白藜芦醇衍生物TMS对脂多糖诱导血管内皮细胞表达NO、ICAM-1和NF-κB的影响  

Effects of trans-3,5,4'-trimethoxystilbene on the expressions of NO,ICAM-1 and NF-κB in human umbilical vein endothelial cells induced by lipopolysaccharide in vitro

作　　者：付海燕[1] 胡占升[1] 杜红阳[2] 

机构地区：[1]辽宁医学院附属第一医院重症医学科,辽宁锦州121001 [2]辽宁医学院附属第一医院皮肤科,辽宁锦州121001

出　　处：《山东大学学报（医学版）》2015年第11期10-15,20,共7页Journal of Shandong University:Health Sciences

基　　金：辽宁省科技厅自然科学基金(2013022059);中国博士后科学基金(2015M572776);辽宁医学院青年科技启动基金(Y2011Z010)

摘　　要：目的探讨白藜芦醇衍生物反式-3,5,4'-三甲氧基二苯乙烯(TMS)对脂多糖(LPS)诱导的人脐静脉内皮细胞(HUVEs)表达NO、细胞间黏附分子-1(ICAM-1)和核转录因子-κB(NF-κB)的影响。方法采用CCK-8检测不同浓度TMS对HUVEs存活率的影响。细胞分为正常对照组(CON组)、LPS组、低浓度TMS+LPS组、中浓度TM S+LPS组、高浓度TM S+LPS组和吡咯烷二硫代氨基甲酸铵(PDTC)+LPS组,不同浓度TM S和10μmol/L PDTC预处理细胞,0.1μg/m L的LPS进行诱导后,Griess法检测各组细胞产生的NO浓度;Real-time PCR检测ICAM-1、NF-κB p65的m RNA表达;Western blotting检测ICAM-1、NF-κB p65和IκBα的蛋白表达;免疫细胞化学染色检测ICAM-1及NF-κB p65的蛋白表达。结果较低浓度(5、10μmol/L)TMS对细胞的存活率影响较小,而较高浓度(50、100μmol/L)TMS处理后,细胞存活率明显下降,并呈时间、剂量依赖性。Griess法结果显示低、中、高浓度TMS+LPS组与PDTC+LPS组的NO表达量均较LPS组降低(P<0.05)。Real-time PCR和Western blotting检测结果均显示,与LPS组、CON组相比,中浓度TM S+LPS组和PDTC+LPS组中ICAM-1、NF-κB p65的m RNA和蛋白表达差异均有统计学意义(P<0.05,P<0.01)。Western blotting结果还显示,除CON组外,NF-κB p65蛋白在LPS组、中浓度TM S+LPS组和PDTC+LPS组中均有核表达;IκBα蛋白在LPS组中表达量较CON组降低(P<0.01),其在中浓度TMS+LPS组和PDTC+LPS组的表达低于CON组(P均<0.05)。细胞免疫荧光检测结果显示,ICAM-1蛋白在LPS组中表达强荧光,在中浓度TMS+LPS组和PDTC+LPS组中表达均减弱;NF-κB p65蛋白在CON组中无表达,在LPS组中为细胞核荧光强表达,在中浓度TM S+LPS组和PDTC+LPS组中荧光表达相对减弱,且无核表达。结论白藜芦醇衍生物TMS抑制LPS诱导血管内皮细胞的NO、ICAM-1和NF-κB p65表达,且其对ICAM-1的抑制可能是通过NF-κB细胞信号通路起作用。Objective To explore the effects of trans-3,5,4＇-trimethoxystilbene （TMS） on the expressions of NO, intercellular adhesion molecule-1 （ICAM-1） and nuclear factor-κB （NF-κB） in human umbilical vein endothelial cells （HUVEs） induced by lipopolysaccharide （LPS） in vitro. Methods The cell viabilities influenced by the different concentrations of TMS were assessed by CCK-8 assay. The cells were divided into the control group （ CON group）, LPS group, low-concentration TMS plus LPS group, medium-concentration TMS plus LPS group, high-concentration TMS plus LPS group and ammonium pyrrolidine dithiocarbamate （PDTC） plus LPS group. HUVEs were pretreated with the different concentrations of TMS and 10 i^mol/L PDTC, and then were stimulated with 0.1 ~g/mL LPS. After incubation, the level of NO was determined by Griess assay. The mRNA expressions of ICAM-1 and NF-κB p65 were detected by Real-time PCR, the protein expressions of ICAM-1, NF-κB 1065 and IKBct by Western blotting assay, and the protein expressions of ICAM-1 and NF-κB 1065 by immunocytochemetry assay. Results There was little effect of low-concentration TMS （5 or 10μmol/L） on the cell viability, but the cell viability decreased significantly when trea- ted with high-concentration TMS （50 or 100 μmol/L） in time- or concentration-dependent manners. Griess results showed that the level of NO in the low-, medium- and high-concentration TMS plus LPS groups and PDTC plus LPS group decreased compared with that in LPS group （ P 〈 0.05 ）. The results of Real-time PCR and Western blotting showed that compared with LPS group and CON group, there were significant difference of mRNA and protein expressions of ICAM-1 and NF-κB 1065 in medium-concentration TMS plus LPS group and PDTC plus LPS group （ P 〈 0. 05 ; P 〈 0.01 ）. Furthermore, there were nuclear expressions of NF-κB p65 protein in medium-concentration TMS plus LPS group, PDTC plus LPS group and LPS group except for CON group. The protein expression of IKBct d

关 键 词：反式-3 5 4'-三甲氧基二苯乙烯 脂多糖 血管内皮细胞 NO 细胞间黏附分子-1 核转录因子-ΚB 

分 类 号：R965.1[医药卫生—药理学]