内皮源性降钙素基因相关肽抗血管紧张素Ⅱ诱导的内皮细胞凋亡作用  

Role of endothelial cell-derived calcitonin gene-related peptide in angiotensin II-induced endothelial cell apoptosis

作　　者：张毅民 彭军[2] 李元建[2] 

机构地区：[1]长沙市第一医院药剂科,湖南长沙410005 [2]中南大学药学院药理学系,湖南长沙410078

出　　处：《中国现代医学杂志》2013年第24期25-32,共8页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:30873061);全国优秀博士专项基金(No:2007B7)

摘　　要：目的探讨内皮源性降钙素基因相关肽对血管紧张素II诱导的内皮细胞抗凋亡作用。方法培养HUVEC-12,分别加入不同剂量的AngII孵育内皮细胞,研究其量效和时效关系,并给予特异性AT1受体阻断剂氯沙坦研究其可能的机制。Real time-PCR检测内皮细胞中CGRP mRNA水平;放射免疫法检测内皮细胞CGRP的含量。在培养HUVEC-12给予AngII诱导细胞凋亡,外源性给予CGRP和辣椒素。Annexin V-FITC流式细胞检测细胞凋亡;比色法测定caspase-3活性;RT-PCR检测内皮细胞中CGRP、Bcl-2和Bax mRNA的表达。结果①AngII孵育内皮细胞可剂量和时间依赖性地降低α-和β-CGRP mRNA的表达水平和CGRP含量,此作用可被预先给予的氯沙坦阻断。②CGRP可剂量依赖性降低AngII诱导的内皮细胞凋亡,减弱AngII增加内皮细胞caspase-3活性,此作用能被预先给予的CGRP受体阻断剂CGRP8-37取消。③辣椒素可剂量依赖性上调内皮源性α-和β-CGRP mRNA的表达水平,此作用能被预先给予的VR1受体阻断剂capsazepine取消。④辣椒素可剂量依赖性降低AngII诱导的内皮细胞凋亡,减弱AngII增加内皮细胞caspase-3活性,此作用能被预先给予的capsazepine和CGRP8-37取消。⑤辣椒素可以上调抗凋亡分子Bcl-2 mRNA的表达,下调促凋亡分子Bax mRNA的表达,此作用能被预先给予的capsazepine和CGRP8-37取消。结论 AngⅡ能抑制内皮细胞合成与释放CGRP;用辣椒素促进内皮源性CGRP的表达能对抗AngⅡ诱导的内皮细胞凋亡。[ Objective ] To explore the role of endothelial cell - derived calcitonin gene-related peptide in an- giotensin-Ⅱ induced endothelial cell apoptosis. [Methods ] Endothelial cells were treated with AngⅡ to evaluate the dose-effect relationship or the time-effect relationship on CGRP expression. Next, the ceils were incubated with AngⅡ （10-5 M） for 24 h after the pretreatment with Losartan for 30 min to determine whether AT1 receptor was in- volved. The level of CGRP mRNA was detected by Real time-PCR and the protein level was measured by radioim- munoassay. To explore the effect of CGRP on the AngⅡ-induced apoptosis, endothelial cells were pretreated with CGRP or capsaicin before Ang Ⅱ treatment. Cell apoptosis was examined by FITC-Annexin V flow cytometry. Activ- ity of Caspase-3 was measured by colorimetry. The level of CGRP, Bcl-2 and Bax mRNA were detected by RT- PCR. [ Results ] AngⅡ significantly decreased α- and 15-CGRP mRNA expression and the content of CGRP in cul-tured endothelial cell in a dose-dependent manner, which was inhibited by pretreatment with Losartan. Pretreatment the cells with exogenous CGRP decreased AngⅡ-induced endothelial cell apoptosis accompanied by the decreased caspase-3 activity, which was attenuated in the presence of CGRP8-37, the antagonist of CGRP receptor. Treating the cells with capsaicin at concentration of 10^-8, 10^-7 or 10^-6 M for 24 h, the expression level of CGRP （α andβ ） mRNA increased in a dose-dependent manner, which was inhibited by capsazepine, the antagonist of VR1 receptor. Pre- treatment the cells with capsaicin decreased AngⅡ-induced endothelial cell apoptosis accompanied by the increased Bcl-2 mRNA expression, the decreased Bax mRNA expression and caspase-3 activity which was attenuated in the presence of capsazepine or CGRPs-37. [ Conclusions ] Ang Ⅱ is able to inhibit the synthesis and release of CGRP in the cultured HUVECs, which may contribute to Ang Ⅱ-induced endothelial cell apoptosis. Exogenous administration of CGRP or

关 键 词：内皮源性降钙素基因相关肽 人脐静脉内皮细胞 血管紧张素Ⅱ 凋亡 

分 类 号：R346[医药卫生—基础医学] R363