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

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

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-kB 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1β（IL-1β）、肿瘤坏死因子α（TNF-α ）和基因表达水平，免疫印迹法（Western blot）检测不同组VSMCs 中Toll 样受体4（TLR4）、I资Ba及p65蛋白表达水平。结果①LPS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）；②CGRP 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）；③CGRP能抑制LPS 诱导的细胞TLR4 蛋白的积累（p <0.05），抑制IkBa 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-kB 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IkBa 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。

Abstract:

Objective To investigate the effect of calcitonin gene -related peptide (CGRP) on lipopolysaccharide (LPS)-induced expressions of Toll-like receptor 4 (TRL4)/NK-kB 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-1βand TNF-α. Western blot was used to detect the expression of TLR4, IkBa 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 IkBa 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-kB signaling pathway.