Relationship between GRP78 and diabetes mellitus-induced influence on myocardial protection provided by remifentanil postconditioning in vitro

Abstract

Objective To evaluate the relationship between glucose-regulated protein 78 (GRP78) and diabetes mellitus-induced influence on myocardial protection provided by remifentanil postconditioning in vitro. Methods H9c2 cells were cultured in DMEM/F12 culture medium supplemented with 10% fetal bovine serum. The cells were seeded in 96-well (100 μl/well) or 6-well (2 ml/well) plates at the density of 105 cells/ml. The cells were then randomly divided into 6 groups (n = 24 each) using a random number table: normoglycemic control group (group NC), normoglycemic hypoxia/reoxygenation (H/R) group (group NHR), normoglycemic remifentanil postconditioning group (group NRP), hyperglycemic control group (group HC), hyperglycemic H/R group (group HHR), and hyperglycemic remifentanil postconditioning group (group HRP). In NC, NHR and NRP groups, the cells were cultured in normoglycemic culture medium (5.5 mmol/L) for 48 h. In HC, HHR and HRP groups, the cells were incubated in hyperglycemic culture medium (25.0 mmol/L) for 48 h. In NHR, NRP, HHR and HRP groups, after changing the culture medium for Tyrode solution, the cells were exposed to 95% N2-5% CO2 in an incubator at 37 ℃ for 5 h. Subsequently, in NHR and HHR groups, the culture medium was changed to DMEM/F12 culture medium supplemented with 10% fetal bovine serum and glucose at the corresponding concentration, and the cells were incubated for 1 h; in NRP and HRP groups, the cells were incubated for 1 h in the DMEM culture medium containing remifentanil at the final concentration of 1 μmol/L. At 1 h of reoxygenation, the cells of 9 wells in each group were selected to measure the cell viability by CCK8 assay, the cells of 12 wells in each group were selected to determine the activity of lactic dehydrogenase (LDH) released in the supernatant using colorimetric method, and the cells of 3 wells in each group were selected to detect the expression of GRP78 by Western blot. Results Compared with group NC, the cell viability was significantly decreased, the LDH activity was increased, and the expression of GRP78 was up-regulated in group NHR. Compared with group NHR, the cell viability was significantly increased, the LDH activity was decreased, and the expression of GRP78 was down-regulated in group NRP, and the cell viability was significantly decreased, the LDH activity was increased, and the expression of GRP78 was up-regulated in group HRP. Compared with group HC, the cell viability was significantly decreased, the LDH activity was increased, and the expression of GRP78 was up-regulated in group HHR. There was no significant change in the parameters mentioned above between group HRP and group HHR. Compared with group NRP, the cell viability was significantly decreased, the LDH activity was increased, and the expression of GRP78 was up-regulated in group HRP. Conclusion Up-regulation of GRP78 expression may be involved in the mechanism by which diabetes mellitus negates myocardial protection induced by remifentanil postconditioning in vitro. Key words: Heat-shock proteins; Diabetes mellitus; Piperidines; Myocardial reperfusion injury; Ischemic postconditioning