ROLE OF OXIDATIVE STRESS IN THE MECHANISM OF HEPATIC DAMAGE

采用双氧水荧光探针二氯荧光素（DCFH）对灌注大鼠肝脏的氧化应激进行了数字显微荧光分析。已知DCFH被过氧化氢氧化为二氯荧光素（DCF）。肝损伤也通过碘化丙啶（PI）来评估，已知它可以标记非活细胞的细胞核。利用配备硅强化靶相机的荧光显微镜观察探针的荧光，并通过图像处理器（ARGUS-200, Hamamatsu photonicus, Japan）进行数字处理。DCF荧光显示四氯化碳诱导的氧化应激在3区，随后PI评估细胞损伤。细胞色素P450抑制剂SKF525A或自由基清除剂异丙嗪可阻止这些变化。低流量缺氧先使2区DCF荧光增强，然后向3区扩散。DCF荧光增加后，3区出现PI荧光，提示中区氧化应激先于细胞死亡。这些变化可以通过添加别嘌呤醇（一种黄嘌呤氧化酶抑制剂）或PGE1来预防。采用线粒体激活指标罗丹明123（Rh123）评价脂多糖诱导的肝损伤。LPS灌注降低了Rh123的荧光，LNMA阻断了这种变化，并抑制了一氧化氮合成酶。LPS诱导的肝细胞线粒体功能障碍仅在Kupffer细胞存在时观察到，提示在LPS诱导的肝损伤中，活化的Kupffer细胞释放的一氧化氮介导了肝脏线粒体功能障碍。

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.