Wu Jianqun, Liu Song, Wang Zhao, Ma Shenghui, Meng Huan, Hu Jijie
Department of Spine Surgery, Huadu District People's Hospital, Guangzhou, Guangzhou, 510800 Guangdong Province China.
2Department of Orthopedics, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou City, 510150 Guangdong Province China.
Proteome Sci. 2018 Nov 14;16:18. doi: 10.1186/s12953-018-0146-4. eCollection 2018.
Calcitonin gene-related peptide (CGRP) contributes to bone formation by stimulating bone marrow stromal cell (BMSC) proliferation and differentiation. However, the proliferative and apoptotic effects of CGRP on bone marrow-derived endothelial progenitor cells (EPCs) have not been investigated.
We tested the effects of CGRP on EPC proliferation and apoptosis by Cell Counting Kit-8, flow cytometry, and studied the effects of CGRP on the expression of proliferation- and apoptosis-related markers in EPCs and the underlying mitogen-activated protein kinase (MAPK) signalling pathway by quantitative polymerase chain reaction and western blotting.
We detected EPC markers (CD34, CD133 and VEGFR-2) in 7-day cultures and found that CGRP (10-10 M) promoted the proliferation of cultured EPCs, with a peak increase of 30% at 10 M CGRP. CGRP also upregulated the expression of proliferation-associated genes, including cyclin D1 and cyclin E, and increased the percentages of G2/M-phase and S-phase cells after incubation 72 h. CGRP inhibited serum deprivation (SD)-induced apoptosis in EPCs after 24 and 48 h and downregulated the expression of apoptosis-related genes, including caspase-3, caspase-8, caspase-9 and Bax. Phosphorylated (p-)ERK1/2, p-p38 and p-JNK protein levels in EPCs treated with CGRP were significantly lower than those in untreated EPCs. Pre-treatment with the calcitonin receptor-like receptor (CRLR) antagonist CGRP8-37 or a MAPK pathway inhibitor (PD98059, SB203580 or SP600125) completely or partially reversed the pro-proliferative and anti-apoptotic effects and the reduced p-ERK1/2, p-p38 and p-JNK expression induced by CGRP.
Our results show that CGRP exerts pro-proliferative and anti-apoptotic effects on EPCs and may act by inhibiting MAPK pathways.
降钙素基因相关肽（CGRP）通过刺激骨髓基质细胞（BMSC）增殖和分化促进骨形成。然而，CGRP对骨髓来源的内皮祖细胞（EPCs）增殖和凋亡的影响尚未得到研究。
我们通过细胞计数试剂盒-8、流式细胞术检测CGRP对EPCs增殖和凋亡的影响，并通过定量聚合酶链反应和蛋白质印迹法研究CGRP对EPCs中增殖和凋亡相关标志物表达以及潜在的丝裂原活化蛋白激酶（MAPK）信号通路的影响。
我们在7天培养物中检测到EPCs标志物（CD34、CD133和VEGFR-2），发现CGRP（10-10 M）促进培养的EPCs增殖，在10 M CGRP时增殖峰值增加30%。CGRP还上调了包括细胞周期蛋白D1和细胞周期蛋白E在内的增殖相关基因的表达，并在孵育72小时后增加了G2/M期和S期细胞的百分比。CGRP在24小时和48小时后抑制血清剥夺（SD）诱导的EPCs凋亡，并下调包括半胱天冬酶-3、半胱天冬酶-8、半胱天冬酶-9和Bax在内的凋亡相关基因的表达。用CGRP处理的EPCs中磷酸化（p-）ERK1/2、p-p38和p-JNK蛋白水平显著低于未处理的EPCs。用降钙素受体样受体（CRLR）拮抗剂CGRP8-37或MAPK途径抑制剂（PD98059、SB203580或SP600125）预处理可完全或部分逆转CGRP诱导的促增殖和抗凋亡作用以及p-ERK1/2、p-p38和p-JNK表达的降低。
我们的结果表明，CGRP对EPCs具有促增殖和抗凋亡作用，可能通过抑制MAPK途径发挥作用。
