Calcitonin Gene-Related Peptide Improves Hypoxia-Induced Inflammation and Apoptosis via Nitric Oxide in H9c2 Cardiomyoblast Cells

Abstract

Objectives: 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 (<smlcap>L</smlcap>-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 <smlcap>L</smlcap>-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 <smlcap>L</smlcap>-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 <smlcap>L</smlcap>-NAME and CGRP cotreatment following 2 h of acute hypoxia, but the apoptotic factors (p < 0.05) were increased compared with only CGRP pretreatment. Conclusion: CGRP protects cardiomyocytes from hypoxia-induced inflammation and apoptosis by modulating NO production.