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1、外源性一氧化碳释放分子抑制脓毒症炎症反应的实验研究 作者：孙炳伟, 陈曦, Kazuhiro Katada, Richard F Potter, Gediminas Cepinskas【摘要】 目的： 探讨外源性一氧化碳释放分子对脓毒症炎症反应的抑制作用及可能的机制。方法： 应用盲肠结扎及穿孔脓毒症小鼠模型，使用外源性一氧化碳释放分子（CORM2, 8 mg/kg 体质量，尾静脉注射）进行干预。检测肝、肺脏髓过氧化物酶(MPO) 活性。应用内毒素(LPS，10 g/ml)刺激的人脐静脉内皮细胞炎症模型，使用外源性一氧化碳释放分子（ CORM2,10100 mol/L）进行干预。检测核因子B （
2、NFB）活性, 内皮细胞黏附分子的表达，氧化产物、NO产物以及多形核白细胞对内皮细胞的黏附作用。 结果： 盲肠结扎及穿孔脓毒症小鼠模型使用外源性一氧化碳释放分子干预后肝、肺组织MPO活性明显下降。CORM2 抑制了LPS刺激导致的 NFB活性上调。 同时，NO产物下降，内皮细胞ICAM1的表达抑制，白细胞对内皮细胞的黏附作用明显抑制。结论： 外源性一氧化碳释放分子通过抑制NFB 活性，抑制ICAM1 蛋白和NO的表达，抑制白细胞对内皮细胞的黏附作用，进而有效抑制脓毒症炎症反应。 【关键词】 一氧化碳； 盲肠结扎及穿孔； 炎症反应； 核因子BCLP (cecal ligation and pun
3、cture) may induce the activation of an inflammatory cascade, cause damage to multiple organs distant from the original burn wound and may lead to sepsis and multiple organ failure1.There have been several reports indicating that the inflammatory response syndrome, which contributes to oxidative cell
4、/tissue damage, might frequently be accompanied by leukocyte sequestration in many important organ systems in the body2.The increase of production of proinflammatory mediators such as interleukin (IL)1 and tumor necrosis factor (TNF) is closely associated with activation of leukocytes and macrophage
5、s which were sequestrated in the tissue3,4. Leukocytes sequestration and their subsequent infiltration in organ tissue can cause leukocyte activation and contribute to vascular damage and the development of systemic inflammatory reaction.As the prerequisite, activation of leukocytes and endothelial
6、cells results in aggregation of leukocytes, platelets and erythrocytes in vivo.This may favor disseminated intravasal coagulation and further multiple organ failure. Carbon monoxide (CO) has long been known in biology and medicine as a toxic compound, due to its ability to bind hemoglobin with a muc
7、h higher affinity than oxygen5.Evidence accumulated to date suggests that endogenous carbon monoxide (CO), a biproduct of inducible heme oxygenase (HO1) can modulate inflammation, inhibits lipopolysaccharide (LPS)induced production of cytokines both in vivo and in vitro, and consequently exhibits im
8、portant cytoprotective function and antiinflammatory properties that are beneficial for the resolution of acute inflammation6-8.Inhaled CO at concentrations of 250500 parts per million (ppm) has also been shown to be beneficial in a number of lung injury models, including hyperoxic injury9,10 allerg
9、eninduced inflammation11. Recently, transitional metal carbonyls have been identified as potential COreleasing molecules (CORMs) with the potential to facilitate the pharmaceutical use of CO by delivering it to tissues and organs12.CORMs have been shown to act pharmacologically in rat aortic and car
10、diac tissue where liberation of CO produced vasorelaxant effects13-16 and decreased myocardial ischemiareperfusion damage17,18 in the absence of dramatic changes in blood carboxyhemoglobin (COHb) levels. On the basis of these data, the present study was, therefore, designed as a prospective laborato
11、ry experiment to investigate the effects of tricarbonyldichlororuthenium () dimer (CORM2), one of the novel group of CORMs, on attenuation of leukocyte sequestration and decrease of inflammatory responses and oxidative stress in the organs of CLPinduced mice and LPSinduced HUVEC (human umbilical vei
12、n endothelial cell), and discussed the possible molecular mechanisms. 1 Material and methods 1.1 Materials Medium 199 (M199), fetal calf serum (FCS), penicillin, and streptomycin were purchased from GIBCO BRL (Gland Island, NY).Tricarbonyldichlororuthenium(II) dimer (CORM2) was obtained from Sigma A
13、ldrich and solubilized in dimethyl sulfoxide (DMSO) to obtain a 10 mmol/L stock.LPS(Escherichia coli serotype 055:B5) was purchased from Sigma.AntiICAM1 polyclonal antibody was purchased from Transduction Laboratories (Lexington, KY).Antimouse IgG conjugated to horseradish peroxidase was purchased f
14、rom Kirkegaard and Perry Laboratories (Gaithersburg, MD). 1.2 Animals The C57BL/6 micemale, N=21; bw (20 2)g were fed a standard laboratory diet and water ad libitum.Mice were assigned to three groups in three respective experiments.In each experiment, mice in sham group (n=7) were underwent sham pr
15、ocedure, whereas mice in CLP group (n=7) received cecal ligation and puncture and mice in CORM2 group (n=7) underwent the same injury with immediate administration of CORM2 (8 mg/kg, i.v.).The concentration of CORM2 used in the present study was based on a previous report in of the use of this compo
16、und in mice19 and the preliminary experiments in our lab by measuring dynamic COHb levels and peak levels which did not averaged 15%5% above normal levels.The experimental protocol was approved by The Council on Animal Care at Jiangsu University on the protection and the welfare of animals and met National Institutes of Health guidelines for the care and use of experimental anim
