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

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 decreased significantly in LPS group compared with that in CON group （P 〈 0.01 ）, and those in medium-concentration TMS plus LPS group and PDTC plus LPS group were lower than those in CON group （ P 〈 0.05 ）. Immunofluorescence assay showed there were hyperfluorescence of ICAM-1 in LPS group but weakened fluorescence in medium-concentra- tion TMS plus LPS group and PDTC plus LPS group. There were no fluorescence of NF-KB p65 in CON group, cell nucleus hyperfluorescence in LPS group, and weakened fluorescence in medium-concentration TMS plus LPS group and PDTC plus LPS group with no cell nucleus expression. Conclusion TMS can inhibit LPS-mediated NO, ICAM-1 and NF-κB p65 expressions in HUVEs in vitro, and the inhibition of ICAM-1 may be affected through NF-κB cell pathway.