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目的评价马兜铃酸的线粒体毒性,从而探讨马兜铃酸的可能毒性机制。方法 HepG2细胞培养基中分别加入齐多夫定8 000~20 000μmol/L,或加入马兜铃酸25~500μmol/L,均培养24 h,以CCK8细胞计数法测定细胞存活率。同时比较不同浓度齐多夫定(8 000、16 000和20 000μmol/L)和马兜铃酸(25、200和500μmol/L)培养24 h的胞内ATP合成水平和胞内钙离子浓度、活性氧及线粒体膜通透性转换孔(MPTP)变化,透射电镜观察线粒体超微结构。结果齐多夫定8 000~20 000μmol/L可抑制细胞存活,IC50为12 713μmol/L;马兜铃酸25~500μmol/L可抑制细胞存活,IC50为214.6μmol/L。与溶媒对照组(DMEM)相比,齐多夫定≥8 000μmol/L细胞内活性氧水平显著升高(P<0.01);≥16 000μmol/L线粒体ATP显著下降(P<0.01)、钙离子浓度明显升高(P<0.01),并可见线粒体结构发生病理改变;20 000μmol/L组MPTP开放水平显著升高(P<0.01)。与溶媒对照组(DMSO)相比,马兜铃酸≥25μmol/L,ATP合成水平明显下降(P<0.01)、MPTP开放水平显著升高(P<0.01);≥200μmol/L细胞内钙离子浓度明显升高(P<0.01);500μmol/L细胞内活性氧水平显著升高(P<0.01)、并可见线粒体结构发生病变。结论马兜铃酸可以通过干扰破坏线粒体代谢功能和结构而诱导线粒体损伤。
Objective To evaluate the mitochondrial toxicity of aristolochic acid and to explore the possible toxic mechanism of aristolochic acid. Methods HepG2 cells were treated with zidovudine 8 000 ~ 20 000 μmol / L or aristolochic acid 25 ~ 500 μmol / L for 24 h, respectively. Cell viability was determined by CCK8 cell counting. At the same time, intracellular ATP synthesis level and intracellular Ca2 + concentration of zidovudine (8 000, 16 000 and 20 000 μmol / L) and aristolochic acid (25, 200 and 500 μmol / L) Reactive oxygen species and mitochondrial permeability transition pore (MPTP) changes, transmission electron microscopy of mitochondrial ultrastructure. Results Zidovudine 8 000 ~ 20 000 μmol / L inhibited cell survival with an IC50 of 12 713 μmol / L. Aristolochic acid 25 ~ 500 μmol / L inhibited cell survival with an IC50 of 214.6 μmol / L. Compared with vehicle control group (DMEM), the level of reactive oxygen species (ROS) in zidovudine≥8 000 μmol / L was significantly increased (P <0.01); the mitochondrial ATP concentration in ≥16 000 μmol / L was significantly decreased (P <0.01). The pathological changes of mitochondrial structure were observed. The MPTP opening in 20 000 μmol / L group was significantly increased (P <0.01). Compared with vehicle control group, aristolochic acid ≥25μmol / L, ATP synthesis level decreased significantly (P <0.01), MPTP opening level increased significantly (P <0.01); ≥200μmol / L intracellular calcium (P <0.01). The level of reactive oxygen species in 500 μmol / L cells was significantly increased (P <0.01), and mitochondrial structure was observed. Conclusion Aristolochic acid induces mitochondrial damage by disrupting mitochondrial metabolic function and structure.