Yerushalmi B, Dahl R, Devereaux M W, Gumpricht E, Sokol R J
Pediatric Liver Center and Liver Transplantation Program, Section of Pediatric Gastroenterology, Hepatology and Nutrition, Department of Pediatrics, University of Colorado School of Medicine and The Children's Hospital, Denver, CO, USA.
Hepatology. 2001 Mar;33(3):616-26. doi: 10.1053/jhep.2001.22702.
The accumulation of hydrophobic bile acids plays a role in the induction of apoptosis and necrosis of hepatocytes during cholestasis. The aim of this study was to determine in freshly isolated rat hepatocytes the roles of oxidant stress and the mitochondrial permeability transition (MPT) in bile acid-induced apoptosis. Hepatocytes isolated from adult male Sprague-Dawley rats were incubated for 4 hours in buffer containing the hydrophobic bile acid, glycochenodeoxycholic acid (GCDC, 0-500 micromol/L) or the hydrophilic bile acid, glycocholic acid (GCA), and either the antioxidants, alpha tocopherol, ebselen, or idebenone (a coenzyme Q analogue); or the MPT blockers, cyclosporin A, or bongkrekic acid, or a caspase-8 inhibitor. Apoptosis was assessed hourly by nuclear morphologic changes of fixed cells by DAPI fluorescence microscopy and reactive oxygen species (ROS) generation by dichlorofluorescein fluorescence of hepatocytes. The percent of cells undergoing apoptosis increased in a time- and concentration-dependent manner in cells exposed to GCDC, and to a much lesser extent to GCA. ROS generation preceded the onset of apoptosis. MPT blockers, caspase-8 inhibition, and antioxidants prevented apoptosis and reduced ROS generation by hepatocytes. Flow cytometry analysis showed that MPT occurred within 1 hour of exposure of cells to 100 micromol/L GCDC, prior to onset of significant apoptosis. In conclusion, ROS generation, MPT induction, and cytochrome c release are critical steps in the induction of apoptosis by bile acids. Antioxidants may reduce liver injury caused by low levels of bile acids by preventing the generation of oxidant stress and subsequent stimulation of the MPT and release of cytochrome c from mitochondria.
在胆汁淤积过程中，疏水性胆汁酸的蓄积在诱导肝细胞凋亡和坏死中起作用。本研究的目的是确定在新鲜分离的大鼠肝细胞中，氧化应激和线粒体通透性转换（MPT）在胆汁酸诱导的凋亡中的作用。从成年雄性Sprague-Dawley大鼠分离的肝细胞在含有疏水性胆汁酸甘氨鹅去氧胆酸（GCDC，0 - 500 μmol/L）或亲水性胆汁酸甘胆酸（GCA）以及抗氧化剂α-生育酚、依布硒啉或艾地苯醌（一种辅酶Q类似物）；或MPT阻滞剂环孢素A、或邦克酸、或半胱天冬酶-8抑制剂的缓冲液中孵育4小时。通过DAPI荧光显微镜观察固定细胞的核形态变化以及通过肝细胞二氯荧光素荧光检测活性氧（ROS）生成，每小时评估一次凋亡情况。在暴露于GCDC的细胞中，发生凋亡的细胞百分比呈时间和浓度依赖性增加，而在暴露于GCA的细胞中增加程度要小得多。ROS生成先于凋亡开始。MPT阻滞剂、半胱天冬酶-8抑制和抗氧化剂可防止凋亡并减少肝细胞的ROS生成。流式细胞术分析表明，在细胞暴露于100 μmol/L GCDC后1小时内发生MPT，早于明显凋亡的开始。总之，ROS生成、MPT诱导和细胞色素c释放是胆汁酸诱导凋亡的关键步骤。抗氧化剂可通过防止氧化应激的产生以及随后刺激MPT和细胞色素c从线粒体释放来减少低水平胆汁酸引起的肝损伤。
