The effect of ketamine on lipopolysaccharide induced activation of human umbilical vein endothelial cells WANG Hai-yun，WANG Guo-lin. Department of Anesthesiology，General Hospital of Tianjin Medical University，Tianjin 300052，China
       【Abstract】 Objective To study the effect of ketamine and MK-801 on lipopolysaccharide（LPS）induced the expression of intercellular adhesion molecule-1（ICAM-1）and the translocation of nuclear factor-kappa B（NF-κB）into nuclei of human umbilical vein endothelial cells（HUVECs）.

         Methods The cultured HUVECs were randomly divided into ten groups: control group（group C，RPMI-1640），LPS group（group L，LPS 1μg/ml），ketamine group（group K，and divided into KⅠ-KⅣ according the varying concentrations of ketamine，ketamine 12.5、25.0、100、300μmol/L +LPS 1μg/ml），MK-801 group（group M，and divided into MⅠ-MⅣ according the different concentrations of MK-801，MK-801 1.25、2.50、10、30μmol/L +LPS 1μg/ml）. After 18h of incubation at 37℃ in a 95% air / 5% CO2 incubator , the HUVECs were harvested for flow cytometric analysis of ICAM-1. In another set of experiments, the cultured HUVECs were stimulated with LPS 1μg/ml for 2 hours in the absence or presence of ketamine(12.5, 25.0, 100 and 300μmol/L) or MK-801(1.25, 2.50, 10 and 30μmol/L). The translocation of NF-κB p65 into nuclei was stained with immunohistochemistry (Streptavidin/Peroxidase)technique. Results Group KⅡ、KⅢ、KⅣsignificantly reduced the up-regulation of ICAM-1 and the translocation of NF-κB into nuclei of HUVECs caused by LPS（P＜0.05）,and there was positive correlation between them（r=0.985, P<0.01）. Group MⅠ、MⅡ、MⅢ、MⅣ had no effect on the expression of ICAM-1 and the translocation of NF-κB into nuclei（P＞0.05）.   

        Conclusions At the higher concentrations(≥25μmol/L), ketamine could suppressed LPS induced HUVECs activation. The inhibitory effects of ketamine is most likely not mediated through NMDA receptor interactions.
       【Key words】 Ketamine；MK-801；Intercellular adhesion molecule-1；Nuclear factor-kappa B

        

 

        近年研究发现，氯胺酮（ketamine）可以抑制炎症反应中白细胞的活化[1]。但氯胺酮是否能影响内皮细胞的活化尚无定论[2，3]，其抗炎作用的确切机制也不清楚。本实验拟采用脂多糖（lipopolysaccharide,LPS）刺激人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)模拟体内的炎症反应过程，通过检测HUVECs胞间黏附分子-1（intercellular adhesion molecule-1, ICAM-1）表达及核因子-kappa B （nuclear factor-kappa B，NF-κB）易位活化的变化，观察氯胺酮及MK-801对炎症反应中内皮细胞活化的影响，初步探讨其抗炎机制。
        材料与方法
        试剂与仪器 RPMI-1640培养液、LPS、MK-801（Sigma，美国）；氯胺酮（江苏恒瑞医药股份有限公司，批号011110）；ICAM-1 FITC标记单克隆抗体、IgG1同型对照抗体（Immunotech Coulter，美国）；NF-κB p65单克隆抗体、过氧化物酶标记的链霉卵白素染色试剂盒（北京中山生物技术有限公司）。Olympus CK 40显微镜（日本）；EPICS-XL Bechman Coulter流式细胞仪（美国）。
        HUVECs的培养 参照Jaffe［4］的方法。取新鲜脐带，注入0.1%Ⅰ型胶原酶消化内皮细胞，培养于20%胎牛血清、100ku/L青霉素G、100mg/L链霉素、90mg/L肝素的RPMI-1640培养液中，置入37℃、5%CO2培养箱中，约7d长成单层，传代后用于实验。
        ICAM-1表达的测定 体外培养的HUVECs随机分为十组，（每组细胞孔数 n=8）：对照组（C组，RPMI-1640）、LPS组（L组，LPS 1μg/ml）、氯胺酮组（K组，依浓度不同分为KⅠ、KⅡ、KⅢ、KⅣ亚组，即Ketamine 12.5、25.0、100、300μmol/L + LPS 1μg/ml）、MK-801组（M组，依浓度不同分为MⅠ、MⅡ、MⅢ、MⅣ亚组，即MK-801 1.25、2.50、10、30μmol/L + LPS 1μg/ml）。K组和M组各组加入不同浓度的氯胺酮或MK-801后，在37℃、5%CO2培养箱中孵育30min，再加入LPS 1μg/ml，放入培养箱中孵育18h；L组不加入氯胺酮或MK-801，仅在孵育30min后加入LPS 1μg/ml作用18h；而C组在孵育30min后加入与LPS等体积的RPMI-1640作用18h。随后每孔中加入0.25%胰酶-0.2% EDTA消化贴壁的内皮细胞，将细胞清洗后每份样品中加入FITC标记的ICAM-1单克隆抗体20μl，4℃下避光30min，同时设立同型对照，每份加入FITC标记的IgG1单克隆抗体20μl。用流式细胞仪（EPICS-XL Bechman Coulter，美国）检测ICAM-1的表达。

NF-κB p65表达的测定 实验分组方法同上。K组和M组每组加入不同浓度的氯胺酮或MK-801，在37℃、5%CO2培养箱中孵育30min，再加入LPS 1μg/ml孵育2h；L组仅在孵育30min后，加入LPS 1μg/ml作用2h；而C组在孵育30min后，加入与LPS等体积的RPMI-1640作用2h。用免疫组化（SP）方法测定内皮细胞中NF-κB p65亚基的表达。在200×高倍视野中每个培养孔随机选取10个视野，计算阳性细胞在整个视野所有细胞中所占比例。采用双盲法以减少人为误差。单位以百分率（%）表示。
        统计学处理 数据以均数 标准差（ S）表示，应用SPSS11.0 FOR WIN统计软件，多个均数的比较采用单因素方差分析、均数间的两两比较采用q检验，ICAM-1和NF-κB两变量间的关系采用直线相关分析，P＜0.05为差异有显著性。
        结 果
        HUVECs表面ICAM-1表达的变化 L组HUVECs在LPS作用后ICAM-1的表达明显高于C组（P＜0.05）。KⅠ亚组ICAM-1的表达与L组比较差异无显著性 (P＞0.05)，KⅡ、KⅢ、KⅣ亚组与L组相比ICAM-1的表达明显受抑制（P＜0.05），并呈剂量依赖性关系。M组各亚组ICAM-1的表达与L组比较差异无显著性（P＞0.05) 

 

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