| Objective;To stady whether 14-3-3 protein involves in the delayed protection 24 hr after anoxic preconditioning. Methods; Cultured myocardial cells of neonatal SD rats have been randomly divided into six groups: Control group; PD98059+Control group; anoxia reoxygenation (A/R)group;anoxia preconditioning group(APC); DMSO+APC group; PD98059 +APC group. In each group, the cardiomyocytes were subjected to anoxia (3 hr)followed by reoxygenation (1 hr) 24 hr after anoxic preconditioning on the model of the cultured cardiomyocytes. At the end of experiment, myocardial cells beating rate, cell viability and the activity of LDH in culture were measured,furthermore, The morphology or ultrastructure assessment of ventricular myocytes was observed by inverted microscope or transmission electron microscope, the expression of 14-3-3 protein were measured by western blotting techniques, respectively. Results: ①Beating rate of ventricular myocytes: There was no significance in PD98059+Control group (95±13beat/min)(p>0.05),compared with that of control group(98±16 beat/min);The beating rate was significantly slow in A/R group(13±3 beat/min) compared with that of control group(p<0.01); Between APC group(92±10 beat/min) and DMSO+APC group (90±8 beat/min), the beating rate was similar to each other(p>0.05),and Compared with A/R , The beating rate was significantly increased(p<0.01); The beating rate of PD98059+APC group (18±6 beat/min) was significantly lower than APC groups.②the cell viability: There was no significance in PD98059+Control group(91.2±12.5%) compared with that of control group(89.6±10.8%)(p>0.05)；The cell viability of A/R(38.9±7.2%) was significantly decreased when compared with that of control group(p<0.01);The viability of APC (78.2±10.6%) , DMSO+APC (80.6±12.4%) was significantly increased when compared with A/R respectively,(p<0.01),between APC and DMSO+APC, The cell viability was similar to each other(p>0.05);The viability of PD98059+APC (57.9±6.8%) was significantly decreased when compared with APC group.③LDH activity:LDH activity of PD98059+Control(3.45±0.42 IU/L) was similar to that of Control group(3.06±0.51 IU/L)(p>0.05)；LDH activity of A/R (30.26±4.88 IU/L) was significantly increased when compared with Control group(p<0.01)； LDH activity of APC(14.12±2.06 IU/L)or DMSO+APC (12.65±2.81 IU/L )was significantly decreased when compared with A/R respectively (p<0.01),but LDH activity of DMSO+APC was similar to that of APC group(p>0.05)；LDH activity of PD98059+APC (28.52±4.62 IU/L) was similar to that of A/R(p>0.05)but was dramatically increased when compared with APC group(p<0.01)；④Expression of 14-3-3 protein; Western blotting revealed 14-3-3 protein showed a basic expression in Contral group.The expression quantity of 14-3-3 protein in PD98059+Control group was similar to that of Control and 0.98±0.12 fold of Control group(p>0.05).14-3-3 protein expression of A/R was increased when compared with Control group and 3.52±0.46 fold of Control group(p<0.01).The 14-3-3 protein contents were increased significantly at APC and DMSO+APC,compared with those in Control and A/R group(p<0.01),they were 5.41±0.69, 5.56±0.72 fold of Control group respectively.but 14-3-3 protein expression of DMSO+APC was similar to that of APC group(p>0.05). 14-3-3 protein in PD98059+APC group was 4.06±0.62 fold of Control group ,and was dramatically decreased when compared with APC group(p<0.01)but was similar to that of A/R(p<0.05),which demonstrated PD98059 suppressed upregulation of 14-3-3 protein;⑤ Ultrastructural assessment by the transmission electron microscope: For Control group, the cell membrane was intact, mitochondria scattered loosely throughout the cytoplasm, mitochondria membrane was intact and clear, nuclear chromatin material was uniformly dispersed. The myocytes subjected to A/R injury were characterized by calcified and elongated mitochondria,dispersed nuclear chromatin material,ruptured nuclear membrane,disappeared myo-fibers and... |
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