Sokol R J, Straka M S, Dahl R, Devereaux M W, Yerushalmi B, Gumpricht E, Elkins N, Everson G
Pediatric Liver Center, Section of Pediatric Gastroenterology, Hepatology and Nutrition, The Children's Hospital, Denver, Colorado 80218, U.S.A.
Pediatr Res. 2001 Apr;49(4):519-31. doi: 10.1203/00006450-200104000-00014.
Hydrophobic bile acids may cause hepatocellular necrosis and apoptosis during cholestatic liver diseases. The mechanism for this injury may involve mitochondrial dysfunction and the generation of oxidant stress. The purpose of this study was to determine the relationship of oxidant stress and the mitochondrial membrane permeability transition (MMPT) in hepatocyte necrosis induced by bile acids. The MMPT was measured spectrophotometrically and morphologically in rat liver mitochondria exposed to glycochenodeoxycholic acid (GCDC). Freshly isolated rat hepatocytes were exposed to GCDC and hepatocellular necrosis was assessed by lactate dehydrogenase release, hydroperoxide generation by dichlorofluorescein fluorescence, and the MMPT in cells by JC1 and tetramethylrhodamine methylester fluorescence on flow cytometry. GCDC induced the MMPT in a dose- and Ca(2+)-dependent manner. Antioxidants significantly inhibited the GCDC-induced MMPT and the generation of hydroperoxides in isolated mitochondria. Other detergents failed to induce the MMPT and a calpain-like protease inhibitor had no effect on the GCDC-induced MMPT. In isolated rat hepatocytes, GCDC induced the MMPT, which was inhibited by antioxidants. Blocking the MMPT in hepatocytes reduced hepatocyte necrosis and oxidant stress caused by GCDC. Oxidant stress, and not detergent effects or the stimulation of calpain-like proteases, mediates the GCDC-induced MMPT in hepatocytes. We propose that reducing mitochondrial generation of reactive oxygen species or preventing increases in mitochondrial Ca(2+) may protect the hepatocyte against bile acid-induced necrosis.
在胆汁淤积性肝病期间，疏水性胆汁酸可能导致肝细胞坏死和凋亡。这种损伤的机制可能涉及线粒体功能障碍和氧化应激的产生。本研究的目的是确定氧化应激与胆汁酸诱导的肝细胞坏死中线粒体膜通透性转换（MMPT）之间的关系。通过分光光度法和形态学方法测定了暴露于甘氨鹅去氧胆酸（GCDC）的大鼠肝线粒体中的MMPT。将新鲜分离的大鼠肝细胞暴露于GCDC，并通过乳酸脱氢酶释放评估肝细胞坏死，通过二氯荧光素荧光测定过氧化氢生成，并通过流式细胞术上的JC1和四甲基罗丹明甲酯荧光测定细胞中的MMPT。GCDC以剂量和Ca（2+）依赖性方式诱导MMPT。抗氧化剂显著抑制了GCDC诱导的MMPT以及分离线粒体中过氧化氢的生成。其他去污剂未能诱导MMPT，并且一种钙蛋白酶样蛋白酶抑制剂对GCDC诱导的MMPT没有影响。在分离的大鼠肝细胞中，GCDC诱导了MMPT，而抗氧化剂抑制了该过程。阻断肝细胞中的MMPT可减少GCDC引起的肝细胞坏死和氧化应激。氧化应激而非去污剂作用或钙蛋白酶样蛋白酶的刺激介导了GCDC诱导的肝细胞MMPT。我们提出，减少线粒体活性氧的产生或防止线粒体Ca（2+）增加可能保护肝细胞免受胆汁酸诱导的坏死。
