Mitochondrial permeability transition in acetaminophen-induced necrosis and apoptosis of cultured mouse hepatocytes
Mitochondrial permeability transition in acetaminophen-induced necrosis and apoptosis of cultured mouse hepatocytes
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DOI：
10.1002/hep.20437
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发表时间：
2004-11-01
期刊：
影响因子：
13.5
通讯作者：
Lemasters, JJ
中科院分区：
文献类型：
--
作者：
Kon, K;Kim, JS;Lemasters, JJ
对乙酰氨基酚过量会通过谷胱甘肽耗竭、氧化应激和线粒体功能障碍等机制导致严重肝功能衰竭。对乙酰氨基酚导致细胞死亡的最终目标仍不确定，细胞凋亡在对乙酰氨基酚诱导的细胞杀伤中的作用仍存在争议。我们的目的是评估线粒体通透性转变（MPT）在对乙酰氨基酚诱导的小鼠原代培养肝细胞坏死和凋亡中的关键作用。给药10 mmol/L对乙酰氨基酚后，6 h和16 h的坏死杀灭率分别达到49%和74%以上。MPT抑制剂、环孢素A （cyclosporin A, CsA）和NIM811在6小时后将坏死杀伤暂时降低至26%，但在16小时后细胞保护丧失。对乙酰氨基酚给药后约4.5小时，共聚焦显微镜显示线粒体去极化和内膜通透。CsA将这些变化延迟至对乙酰氨基酚给药后约11小时，表明MPT。对乙酰氨基酚后，细胞核改变、末端脱氧核苷酸转移酶介导的dUTP缺口末端标记和caspase-3激活也增加。果糖（20 mmol/L，一种产生三磷酸腺苷的糖酵解底物）和甘氨酸（5 mmol/L，一种膜稳定氨基酸）几乎阻止了所有坏死细胞的死亡，但在16小时后却自相矛盾地使凋亡从37%增加到59%。在果糖加甘氨酸存在的情况下，CsA减少了细胞凋亡，延迟了MPT，但没有阻止MPT。总之，对乙酰氨基酚后3 ~ 6小时发生csa敏感的MPT，对乙酰氨基酚后9 ~ 16小时发生csa不敏感的MPT。然后MPT诱导ATP消耗依赖性坏死或半胱天冬酶依赖性凋亡，部分由糖酵解产生的ATP可用性决定。
Acetaminophen overdose causes massive hepatic failure via mechanisms involving glutathione depletion, oxidative stress, and mitochondrial dysfunction. The ultimate target of acetaminophen causing cell death remains uncertain, and the role of apoptosis in acetaminophen-induced cell killing is still controversial. Our aim was to evaluate the mitochondria( permeability transition (MPT) as a key factor in acetaminophen-induced necrotic and apoptotic killing of primary cultured mouse hepatocytes. After administration of 10 mmol/L acetaminophen, necrotic killing increased to more than 49% and 74%, respectively, after 6 and 16 hours. MPT inhibitors, cyclosporin A (CsA), and NIM811 temporarily decreased necrotic killing after 6 hours to 26%, but cytoprotection was lost after 16 hours. Confocal microscopy revealed mitochondrial depolarization and inner membrane permeabilization approximately 4.5 hours after acetaminophen administration. CsA delayed these changes, indicative of the MPT, to approximately 11 hours after acetaminophen administration. Apoptosis indicated by nuclear changes, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and caspase-3 activation also increased after acetaminophen administration. Fructose (20 mmol/L, an adenosine triphosphate-generating glycolytic substrate) plus glycine (5 mmol/L, a membrane stabilizing amino acid) prevented nearly all necrotic cell killing but paradoxically increased apoptosis from 37% to 59% after 16 hours. In the presence of fructose plus glycine, CsA decreased apoptosis and delayed but did not prevent the MPT. In conclusion, after acetaminophen a CsA-sensitive MPT occurred after 3 to 6 hours followed by a CsA-insensitive MPT 9 to 16 hours after acetaminophen. The MPT then induces ATP depletion-dependent necrosis or caspase-dependent apoptosis as determined, in part, by ATP availability from glycolysis.