蒙花苷对TNF-α诱导的血管内皮细胞炎症损伤及TLR4/IκBα/NF-κB信号通路的影响

Buddleoside Prevents TNF-α-induced Human Aortic Endothelial Cells Inflammatory Injury Through Inhibiting TLR4/IκBα/NF-κB Signaling Pathway

目的探讨蒙花苷对血管内皮细胞的保护作用和机制。方法 MTT法检测肿瘤坏死因子(TNF-?)和蒙花苷对人脐静脉内皮细胞(EA.hy926)活性的影响;采用TNF-?(50 ng·m L-1)刺激EA.hy926,加入不同浓度(5,15和25?mol·L-1)蒙花苷处理24 h,DAPI染色法测定EA.hy926细胞与单核细胞(THP-1)的黏附能力;Western blot法检测细胞中Toll样受体(TLR4)、核因子-?B抑制蛋白(IκBα)、核因子-?B(NF-?B)、细胞间黏附分子-1(ICAM-1)和血管细胞黏附分子-1(VCAM-1)蛋白的表达;RT-PCR法检测ICAM-1、VCAM-1 m RNA的表达;ELISA法检测细胞上清液中白介素-1(IL-1)、白介素-6(IL-6)和白介素-8(IL-8)的含量。结果蒙花苷可显著抑制TNF-?刺激EA.hy926细胞与THP-1细胞间黏附作用,降低EA.hy926细胞分泌的炎症因子IL-1、IL-6和IL-8水平。Western blot和RT-PCR结果表明蒙花苷能减少EA.hy926细胞中TLR4、NF-?B、ICAM-1、VCAM-1的蛋白表达量及ICAM-1、VCAM-1 m RNA的表达量,同时能升高细胞中IκBα的蛋白表达量。结论蒙花苷能缓解血管内皮细胞炎症损伤,其作用机制可能主要是通过抑制TLR4/IκBα/NF-κB信号通路发挥效用。

OBJECTIVE To study the protective effect and mechanism of buddleoside on human aortic endothelial cells. METHODS MTT assay was used to detect the viability of EA.hy926 in TNF-α and buddleoside. EA.hy926 cells were treated with TNF-α（50 ng·mL^-1） for inflammatory injury model. After being pretreated with different concentrations（5, 15, 25 mmol·L-1） of buddleoside for 24 h, DAPI staining was used to evaluate the effect of buddleoside on adhesion of EA.hy926 and THP-1. The protein expression of TLR4, IκBα, NF-κB, ICAM-1 and VCAM-1 were tested by Western blot method respectively. The mRNA expression of ICAM-1 and VCAM-1 were tested by RT-PCR method respectively. ELISA method was used to measure the levels of IL-1, IL-6 and IL-8. RESULTS Buddleoside could inhibit the adhesion of TNF-α stimulated EA.hy926 cell and THP-1 cell. ELISA results showed that buddleoside reduced IL-1, IL-6 and IL-8 production in TNF-α-stimulated EA.hy926. Buddleoside significantly decreased TLR4, NF-κB, ICAM-1 and VCAM-1 protein expression in TNF-α-stimulated EA.hy926 cell. In addition, buddleoside increased IκBα protein expression. RT-PCR result showed buddleoside decreased ICAM-1 and VCAM-1 mRNA expression in TNF-α-stimulated EA.hy926 cell. CONCLUSION Buddleoside can alleviate the inflammatory injury of vascular endothelial cells, and its mechanism may be mainly through the inhibition of TLR4/IκBα/NF-κB signaling pathway.