Hong Ji-Young, Lebofsky Margitta, Farhood Anwar, Jaeschke Hartmut
Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd, MS 1018, Kansas City, KS 66160, USA.
Am J Physiol Gastrointest Liver Physiol. 2009 Mar;296(3):G572-81. doi: 10.1152/ajpgi.90435.2008. Epub 2009 Jan 8.
Oxidant stress is critically involved in various liver diseases. Superoxide formation causes c-Jun NH2-terminal kinase (JNK)- and caspase-dependent apoptosis in cultured hepatocytes. To verify these findings in vivo, male Fisher rats were treated with diquat and menadione. The oxidant stress induced by both compounds was confirmed by increased formation of glutathione disulfide and 4-hydroxynonenal protein adducts. Plasma alanine aminotransferase activities increased from 46+/-4 U/l in controls to 955+/-90 U/l at 6 h after diquat treatment. Hematoxylin and eosin staining of liver sections revealed large areas of necrotic cells at 3 and 6 h. DNA strandbreaks, evaluated with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, showed clusters of TUNEL-positive cells, where the staining was predominantly cytosolic and the cells were swollen, indicating oncotic necrosis. There was no significant increase in caspase-3 activities or relevant release of DNA fragments into the cytosol at any time between 0 and 6 h after diquat treatment. Despite the activation of JNK after high doses of diquat, the JNK inhibitor SP-600125 did not protect against diquat-induced necrosis. Menadione alone did not cause liver injury, but, in combination with phorone and FeSO4, induced moderate oncotic necrosis. On the other hand, if animals were treated with galactosamine/endotoxin as positive control for apoptosis, caspase-3 activities were increased by 259%, the number of TUNEL-positive cells with apoptotic morphology was increased 103-fold, and DNA fragmentation was enhanced 6-fold. The data indicate that liver cell death initiated by diquat-induced superoxide formation in vivo is mediated predominantly by oncotic necrosis and is independent of JNK activation.
氧化应激与多种肝脏疾病密切相关。超氧化物的形成会导致培养的肝细胞中c-Jun氨基末端激酶（JNK）和半胱天冬酶依赖性凋亡。为了在体内验证这些发现，对雄性Fisher大鼠进行了敌草快和甲萘醌处理。两种化合物诱导的氧化应激通过谷胱甘肽二硫化物和4-羟基壬烯醛蛋白加合物形成增加得到证实。血浆丙氨酸转氨酶活性从对照组的46±4 U/L在敌草快处理后6小时增加到955±90 U/L。肝切片苏木精和伊红染色显示在3小时和6小时时有大面积坏死细胞。用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）试验评估DNA链断裂，显示TUNEL阳性细胞簇，染色主要在细胞质中，细胞肿胀，表明为胀亡性坏死。在敌草快处理后0至6小时的任何时间，半胱天冬酶-3活性均无显著增加，也没有相关的DNA片段释放到细胞质中。尽管高剂量敌草快后JNK被激活，但JNK抑制剂SP-600125并不能预防敌草快诱导的坏死。单独的甲萘醌不会引起肝损伤，但与佛波酯和硫酸亚铁联合使用会诱导中度胀亡性坏死。另一方面，如果用半乳糖胺/内毒素作为凋亡的阳性对照处理动物，半胱天冬酶-3活性增加259%，具有凋亡形态的TUNEL阳性细胞数量增加103倍，DNA片段化增强6倍。数据表明，体内敌草快诱导的超氧化物形成引发的肝细胞死亡主要由胀亡性坏死介导，且与JNK激活无关。
