降钙素基因相关肽对脂多糖诱导血管平滑肌细胞TLR4/NK-κB及炎症因子表达的影响  

Effect of CGRP on lipopolysaccharide-induced expressions of TLR4/NK-κB and inflammatory factors of vascular smooth muscle cells

作　　者：王芳[1] 周祥群[1] 付洁[2] 

机构地区：[1]海南省第三人民医院心血管内科,海南三亚572000 [2]新乡医学院组织胚胎学教研室,河南新乡453003

出　　处：《中国现代医学杂志》2017年第21期12-17,共6页China Journal of Modern Medicine

基　　金：海南省卫生厅课题(No:1421320.24A1004)

摘　　要：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、CGR P组、(LPS+CGRP)组及(LPS+CGRP+C8-37)组;ELISA检测不同组VSMCs中白介素1β(IL-1β)、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 (1)LPS处理VSMCs后,随着LPS浓度的升高,IL-1β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8 h时达到峰值(P<0.05);(2)CGRP剂量依赖性降低LPS诱导IL-1β和TNF-α的表达(P<0.05),并于100 nmol/L时达到低峰点(P<0.05);(3)CGRP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);(4)CGRP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-1β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.

关 键 词：降钙素基因相关肽 血管平滑肌细胞 TOLL样受体4 NF-ΚB 

分 类 号：R685.6[医药卫生—骨科学]