Aim To investigate protective effects and mechanism of pinacidil, metoprolol, glutamine, insulin on H9c2 cardiac myocytes exposed to simulated hy-poxia/reoxygenation. Methods H9c2 cardiac myocytes were randomly divided into seven groups: ①Control group; ② Hypoxia/reoxygenation ( H/R ) group; ③ Pinacidil ( Pin ) group; ④ Metoprolol ( Met ) group; ⑤Glutamine ( Glu ) group; ⑥ Insulin ( Ins ) group; ⑦ ) Pinacidil + Metoprolol + Glutamine + Insulin ( PMGI ) group. The survival rates of cardiomyocytes were detected. The activity of lactate dehydrogenase ( LDH ) in the culture medium was measured. The mitochondria were labeled by Rhodamine123 fluorescence probe and the fluorescent intensity produced by Rhl23 was measured, which reflected changes of mitochondria! Mem-brane potential ( MMP, ΔΨm ). The apoptotic percentage was measured with flow cytometry. Results The combined use of pinacidil, metoprolol, glutamine, and insulin could protect the H9c2 cardiac cell membrane caused by hypoxia/reoxygenation injury, increase cell survival rate,decrease LDH leakage, decrease the apoptotic rate and increase the mitochondria! Membrane potential. Conclusions Compared to the individual drug, the combination of the different mechanisms protects the mitochondria of cardiac myocyte, decreases the injury and enhances antiapoptotic effection of myocar-dial cells.%目的 研究吡那地尔(pinacidil,Pin)、美托洛尔(metoprolol,Met)、谷氨酰胺(glutamine,Glu)、胰岛素(insulin,Ins)4药联用对缺氧/复氧(hypoxia/reoxygenation,H/R)所致H9c2心肌细胞损伤的抗凋亡保护作用并探讨其作用机制.方法 将培养的H9c2心肌细胞随机分为7组,即①正常对照(Con)组;② 缺氧/复氧(H/R)组;③吡那地尔(Pin)组;④美托洛尔(Met)组;⑤谷氨酰胺(Glu)组;⑥胰岛素(Ins)组;⑦ 吡那地尔、美托洛尔、谷氨酰胺和胰岛素4药联用(PMGI)组.细胞经药物处理建立H/R损伤模型,并测定各组H9c2心肌细胞的存活率;收集培养液测定乳酸脱氢酶(lactate dehydrogenase,LDH)活性;流式细胞仪检测心肌细胞凋亡百分率;罗丹明123 (Rhodamine123,Rh123)荧光探针标记线粒体,检测荧光强度以反映心肌细胞线粒体膜电位(mitochondrial membrane potential,△Ψm)变化.结果 对于H/R损伤的H9c2心肌细胞,吡那地尔、美托洛尔、谷氨酰胺与胰岛素4药联用组与药物单用组或H/R组相比能明显提高细胞存活率,保护其细胞膜,减少LDH渗漏;减少细胞凋亡率;提高△Ψm.结论 吡那地尔、美托洛尔、谷氨酰胺和胰岛素4药联用后,通过不同途径保护心肌细胞,达到协同抗凋亡作用.
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