| [Background]Ischemic heart disease (IHD) is the number one killer for human health. There are more than 20 million IHD patients in China and 1 million people are affected with this disease every year. The core of its treatment is to restore the myocardial blood supply. However, restoration of myocardial blood supply can lead to complex pathophysiological changes, which may increase myocardial ischemia/reperfusion injury(I/RI) and easily lead to death and other serious complications. I/RI is the reason for the poor clinical outcomes of ischemic heart disease. Notch signaling pathway plays a basic role in the occurrent, developmental, pathological and physiological processes of cardiovascular system. Studies have shown that (1) Notch1 was activated in border zone cardiomyocytes following cardiac infarction, and (2)infarcted hearts injected with adenoviral vector expressing the NICD exhibited improved hemodynamic function; these results, from a mouse model, suggested that Notch signaling has a cardioprotective role following cardiac injury. So far, it has not been reported that Notch signaling pathway plays a role in acute phase of myocardial I/RI.[Objectives]1. To investigate the changes of Notch signaling pathway molecules (NICD and Hes1) before and after myocardial ischemia and reperfusion.2. To explore the specific mechanisms of Notch signaling pathway involved in the modulation of myocardial I/RI process.[Methods]1. Twenty patients underwent cardiac surgery with cardiopulmonary bypass (CPB) were involved in this study. A small piece of right atrial tissue was obtained before CPB setup, and another piece of right atrial tissue was obtained at the same site of the first sample 15 minutes after heart beating resumed. NICD and Hes1 were detected by western blotting.2. Neonatal rat cardiomyocytes were cultured ex vivo, and subjected to simulated ischemia and reperfusion (SI/R) protocol. DAPT (the specific inhibitor of Notch signaling pathway) or Jagged1 protein (one ligand of Notch signaling pathway, which can active Notch signaling pathway) were used before SI/R, to observe the influence of inhibition or activation of Notch signaling pathway on cardiomyocytes IR/I through following ways: 1) NICD and Hes1 were detected with immunofluorescence; 3) Cardiomyocytes viability was evaluated with MTT; 4) Lactate dehydrogenase (LDH) release were detected; 5) Cardiomyocyte apoptosis assay detected with TNUEL; 6) Bcl-2, caspase 3, NICD and Hes1 expressions were detected with Western blotting. [Results]1. NICD and Hes1 in human cardiac tissue decreased significantly after I/RI processes during cardiac surgery with CPB. The expression of NICD decreased to 21.97%±8.45% compared to pre-ischemia value, and the expression of Hes1decreased to 26.56%±6.59% compared to pre-ischemia value, P<0.01.2. NICD and Hes1 in cardiomyocytes decreased significantly after SI/R process. The expression of NICD decreased to 25.39%±7.29% compared to pre-ischemia value, and the expression of Hes1 decreased to 30.97%±8.45% compared to pre-ischemia value, P<0.01.Jagged1 pretreatment significantly increased NICD and Hes1 expressions which also associated with the increased cell viability, the decreased LDH release and cell apoptosis. DAPT pretreatment significantly decreased NICD and Hes1 expressions, which also associated with the decreased cell viability, increased LDH release and cell apotosis.The data of Jagged1 pretreatment + SI/R group was compared with I / R group:1) Cell activity was significantly increased (0.55±0.05 vs 0.42±0.06, P<0.05); 2) LDH release was significantly decreased (28.32%±3.15% vs 36.25±3.29%, P<0.01); 3) Apoptosis index was significantly decreased (23.52%±13.47% vs 34.18%±3.51%, P<0.05); 4) The expression of NICD was significantly increased (61.12%±5.71% vs 45.27±6.92%, P<0.05), the expression of Hes1 was significantly increased (59.46%±3.85% vs 44.98%±5.83%, P<0.05); 5) The expression of Bcl2 was significantly increased (69.25%±4.82% vs 53.65±3.85%, P<0.05), the expression of Caspase3 was decreased significantly (126.21%±5.14% vs 216.64±8.21%, P<0.01).Jagged1 + DAPT + I / R group was compared with Jagged1 + I / R group: 1) cell viability decreased significantly (0.23±0.05 vs 0.55±0.05, P<0.01); 2) LDH release increased significantly (49.63%±3.89% vs 28.32%±3.15%, P<0.01); 3) The apoptotic index was significantly increased (46.29±4.08% vs 23.52%±13.47%, P<0.01); 4) The expression of NICD was significantly decreased (22.54±6.30% vs 61.12%±5.71%, P<0.01), the expression of Hes1 was significantly decreased (31.24%±4.33% vs 59.46%±3.85%, P<0.01); 5) The expression of Bcl2 was significantly decreased (39.45±4.12% vs 69.25%±4.82%, P<0.01), the expression of Caspase3 was significantly higher (165.64%±8.21% vs 126.21%±5.14%, P<0.01).[Conclusion]1. This is the first report showing that NICD and Hes1 in human cardiac tissue decreased significantly after I/RI processes during cardiac surgery with CPB, which suggests that Notch signaling pathway may be involved in the modulation of myocardial I/RI process.2. NICD and Hes1 in cardiomyocytes decreased significantly after SI/R process. Jagged1 pretreatment significantly increased NICD and Hes1 expressions which also associated with the increased cell viability, the decreased LDH release and cell apoptosis. DAPT pretreatment significantly decreased NICD and Hes1 expressions, which also associated with the decreased cell viability, increased LDH release and cell apotosis. Our findings imply that the Notch signaling pathway plays a regulatory role in myocardial IR injury and the up-regulation of the Notch signaling pathway favors a protective effect... |
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