 and Cy5 were used to marked different groups, thereinto Cyt2 were as inner mark. Differences point analysis were did by using the software DeCyder2D6.5. The difference proteins points which occurred above 3 or more groups were farther analysed by using MALDI-TOF/TOF. The proteins were identified by PMF using SWISS-PROT database.Result:We collected 13 subgroups brain tissues from different cerebral regions and different surgery groups. We compared 6 subgroups'brain tissues according ischemia and RPC pathophysiology, included sham control group(A),1 hour ischemia core (B),1 hour ischemia penumbra(C),6 hours ischemia penumbra(D), RPC 1 hours penumbra(E) and RPC 6 hours penumbra(F). Through 2-D-DIGE electrophoresis and DeCyder2D6.5 difference points analyses, we got 126 different proteins points from 15 difference-groups through compared 6 tissure-subgroups.10 different proteins points was successfully founded between group A and B,8 points between A and C,23 points between A and D,16 points between A and E,28 points between A and F,16 points between B and C,18 points between B and D,20 points between B and E,17 points between B and F,10 points between C and D,11 points between C and E,14 points between C and F,10 points between D and E,16 points between D and F,22 points between E and F. There are 52 different proteins points occurred above three or more subgroups. We then choosed these different proteins points to do Mass spectrometry.16 kinds of proteins were identified by MALDI-TOF Mass Spectrometry and SWISS-PROT database checking.5 protein were identified successfully between sham control group and ischemic core group, of which,3 proteins were lower expression and 2 were higher expression in ischemic group.14 proteins were identified successfully between ischemic group and RPC group, of which,8 were higher expression and 6 were lower expression in RPC group. These identified proteins were functionally involved in the cellular processes of energy metabolism, anti-oxidation and anti- apoptosis.Conclusion:The results showed that many proteins changed after ischemia and cerebral protection induced by RPC. These changed proteins involved cellular energy metabolism,anti-oxidation and anti-apoptosis. Our results suggest that functionally proteins changing may be involved in cerebral protections induced by RPC.