DCA干预对缺氧复氧肥大心肌细胞能量代谢及细胞凋亡的影响

Effects of Dichloroacetate on Changes of Energy Metabolic-pathway of Hypertrophic Cardiomyocytes and Antapoptosis

目的:探讨缺氧复氧损伤诱导体外培养的新生大鼠肥大心肌细胞凋亡及能量代谢途径变化及药物干预的作用。方法:取体外培养的新生大鼠心肌细胞,以血管紧张素Ⅱ诱导其肥大,分4组:一组于3%O_2、5%CO_2、92%N_2三气培养箱中培养12 h,再恢复正常条件培养4 h,建立缺氧复氧损伤的肥大心肌细胞模型;另三组加入二氯乙酸盐(DCA)使其终浓度分别为10^(-3)mmol/L、10^(-4) mmol/L和10^(-5)mmol/L,再缺氧复氧相同时间。电镜观察肥大心肌细胞及凋亡细胞的超微结构变化,Hochest33342/PI荧光染色识别凋亡细胞;TUNEL法观察心肌细胞凋亡形态学特征,并记数凋亡心肌细胞数,检测心肌细胞凋亡率;以同位素液闪计数法测定丙酮酸脱氢酶(PDH)肉碱脂酰转移酶-1(CPT-1)活性,以及葡萄糖有氧氧化率,葡萄糖酵解率和脂肪酸有氧氧化率。结果:肥大心肌细胞在缺氧12 h复氧4 h,TUNEL法可检测到阳性的凋亡细胞,凋亡率为(19.99±4.88)%,肥大心肌细胞缺氧培养12h后加入DCA10^(-3)mmol/L、10^(-4)mmol/L和10^(-5)mmol/L再复氧4 h检测其凋亡率分别为(16.5±3.24)%、(17.4±3.72)%和(18.4±3.44)%;与正常心肌细胞比较,肥大心肌细胞总的PDH活性没有明显改变,但活化型PDH活性和葡萄糖氧化代谢率(GOR)显著增强,CPT-1活性和脂肪酸有氧氧化代谢率(FOR)显著降低;与对照肥大心肌细胞比较,二氯乙酸(DCA 10^(-3)mmol/L～DCA 110^(-3)mmol/L)呈剂量依赖性的升高PDH活性和GOR,抑制CPT21活性,FOR和葡萄糖酵解率(glucolysis rate,GLR)。结论:DCA对缺氧复氧损伤引起的肥大心肌细胞凋亡有抑制作用。肥大心肌细胞能量代谢向糖代谢转化,DCA可进一步增强糖有氧氧化代谢抑制脂肪酸代谢。

Objectives： To explore effects of dichloroacetate on changes of energy metabolic pathway of rat hypertrophic cardiomyocytes induced with hypoxia/reoxygenation and its apoptosis. Methods： Hypertrophied cardiomyocyte model induced by Angiotensin Ⅱ was set up. By [3H]-Leu incorporation, the model of hypertrophied cardiomyocytes was assessed. The cultured media were replaced by low glucose DMEM supplemented before hypoxia.The cardiomyocytes were incubated at 37 ℃ in an air-tighe incubator where normal air was replaced by 92% N2, 5% CO2, 3% O2 for 24 h to produce hypoxia, and then the air was replaced by 23% O2, 5% CO2 for 4 h to produce reoxygenation. Apoptotic cell death was evaluated using a modified TUNEL （TdT-mediated Dutp Nick-EndL abeling） assay （DeadEndTMColorimetric TUNEL）. The activity of pyruvat dehydrogenase （PDH）, carnitine palmitoyltrans cerase 1 （ CPT- 1 ）, glucoseoxi dationrate （GOR）, glucolysisrate （GLR） and fatyy acid oxidation rate （FOR） were determined by liquid scintillation counting. Results： The apoptosis rate ofcardiomyocytes increased with the duration of hypoxia/reoxygenation （H/R）. The model for H/R induced apoptosis of culturing rat hypertrophic cardiomyocytes was established successfully. When cardiomyocytes were pretreated with dichloroacetate, the rates of apoptosis were decreased in a dose-dependent way. Changing metabolic pathway has effect on apoptosis of rat hypertrophic cardiomyocytes. The activity of the active PDH and GOR of hypertrophic cardiocytes increased but their activity of CPT-1 and FOR decreased significantly compared with thenormal cardiocytes. Compared with the control hypertrophic cardiomyocytes, the activity of the active PDH and GOR of hypertrophic cardiocytes were stimulated and their activity of CPT-1 and FOR and GLR were inhibited by DCA （10^-5-10^-3） respectively in a dose-dependent manner. Conclusion： Heglucose oxidation increased and fatty oxidation decreased inhypertrophic cardiomyocytes, and it could be enhanced deffectively byDCA.