降钙素基因相关肽对人脐静脉内皮细胞增殖和迁移的作用及其机制

The effect of calcitonin gene-related peptide on proliferation and migration of human umibilical vein and its mechanisms

目的探讨降钙素基因相关肽(calcitonin gene-related peptide,CGRP)对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)增殖、迁移的作用及其机制。方法培养HUVECs,随机分为对照组(Control组)、CGRP组、VEGF抑制剂组(Bevacizumab组)、一氧化氮合成酶抑制剂组(L-NMMA组)。MTT法检测CGRP、贝伐珠单抗、L-NMMA对HUVECs增殖的影响;划痕实验测定HUVECs迁移情况;Western blot和细胞免疫荧光检测相关蛋白表达情况。结果CGRP与降钙素受体样受体(calcitonin receptor-like receptor,CRLR)结合能显著促进HUVECs增殖(t=3.434,P<0.05),抑制血管内皮生长因子(VEGF)或内皮型一氧化氮合酶(eNOS)能显著抑制CGRP诱导的HUVECs增殖,主要表现在HUVECs存活率(P<0.05)和内皮细胞标志物(P<0.05)的表达显著降低。划痕实验结果显示抑制VEGF或eNOS可显著抑制CGRP诱导的HUVECs迁移(P<0.05)。抑制VEGF或eNOS能显著降低与增殖密切相关的激酶Raf-1的表达(P<0.05),同时显著抑制其下游ERK(P<0.05)和JNK(P<0.05)信号通路激活。结论CGRP通过激活VEGF/eNOS-Raf-1-ERK/JNK信号通路诱导HUVECs增殖和迁移,抑制VEGF或eNOS可能是抗血管生成的潜在靶点。

Objective To investigate the effect of calcitonin gene-related peptide(CGRP)on proliferation and migration of human umbilical vein endothelial cells(HUVECs)and the underlying mechanisms.Methods HUVECs were cultured and randomly divided into control group,CGRP group,bevacizumab(VEGF inhibitor)group,and L-NMMA(nitric oxide synthase inhibitor)group.The MTT assay was employed to evaluate the effects of CGRP,bevacizumab and L-NMMA on the viability of HUVECs.The scratch assay was conducted to assess the migration of HUVECs and the expression levels of relevant proteins were detected by immunocytochemistry assay and Western blot.Results CGRP treatment markedly promoted the proliferation of HUVECs by binding to its receptor,calcitonin receptor-like receptor(CRLR)(t=3.434,P<0.05).When VEGF or eNOS was inhibited,the CGRP-induced endothelial cell proliferation was significantly suppressed,as indicated by decreased cell viabilities(P<0.05)and the expression of cell makers(P<0.05).In the CGRP stimulated HUVECs,inhibition of VEGF or eNOS could also suppress cell migration as detected by the scratch assay(P<0.05).Additionally,inhibition of VEGF or eNOS decreased the expression of the Raf-1,a kinase that is closely related to proliferation(P<0.05).It also reduced the activities of its downstream extracellular-signal-regulated kinase(ERK)(P<0.05)and c-Jun N-terminal kinase(JNK)(P<0.05)signaling pathways in HUVECs stimulated by CGRP.Conclusion CGRP may induce HUVECs proliferation and migration through activating the VEGF/eNOS-Raf-1-ERK/JNK signaling pathway,suggesting that inhibition of VEGF or eNOS may serve as a potential target for anti-angiogenic therapy.