降钙素基因相关肽抑制动脉损伤后NF-κB表达及其对新生内膜增殖的作用

发布时间：2019-05-09 03:57

【摘要】：目的通过降钙素基因相关肽(calcitonin gene related peptide,CGRP)处理血管平滑肌细胞(vascular smooth muscle cells,VSMCs)和颈动脉损伤大鼠模型,探讨NF-κB在CGRP调节VSMCs表型改变和动脉损伤后新生内膜增殖中的作用。方法建立颈动脉损伤大鼠模型,并分为对照组和CGRP组(n=24),通过HE染色观察新生内膜形成情况,并Western blot检测两组动物损伤动脉中NF-κB p65蛋白表达情况。以组织块贴壁培养法获得VSMCs,并分为对照组、IL-1β组(给予IL-1β处理)、CGRP组(给予IL-1β+CGRP处理)和CGRP8-37组(在IL-1β+CGRP基础上,加CGRP8-37)(n=24),Western blot法检测细胞内NF-κB p65蛋白和细胞表型标志蛋白表达变化,MTT法检测细胞增殖能力。结果①对照组大鼠的颈动脉损伤后7 d时新生内膜明显增加,至28 d时基本稳定,而CGRP组7 d和28 d时新生内膜面积较对照组同时间点显著降低[7d:(2.09±0.26)vs(3.76±0.33);14 d:(3.52±0.41)vs(6.38±0.61),P0.05];且与对照组比较,CGRP组1周时NF-κB p65蛋白表达水平明显降低(P0.05),而28 d时两组NF-κB p65表达水平无差异(P0.05)。②在体外培养VSMCs中,IL-1β处理显著激活细胞NF-κB p65蛋白表达(P0.05),伴随着VSMCs收缩表现标志蛋白SMα-actin表达减少和OPN表达增加(P0.05);同时再给予CGRP处理后,则显著减弱IL-1β诱导的NF-κB p65蛋白表达(P0.05),且使VSMCs合成表现标志蛋白OPN表达减少。RT-PCR分析获得相一致的结果。同时,CGRP显著抑制IL-1β引起的VSMCs增殖。③CGRP受体阻断剂CGRP8-37可以阻断CGRP减弱IL-1β诱导的NF-κB p65蛋白表达作用,并抑制CGRP对VSMC表达蛋白调节作用。结论 CGRP与受体结合后抑制NF-κB激活,促进VSMCs分化标志基因表达,从而抑制细胞增殖,降低动脉损伤后新生内膜增殖,这对于防治动脉粥样硬化和血管成形术后再狭窄可能具有重要应用价值。
[Abstract]:Objective to treat the rat model of vascular smooth muscle cell (vascular smooth muscle cells,VSMCs) and carotid artery injury by calcitonin gene-related peptide (calcitonin gene related peptide,CGRP). To investigate the role of NF- 魏 B in the regulation of VSMCs phenotypic changes by CGRP and neointimal proliferation after arterial injury. Methods A rat model of carotid artery injury was established and divided into control group and CGRP group (n = 24). Neointimal formation was observed by HE staining, and expression of NF- kappa B p65 protein in injured arteries of two groups was detected by Western blot. VSMCs, was obtained by tissue adherent culture and divided into control group, IL-1 尾 group (IL-1 尾 treated), CGRP group (treated with IL-1 尾 CGRP) and CGRP8- 37 group (IL-1 尾 CGRP plus CGRP8-37) (n = 24), and IL-1 尾 treated), CGRP group (treated with IL-1 尾 CGRP) and CGRP8- 37 group (on the basis of IL-1 尾 CGRP) (n = 24). The expression of NF- 魏 B p65 protein and phenotypic marker protein in cells was detected by Western blot assay, and the proliferation ability of cells was detected by MTT assay. Results 1 the neointima of the control group increased significantly at 7 days after carotid artery injury, and was basically stable at 28 days after carotid artery injury. The neointimal area in CGRP group was significantly lower than that in control group on the 7th and 28th day [(2.09 卤0.26) vs (3.76 卤0.33); 14 d: (3.52 卤0.41) vs (6.38 卤0.61), P 0.05]; Compared with the control group, the expression level of NF- kappa B p65 protein in CGRP group was significantly lower than that in control group at 1 week (P0.05), but there was no difference in NF- kappa B p65 expression level between the two groups on the 28th day (P0.05). IL-1 尾 treatment significantly activated the expression of NF- kappa B p65 protein (P0.05), accompanied by a decrease in the expression of SM 伪-actin and an increase in OPN expression in VSMCs (P0.05). When treated with CGRP, the expression of NF- kappa B p65 protein induced by IL-1 尾 was significantly decreased (P0.05), and the expression of OPN, a marker of VSMCs synthesis, was decreased. The results of RT-PCR analysis were consistent. At the same time, CGRP significantly inhibited the proliferation of VSMCs induced by IL-1 尾. 3CGRP receptor blocker CGRP8-37 could block the effect of CGRP on IL-1 尾-induced NF- kappa B p65 protein expression and the regulation of CGRP on VSMC expression protein. Conclusion CGRP can inhibit the activation of NF- 魏 B, promote the expression of VSMCs differentiation marker gene, inhibit cell proliferation and decrease neointimal proliferation after arterial injury. This may have important application value in the prevention and treatment of atherosclerosis and restenosis after angioplasty.
【作者单位】：遵义医学院第一附属医院心血管内科;
【基金】：国家自然科学基金(30860100) 贵州省国际合作项目(黔科合外G字[2010]0732)~~
【分类号】：R563.8

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