Aziz S M, Toborek M, Hennig B, Mattson M P, Guo H, Lipke D W
Division of Pharmacology and Experimental Therapeutics, University of Kentucky, Lexington 40536, USA.
Int J Biochem Cell Biol. 1997 May;29(5):775-87. doi: 10.1016/s1357-2725(97)00010-1.
Oxidative stress may be involved in monocrotaline (MCT)-induced endothelial cell injury and upregulation of extracellular matrix proteins in the pulmonary vasculature. To test this hypothesis, cytotoxicity, expression and distribution of tenascin (TN) as well as cellular oxidation were determined in porcine pulmonary artery endothelial cells (PAECs) exposed to MCT and/or to an oxygen radical scavenger, dimethylthiourea (DMTU). Relative to controls, treatment with 2.5 mM MCT for 24 hr produced cytotoxicity as evidenced by changes in cellular morphology, cell detachment, hypertrophy, reduction in cellular proliferation and severe cytoplasmic vacuolization. Parallel studies showed that MCT markedly altered the expression and distribution of TN in PAEC as determined by immunocytochemistry. Western analysis showed that MCT increased cellular TN content and promoted the appearance of an additional, smaller TN isoform. Northern analysis demonstrated an increase in the steady-state level of TN-specific mRNA in response to MCT treatment. Exposure to MCT also increased the synthesis of cell-associated and media-associated TN as determined by immunoprecipitation. In addition, MCT increased the intensity of cellular oxidative stress as measured by 2,7-dichlorofluorescein fluorescence. Co-treatment with DMTU prevented MCT-induced cytotoxicity, alterations in TN distribution and content, and reduced the increase in DCF fluorescence. These results suggest that MCT-induced cytotoxicity and upregulation of TN are mediated, at least in part, by induction of cellular oxidative stress.
氧化应激可能参与了野百合碱（MCT）诱导的肺血管内皮细胞损伤及细胞外基质蛋白上调。为验证这一假说，我们检测了暴露于MCT和/或氧自由基清除剂二甲基硫脲（DMTU）的猪肺动脉内皮细胞（PAECs）的细胞毒性、腱糖蛋白（TN）的表达与分布以及细胞氧化情况。与对照组相比，用2.5 mM MCT处理24小时产生了细胞毒性，表现为细胞形态改变、细胞脱落、肥大、细胞增殖减少以及严重的细胞质空泡化。平行研究表明，免疫细胞化学检测显示MCT显著改变了PAEC中TN的表达与分布。蛋白质免疫印迹分析表明，MCT增加了细胞TN含量，并促使出现一种额外的、较小的TN异构体。Northern印迹分析表明，MCT处理后TN特异性mRNA的稳态水平增加。免疫沉淀法检测显示，暴露于MCT还增加了细胞相关和培养基相关TN的合成。此外，用2,7 - 二氯荧光素荧光法测定，MCT增加了细胞氧化应激强度。与DMTU共同处理可防止MCT诱导的细胞毒性、TN分布和含量的改变，并减少DCF荧光的增加。这些结果表明，MCT诱导的细胞毒性和TN上调至少部分是由细胞氧化应激诱导介导的。
