Antagonism of cortistatin against cyclosporine-induced apoptosis in rat myocardial cells and its effect on myocardial apoptosis gene expression

L. Geng, X.-T. Wang, J. Yu, Y.-L. Yang

Department of International Medical, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China. youbang0918@163.com

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• Pharmacology

OBJECTIVE: To investigate the role of cortistatin (CST) on cyclosporine A (CsA)-induced myocardial apoptosis in rats and determine its effect on the expressions of myocardial apoptosis genes.

MATERIALS AND METHODS: H9C2 cells were treated with different concentrations of CsA solution (0.04, 0.2, 1 and 5 μM) for 24, 48 and 72 h, respectively. The cell viability was detected via methyl thiazolyl tetrazolium (MTT) assay, and the appropriate dose and time were compared and determined. At the same time, CST in different concentrations (0.08, 0.04, 0.2, 1, 5 and 25 μM) was added into cell culture, and the appropriate dose was identified using MTT assay. The cellular morphology in each group was observed, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed for the detection of cell apoptosis. Moreover, in molecular mechanism research, the apoptosis-associated factors, factor associated suicide (Fas), Fas ligand (FasL) and B-cell lymphoma-2-associated X protein (Bax), were detected via quantitive Real-time polymerase chain reaction (qPCR). Finally, the levels of a protein related to myocardial apoptosis in rats were investigated via Western blotting.

RESULTS: The treatment with 1 μM CsA for 48 h caused significant apoptosis. The results of TUNEL staining showed the inhibitory role of CST on the myocardial apoptosis in rats induced by CsA. The detection of apoptosis factors via Real-time PCR revealed that after the induction of CsA, the expressions of Fas, FasL and Bax mRNA in cells were significantly higher than those in control group, but were significantly decreased after administration of CST. Western blotting showed that the protein expressions of Caspase 3 and Caspase 9 were remarkably elevated in cells after the use of CsA, but were significantly reduced after administration of CST (p < 0.01).

CONCLUSIONS: CST contributes to antagonistic function against the CsA-induced apoptosis of rat myocardial cells, and its effect is related to the down-regulation of expressions of apoptotic factors, Fas, FasL, Bax, Caspase 3, and Caspase 9.

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