Duan Lixiao, Lei Han, Zhang Yi, Wan Biao, Chang Jing, Feng Qingping, Huang Wei
Cardiovascular Laboratory, Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, PR China.
Cardiology. 2016;133(1):44-53. doi: 10.1159/000439123. Epub 2015 Oct 3.
The aim of this work was to investigate whether calcitonin gene-related peptide (CGRP) plays a protective role in cardiomyocytes against hypoxia-induced inflammation and apoptosis via an NO-mediated pathway.
H9c2 cardiac cells were exposed to hypoxia for 2 h to establish a model of myocardial hypoxic-ischemic injury. The cells were pretreated with either CGRP or nitric oxide synthase (NOS) inhibitor (L-NAME) before being exposed to hypoxia for 30 min. Cell viability was analyzed using a cell counter kit 8 (CCK-8). The levels of IL-6 and TNF-α were determined by the corresponding enzyme-linked immunosorbent assay. The expression levels of several apoptosis proteins (p53, caspase-3, cytochrome C) and NOS were detected by Western blot assays. An NO kit was used to evaluate the production of NO.
Pretreatment of H9c2 cardiac cells with CGRP for 30 min prior to exposure to hypoxia markedly improved cell viability (83.57 ± 3.21 vs. 62.83 ± 8.30%, p < 0.001); the same effect was observed following pretreatment with the NOS inhibitor L-NAME (89.34 ± 5.95 vs. 75.01 ± 5.61%, p < 0.01). Pretreatment with CGRP also significantly attenuated the inflammatory responses induced by hypoxia, as evidenced by decreases of the levels of both IL-6 (193.21 ± 13.54 vs. 293.38 ± 56.49%, p < 0.001) and TNF-α (207.71 ± 44.27 vs. 281.46 ± 64.88%, p < 0.001). Additionally, CGRP significantly decreased the hypoxia-induced overexpression of the apoptotic proteins (p53: 0.27 ± 0.10 vs. 0.87 ± 0.30, p < 0.001; caspase-3: 0.65 ± 0.15 vs. 0.98 ± 0.26, p < 0.001; cytochrome C: 1.51 ± 0.39 vs. 2.80 ± 0.69, p < 0.001) and enhanced the expression of both endothelial NOS (eNOS; 0.59 ± 0.24 vs. 0.37 ± 0.14, p < 0.05) and phosphorylated eNOS (0.60 ± 0.13 vs. 0.40 ± 0.07, p < 0.05). Furthermore, the application of both L-NAME and CGRP attenuated the hypoxia-induced expression of inducible NOS (iNOS; p < 0.05) and enhanced a hypoxia-mediated decrease in NO (p < 0.01). Interestingly, the expression levels of cell apoptosis (p < 0.05), iNOS and eNOS (p < 0.05) were decreased with L-NAME and CGRP cotreatment following 2 h of acute hypoxia, but the apoptotic factors (p < 0.05) were increased compared with only CGRP pretreatment.
CGRP protects cardiomyocytes from hypoxia-induced inflammation and apoptosis by modulating NO production.
本研究旨在探讨降钙素基因相关肽（CGRP）是否通过一氧化氮（NO）介导的途径对心肌细胞缺氧诱导的炎症和凋亡发挥保护作用。
将H9c2心肌细胞暴露于缺氧环境2小时，以建立心肌缺氧缺血损伤模型。在暴露于缺氧环境30分钟之前，用CGRP或一氧化氮合酶（NOS）抑制剂（L-NAME）对细胞进行预处理。使用细胞计数试剂盒8（CCK-8）分析细胞活力。通过相应的酶联免疫吸附测定法测定白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的水平。通过蛋白质印迹法检测几种凋亡蛋白（p53、半胱天冬酶-3、细胞色素C）和NOS的表达水平。使用NO试剂盒评估NO的产生。
在暴露于缺氧环境之前，用CGRP预处理H9c2心肌细胞30分钟可显著提高细胞活力（83.57±3.21对62.83±8.30%，p<0.001）；用NOS抑制剂L-NAME预处理后也观察到相同的效果（89.34±5.95对75.01±5.61%，p<0.01）。CGRP预处理还显著减轻了缺氧诱导的炎症反应，IL-6水平（193.21±13.54对293.38±56.49%，p<0.001）和TNF-α水平（207.71±44.27对281.46±64.88%，p<0.001）均降低证明了这一点。此外，CGRP显著降低了缺氧诱导的凋亡蛋白的过表达（p53：0.27±0.10对0.87±0.30，p<0.001；半胱天冬酶-3：0.65±0.15对0.98±0.26，p<0.001；细胞色素C：1.51±0.39对2.80±0.69，p<0.001），并增强了内皮型NOS（eNOS；0.59±0.24对0.37±0.14，p<0.05）和磷酸化eNOS（0.60±0.13对0.40±0.07，p<0.05）的表达。此外，L-NAME和CGRP的应用均减轻了缺氧诱导的诱导型NOS（iNOS）的表达（p<0.05），并增强了缺氧介导的NO降低（p<0.01）。有趣的是，在急性缺氧2小时后，L-NAME和CGRP联合处理可降低细胞凋亡（p<0.05）、iNOS和eNOS的表达水平（p<0.05），但与仅用CGRP预处理相比，凋亡因子（p<0.05）增加。
CGRP通过调节NO的产生保护心肌细胞免受缺氧诱导的炎症和凋亡。
