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目的探讨左旋尼汀对心肌细胞凋亡的抑制作用及其可能的作用机制。方法采用培养的新生乳大鼠心肌细胞制备H2O2200μmol.L-1氧化损伤模型。实验分为正常对照组、H2O2200μmol.L-1模型组、左卡尼汀(0.3,0.6及1.2 mmol.L-1)组、左卡尼汀1.2 mmol.L-1+H2O2200μmol.L-1组。左卡尼汀0.3,0.6和1.2 mmol.L-1作用1 h后加入H2O2200μmol.L-1培养12 h,MTT法测定心肌细胞存活率,流式细胞仪测定心肌细胞的凋亡率,Western印迹法测定胱天蛋白酶3表达;用试剂盒方法检测过氧化物歧化酶(SOD)活性和丙二醛(MDA)水平。左卡尼汀1.2 mmol.L-1作用5 min后加入H2O2200μmol.L-1,采用Till阳离子测定系统监测5 min的心肌细胞[Ca2+]i瞬间变化。结果 H2O2200μmol.L-1作用于心肌细胞12 h能引起心肌细胞凋亡。左卡尼汀0.3,0.6和1.2 mmol.L-1能够不同程度的逆转由H2O2所致的损伤,使SOD活性明显增加,MDA水平明显降低(P<0.01)。左卡尼汀1.2 mmol.L-1作用最强,能够明显降低心肌细胞胱天蛋白酶3表达(P<0.01),明显降低H2O2引起的[Ca2+]i瞬间变化升高(P<0.01),但对于H2O2引起无钙血清培养的心肌细胞静息钙升高无影响。结论左卡尼汀能够抑制由H2O2引起的心肌细胞凋亡,该抑制作用可能与其保护细胞膜和减轻钙通道的损害、纠正[Ca2+]i瞬变失调及其抗氧化作用有关。
Objective To investigate the inhibitory effect of levosidine on cardiomyocyte apoptosis and its possible mechanism. Methods Cultured neonatal rat cardiomyocytes were used to prepare oxidative damage model of H2O2 at 200μmol.L-1. The experiment was divided into normal control group, H2O2200μmol.L-1 model group, levocarnitine (0.3,0.6 and 1.2 mmol.L-1) group, levocarnitine 1.2 mmol.L-1 + H2O2200μmol.L-1 group . L-carnitine 0.3,0.6 and 1.2 mmol.L-1 for 1 h and then added H2O22200μmol.L-1 cultured for 12 h. The survival rate of cardiomyocytes was determined by MTT method. The apoptosis rate of cardiomyocytes was measured by flow cytometry. Western blot Method was used to determine the expression of caspase-3. The activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) were detected by kit method. After 2 min L-1 levocarnitine treatment, H2O2200 μmol·L-1 was added for 5 min. The transient changes of [Ca2 +] i in cardiomyocytes were detected by Till cationization system for 5 min. Results H202200μmol.L-1 induced cardiomyocyte apoptosis at 12 h. L-carnitine 0.3, 0.6 and 1.2 mmol.L-1 could reverse the damage induced by H2O2 to some extent, which increased the activity of SOD and decreased the level of MDA significantly (P <0.01). The effect of L-carnitine 1.2 mmol.L-1 was the strongest, which could significantly decrease the expression of caspase 3 (P <0.01) and the transient increase of [Ca2 +] i induced by H2O2 (P <0.01) There was no effect on the resting calcium in cardiomyocytes induced by H2O2 without Ca2 +. Conclusion L-carnitine can inhibit the apoptosis of cardiomyocytes induced by H2O2, which may be related to protecting the cell membrane and alleviating the damage of calcium channels and correcting the transient disturbance of [Ca2 +] i and its antioxidation.