降钙素基因相关肽对血管平滑肌细胞的心肌素表达及细胞炎症激活的作用

Effect of calcitonin gene-related peptide on myocardin expression and inflammatory activation in vascular smooth muscle cells

目的探讨降钙素基因相关肽(calcitonin gene-related peptide,CGRP)对体外培养的血管平滑肌细胞(vascular smooth muscle cells,VSMCs)心肌素表达及细胞炎症激活的影响。方法取大鼠胸主动脉,以组织块贴壁培养法获得VSMCs,根据不同的处理方案,分为对照组、IL-1β组、CGRP组、心肌素高表达组、心肌素干扰组和CGRP8-37组(CGRP受体拮抗剂组)。Western blot检测不同组VSMCs中心肌素和IL-6蛋白表达水平。结果以IL-1β处理VSMCs后,细胞培养24、48、72 h时IL-6表达水平均高于基线水平(P<0.05),而心肌素水平无显著变化;以高表达心肌素的腺病毒载体转染VSMCs并给予IL-1β处理后,与单纯IL-1β处理比较,各时间点IL-6水平显著下降(P<0.05)。CGRP组中各个时间点VSMCs内心肌素水平较同时间点IL-1β组心肌素水平显著增加[24 h:(1.17±0.03)vs(0.25±0.03);48 h:(2.04±0.05)vs(0.19±0.01);72 h:(0.96±0.02)vs(0.28±0.02);P<0.05],而细胞内IL-6表达水平较同时间点IL-1β组水平显著降低[24 h:(0.23±0.01)vs(0.77±0.03);48 h:(0.28±0.02)vs(1.35±0.04);72 h:(0.20±0.02)vs(1.04±0.04);P<0.05]。心肌素干扰组VSMCs中CGRP处理后各个时间点细胞内IL-6水平较CGRP组显著升高[24 h:(1.74±0.04)vs(0.51±0.01);48 h:(1.68±0.03)vs(0.29±0.01);72 h:(0.98±0.02)vs(0.31±0.01);P<0.05],且CGRP8-37组与CGRP组比较也获得类似结果,而心肌素干扰组和CGRP8-37组中同时间点的细胞内IL-6水平无明显差异。结论 CGRP通过促进VSMCs心肌素的表达而抑制IL-1β诱导的细胞炎症激活蛋白IL-6表达,且这一作用是CGRP通过与其受体结合后实现的。

Objective To investigate the effect of calcitonin gene-related peptide（ CGRP） on myocardin expression and inflammatory activation in vascular smooth muscle cells（ VSMCs） in vitro.Methods VSMCs were obtained by aortic tissue adherent culture and treated with interleukin-1β（ IL-1βgroup）,IL-1β ＋ CGRP（ CGRP group）,and IL-1β ＋ CGRP ＋ CGRP8-37（ CGRP receptor antagonist,CGRP8-37group） or transfected with recombinant adenovirus vector over-expressing myocardin（ myocardin group） or siRNA-myocardin（ siRNA-myocardin group）. The expression of myocardin protein and interleukin-6（ IL-6）protein at different time points in the VSMCs were detected by Western blotting. Results In the in vitro cultured VSMCs of the IL-1β group,the expression of IL-6 at 24,48 and 72 h were significantly higher than the baseline（ P〈0. 05）,and the expression of myocardin was similar. In the myocardin group,the level of IL-6 at any time point was obviously lower than that in the IL-1β group（ P〈0. 05）. The CGRP group showed higher myocardin expression and lower IL-6 level at any time point than the IL-1β group（ myocardin： 1. 17 ±0. 03 vs 0. 25 ± 0. 03 at 24 h; 2. 04 ± 0. 05 vs 0. 19 ± 0. 01 at 48 h; 0. 96 ± 0. 02 vs 0. 28 ± 0. 02 at 72 h; P〈0. 05; IL-6： 0. 23 ± 0. 01 vs 0. 77 ± 0. 03 at 24 h; 0. 28 ± 0. 02 vs 1. 35 ± 0. 04 at 48 h; 0. 20 ± 0. 02 vs1. 04 ± 0. 04 at 72 h; P〈0. 05）. The level of IL-6 in the siRNA-myocardin group was higher than that in the IL-1β group at any time point（ 1. 74 ± 0. 04 vs 0. 51 ± 0. 01 at 24 h; 1. 68 ± 0. 03 vs 0. 29 ± 0. 01 at 48 h;0. 98 ± 0. 02 vs 0. 31 ± 0. 01 at 72 h; P〈0. 05）. The comparison had similar results between the CGRP8-37 group and the CGRP group. Also,there was no difference in the IL-6 level at the same time point between the siRNA-myocardin group and the CGRP8-37 group. Conclusion CGRP may inhibit IL-1β-induced IL-6 protein expression through up-regulating the expression of myocardin protein in the VSMCs, which may be accomplished by binding CGRP and its receptor.