| Objective:To investigate protective effects and mechanism of pinacidil, metoprolol, glutamine, insulin on rat H9c2 cardiac myocytes exposed to simulated hypoxia/reoxygenation.Methods:Rat H9c2 cardiac myocytes were randomly divided into seven groups:(1) Control group:cardiomyocytes were seeded in normoxic incubation; (2) Hypoxia/reoxygenation (H/R) group, in which the culture dishes were transferred to a hypoxic incubator in a humidified atmosphere that contained 95% N2-5% CO2 for 180 min of hypoxia, and, subsequently, the dishes were transferred to a normoxic incubator (95% air-5% CO2) for 150 min of reoxygenation; (3) Pinacidil (Pin) group:cardiomyocytes were incubated with pinacidil (1×10-6 mol/L) for 30 minutes before hypoxia; (4) Metoprolol (Met) group:cardiomyocytes were incubated with metoprolol (2×10-5 mol/L) for 30 minutes before hypoxia; (5) Glutamine (Glu) group: cardiomyocytes were incubated with glutamine (8×10-3 mol/L) for 30 minutes before hypoxia; (6) Insulin (Ins) group:cardiomyocytes were incubated with insulin (0.01 U/L) for 30 minutes before hypoxia; (7) Pinacidil+Metoprolol+ Glutamine+Insulin (PMGI) group:cardiomyocytes were incubated with pinacidil (1×10-6 mol/L), metoprolol (2×10'5 mol/L), glutamine (8×10-3 mol/L), insulin (0.01 U/L) for 30 minutes before hypoxia. The survival rates of cardiomyocytes were detected. The activity of lactate dehydrogenase (LDH) and superoxide dismutase (SOD) in the culture medium were measured. The mitochondria were labeled by Rhodaminel23 fluorescence probe and the fluorescent intensity produced by Rh123 was measured. which reflects changes of mitochondrial membrane potential (MMP,△ψm). The apoptosis rates were measured with flow cytometry. Western blot was performed to exam expression of heat shock protein 70 (HSP70) in cardiomyocytes respectively.Results:1. Cell survival rate:compared with control group, the cell survival rate of hypoxia/reoxygenation's group was reduced significantly (96.0±4.0% vs 48.3.±10.3%, and P<0.01); and compared with H/R group, the cell survival rate of Pin,Met,Glu,Ins,PMGI was increased significantly (72.5±5.4% and 71.2±5.2% and 70.2±5.5% and 68.4±10.8% and 68.4±10.8% vs 48.3.±10.3 %, and P<0.01), there was no significance between Pin,Met,Glu,Ins,PMGI and Con(P>0.05); Compared with Pin,Met,Glu,Ins,PMGI, the cell survival rate of PMGI was increased significantly (P<0.01), there was no significance between PMGI and Con (P>0.05).2. Lactate dehydrogenase activity:compared with control group, the LDH activity of hypoxia/reoxygenation's group was increased significantly (584.1±17.0 U/L vs 838.1±28.2 U/L, and P<0.01); and compared with H/R group, the LDH activity of Pin,Met,Glu,Ins,PMGI was decreased significantly (637.8±25.4 U/L,656.3±21.8 U/L,667.5±26.5 U/L,703.0±36.6 U/L,597.0±12.7 U/L vs 834.8±25.4 U/L and P<0.01), there was no significance between Pin,Met,Glu,Ins,PMGI and Con (P>0.05); Compared with Pin,Met,Glu,Ins,PMGI, the LDH activity of PMGI was decreased significantly (P<0.01), there was no significance between PMGI and Con (P>0.05).3. Total superoxide dismutase activity and Mn-SOD activity:compared with control group, the T-SOD activity of hypoxia/reoxygenation's group was decreased significantly (53.44±1.22 U/ml vs 35.90±1.64 U/ml, and P<0.01), the Mn-SOD activity was also decreased significantly (20.22±1.47 U/ml vs 12.66±0.54 U/ml, and P<0.01); and compared with H/R group, the T-SOD activity of Pin,Met,Glu,Ins,PMGI was increased significantly (50.02±3.76 U/ml,44.45±4.83 U/ml,48.38±3.87 U/ml,42.39±2.30 U/ml,53.19±1.57 U/ml vs 35.90±1.64 U/ml, and P<0.01), and the Mn-SOD activity was also increased significantly(18.02±2.17U/ml,15.24±1.87 U/ml.17.34±1.69 U/ml,14.19±1.08 U/ml,19.78±1.49 U/ml vs 12.66±0.54 U/ml, and P<0.01), there was no significance between Pin,Met,Glu,Ins,PMGI and Con (P>0.05); Compared with Pin,Met,Glu,Ins,PMGI, the T-SOD activity and Mn-SOD activity of PMGI was decreased significantly (P<0.05), there was no significance between PMGI and Con (P>0.05). 4.△ψm:compared with control group, the mitochondrial fluorescence intensity of hypoxia/reoxygenation's group was decreased significantly (248712±12377 vs 153851±5498, and P<0.01); and compared with H/R group, the mitochondrial fluorescence intensity of Pin,Met,Glu,Ins,PMGI was increased significantly (190514±10428,160514±7784,166656±6150.169384±7696,224103±11731 vs 153851±5498, and P<0.01), there was no significance between Pin,Met,Glu,Ins,PMGI and Con (P>0.05); Compared with Pin,Met,Glu,Ins,PMGI, the mitochondrial fluorescence intensity of PMGI was increased significantly (P<0.01), there was no significance between PMGI and Con (P>0.05).5. Cell apoptosis rate:compared with control group, the apoptosis rate of hypoxia/reoxygenation's group was increased significantly (0.11±0.03% vs 10.95±1.82%, and P<0.01); and compared with H/R group, the apoptosis rate of Pin,Met,Glu,Ins,PMGI was decreased significantly (2.85±0.19%, 6.83±0.92%,3.47±0.53%,4.90±0.70%,1.44±0.11% vs 10.95±1.82%, and P<0.01); Compared with Pin,Met,Glu,Ins, the apoptosis rate of PMGI was increased significantly (P<0.05).6. Heat shock protein 70:compared with control group, the expression of HSP70 of hypoxia/reoxygenation's group was increased significantly (10.2±1.2 vs 20.7±2.1, and P<0.01); and compared with H/R group, the expression of HSP70 of Pin,Met,Glu,Ins,PMGI was increased significantly (42.2±4.7,43.2±4.9,56.8±5.2,44.1±3.9,71.7±6.3 vs 20.7±2.1, and P<0.01); Compared with Pin,Met,Glu,Ins, the expression of HSP70 of PMGI was increased significantly (P<0.01).Conclusion:The combination therapy of pinacidil, metoprolol, glutamine. insulin can protect the rat H9c2 cardiac cell membrane caused by hypoxia/reoxygenation injury; increase cell survival rate, derease LDH leakage, protect cardiac cell against H/R injury through elevating T-SOD and Mn-SOD activity, decrease the apoptosis rate, increase the mitochondrial membrane potential and the expression of HSP70. |
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