title: Calcitonin Gene-Related Peptide Improves Hypoxia-Induced Inflammation and Apoptosis via Nitric Oxide in H9c2 Cardiomyoblast Cells.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  CGRP protects cardiomyocytes from hypoxia-induced inflammation and apoptosis by modulating NO production.
authors.affiliation: Cardiovascular Laboratory, Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, PR China.
authors.forename: Lixiao
authors.initials: L
authors.lastname: Duan
authors.forename: Han
authors.initials: H
authors.lastname: Lei
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Biao
authors.initials: B
authors.lastname: Wan
authors.forename: Jing
authors.initials: J
authors.lastname: Chang
authors.forename: Qingping
authors.initials: Q
authors.lastname: Feng
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D015740:Calcitonin Gene-Related Peptide / Q000494:pharmacology*; D015687:Cell Hypoxia; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007249:Inflammation / Q000378:metabolism*; D015850:Interleukin-6 / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; C508600:IL6 protein, human; D015850:Interleukin-6; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D015740:Calcitonin Gene-Related Peptide