Role of GRP78 in sevoflurane preconditioning-induced inhibition of apoptosis in cardiomyocytes of rats / 中华麻醉学杂志

ABSTRACT

Objective To investigate the role of glucose-regulated protein 78 (GRP78) in sevoflurane preconditioning-induced inhibition of apoptosis in cardiomyocytes of rats.Methods The cultured cardiomyocytes of Sprague-Dawley rats were randomly divided into 5 groups (n =30 each) using a random number tablecontrol group (group C),hypoxia-reoxgenation (H/R) group,sevoflurane preconditioning group (group S),siRNA-GRP78 group and siRNA control group.H/R was produced by 2 h exposure of cells to 95％ N2-5％ CO2 in an air-tight chamber at 37 ℃,followed by reoxygenation with 95％ O2-5％ CO2 in an air-tight chamber at 37 ℃ for 1 h.In group S,the cells were incubated with 2.5％ sevoflurane for 20 min,followed by 10-min washout before H/R.In siRNA-GRP78 group,the cells were transfected with siRNA-GRP78 100 nmol/L,and 24 h later preconditioning with sevoflurane was performed and H/R was produced.In siRNA group,cells were transfected with siRNA,and the other treatments were similar to those previously described in siRNA-GRP78 group.After treatment in each group,the expression of GRP78 in myocardial cells and cytochrome c in cytoplasm and mitochondria was detected by Western blot.Lactic dehydrogenase (LDH) and creatine kinase (CK) activities in the culture medium of each group were determined by ELISA.The apoptosis in myocardial cells was assessed by flow cytometry.Apoptotic rate was calculated.Intracellular free Ca2+ concentration ([Ca2+]i) was measured with the fluorescent probe Fura-2/ AM.The opening of mPTP was measured by fluorescence spectrophotometry.Results Compared to group C,the expression of GRP78 in myocardial cells and cytochrome c in cytoplasm was significantly up-regulated,LDH and CK activities in the culture medium,apoptotic rate and [Ca2+]i were increased,and the expression of cytochrome c in mitochondria was down-regulated in H/R group.Compared to group H/R,the expression of GRP78 in myocardial cells and cytochrome c in mitochondria was significantly up-regulated,LDH and CK activities in the culture medium,apoptotic rate,[Ca2+] i and opening of mPTP were decreased,and the expression of cytochrome c in cytoplasm was down-regulated in group S,and no significant change was found in the parameters mentioned above in siRNA group.Compared to group S,the expression of GRP78 in myocardial cells and cytochrome c in mitochondria was significantly down-regulated,LDH and CK activities in the culture medium,apoptotic rate,[Ca2+] i and opening of mPTP were increased,and the expression of cytochrome c in cytoplasm was up-regulated in group siRNA-GRP78.Conclusion GRP78 is involved in sevoflurane preconditioning-induced inhibition of apoptosis in cardiomyocytes of rats,and the mechanism is related to maintenance of intracellular Ca2+ stability and inhibition of opening of mPTP.