Study On The Expression Of Calpain-1 And Apoptosis In Myocardial Of Neonatal Rats After Hypoxic-ischemic Brain Damage

Hypoxic ischemic brain damage of neonates, HIBD of neonates, is caused by newborn suffocation in the perinatal period, which is the common reason of neonatal deaths and neurological sequelae. The pathogenesis of HIBD is quite complicated. Hypoxia is basic pathophysiological change, Hemodynamics disorder causes multiple organs exhaustion. HIBD is a systemic pathological process, hypoxia and the cumulation of acid metabolites lead cardiac myocytes to energy metabolic dysfunction, the lose of ATP, so that it will cause myocardial damage. Besides necrosis (non-viable non-apoptotic cell, NVNA), cardiomyocytes may also undergo apoptosis, and it is more than NVNA in hypoxic ischemic atmosphere.The aim of this study was to observe the effectiveness that hypoxic ischemic (HI) took on apoptosis of rats cardiomyocytes, and to detect the expression of Calpain -1 mRNA and protein level in myocardial tissue, and to detect the variation laws of the expression of Bcl-2, Bax, Fas, and Caspase-3 mRNA and study the relationship among Calpain-1, related genes of apoptosis, with cardiomyocyte apoptosis (CMA).Hypoxic-ischemic brain damage model was built up using 7-day-year old newborn Wistar rats. All rats in the study were divided into 8 groups as sham operation control group,2hr,12 hr,24hr,2d,3d,5d, and 7days after HI group, with 8 rats each. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to detect the expression of Calpain -1, Bcl-2, Bax, Fas and Caspase-3 mRNA in myocardial tissue of rats. Western blot technique was used to detect calpain-1 protein activation. Apoptosis index (AI) was detected by terminal deoxynucleotidyl transferase mediated d-UTP nick end labeling staining (TUNEL).1.2 days after HI, the number of apoptotic cells of newborn rats began to increase. From 12hours to 3days, the AI was significantly (P<0.01) higher in study group than in control group. The number peaks at 3d, and a decrease at 5d, but it was still higher than that in control group. 2. Calpain-1 mRNA of myocardial tissues began to increase 12h after H1, peak expression was shown at 2d(P<0.01), its level decreased during 3d-5d and was still more than that of the control(P<0.01). Calpain-1 schizolysis into two section of 76 and 80, activated calpain-1 protein increased 2h after H1 (P<0.05), reaehed its peak at 3d(P<0.01) and was stillin high level 5d after HIBD(P<0.01),and was same as the control at 7d. Calpain-1 protein activity (76KD/80KD) had a relationship with AI (r =-0.853; p〈0.01).3. Compared with control group, Bax mRNA began to increase at 2h after HI, reached the peak at 2d, its level decreased at 3d-5d. Bcl-2 mRNA began to decreased at 2h after HI, and the expression drastically decreased at 12h-24h (p<0.01), and reached the minimum level at 2d then began to increase, at last stays the same as the control at 7d. In the HI group, the ratio of Bcl-2 to Bax gradually began to decreases at 2h after HI, and gated to the minimum at 2d, and its level decreased during 3d-5d, and staid the same as the control at 7d finally, no statistically significant. So, the ratio of Bcl-2 to Bax has a negative correlation with AI(r=-0.601; p<0.01).Expression of Caspase-3 mRNA began to increase at 2h after HI, and its level increased from 12h to 2d (p<0.01), and reached the peak at 2d. Its level decreased during 3-5d, and still more than that of control (p<0.01). Without difference was with control group at 7d.4. Compared with control group, Fas mRNA increased 2h after HI (p<0.01), and added up significantly from 12h to 2d (p<0.01) compared with the control, and reached the peak at 3d. It decreased at 5d, and was still higher than control group at 7d(p<0.01).1.2 hours after HI apoptosis cells began increasing, the peak of which at 3d after HI. It proved that myocardial cells apoptosis of HIBD newborn rats really existed.2. After HIBD, the expression of Calpain -1 mRNA and protein level in myocardial tissue were increased significantly. It has obvious differences compared with control group, which was a sign that Calpain-1 may be a reason of myocardial damage in the condition of hypoxic and ischemic. 3. The expression of gene Bax in myocardial tissue increases after HI, gene Bcl-2 decreases. The ratio of Bcl-2 and Bax reduced after HIBD, the result showed that Bax can activate the process of apoptosis and the effect of Bcl-2 was inverse.4. Expression of caspase-3 and Fas was obviousiy higher, it showed that level of Caspase-3 and Fas had the positive correlation with cell apoptosis index. It proved that Caspase-3 and Fas participated and mediated the CMA.