川芎嗪、缬沙坦及曲美他嗪对乳鼠肥大心肌细胞线粒体结构和能量代谢的影响

目的通过大鼠原代心肌细胞培养,探讨心肌肥大过程中有关心肌细胞线粒体结构和功能的病理改变以及川芎嗪、缬沙坦和曲美他嗪对其的药理作用。方法分离和培养大鼠原代心肌细胞,并与血管紧张素Ⅱ(AngⅡ)共培养72h、96h。BCA法检测细胞总蛋白含量,倒置显微镜拍摄并测量细胞直径,反映心肌细胞增殖情况;荧光显微镜测量线粒体内膜膜电位(ΔΨm)、酶标仪检测线粒体单胺氧化酶(MAO)活性、分光光度计检测线粒体细胞色素C氧化酶(COX)活性和线粒体损伤百分率,高效液相色谱法检测心肌细胞内ATP、ADP、AMP含量,反映心肌细胞线粒体结构和功能的损伤情况。在此基础上给予川芎嗪、缬沙坦和曲美他嗪,观察对心肌细胞重构中线粒体结构和功能的药理作用。结果在细胞肥大过程中,心肌细胞MAO活性和线粒体外膜损伤百分率均显著升高(P<0.01),线粒体COX活性和线粒体ΔΨm均显著减低(P<0.01),ATP、ADP含量显著减少(P<0.01),AMP含量显著增加(P<0.01)。川芎嗪能抑制AngⅡ引起的心肌细胞蛋白质合成增加和细胞直径增大,改善线粒体外膜损伤,提高线粒体内膜膜电位及COX活性,降低MAO活性(72h时P<0.01,96h时P<0.05),增加ATP、ADP含量、降低AMP含量。缬沙坦能抑制AngⅡ引起的心肌细胞蛋白质合成增加和细胞直径增大,改善线粒体外膜损伤,提高线粒体内膜膜电位(P<0.05)及MAO活性(72h时P<0.01,96h时P<0.05),增加ATP、ADP含量、降低AMP含量(P<0.01)。曲美他嗪能在72h升高线粒体膜电位(P<0.05),增加ATP、ADP含量(P<0.05)。结论在心肌肥大过程中,存在线粒体结构和功能损害。川芎嗪和缬沙坦在逆转心肌细胞肥大过程中具有保护心肌细胞线粒体结构和功能的作用,曲美他嗪无显著逆转心肌细胞重构作用,对能量代谢的改善作用亦有限。

The Influence of Ligustrazine,Valsartan,and Trimetazidine on Mitochondrial Structure and Energy Metabolism in Rats with Hypertrophiciu

Objective To explore the effect of ligustrazine,valsartan,and trimetazidine on mitochondrial structure and energy metabolism in rats of myocardial cell hypertrophy.Methods Neonatal myocardial cells were isolated and cultured with angiotensinⅡ(AngⅡ) for 72 h or 96 h.The total protein content was detected with BCA method.The cell diameter was measured with inverted microscope to analyze the situation of cardiomyocyte hypertrophy.Mitochondrial membrane potential(ΔΨm) was detected with fluorescence microscope.Mitochondrial single amine oxidase(MAO) activity was tested with spectrophotometer.Mitochondrial cytochrome oxidase(COX) activity and the damage percentage of mitochondrial outer membrane tested with microplate reader.The content of adenosine triphosphate(ATP),adenosine diphosphate(ADP) and adenosine monophosphate(AMP) were tested with high performance liquid chromatography to observe the damage of mitochondrial construction and function and energy metabolism.The cells were treated with ligustrazine,valsartan,and trimetazidine.The pharmacological effects on mitochondrial structure and function in the myocardial cells treated with AngⅡ were observed.Results Total protein content and diameter in cells treated with AngⅡ were increased at 72 h and 96 h compare with control(P<0.01).Myocardial cell MAO activity and the damage percentage of mitochondrial outer membrane were significantly increased(P<0.01).Mitochondrial COX activity and mitochondrial Δ Ψ m were significantly reduced(P<0.01).The content of ATP and ADP were significantly decreased(P<0.01) and the content of AMP was increased(P<0.01).Ligustrazine reduced myocardial cell total protein content(P<0.05) and cell diameter(P<0.01),improved the outer membrane injure(P<0.01),increased mitochondrial Δ Ψ m and mitochondrial COX activity(P<0.05),decreased myocardial cell MAO activity(P<0.01 at 72 h,P<0.05 at 96 h),significantly increased the content of ATP,ADP(P<0.01),decreased the content of AMP(P<0.01).Valsartan reduced myocardial cell total protein content(P<0.01),and cell diameter(P<0.01),improved the outer membrane injure(P<0.01),increased mitochondrial Δ Ψ m(P<0.05),decreased myocardial cell MAO activity(P<0.01 at 72 h,P<0.05 at 96 h),significantly increased the content of ATP,ADP(P<0.01),decreased the content of AMP(P<0.01).At 72 h,trimetazidine increased mitochondrial Δ Ψ m(P<0.05),and increased the content of ATP and ADP(P<0.05).Conclusion During the process of myocardial hypertrophy,damages of mitochondrial structure and function occurred,and changes of myocardial cell energy metabolism occurred secondary to the damage of mitochondria.Ligustrazine and valsartan could reverse myocardial hypertrophy.Trimetazidine could not reverse myocardial cell hypertrophy,and its effect on energy metabolism was limited.Thus,reversing myocardial hypertrophy and protecting mitochondria had benefit to energy metabolism of the myocardial cell.