浅论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.docx

浅论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
【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进行干预。检测核因子κB 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。CORM2 抑制了LPS刺激导致的 NFκB活性上调。 同时，NO产物下降，内皮细胞ICAM1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NFκB 活性，抑制ICAM1 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κB
CLP (cecal ligation and puncture) 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 failure［1］.There have been several reports indicating that the inflammatory response syndrome, which contributes to oxidative cell/tissue damage, might frequently be accompanied by leukocyte sequestration in many important organ systems in the body［2］.The increase of production of proinflammatory mediators such as interleukin (IL)1β and tumor necrosis factor (TNF)α is closely associated with activation of leukocytes and macrophages which were sequestrated in the tissue［3,4］.
Leukocytes sequestration and their subsequent infiltration in organ tissue can cause leukocyte activation and contribute to vascular damage and the development of systemic inflammatory　the prerequisite, activation of leukocytes and endothelial cells results in aggregation of leukocytes, platelets and erythrocytes in　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 much higher affinity than oxygen［5］.Evidence accumulated to date suggests that endogenous carbon monoxide (CO), a biproduct of inducible heme oxygenase (HO1) can modulate inflammation, inhibits lipopolysaccharide (LPS)induced production of cytokines both in vivo and in vitro, and consequently exhibits important cytoprotective function and antiinflammatory properties that are beneficial for the resolution of acute inflammation［6-8］.Inhaled CO at concentrations of 250～500 parts per million (ppm) has also been shown to be beneficial in a number of lung injury models, including hyperoxic injury［9,10］ allergeninduced inflammation［11］.
Recently, transitional metal carbonyls have been identified as potential COreleasing molecules (CORMs) with the potential to facilitate the pharmaceutical use of CO by delivering it to tissues and organs［12］.CORMs have been shown to act pharmacologically in rat aortic and cardiac tissue where liberation of CO produced vasorelaxant effects［13-16］ and decreased myocardial ischemiareperfusion damage［17,18］ in the absence of dramatic changes in blood carboxyhemoglobin (COHb) levels.
On the basis of these data, the present study was, therefore, designed as a prospective laboratory experiment to investigate the effects of tricarbonyldichlororuthenium (Ⅱ) dimer (CORM2), one of the novel group of CORMs, on attenuation of leukocyte sequestration and decrease of inflammatory responses and oxidative stress in the organs of CLPinduced mice and LPSinduced HUVEC (human umbilical vein endothelial cell), and discussed the possible molecular mechanisms.
1 Material and methods
　Materials
Medium 199 (M199), fetal calf serum (FCS), penicillin, and streptomycin were purchased from GIBCO BRL (Gland Island, NY).Tricarbonyldichlororuthenium(II) dimer (CORM2) was obtained from Sigma Aldrich and solubilized in dimethyl sulfoxide (DMSO) to obtain a 10 mmol/L (Escherichia coli serotype 055:B5) was purchased from ICAM1 polyclonal antibody was purchased from Transduction Laboratories (Lexington, KY).Antimouse IgG conjugated to horseradish peroxidase was purchased from Kirkegaard and Perry Laboratories (Gaithersburg, MD).
　Animals
The C57BL/6 mice［male, N=21; bw (20± 2)g］ were fed a standard laboratory diet and water ad　were assigned to three groups in three respective　each experiment, mice in sham group (n=7) were underwent sham procedure, whereas mice in CLP group (n=7) received cecal ligation and puncture and mice in CORM2 group (n=7) underwent the same injury with immediate administration of CORM2 (8 mg/kg, ).The concentration of CORM2 used in the present study was based on a previous report in of the use of this compound in mice［19］ and the preliminary experiments in our lab by measuring dynamic COHb levels and peak levels which did not averaged 15%±5% above normal　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 animals.
　CLP Mice were anesthetized with 2% isoflurane in oxygen via a　1to 2 cm midline incision was made through the abdominal wall; the cecum was identified and ligated with a 3-0 silk tie 1 cm from the　was taken not to cause bowel　single puncture of the cecal wall was performed with a 20gauge　cecum was lightly squeezed to express a small amount of stool from the puncture site to assure a fullthickness　care was taken to preserve continuity of flow between the small and large　of mice at various intervals after CLP did not reveal evidence of bowel　cecum was returned to the abdominal cavity, and the incision was closed with　mice underwent anesthesia and midline laparotomy; the cecum was exteriorized and returned to the abdomen, and the wound was closed with　received injection of CORM2 ( mg/kg, ) immediately after　mice received 160 μl % DMSOnormal saline in the same　of MPO in the liver was performed at 24 h after CLP.
　MPO activity MPO activity as an assessment of neutrophil influx was measured according to established protocols［20］.In brief, tissue was homogenized in　ml of 50 mmol/L potassium phosphate buffer (pH ) and centrifuged at 10,000 r/min at 4℃ for 30　remaining pellet was resuspended in　ml of 50 mmol/L potassium buffer pH　with % hexadecyltrimethylammonium bromide, sonicated on ice, and then centrifuged at 12,000 r/min at 4℃ for 10　were then assayed at a 1∶20 dilution in reaction buffer containing 50 mmol/L PB, 530 mmol/L odianisidine, and 20 mmol/L H2O2　unit of enzyme activity was defined as the amount of MPO present that caused a change in absorbance measured at 460 nm for 3　activity was expressed as U/g tissue.
　Cells
　Isolation and culture of human umbilical vein endothelial cells (HUVEC) Human umbilical vein endothelial cells (HUVEC) were harvested from the fresh human umbilical vein of newborns by collagenase treatment (Worthington Biochem, Freehold, NJ) as previously described［21］.The cells were grown in medium 199(M199; GIBCO, Burlington, Canada) supplemented with 10% heatinactivated FCS (Intergen, Purchase, NY),　mg/L thymidine (Sigma Chemical, Oakville, Canada), 10 IU/ml heparin sodium, antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin; GIBCO),　μg/ml fungizone (GIBCO),and 80 μg/ml endothelial mitogen (Biomedical chnologies, Stoughten, MA).The cell cultures were incubated in room air with 5% CO2 at 37℃ and 95% humidity and were expanded by brief trypsinization with % trypsin in PBS containing %　experiments were conducted on passage 3　18 h, the medium was changed to 500 μl of fresh complete　were stimulated with LPS (10 g/ml).After treatment for 4 h, the cells and medium were harvested separately.
　PMN adhesion assays Human neutrophilic PMN were isolated from the venous blood of healthy adults using standard dextran sedimentation and gradient separation on Histopaque procedure yields a PMN population that is 95%～98% viable (trypan blue exclusion) and 98% pure (acetic acidcrystal violet staining).
For the static adhesion assay, isolated neutrophils were suspended in PBS buffer and radiolabeled by incubating the cells at 5×107 cells/ml with 50 μCi Na51CrO4/ml PMN suspension at 37℃ for 60　cells were then washed with cold PBS to remove unincorporated　PMN (5×105/well) were added to HUVEC monolayers grown in 48well plates (Costar), and 30 min later the percentage of added PMN that remained adherent after a wash procedure was quantitated as follows: %PMN adherence = lysate (cpm)/［supernatant (cpm) + wash (cpm) + lysate (cpm)］, where cpm is counts per minute.
　Oxidant production Oxidant production within HUVEC was assessed by measuring the oxidation of intracellular dihydrorhodamine 123 (DHR 123; Molecular Probes, Inc.), an oxidantsensitive fluorochrome, as described previously［22］.Briefly, the cells were treated with DHR 123 (5 mmol/L) for 1 h before being subjected to LPS　LPS stimulation the cells were washed with PBS, lysed, and DHR 123 oxidation was assessed spectrofluorometrically at excitation and emission wavelengths of 502 and 523 nm, respectively.
　Nitric oxide production NO production by HUVEC was assessed by measuring the fluorescence of 4amino5methylamino2′,7′difluorofluorescein diacetate (DAFFM diacetate), a specific NO probe (Molecular Probes, Inc.)［23］.Briefly, DAFFM diacetate (10 mmol/L) in M199 was added to the HUVEC 1h before the LPS　LPS stimulation, the HUVEC and supernatants were collected and analyzed spectrofluorometrically at excitation and emission wavelengths of 495 nm and 515 nm, respectively.
　SDSpolyacrylamide gel electrophoresis and Western blotting SDSpolyacrylamide gel electrophoresis and Western blotting were performed as described previously［24］.Samples (10 μg of protein) were subjected to electrophoresis on 7% (for ICAM1) SDSpolyacrylamide gels, with the use of the discontinuous system and transferred onto nitrocellulose　membranes were probed with antiICAM1 monoclonal antibody (1∶2 500).Antimouse IgG conjugated to horseradish peroxidase (1∶2 500) was used as a secondary　bands were visualized by the use of ECL reagent and Hyperfilm ECL (Amersham, Arlington Heights, IL) as described by the　were scanned using a flatbed scanner and the bands were quantified using Basic Quantifier software (Bio Image, Ann Arbor, MI), an image analysis program, on computer.
　HUVEC nuclear protein extraction and Electrophoretic mobility shift assay(EMSA) Nuclear protein was extracted from HUVEC as previously described［25］.Cells were grown to confluence in Petridish, scraped, washed with cold PBS, and incubated in 150 μl of buffer E(+) (% Nonidet P40, 10 mmol/L Tris (pH ), 60 mmol/L NaCl, 1 mmol/L EDTA,　mmol/L dithiothreitol (DTT), 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride ) for 5 min on　were centrifuged at 4℃ for 5 min at 500 r/ supernatant was then removed, and the pellets (nuclei) were resuspended in 150 μl of buffer E (10 mmol/L Tris (pH ), 60 mmol/L NaCl, 1 mmol/L EDTA, and　mmol/L DTT) and centrifuged at 500 r/min for 5 min at 4℃.The nuclei were then extracted in 30～50 μl of buffer E(c)(20 mmol/L HEPES,　mmol/L spermidine,　mmol/L spermine,　mmol/L EDTA, 2 mmol/L EGTA, 2 mmol/L DTT, 20% glycerol, and 1 mmol/L PMSF (4 ℃) in the presence of　mol/L NaCl) and were incubated on ice for 20 , the samples were centrifuged for 10 min at 500 r/min (4℃), and the supernatants were collected and saved as the nuclear protein　were stored at -80℃.
The doublestranded oligonucleotide containing consensus (58AGGGACTTCCGCTGGGGACTTTCC38) binding sites for NFκB (synthesized on site; BeckmanOligo 1 000 mol/L DNA synthesizer) were endlabeled with［γ-32P］ATP (Amersham) by using T4polynucleotide kinase (MBI Fermentas, Flamborough, ON), as described previously［25］ .One picomole of the labeled oligonucleotide was incubated with 5 μg of nuclear extract protein in the presence or absence of 50×excess of cold　were incubated for 30 min at room temperature and then run through a 4% nondenaturing polyacrylamide gel at 280 V for 45～60　gel was dried and then exposed to Xray film (Kodak) in cassettes for 2～4 h at -80℃ with intensifying screens.
　Cell ELISA For assessment of ICAM1 surface expression level, an ELISA was performed［26］ on HUVEC grown in 96well cell culture plates (Corning).HUVEC were fixed in 4% paraformaldehyde at 4℃ for 30　cells were then washed two times with cold PBS and were incubated with the mouse primary monoclonal antibody (MAb) against human ICAM1 (Dako) at a concentration of 10 μg/ml for 1 h at room　this treatment, immunocytochemical staining of HUVEC monolayers was performed using an avidinbiotinconjugated peroxidase mouse IgG staining kit (Vectastain), and MAb binding was subsequently quantified with a microplate reader (model 3550UV; BioRad) at 450 nm wavelength.
　Statistical Analysis
All of the values are presented as means ±　analysis was performed with the use of ANOVA and Student′s ttest for the　value of　was considered to be statistically significant.
2 Results
　Effect of CORM2 on MPO activity in lung and liver of CLPchallenged mice
To determine whether CLPinduced increase in PMN accumulation in the lung and liver was effectively prevented by CORM2, the activity of MPO, an enzyme in azurophilic granules of neutrophils, was　of the organs samples were examined for content of MPO at 24 h after CLP　activity in organs obtained from CLPinduced mice was markedly increased compared to sham (P ), while it significantly decreased by treatment with CORM2 ().
Fig 1 Effects of CORM2 on MPO activity in the
lung and liver of CLPchallenged mice
Mice were injected () with CORM2 (8 mg/kg) immediately after CLP　mice received 160 μl bolus injection of % DMSO/ activities in the lung and liver were assessed 24 h following　are mean ± SE of three experiments (three mice per group), * compared to sham mice.﹟P　compared to CLP mice.
　Effect of CORM2 on expression of ICAM1 in the lung and liver of CLPchallenged mice
At 24 h after CLP induction, the expression of ICAM1 in lung and liver tissues significantly increased compared to the sham　vivo administration of CORM2 (8 mg/kg, ), expression of ICAM1 was significantly decreased ()
Fig 2 Effects of CORM2 on protein expression of ICAM1
in the liver and lung of CLPinduced mice
Mice were challenged with CLP and treated with CORM2 as described in　expression of ICAM1 was performed by western blotting at 24 h after　are mean ± SE.* compared to sham mice.﹟P　compared to CLP mice.
　Effect of CORM2 on activities of NFκB in lung and liver tissue of CLPchallenged mice
Binding activities of nuclear protein to the radiolabeled consensus binding sequences of NFκB was assessed by　24 h after CLP induction, the NFκB activation in lung and liver was markedly increased and this activity was inhibited by in vivo administration of CORM2 (8 mg/kg,) ().
Fig 3 Effects of CORM2 on NFκB activation in the lung
and liver of CLPinduced mice (EMSA)
Mice were challenged with CLP and treated with CORM2 as described in　of NFκB activity was performed by mobility shift assay (EMSA) with 32Plabeled NFκB probe and 5 μg of nuclear extract from liver of sham, CLP and CLP+CORM