目的 探讨降钙素基因相关肽(CGRP)对脂多糖(LPS)诱导的血管平滑肌细胞(VSMCs)TLR4/NK-κB及炎症因子表达的影响.方法 贴壁法培养VSMCs,根据不同的处理方案,分为对照组、LPS处理组、CGRP组、(LPS+CGRP)组及(LPS+CGRP+C8-37)组;ELISA检测不同组VSMCs中白介素1 β(IL-1 β)、肿瘤坏死因子α(TNF-α)和CGRP基因表达水平,免疫印迹法(Western blot)检测不同组VSMCs中Toll样受体4(TLR4)、IκBa及p65蛋白表达水平.结果 ①LPS处理VSMCs后,随着LPS浓度的升高,IL-1 β和TNF-α表达水平成梯度增加,并于1 000 ng/ml时分泌水平最高(P＜0.05),在8h时达到峰值(P＜0.05);②CGRP剂量依赖性降低LPS诱导IL-1 β和TNF-α的表达(P＜0.05),并于100 nmol/L时达到低峰点(P＜0.05);③CGRP能抑制LPS诱导的细胞TLR4蛋白的积累(P＜0.05),抑制IκBa与p65蛋白的磷酸化水平(P＜0.05);④CGRP阻断剂C8-37可以逆转CGRP对LPS刺激的VSMCs中IL-1β和TNF-α表达(P＜0.05),拮抗CGRP对TLR4和NK-κB的抑制(P＜0.05).结论 CGRP通过抑制TLR4蛋白的积累,阻断NF-κB信号蛋白IκBa与p65的磷酸化,进而削弱LPS诱导的细胞炎症因子IL-1 β和TNF-α的激活,抑制LPS诱导的VSMCs炎症的激活.%Objective To investigate the effect of calcitonin gene-related peptide (CGRP) on lipopolysaccharide (LPS)-induced expressions of Toll-like receptor 4 (TRL4)/NK-κB and cytokines in vascular smooth muscle cells (VSMCs).Methods VSMCs were cultured and divided randomly into five groups:blank control group,LPS-insulted group,CGRP-stimulated group,LPS+CGRP stimulated group and LPS+CGRP+C8-37 group.Enzyme-linked immunosorbent assay (ELISA) was used to measure concentrations of IL-1β and TNF-α.Western blot was used to detect the expression of TLR4,IκBa and p65.Results LPS upregulated the expressions of IL-1β and TNF-α in a both time-and dose-dependent manner (P＜ 0.05),which reached the peak value when the cells were insulted by LPS at 1,000 ng/ml for 8 hours.CGRP reduced the LPS-induced expressions of IL-1β and TNF-α in a dose-dependent manner,which reached the lowest values when CGRP was under 100 nmol/L (P＜ 0.05).Moreover,LPS-treated cells exhibited significant decrease of TLR4 and phosphorylation of IκBa and p65,which was attenuated significantly by CGRP (P＜ 0.05).As a specific CGRP antagonist,C8-37 reversed the expression levels of IL-1β and TNF-α in VSMCs stimulated by LPS.Conclusions The present study demonstrates that CGRP can significantly block LPS-induced inflammatory response in VSMCs through inhibition of TLR4-mediated NF-κB signaling pathway.
展开▼
