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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 investigate the effect of trans-3,5,4’-trimethoxy-stilbene (TMS), a resveratrol derivative, on lipopolysaccharide (LPS) induced human umbilical vein endothelial cells (HUVEs) -1 (ICAM-1) and nuclear factor-κB (NF-κB). Methods CCK-8 was used to detect the effect of different concentrations of TMS on the survival rate of HUVEs. The cells were divided into normal control group (CON group), LPS group, low concentration of TMS + LPS group, medium concentration of TM S + LPS group, high concentration of TM S + LPS group and PDTC + LPS The cells were pretreated with different concentrations of TM S and 10 μmol / L PDTC, and induced by 0.1 μg / mL LPS. The concentrations of NO produced by each group were detected by Griess method. The expressions of ICAM-1, NF-κB p65 The protein expression of ICAM-1, NF-κB p65 and IκBα were detected by Western blotting. The protein expressions of ICAM-1 and NF-κB p65 were detected by immunocytochemical staining. Results The survival rates of cells treated with TMS at a lower concentration (5 and 10 μmol / L) had less effects on cell viability. However, the survival rates of cells treated with TMS at a higher concentration (50 and 100 μmol / L) decreased significantly and were time-and dose-dependent. The results of Griess assay showed that NO expression in low, middle and high concentrations of TMS + LPS group and PDTC + LPS group was lower than that of LPS group (P <0.05). The results of Real-time PCR and Western blotting showed that compared with LPS group and CON group, the mRNA and protein expressions of ICAM-1 and NF-κB p65 in TM S + LPS group and PDTC + LPS group were significantly lower than those in LPS group and CON group There was statistical significance (P <0.05, P <0.01). The Western blotting results also showed that in addition to the CON group, NF-κB p65 protein was expressed in the LPS group, the medium concentration TM S + LPS group and the PDTC + LPS group; the expression of IκBα protein in the LPS group was lower than that in the CON group (P <0.01). The expression levels of TMS + LPS group and PDTC + LPS group were lower than that of CON group (all P <0.05). The results of immunofluorescence showed that ICAM-1 protein was strongly expressed in LPS group and decreased in medium concentration TMS + LPS group and PDTC + LPS group. NF-κB p65 protein was not expressed in CON group, In the group, the fluorescence intensity of nucleus was strongly expressed. The fluorescence intensity of TM S + LPS group and PDTC + LPS group were relatively weakened, and there was no nuclear expression. CONCLUSION: Resveratrol derivative TMS can inhibit the expression of NO, ICAM-1 and NF-κB p65 in LPS-induced vascular endothelial cells, and its inhibitory effect on ICAM-1 may be through the NF-κB signaling pathway.