附子对H9c2心肌细胞系线粒体的毒性作用机制

Mitochondrial toxicity effect of Radix Aconiti Lateralis Praeparata on H9c2 cardiomyocytes

目的研究附子对H9c2心肌细胞的线粒体毒性作用与机制。方法附子水提取物6.25,12.5,25,50和100 g·L-1作用于H9c2细胞24 h,荧光染色和CCK-8法检测细胞存活率;附子水提取物6.25,12.5和25 g·L-1作用于H9c2细胞24 h,流式细胞仪检测线粒体膜电位和活性氧（ROS）生成的变化;使用相应的荧光探针,结合激光扫描共聚焦显微镜观察细胞内Ca2＋、线粒体内Ca2＋以及线粒体内超氧化物水平的变化;萤火虫荧光素法检测细胞内ATP浓度变化;实时荧光定量PCR检测过氧化物酶体增殖活化受体共激活因子-1α（Pgc-1α）,Bcl-2和Bax m RNA的表达;Western蛋白印迹法检测Pgc-1α蛋白的表达。结果附子水提取物12.5~100 g·L-1作用于H9c2细胞,可显著抑制细胞活性（P〈0.05）,IC50值为47.4669 g·L-1,95%的可信限32.5997~69.1145 g·L-1。附子水提取物25 g·L-1处理后,ROS的荧光强度由正常对照组的204±67升高到454±78（P〈0.05）,线粒体超氧化物的荧光强度由5.4±1.8升高到26.8±8.5（P〈0.01）,线粒体膜电位由1.7±0.5下降到0.8±0.4（P〈0.05）,线粒体内Ca2＋的荧光强度由38.0±4.3下降到9.2±1.6（P〈0.01）,细胞内Ca2＋的荧光强度由7.8±0.8升高到22.1±0.5（P〈0.05）,细胞内ATP浓度由（10.6±0.4）μmol·g-1下降到（5.3±1.1）μmol·g-1蛋白（P〈0.05）。实时荧光定量PCR和Western蛋白印迹检测结果显示,与正常对照组相比,附子水提取物25 g·L-1组Pgc-1α和Bcl-2的m RNA表达分别由正常对照组的1.00±0.10和1.00±0.10下降到0.09±0.06（P〈0.01）和0.43±0.06（P〈0.01）,Bax m RNA表达由1.00±0.03升高到1.17±0.06（P〈0.05）;Pgc-1α蛋白表达由正常对照组的0.906±0.034下降到0.541±0.003（P〈0.01）。结论附子对心肌细胞具有一定的线粒体毒性,其作用是多种途径共同作用的结果。附子的心肌线粒体毒性可能是附子造成心肌毒性的原因。

OBJECTIVE To study the mitochondrial toxicity effect of Radix Aconiti Lateralis Praeparata（Fuzi）on H9c2 cardiomyocytes. METHODS H9c2 cells were exposed to Fuzi decoction 6.25,12.5,25,50 and 100 g·L-1for 24 h. Fluorescence staining and CCK-8 assay were used to detect cell viability. H9c2 cells were exposed to Fuzi decoction 6.25,12.5 and 25 g ·L-1for 24 h,while the effect on mitochondrial membrane potential and reactive oxygen species（ROS）was detected by flow cytometry.The fluorescence molecular probe and laser scanning confocal microscope were used to observe the effect on Ca2 ＋in cells,Ca2 ＋and superoxide in mitochondria. The effect on ATP concentration in cells was detected via firefly luciferin and the expression of Pgc-1α,Bcl-2 and Bax m RNA evaluated by realtime PCR,while the expression of Pgc- 1α protein was measured by Western blotting. RESULTS H9c2 cell viability was significantly inhibited by Fuzi decoction 12.5-100 g·L-1（P〈0.05,P〈0.01）. The IC50 value was 47.4669 g·L-1,while the 95% confidence limit was 32.5997-69.1145 g·L-1. After treatment with Fuzi decoction 25 g·L-1,the fluorescence intensity of ROS in the normal control group increased from 204±67 to 454±78（P〈0.05）,that of mitochondrial superoxide increased from 5.4±1.8 to 26.8±8.5（P〈0.01）,mitochondrial membrane potential decreased from 1.7±0.5 to 0.8±0.4（P〈0.05）,the fluorescence intensity of intracellular Ca2＋increased from 7.8±0.8 to 22.1±0.5（P〈0.05）while that of mitochondrial Ca2＋decreased from 38.0±4.3 to 9.2±1.6（P〈0.01）,and intracellular ATP concentration decreased from（10.6±0.4）μmol·g- 1to（5.3±1.1）μmol·g- 1protein（P〈0.05）. q PCR and Western blotting test results showed that compared with the normal control group,Pgc-1α and Bcl-2 m RNA relative expression level in Fuzi decoction 25 g·L-1group was decreased from 1.00±0.10 and 1.00±0.10 to 0.09±0.06（P〈0.01）and 0.43±0.06（P〈0.01）,respectively, while the relative expression of Bax m RNA was increased from 1.00±0.03 to 1.17±0.06（P〈0.05）,and the expression of Pgc-1α protein was decreased from0.906±0.034 to 0.541±0.003（P〈0.01）. CONCLUSION Fuzi has some mitochondrial toxicity to cardiomyopathy. This effect arises from the combined action of different mechanisms. Mitochondrial toxicity of myocytes may account for the cardiac toxicity of Fuzi.