Mitchell J, Jiang H, Berry L, Meyrick B
Department of Medicine, Vanderbilt University Medical Center, Nasville, Tennessee 37232-2650, USA.
J Cell Physiol. 1996 Nov;169(2):333-40. doi: 10.1002/(SICI)1097-4652(199611)169:2<333::AID-JCP12>3.0.CO;2-A.
Generation of reactive oxygen species (ROS) is a common event in the pathogenesis of acute lung injury. Endothelial cells may be both a target and a source of the ROS. Exposure of bovine pulmonary endothelial cells (BPAEC) to lipopolysaccharide (LPS) has been shown to result in intracellular generation of both ROS and the antioxidant enzyme, mangano superoxide dismutase (MnSOD). The present study investigates whether alterations in intracellular oxidant state affect LPS-stimulated cytotoxicity and induction of MnSOD mRNA. BPAEC were pretreated with either the free radical scavenger, dimethylsulfoxide (DMSO), the xanthine oxidase inhibitor, allopurinol, or N-acetylcysteine (a cysteine derivate capable of increasing glutathione stores) prior to exposure to LPS (0.1 microgram/ml) for either 4, 8 or 18 hours. We found that pretreatment of BPAEC with DMSO blocked both LPS-induced cytotoxicity and induction of the MnSOD gene. Nuclear run-off experiments demonstrated that LPS-stimulated induction of the MnSOD mRNA occurred at the transcriptional level and that DMSO blocked this event. Pretreatment with allopurinol also prevented the cytotoxicity associated with LPS but, in contrast to DMSO, did not alter induction of MnSOD mRNA. N-acetylcysteine did not affect the LPS-stimulated cytotoxicity but resulted in an early and transient reduction in induction of the MnSOD gene. We conclude that LPS stimulates generation of intracellular ROS that regulate induction of the MnSOD gene at the transcriptional level further, we conclude that LPS-stimulated cytotoxicity involves both the xanthine oxidase pathway and perhaps intracellular generation of hydroxyl radicals. The difference in the protective effect between DMSO, NAC and allopurinol suggest that upregulation of the MnSOD gene does not contribute to LPS-induced cytotoxicity.
活性氧（ROS）的产生是急性肺损伤发病机制中的常见事件。内皮细胞可能既是ROS的靶细胞，也是其来源。已表明，牛肺内皮细胞（BPAEC）暴露于脂多糖（LPS）会导致细胞内ROS和抗氧化酶锰超氧化物歧化酶（MnSOD）的生成。本研究调查细胞内氧化状态的改变是否会影响LPS刺激的细胞毒性以及MnSOD mRNA的诱导。在暴露于LPS（0.1微克/毫升）4、8或18小时之前，用自由基清除剂二甲基亚砜（DMSO）、黄嘌呤氧化酶抑制剂别嘌呤醇或N-乙酰半胱氨酸（一种能够增加谷胱甘肽储备的半胱氨酸衍生物）对BPAEC进行预处理。我们发现，用DMSO预处理BPAEC可阻断LPS诱导的细胞毒性和MnSOD基因的诱导。核转录实验表明，LPS刺激的MnSOD mRNA诱导发生在转录水平，而DMSO可阻断这一过程。用别嘌呤醇预处理也可预防与LPS相关的细胞毒性，但与DMSO不同的是，它不会改变MnSOD mRNA的诱导。N-乙酰半胱氨酸不影响LPS刺激的细胞毒性，但会导致MnSOD基因诱导的早期短暂降低。我们得出结论，LPS刺激细胞内ROS的产生，ROS在转录水平进一步调节MnSOD基因的诱导；此外，我们得出结论，LPS刺激的细胞毒性涉及黄嘌呤氧化酶途径以及可能的细胞内羟基自由基的产生。DMSO、NAC和别嘌呤醇之间保护作用的差异表明，MnSOD基因的上调对LPS诱导的细胞毒性没有贡献。
