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目的探讨白藜芦醇衍生物TMS(trans-3,5,4′-trimethoxystilbene)对脂多糖(lipopolysaccharide,LPS)诱导人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)中一氧化氮(nitric oxide,NO)、血管细胞间黏附分子-1(vascular cell adhesion molecule-1,VCAM-1)及核转录因子-κB(nuclear factor-κB,NF-κB)表达的影响。方法取P3代HUVECs,采用CCK-8法检测不同TMS浓度(0、5、10、20、50、100μmol/L)对HUVECs细胞存活率的影响。将P6代HUVECs分为正常对照组、LPS组、TMS(高、中、低浓度)+LPS组及PDTC+LPS组,Griess法检测各组细胞产生NO浓度;Real-time PCR法检测各组细胞中VCAM-1、NF-κB p65基因m RNA的转录水平;Western blot法检测各组细胞中VCAM-1、NF-κB p65和IκBα蛋白的表达水平;免疫细胞化学染色法检测各组细胞中VCAM-1和NF-κB p65蛋白的表达水平。结果 5、10μmol/L浓度组TMS对细胞存活率影响较小,50、100μmol/L浓度组中细胞生存率明显下降(P<0.05),且呈时间、剂量依赖性。低、中、高浓度TMS+LPS组与PDTC+LPS组细胞中NO浓度显著下降(P<0.05);中浓度TMS+LPS组和PDTC+LPS组细胞中VCAM-1、NF-κB p65基因m RNA转录和蛋白表达水平均明显低于LPS组(P<0.05),IκBα蛋白表达水平明显高于LPS组(P<0.05);正常对照组细胞胞质中有弱VCAM-1蛋白荧光表达,未见NF-κB p65蛋白荧光表达,LPS组可见VCAM-1和NF-κB p65蛋白的强荧光表达,中浓度TMS+LPS组和PDTC+LPS组中VCAM-1和NF-κB p65蛋白荧光表达均弱于LPS组。结论白藜芦醇衍生物TMS可抑制LPS诱导HUVECs表达NO、VCAM-1和NF-κB p65,且其抑制VCAM-1效应可能是通过NF-κB细胞信号通路而发挥作用的,本研究为临床治疗血管内皮功能紊乱相关疾病提供了实验依据。
Objective To investigate the effect of trans-3,5,4’-trimethoxystilbene (TMS) on the expression of nitric oxide (NO) in human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS) (NO), vascular cell adhesion molecule-1 (VCAM-1), and nuclear factor-κB (NF-κB) Methods P3 HUVECs were obtained and the effects of different TMS concentrations (0, 5, 10, 20, 50 and 100 μmol / L) on the cell viability of HUVECs were detected by CCK-8 assay. The P6 generation of HUVECs were divided into normal control group, LPS group, TMS (high, medium and low concentration) + LPS group and PDTC + LPS group. The concentration of NO was detected by Griess method. The expression of VCAM-1, NF-κB p65 gene m RNA was detected by Western blot. The expression of VCAM-1, NF-κB p65 and IκBα in each group were detected by Western blot. The expression of VCAM- -1 and NF-κB p65 protein expression levels. Results TMS at 5 and 10 μmol / L had little effect on cell viability. The cell viability in 50 and 100 μmol / L groups decreased significantly (P <0.05) in a time and dose dependent manner. The concentration of NO in cells in low, medium and high concentrations of TMS + LPS group and PDTC + LPS group was significantly decreased (P <0.05). The levels of VCAM-1 and NF-κB p65 in medium concentrations of TMS + LPS and PDTC + (P <0.05). The expression level of IκBα was significantly higher in LPS group than in LPS group (P <0.05). Fluorescent expression of weak VCAM-1 protein in cytoplasm of normal control group was not observed The fluorescence of NF-κB p65 protein was observed in LPS group. The expression of VCAM-1 and NF-κB p65 protein in LPS group was stronger than that in LPS group. The fluorescence intensity of VCAM-1 and NF-κB p65 in medium concentration TMS + LPS group and PDTC + LPS group Weaker than LPS group. CONCLUSION: Resveratrol derivative TMS can inhibit LPS-induced NO, VCAM-1 and NF-κB p65 expression in HUVECs. The effect of VCAM-1 on VCAM-1 expression may be through NF-κB signaling pathway. Treatment of vascular endothelial dysfunction-related diseases provide experimental evidence.