AIM:To investigate the effects of extracellular cysteine/cystine redox potential (EhCys/CySS) on the mitochondrial function of nonalcoholic fatty liver disease ( NAFLD) hepatocytes.METHODS:LO2 cells were incuba-ted with EhCys/CySS of the oxidized (0 mV), the normal (-80 mV), or the reduced (-150 mV) status medium, then treated with oleic acid to establish NAFLD model in vitro.DCFH-DA and MitoSOX were used as the fluorescent probes for determining reactive oxygen species (ROS).Apocynin (NADPH oxidase inhibitor), MitoQ10 (mitochondria-targeted an-tioxidant), rotenone (mitochondrial respiratory chain complex I inhibitor) and antimycin A (mitochondrial respiratory chain complex III inhibitor) were used to investigate the sources of ROS.RESULTS:An increase in ROS in LO2 cells by oleic acid was aggravated by the oxidized extracellular EhCys/CySS (0 mV), which was removed by the reduced EhCys/CySS (-150 mV) .ROS generation by 0 mV was significantly eliminated by MitoQ10 .ROS levels were dependent on ex-tracellular Eh Cys/CySS in rotenone treated LO2 cells.A decline of mitochondrial membrane potential in the cells with NAFLD was aggravated by 0 mV and reversed by -150 mV.CONCLUSION:The oxidized extracellular Eh Cys/CySS via inhibitiing of complex I intensifies ROS generation and reducing the mitochondrial membrane potential in the NAFLD hepa-tocytes, which were reversed by reduced Eh Cys/CySS.%目的：研究细胞外半胱氨酸／胱氨酸氧化还原电位（EhCys／CySS）对非酒精性脂肪肝（NAFLD）肝细胞线粒体功能的影响。方法：用Eh Cys／CySS分别为0 mV（氧化）、－80 mV（正常）和－150 mV（还原）的氧化还原培养基培养肝细胞株LO2并采用油酸诱导细胞建立NAFLD模型。荧光探针DCFH-DA和MitoSOX分别检测细胞整体水平和线粒体活性氧簇（ROS）生成，使用apocynin（NADPH氧化酶抑制剂）和MitoQ10（靶向线粒体抗氧化剂）、rotenone（线粒体呼吸链复合体I抑制剂）和antimycin A（线粒体呼吸链复合体Ⅲ抑制剂）作用细胞检测线粒体复合体活性从而探究ROS的来源，JC-1检测细胞线粒体膜电位。结果：油酸诱导的NAFLD细胞模型使肝细胞内ROS增多，线粒体膜电位下降；氧化的Eh Cys／CySS加剧了NAFLD肝细胞ROS的生成以及线粒体膜电位的下降，而还原的Eh Cys／CySS能清除ROS并逆转线粒体膜电位的下降。线粒体ROS清除剂MitoQ10能显著地减少氧化的Eh Cys／CySS所增加的ROS，而apocynin效果不明显。 Rotenone作用于细胞后，ROS的增长率与细胞外Eh Cys／CySS有关，氧化状态下ROS增长率最小且复合体I活性减弱，即氧化的Eh Cys／CySS能通过抑制线粒体复合体I增加ROS的生成。结论：氧化的Eh Cys／CySS能通过抑制线粒体复合体I，从而加剧NAFLD肝细胞ROS的生成，并使线粒体膜电位进一步下降，而还原的Eh Cys／CySS能减少高脂所致ROS生成并减轻线粒体损伤。
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