CO from enhanced HO activity or from CORM-2 inhibits both O2- and NO production and downregulates HO-1 expression in LPS-stimulated macrophages
CO from enhanced HO activity or from CORM-2 inhibits both O2- and NO production and downregulates HO-1 expression in LPS-stimulated macrophages
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DOI：
10.1016/j.bcp.2005.10.042
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发表时间：
2006-01-12
影响因子：
5.8
通讯作者：
Cha, YN
中科院分区：
文献类型：
--
作者：
Srisook, K;Han, SS;Cha, YN
由血红素降解产生的一氧化碳（CO），特别是由应激诱导的血红素加氧酶-1（HO-1）催化的，最近已被证明在细菌脂多糖（LPS）刺激的巨噬细胞中对细胞死亡提供细胞保护。在本研究中，我们确定了CO对LPS刺激的RAW 264.7巨噬细胞产生活性氧（ROS）和一氧化氮（NO）的影响。此外，测定了CO暴露对佛波醇肉豆蔻酸酯乙酸酯（PMA）刺激的PLB-985中性粒细胞中超氧化物（O2）产生的影响。用50 μ M [Ru（CO）（3）Cl-2]（2）（一种CO释放分子（CORM-2））预处理的LPS刺激的巨噬细胞产生的ROS被消除，用CORM-2预处理的PMA刺激的中性粒细胞产生的O-2（-）显著减少。CORM-2（50 μ M）是没有细胞毒性的未刺激和LPS刺激的巨噬细胞时，采用线粒体还原酶功能试验（MTT法）测定。在用增加剂量的CORM-2预处理的巨噬细胞中，LPS衍生的iNOS（NO产生）和HO-1表达（CO产生）的上调均以剂量依赖性方式受到抑制。或者，当巨噬细胞与LPS和CO供体一起处理时，LPS引起的NO产生的增加减少。相反，当对照和LPS刺激的巨噬细胞用锌原卟啉IX（ZnPP）处理以抑制HO活性阻断内源性CO产生（基础和增强）时，巨噬细胞广泛死亡。有趣的是，在LPS刺激的巨噬细胞中产生的NO在ZnPP处理后显著增加。将CORM-2添加到另外用ZnPP处理的LPS处理的细胞中不能防止细胞死亡。然而，通过HO-1（通过用丁硫氨酸亚砜亚胺或氯化血红素预处理巨噬细胞获得）的超诱导而内源性CO过量产生降低了LPS衍生的iNOS表达，而不影响细胞存活。因此，HO-1的上调和CO的过度产生可允许LPS刺激的巨噬细胞存活;首先，通过消除游离血红素以防止芬顿反应，其次，通过限制诱导产生NO的血红素酶所需的游离血红素的可用性（即，第三，通过CO衍生的对血红素酶如NADPH氧化酶和iNOS的活性的抑制来限制O-2（-）和NO的额外产生。CO可以使LPS激活的巨噬细胞恢复到正常的安静状态，对引起氧化应激的额外刺激不敏感。(c)2005年爱思唯尔公司All rights reserved.
Carbon monoxide (CO) arising from heme degradation, catalyzed particularly by the stressinducible heme oxygenase-1 (HO-1), has recently been demonstrated to provide cytoprotection against cell death in macrophages stimulated with bacterial lipopolysaccharide (LPS). In the present study, we determined the effects of CO on the production of reactive oxygen species (ROS) and nitric oxide (NO) by the LPS-stimulated RAW 264.7 macrophages. In addition, effect of CO-exposure on the production of superoxide (02) in the phorbol myristate acetate (PMA)-stimulated PLB-985 neutrophils was determined. Production of ROS by the LPS-stimulated macrophages pretreated with 50 mu M [Ru(CO)(3)Cl-2](2), a CO-releasing molecule (CORM-2), was abolished and the production of O-2(-) by the PMA-stimulated neutrophils pretreated with the CORM-2 was decreased markedly. The CORM-2 (50 mu M) was not cytotoxic to both the unstimulated and LPS-stimulated macrophages when determined by employing mitochondrial reductase function test (MTT assay). In macrophages pretreated with increasing doses of CORM-2, both the LPS-derived upregulations of iNOS (NO production) and HO-1 expression (CO production) were suppressed in a dose-dependent manner. Alternatively, when the macrophages were treated with LPS and CO-donor together, the LPS-derived increase in NO production was decreased. Conversely, when the control and LPS-stimulated macrophages were treated with zinc protoporphyrin IX (ZnPP) to inhibit the HO activity blocking endogenous production of CO (basal and enhanced), macrophages died extensively. Interestingly, production of NO in the LPS-stimulated macrophages increased significantly following the ZnPP treatment. Addition of CORM-2 to the LPS-treated cells that were being treated additionally with ZnPP did not prevent the cell death. However, endogenous overproduction of CO by super-induction of HO-1 (obtained by pretreatment of macrophages with either buthionine sulfoximine or hemin) decreased the LPS-derived iNOS expression without affecting cell survival.Combined, these results indicated that enhanced HO activity is essential for the survival of LPS-stimulated macrophages. Thus, upregulation of HO-1 and overproduction of CO may allow the survival of LPS-stimulated macrophages; first, by eliminating the free heme to prevent Fenton reaction, second, by limiting the availability of free heme required for induction of NO-producing heme enzyme (i.e., iNOS), third, by limiting additional production of O-2(-) and NO via CO-derived inhibition on the activities of heme enzymes like NADPH oxidase and iNOS, respectively. CO may allow the LPS-activated macrophages to return back to the normal quiet state insensitive to additional stimuli causing oxidative stress. (c) 2005 Elsevier Inc. All rights reserved.