Center for Drug Safety Evaluation and Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Acta Pharmacol Sin. 2013 Sep;34(9):1229-36. doi: 10.1038/aps.2013.62. Epub 2013 Jul 29.
Tetrandrine, an alkaloid with a remarkable pharmacological profile, induces oxidative stress and mitochondrial dysfunction in hepatocytes; however, mitochondria are not the direct target of tetrandrine, which prompts us to elucidate the role of oxidative stress in tetrandrine-induced mitochondrial dysfunction and the sources of oxidative stress.
Rat primary hepatocytes were isolated by two-step collagenase perfusion. Mitochondrial function was evaluated by analyzing ATP content, mitochondrial membrane potential (MMP) and the mitochondrial permeability transition. The oxidative stress was evaluated by examining changes in the levels of reactive oxygen species (ROS) and glutathione (GSH).
ROS scavengers largely attenuated the cytotoxicity induced by tetrandrine in rat hepatocytes, indicating the important role of ROS in the hepatotoxicity of tetrandrine. Of the multiple ROS inhibitors that were tested, only inhibitors of CYP450 (SKF-525A and others) reduced the ROS levels and ameliorated the depletion of GSH. Mitochondrial function assays showed that the mitochondrial permeability transition (MPT) induced by tetrandrine was inhibited by SKF-525A and vitamin C (VC), both of which also rescued the depletion of ATP levels and the mitochondrial membrane potential. Upon inhibiting specific CYP450 isoforms, we observed that the inhibitors of CYP2D, CYP2C, and CYP2E1 attenuated the ATP depletion that occurred following tetrandrine exposure, whereas the inhibitors of CYP2D and CYP2E1 reduced the ROS induced by tetrandrine. Overexpression of CYP2E1 enhanced the tetrandrine-induced cytotoxicity.
We demonstrated that CYP450 plays an important role in the mitochondrial dysfunction induced by the administration of tetrandrine. ROS generated by CYP450, especially CYP2E1, may contribute to the mitochondrial dysfunction induced by tetrandrine.
汉防己甲素是一种具有显著药理学特性的生物碱，可诱导肝细胞氧化应激和线粒体功能障碍；然而，线粒体不是汉防己甲素的直接靶点，这促使我们阐明氧化应激在汉防己甲素诱导的线粒体功能障碍中的作用以及氧化应激的来源。
采用两步胶原酶灌注法分离大鼠原代肝细胞。通过分析 ATP 含量、线粒体膜电位（MMP）和线粒体通透性转换来评估线粒体功能。通过检测活性氧（ROS）和谷胱甘肽（GSH）水平的变化来评估氧化应激。
ROS 清除剂在很大程度上减轻了汉防己甲素诱导的大鼠肝细胞毒性，表明 ROS 在汉防己甲素的肝毒性中起重要作用。在所测试的多种 ROS 抑制剂中，只有 CYP450 的抑制剂（SKF-525A 等）降低了 ROS 水平并改善了 GSH 的耗竭。线粒体功能测定表明，SKF-525A 和维生素 C（VC）均可抑制汉防己甲素诱导的线粒体通透性转换（MPT），并挽救 ATP 水平耗竭和线粒体膜电位降低。在抑制特定 CYP450 同工酶后，我们观察到 CYP2D、CYP2C 和 CYP2E1 的抑制剂可减轻汉防己甲素暴露后发生的 ATP 耗竭，而 CYP2D 和 CYP2E1 的抑制剂可降低汉防己甲素诱导的 ROS。CYP2E1 的过表达增强了汉防己甲素诱导的细胞毒性。
我们证明 CYP450 在汉防己甲素引起的线粒体功能障碍中起重要作用。由 CYP450 产生的 ROS，特别是 CYP2E1，可能导致汉防己甲素诱导的线粒体功能障碍。
