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αB-crystallin regulates oxidative stress-induced apoptosis in cardiac H9c2 cells via the PI3K/AKT pathway.
MedLine Citation:
PMID:  23212619     Owner:  NLM     Status:  Publisher    
The present study was carried out to observe the protective effects of αB-crystallin protein on hydrogen peroxide (H(2)O(2))-induced injury in rat myocardial cells (H9c2) and to investigate the mechanisms of these protective effects at the cellular level, which could provide the experimental basis for future applications of αB-crystallin in the treatment of cardiovascular disease. Western blotting was used to measure the expression of αB-crystallin in cultured H9c2 cells in vitro. A αB-crystallin recombinant expression vector, pcDNA3.1-Cryab, was constructed to transfect H9c2 cells for the establishment of cells that stably expressed αB-crystallin. A tetrazolium-based colorimetric assay (MTT test) was used to measure changes in the viability of the H9c2 cells at 1, 2, 3 and 4 h after induced by 150 μM H(2)O(2) to establish a model of H(2)O(2) injury to cells. H(2)O(2) was applied to H9c2 cells that were stably transfected with αB-crystallin, and the effect of αB-crystallin overexpression on the viability of myocardial cells subjected to H(2)O(2)-induced injury was measured by the MTT assay. The effect of αB-crystallin overexpression on the H(2)O(2)-induced injury of H9c2 cells was also analyzed by flow cytometry. The mitochondrial components and cytoplasmic components of H9c2 cells were separated, and western blotting was used to measure the effect of αB-crystallin overexpression on the release of cytochrome c from the mitochondria. Western blotting was also used to measure the effect of αB-crystallin overexpression on the expression of the anti-apoptosis protein Bcl-2 and components of the phosphatidylinositol 3-OH kinase (PI3K)/AKT pathway. The αB-crystallin recombinant expression vector pcDNA3.1-Cryab successfully transfected H9c2 cells, and H9c2 cells that were stably transfected with αB-crystallin were established after G418 selection. The measurements carried out by western blotting showed that αB-crystallin proteins are expressed in normal H9c2 cells, but the proteins' expression was much higher in pcDNA3.1-Cryab transfected cells (P < 0.01). The MTT assays showed that 4 h of H(2)O(2) treatment induced significant injury in H9c2 cells (P < 0.01), but αB-crystallin overexpression can effectively antagonize the H(2)O(2)-induced injury to H9c2 cells (P < 0.05). The results of flow cytometry analysis showed that αB-crystallin overexpression can significantly reduce apoptosis in H(2)O(2)-injured H9c2 cells (P < 0.05). The results of western blotting showed that αB-crystallin overexpression in myocardial cells can reduce the H(2)O(2)-induced release of cytochrome c from the mitochondria (P < 0.05), antagonize the H(2)O(2)-induced downregulation of Bcl-2 (P < 0.05) and magnify the decrease in phosphorylated AKT levels induced by H(2)O(2) injury (P < 0.05). The overexpression of αB-crystallin has a protective effect on H(2)O(2)-injured H9c2 cells, and αB-crystallin can play a protective role by reducing apoptosis, reducing the release of cytochrome c from the mitochondria and antagonizing the downregulation of Bcl-2 expression. The protective effects of αB-crystallin may be related to the PI3K/AKT pathway.
Feng Xu; Haixia Yu; Jinyao Liu; Lu Cheng
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-12-1
Journal Detail:
Title:  Molecular biology reports     Volume:  -     ISSN:  1573-4978     ISO Abbreviation:  Mol. Biol. Rep.     Publication Date:  2012 Dec 
Date Detail:
Created Date:  2012-12-5     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0403234     Medline TA:  Mol Biol Rep     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Department of Cardiovascular Medicine, The First Affiliated Hospital of China Medical University, No. 155, Nanjing Street, Shenyang, 110001, China,
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