αB-crystallin regulates oxidative stress-induced apoptosis in cardiac H9c2 cells via the PI3K/AKT pathway

The present study was carried out to observe the protective effects of alpha B-crystallin protein on hydrogen peroxide (H2O2)-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 alpha B-crystallin in the treatment of cardiovascular disease. Western blotting was used to measure the expression of alpha B-crystallin in cultured H9c2 cells in vitro. A alpha B-crystallin recombinant expression vector, pcDNA3.1-Cryab, was constructed to transfect H9c2 cells for the establishment of cells that stably expressed alpha B-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 mu M H2O2 to establish a model of H2O2 injury to cells. H2O2 was applied to H9c2 cells that were stably transfected with alpha B-crystallin, and the effect of alpha B-crystallin overexpression on the viability of myocardial cells subjected to H2O2-induced injury was measured by the MTT assay. The effect of alpha B-crystallin overexpression on the H2O2-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 alpha B-crystallin overexpression on the release of cytochrome c from the mitochondria. Western blotting was also used to measure the effect of alpha B-crystallin overexpression on the expression of the anti-apoptosis protein Bcl-2 and components of the phosphatidylinositol 3-OH kinase (PI3K)/AKT pathway. The alpha B-crystallin recombinant expression vector pcDNA3.1-Cryab successfully transfected H9c2 cells, and H9c2 cells that were stably transfected with alpha B-crystallin were established after G418 selection. The measurements carried out by western blotting showed that alpha B-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 H2O2 treatment induced significant injury in H9c2 cells (P < 0.01), but alpha B-crystallin overexpression can effectively antagonize the H2O2-induced injury to H9c2 cells (P < 0.05). The results of flow cytometry analysis showed that alpha B-crystallin overexpression can significantly reduce apoptosis in H2O2-injured H9c2 cells (P < 0.05). The results of western blotting showed that alpha B-crystallin overexpression in myocardial cells can reduce the H2O2-induced release of cytochrome c from the mitochondria (P < 0.05), antagonize the H2O2-induced downregulation of Bcl-2 (P < 0.05) and magnify the decrease in phosphorylated AKT levels induced by H2O2 injury (P < 0.05). The overexpression of alpha B-crystallin has a protective effect on H2O2-injured H9c2 cells, and alpha B-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 alpha B-crystallin may be related to the PI3K/AKT pathway.