摘要
目的阐明干预肥大心肌细胞代谢途径转化防治缺血再灌注所致细胞凋亡的作用及其分子机制。方法应用血管紧张素Ⅱ(0.1mol.L-1)诱导培养小鼠心肌细胞肥大,在三气孵箱中进行缺氧/复氧处理模拟缺血再灌注。缺氧/复氧前,分别给予无药物干预及DCA(1mmol.L-1)、TMZ(5μmol.L-1)、LC(50μmol.L-1)、AA(10μmol.L-1)预处理。葡萄糖和脂肪酸氧化代谢率采用放射性计数法测定。RT-PCR和Western Blot法分别测定细胞色素C和凋亡诱导因子mRNA和蛋白质表达水平。分光光度法测定Caspase-3活性。Hoechst33258染色检测细胞凋亡百分数。结果结果表明,缺氧12h及复氧4h后肥大心肌细胞葡萄糖氧化代谢率均显著降低,而脂肪酸氧化代谢率显著升高,DCA、TMZ、LC均可明显抑制缺氧/复氧后葡萄糖氧化代谢的下降,明显抑制缺氧/复氧后葡萄糖氧化代谢的升高,AA使缺氧/复氧后肥大心肌细胞葡萄糖氧化代谢率的进一步降低和脂肪酸氧化代谢率进一步升高。同时,DCA、TMZ、LC均可明显抑制线粒体细胞色素C和AIF mRNA和蛋白质表达水平,明显抑制细胞色素C和AIF蛋白的核转位,抑制caspase-3活性,而AA作用相反。DCA、TMZ、LC明显抑制缺氧/复氧后肥大心肌细胞凋亡率,而AA作用相反。结论上述结果提示,干预肥大心肌细胞代谢途径通过抑制线粒体凋亡蛋白表达、释放有效防治肥大心肌细胞凋亡。
Aim To illustrate the actions and molecular mechanisms of interventions taken to convert the metabolism pathways of cellular apoptosis caused by hypoxia and myocytes. hypoxia-re Methods perfusion in hypertrophic cardioAngiotensin I1 （0. 1 μmol·L^-1） was applied to induce the hypertrophy of mice cardiomyocytes. The cardiomyocytes received the treatment of hypoxia-reperfusion in a tri-gas incubator to simulate the conditions of hypoxiaeperfusion. Before hypoxia/reperfusion, no drug intervention and pre- treatments of DCA （ 1 mmol · L^- 1 ）, TMZ （ 5 μmol·L^-1 ）, LC（50 μmol·L^-1 ） and AA（ 10 μmol·L^-1 ） were given respectively. The glucose and fatty acid oxidative metabolism rates were measured with radioactive counting methods. RT-PCR and Western blot methods were employed respectively to measure the mRNA and protein expression levels of cytochrome C and apoptosis inducers. The spectrophotometry method was used to measure the activity of Caspase-3 and Hoechst 33258 staining to quantify the percentage of cellular apoptosis. Results At post-hypoxia 12 h and post-reperfu- sion 4 h, the glucose oxidative metabolism rates in hypertrophic cardiomyocytes all decreased while the fatty acid oxidative metabolism rates increased. DCA, TMZ and LC all could inhibit both the reduction of glucose oxidative metabolism after hypoxia-reperfusion and the elevation of fatty acid oxidative metabolism after hypox- ia-reperfusion. AA drove the reduction of glucose oxi- dative metabolism rate even lower and the fatty acid ox- idative metabolism rate even higher in hypertrophic cardiomyocytes after ischemia/reperfusion. At the same time, DCA, TMZ and LC could inhibit the expression levels of mitochondrial cytochrome C and AIF mRNA and proteins, the nuclear translocation of cytochrome c and AIF proteins and the activity of caspase-3. And with the opposing actions to AA, DCA, TMZ and LC could inhibit the apoptotic rate Of hypertrophic cardio- myocytes after hypoxia-reperfusion. And AA had the opposite effect. Conclusion Intervening in the metab- olism pathway of hypertrophic cardiomyocytes was an effective way to prevent and control their programmed death through inhibiting the expression of mitochondrial apoptotic proteins.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2008年第6期748-752,共5页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No30100069)
