Objective: To investigate the effect of ischemic postconditioning on protein aggregation caused by transient ischemia and reperfusion and to clarify its underlying mechanism.
Methods: Two-vessel-occluded transient global ischemia rat model was used. The rats in ischemic postconditioning group were subjected to three cycles of 30-s/30-s reperfusion/clamping after 15 min of ischemia. Neuronal death in the CA1 region was observed by hematoxylin-eosin staining, and number of live neurons was assessed by cell counting under a light microscope. Succinyl-LLVY-AMC was used as substrate to assay proteasome activity in vitro. Protein carbonyl content was spectrophotometrically measured to analyze protein oxidization. Immunochemistry and laser scanning confocal microscopy were used to observe the distribution of ubiquitin in the CA1 neurons. Western blotting was used to analyze the quantitative alterations of protein aggregates, proteasome, hsp70 and hsp40 in cellular fractions under different ischemic conditions.
Results: Histological examination showed that the percentage of live neurons in the CA1 region was elevated from 5.21% ± 1.21% to 55.32% ± 5.34% after administration of ischemic postconditioning (P = 0.0087). Western blotting analysis showed that the protein aggregates in the ischemia group was 32.12 ± 4.87, 41.86 ± 4.71 and 34.51 ± 5.18 times higher than that in the sham group at reperfusion 12h, 24h and 48h, respectively. However, protein aggregates were alleviated significantly by ischemic postconditioning to 2.84 ± 0.97, 13.72 ± 2.13 and 14.37 ± 2.42 times at each indicated time point (P = 0.000032, 0.0000051 and 0.0000082). Laser scanning confocal images showed ubiquitin labeled protein aggregates could not be discerned in the ischemic postconditioning group. Further study showed that ischemic postconditioning suppressed the production of carbonyl derivatives, elevated proteasome activity that was damaged by ischemia and reperfusion, increased the expression of chaperone hsp70, and maintained the quantity of chaperone hsp40.
Conclusion: Ischemic postconditioning could rescue significantly neuronal death in the CA1 region caused by transient ischemia and reperfusion, which is closely associated with suppressing the formation of protein aggregation.
Keywords: Chaperone; Ischemic postconditioning; Proteasome; Protein aggregation.