ROLE OF OXIDATIVE STRESS IN THE MECHANISM OF HEPATIC DAMAGE

用数字显微荧光技术，以氢过氧化物二氯荧光素（DCFH）为荧光探针，对大鼠肝脏灌流过程中的氧化应激进行了研究。已知DCFH被过氧化氢氧化成二氯荧光素（DCF）。还通过碘化丙啶（PI）评估肝损伤，PI已知可标记非活细胞的细胞核。通过配备有硅增强靶照相机的荧光显微镜观察这些探针的荧光，并通过图像处理器（ARGUS-200，Hamamatsu photonicus，Japan）进行数字处理。通过DCF荧光在区3中证明四氯化碳诱导的氧化应激，随后通过PI评估细胞损伤。细胞色素P450抑制剂SKF 525 A或自由基清除剂异丙嗪可阻止这些变化。低流量缺氧首先使2区DCF荧光增强，并向3区扩散。在DCF荧光增加后，在3区观察到PI荧光，表明中间区氧化应激先于细胞死亡。用黄嘌呤氧化酶抑制剂别嘌呤醇或前列腺素E1（PGE 1）预防这些变化。用线粒体标记物罗丹明123（Rh 123）评价脂多糖引起的肝损伤。灌流液中加入LPS可降低Rh 123的荧光强度，这种变化可被一氧化氮合成酶抑制剂LNMA阻断。LPS诱导的肝细胞线粒体功能障碍仅在Kupffer细胞存在的情况下观察到，表明激活的Kupffer细胞释放的一氧化氮介导了LPS诱导的肝损伤中的肝线粒体功能障碍。

Oxidative stress in the perfused rat liver was attempted to be analyzed by digital microfluorography using a fluorescent probe for hydroperoxide, dichlorofluorescin (DCFH). DCFH is known to be oxidized by hydorperoxide to dichlorofluorescein (DCF). Hepatic damage was also assessed by propidium iodide (PI), which is known to label the nuclei of non viable cells. Fluorescence of these probes was observed by fluorescence microscope equipped with silicon intensified target camera, and digitally processed by a image processor (ARGUS-200, Hamamatsu photonicus, Japan). Carbon tetrachloride induced oxidative stress was demonstrated in zone 3 by DCF fluorescence, followed by cell damage assessed by PI. These changes were prevented by SKF525A, an inhibitor of cytochrome P450, or promethazine, a radical scavanger. Low flow hypoxia increase DCF fluorescence in zone 2 first, and spread to zone 3. PI fluorescence was observed in zone 3 after increase in DCF fluorescence, suggesting midzonal oxidative stress preceed cell death. These changes were prevented by addition of allopurinol, a xanthine oxidase inhibitor, or PGE1.Rhodamine 123(Rh123), an indicator of mitochondrial energization, was used to assess lipololysaccharide induced liver damage. Addition of LPS to perfusate decreased Rh123 fluorescence, and the change was blocked by LNMA, and in hibitor of nitric oxide synthetase. Mitochondrial dysfunction in hepatocytes induced by LPS was observed only in the presence of Kupffer cell, suggesting that nitric oxide released from activated Kupffer cell mediates hepatic mitochondiral dysfunction in LPS induced liver damage.