Department of Pharmaceutical & Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425, USA.
Free Radic Biol Med. 2012 Jul 15;53(2):250-9. doi: 10.1016/j.freeradbiomed.2012.05.012. Epub 2012 May 15.
This study investigated the role of inducible nitric oxide synthase (iNOS) in failure of ethanol-induced fatty liver grafts. Rat livers were explanted 20 h after gavaging with ethanol (5 g/kg) and storing in UW solution for 24h before implantation. Hepatic oil red O staining-positive areas increased from ∼2 to ∼33% after ethanol treatment, indicating steatosis. iNOS expression increased ∼8-fold after transplantation of lean grafts (LG) and 25-fold in fatty grafts (FG). Alanine aminotransferase release, total bilirubin, hepatic necrosis, TUNEL-positive cells, and cleaved caspase-3 were higher in FG than LG. A specific iNOS inhibitor 1400W (5 μM in the cold-storage solution) blunted these alterations by >42% and increased survival of fatty grafts from 25 to 88%. Serum nitrite/nitrate and hepatic nitrotyrosine adducts increased to a greater extent after transplantation of FG than LG, indicating reactive nitrogen species (RNS) overproduction. Phospho-c-Jun and phospho-c-Jun N-terminal kinase-1/2 (JNK1/2) were higher in FG than in LG, indicating more JNK activation in fatty grafts. RNS formation and JNK activation were blunted by 1400W. Mitochondrial polarization and cell death were visualized by intravital multiphoton microscopy of rhodamine 123 and propidium iodide, respectively. After implantation, viable cells with depolarized mitochondria were 3-fold higher in FG than in LG and 1400W decreased mitochondrial depolarization in FG to the levels of LG. Taken together, iNOS is upregulated after transplantation of FG, leading to excessive RNS formation, JNK activation, mitochondrial dysfunction, and severe graft injury. The iNOS inhibitor 1400W could be an effective therapy for primary nonfunction of fatty liver grafts.
这项研究探讨了诱导型一氧化氮合酶（iNOS）在乙醇诱导的脂肪肝移植物失功中的作用。在给予乙醇（5 g/kg）灌胃 20 小时后，将大鼠肝脏取出，并在 UW 溶液中储存 24 小时，然后进行移植。乙醇处理后，肝油红 O 染色阳性区域从约 2%增加到约 33%，表明存在脂肪变性。移植 lean 移植物（LG）后 iNOS 表达增加约 8 倍，移植 fatty 移植物（FG）后增加约 25 倍。与 LG 相比，FG 中的丙氨酸氨基转移酶释放、总胆红素、肝坏死、TUNEL 阳性细胞和 cleaved caspase-3 更高。冷保存液中浓度为 5 μM 的特异性 iNOS 抑制剂 1400W 使这些变化降低了＞42%，并使 fatty 移植物的存活率从 25%增加到 88%。与 LG 相比，FG 移植后血清中亚硝酸盐/硝酸盐和肝硝基酪氨酸加合物的产生增加更多，表明活性氮物种（RNS）的过度产生。FG 中的磷酸化 c-Jun 和磷酸化 c-Jun N-末端激酶-1/2（JNK1/2）高于 LG，表明 fatty 移植物中 JNK 激活更多。1400W 抑制了 RNS 的形成和 JNK 的激活。通过 rhodamine 123 和碘化丙啶的活体多光子显微镜分别可视化 RNS 形成和 JNK 激活。植入后，FG 中的失活细胞与具有去极化线粒体的细胞数量是 LG 的 3 倍，1400W 将 FG 中的线粒体去极化减少到 LG 的水平。综上所述，FG 移植后 iNOS 上调，导致过量的 RNS 形成、JNK 激活、线粒体功能障碍和严重的移植物损伤。iNOS 抑制剂 1400W 可能是治疗脂肪肝移植物原发性无功能的有效方法。
