Laskin D L, Rodriguez del Valle M, Heck D E, Hwang S M, Ohnishi S T, Durham S K, Goller N L, Laskin J D
Environmental and Occupational Health Sciences Institute, Rugers University, Piscataway, NJ 08855-0789, USA.
Hepatology. 1995 Jul;22(1):223-34.
In the present studies, we analyzed the effects of acute endotoxemia on hepatocyte nitric oxide production and functional activity. Treatment of rats with 5 mg/kg of lipopolysaccharide (LPS), which induces acute endotoxemia, caused an increase in nitric oxide production in the liver, as measured by electron paramagnetic spin trapping, which was evident within 6 hours. This was associated with expression of inducible nitric oxide synthase (iNOS) messenger (m) RNA in hepatocytes and in sinusoidal cells throughout the liver lobule. Acute endotoxemia also caused alterations in hepatic structure, including hypertrophy, vacuolization, and chromosomal emargination, however these changes were not apparent for 24 to 48 hours. Hepatocytes isolated from endotoxemic rats released increased amounts of nitric oxide, measured by nitrite production, in response to interferon gamma (gamma-IFN) alone or in combination with LPS, tumor necrosis factor alpha, macrophage-colony stimulating factor, granulocyte/macrophage-colony stimulating factor, or hepatocyte growth factor. These results show that hepatocytes are sensitized by acute endotoxemia to respond to inflammatory mediators and growth factors. Increased nitrite production by hepatocytes was due to increased expression of iNOS mRNA and protein and was correlated with the time following induction of acute endotoxemia. Thus, cells isolated 48 hours after induction of acute endotoxemia released significantly more nitrite than cells recovered after 6 hours, a response that was not due to alterations in hepatocyte viability. Hepatocytes isolated from endotoxemic rats also exhibited a marked increase in proliferative capacity when compared with cells from control rats. Nitric oxide production by hepatocytes in vitro was associated with inhibition of cell growth and protein synthesis, which was reversed by the nitric oxide synthase inhibitor, NG-monomethyl-l-arginine (L-NMMA). Agarose gel electrophoresis showed extensive cytoplasmic DNA fragmentation in hepatocytes treated with LPS and gamma-IFN, a characteristic of apoptosis, which was also reversed by L-NMMA. These results, together with our findings that treatment of rats with an inhibitor of nitric oxide synthase partially reversed the structural alterations in the liver associated with acute endotoxemia suggest that nitric oxide may contribute to the pathophysiologic response to this bacterially derived toxin.
在本研究中，我们分析了急性内毒素血症对肝细胞一氧化氮生成及功能活性的影响。用5mg/kg脂多糖（LPS）处理大鼠可诱导急性内毒素血症，通过电子顺磁共振自旋捕获法检测发现，肝脏中一氧化氮生成增加，6小时内即很明显。这与肝小叶内肝细胞及窦状隙细胞中诱导型一氧化氮合酶（iNOS）信使（m）RNA的表达有关。急性内毒素血症还导致肝脏结构改变，包括肥大、空泡化和染色体边缘突出，但这些变化在24至48小时内并不明显。从内毒素血症大鼠分离的肝细胞，单独或与LPS、肿瘤坏死因子α、巨噬细胞集落刺激因子、粒细胞/巨噬细胞集落刺激因子或肝细胞生长因子联合，对干扰素γ（γ-IFN）反应时，通过亚硝酸盐生成检测发现释放的一氧化氮量增加。这些结果表明，急性内毒素血症使肝细胞对炎症介质和生长因子敏感。肝细胞亚硝酸盐生成增加是由于iNOS mRNA和蛋白表达增加，且与急性内毒素血症诱导后的时间相关。因此，急性内毒素血症诱导48小时后分离的细胞释放的亚硝酸盐明显多于6小时后回收的细胞，这种反应并非由于肝细胞活力改变。与对照大鼠的细胞相比，从内毒素血症大鼠分离的肝细胞增殖能力也显著增加。体外肝细胞一氧化氮生成与细胞生长和蛋白质合成的抑制有关，一氧化氮合酶抑制剂NG-单甲基-L-精氨酸（L-NMMA）可逆转这种抑制。琼脂糖凝胶电泳显示，用LPS和γ-IFN处理的肝细胞中有广泛的细胞质DNA片段化，这是细胞凋亡的特征，L-NMMA也可逆转。这些结果，连同我们的发现，即用一氧化氮合酶抑制剂处理大鼠可部分逆转与急性内毒素血症相关的肝脏结构改变，提示一氧化氮可能参与了对这种细菌衍生毒素的病理生理反应。
